Diabetologia (1990) 33 : All-A230

Diabetologia 9 Springer-Verlag 1990

26th Annual Meeting of the European Association for the Study of Diabetes Copenhagen, D e n m a r k , 10-14 September 1990

Abstracts

A12

Oral Presentations

OP 1 Hall A-1

Na/Li-Cou ntertransport 1

2

ERYTHROCYTE SODIUM-LITHIUM COUNTERTRANSPORT AND RISK OF NEPHROPATHY IN TYPE i (INSULIN-DEPENDENT) DIABETES. R.Mangili, G.Zerbini, F.Garbetta, D.Cusi, M.R.Pastore, E.Bognetti and G.Pozza. Istituto Scientifico San Raffaele, Milan, Italy. Elevated erythrocyte sodium-lithium oountertransport (Na/Li CT) activity - a marker of essential hypertension with a large genetic component - is found in Type i (insulin-dependent) diabetic patients with overt nephropathy. Whether this Na transport abnormality may help identifying patients at risk for nephropathy early in the course of diabetes is unknown. We measured erythrocyte Na/Li CT activity in two groups of normoalbuminuric Type i (insulin-dependent) diabetic patients with different duration of diabetes (DD), but comparable age at onset of diabetes and sex. Na/Li CT activity clustered at lower values in patients with DD=16-43 (mean 2 1 . 9 ) years (n=30; median: 0.287 mmol/i/hour) than in patients with DD=I-6 (mean 4.3) years (n=43; median: 0.386 mmol/i/hour; 2P<0.05). Glycated haemoglobin levels were similar in the two groups (8.7• vs 8.8• mean• 2P=NS) and did not correlate with Na/Li CT activity. A low risk of nephropathy (as suggested by normoalbuminuria in patients with long DD) is thus associated with lower Na/Li CT activity than that found in patients with short DD (hence at higher risk for nephropathy) independently of giycaemic control. We suggest that elevated Na/Li CT early in Type I (insulin-dependent) diabetes may associate with higher risk for later nephropathy, though this hypothesis awaits proseetive observations.

SODIUM-LITHIUM COUNTERTRANSPORT, INSULIN R E S I S T A N C E A N D H Y P E R L I P I D A E M I A IN D I A B E T I C S . JB L o p e s de Faria, SL Jones, FA M a c D o n a l d , SR F e r r e i r a , C Hill, J M e s s e n t , M M a t t o c k , and GC Viberti. Unit for M e t a b o l i c Medicine, Guy's H o s p i t a l , London, UK. In T y p e 1 ( i n s u l i n - d e p e n d e n t ) d i a b e t i c p a t i e n t s an overactivity of sodium-lithium c o u n t e r t r a n s p o r t (Na+/Li § CT) has b e e n a s s o c i a t e d w i t h the r i s k of n e p h r o p a t h y and h y p e r t e n s i o n , both insulin-resistance conditions. We have investigated the i n s u l i n sensitivity using a hyperinsulinaemic (=I00 ~U/ml) euglycaemic clamp, in Ii (gM:2F) IDDs w i t h h i g h Na*/Li § CT activity (mean 0.464, range 0.40-0.68 mmol/L RBC/h) - g r o u p 1 - a n d 12 (9M:3F) w i t h n o r m a l Na*/Li + CT (mean 0.237, r a n g e 0 . 1 2 - 0 . 3 1 3 m m o l / L RBC/h) , group 2. Both groups Were nonp r o t e i n u r i c and n o n - h y p e r t e n s i v e . T h e two g r o u p s w e r e s i m i l a r in age (35• vs 32• y), d u r a t i o n of d i a b e t e s (19• vs 18• y), BMI (26• vs 24• k g / m 2) and m e a n b l o o d p r e s s u r e (92• vs 94• mmHg). Albumin excretion rate (AER) ranged b e t w e e n 4.7 and 158 ( g e o m e t r i c m e a n 14.7) ~ g / m i n in g r o u p 1 and b e t w e e n 2.7 a n d 93 ( g e o m e t r i c mean 10.7) ~g/min in g r o u p 2. T h e r e w e r e 4 microalbuminuric patients (AER>30 ~g/min) in e a c h group. T r i g l y c e r i d e s (TG) and c h o l e s t e r o l (CHOL) w e r e h i g h e r in g r o u p 1 (TG 0.89 vs 0.52 mmol/L, p=0.028, CHOL 5.22 vs 4.62 mmol/L, p=0.069). Glucose disposal rate was s i g n i f i c a n t l y r e d u c e d in g r o u p 1 c o m p a r e d w i t h group 2 (7.68• vs 8.86• mg/kg/min, p=0.04). In c l i n i c a l l y non-proteinuric, nonh y p e r t e n s i v e IDDs i n c r e a s e d Na*/Li+ CT a c t i v i t y is associated with insulin resistance and elevated triglycerides. These abnormalities e i t h e r s i n g l y or in c o m b i n a t i o n m a y c o n t r i b u t e to v a s c u l a r d a m a g e in a s u b s e t of IDDs.

3 RED CELL S O D I U M - L I T H I U M C O U N T E R T R A N S P O R T A C T I V I T Y IN F A M I L I A L D I A B E T I C NEPHROPATHY. J.H.M. Berden, L.D. Elving, M.J.A. Bergman, J.F.M. W e t z e l s and E. de Nobel. D e p a r t m e n t s of General Internal M e d i c i n e and Nephrology, Univ e r s i t y H o s p i t a l Nijmegen, The Netherlands. An increased incidence of diabetic n e p h r o p a t h y (DNP) is found in diabetic sibs of patients w i t h DNP. An a s s o c i a t i o n of DNP w i t h familial essential h y p e r t e n s i o n has also b e e n suggested. We s t u d i e d red cell s o d i u m - l i t h i u m countert r a n s p o r t (NaLiCT), a m a r k e r for essential hypertension, in type 1 (insulin-dependent) diabetic p a t i e n t s w i t h and w i t h o u t DNP and in their diabetic sibs. DNP was d e f i n e d as a l b u m i n e x c r e t i o n rate >50 pg/min. D u r a t i o n of diabetes in p r o b a n d s and sibs had to be >15 yrs. Of the 6 p r o b a n d s w i t h DNP, 4 out of 6 sibs had DNP, w h e r e a s of the 8 probands w i t h o u t DNP only 1 out of 9 sibs had DNP (p=0.09). N a L i C T correlated s i g n i f i c a n t l y b e t w e e n probands and sibs (rs=0.63; p<0.02). Three groups of sib-pairs w e r e formed: c o n c o r d a n t for DNP (A;n=8), conc o r d a n t for a b s e n c e of DNP (B;n=15), d i s c o r d a n t for DNP (C;n=6). M e d i a n (range) d u r a t i o n of d i a b e t e s was resp 24(15-35), 26 (18-44) and 28(17-39) yrs (NS). M e d i a n (range) N a L i C T did not differ b e t w e e n A (265 (217-338)), B (305 (189-442)) and C (447 (310-649)) p m o l / l . c e l l s / hr. There was no s i g n i f i c a n t d i f f e r e n c e in N a L i C T b e t w e e n the patients w i t h and w i t h o u t DNP. In conclusion: there is a trend to familial c l u s t e r i n g of diabetic nephropathy, however, N a L i C T does not segregate w i t h familial diabetic nephropathy. We even did not find an increased N a L i C T in patients w i t h diabetic nephropathy.

RED CELL SUITABLE

SODIUM-LITHIUM AS A MARKER

COUNTERTRANSPORT

FOR DIABETIC

IS N O T NEPHROPATHY.

L.D. Elving, J.F.M. Wetzels, E. de Nobel and J.H.M. Berden. D e p a r t m e n t s of General Internal M e d i c i n e and Nephrology, U n i v e r s i t y H o s p i t a l Nijmegen, The Netherlands. To e v a l u a t e the value of red cell sodium-lithium c o u n t e r t r a n s p o r t (NaLiCT) as a m a r k e r for diabetic n e p h r o p a t h y (DNP), we m e a s u r e d N a L i C T in 37 type 1 (insulin-dependent) diabetic p a t i e n t s w i t h DNP (DNP+). For c o m p a r i s o n we s t u d i e d 19 diabetic patients w i t h o u t DNP (DNP-), 42 n o n - d i a b e t i c patients w i t h various renal diseases (RD) and 24 h e a l t h y controls (C). All DNP- patients had a diabetes d u r a t i o n >15 yrs and a l b u m i n e x c r e t i o n rate <20 pg/min. N a L i C T was m e a s u r e d in d u p l i c a t e after loading of red cells w i t h LiC03; intra-assay v a r i a t i o n 5%, i n t e r a s s a y v a r i a t i o n 13%. M e d i a n (range) N a L i C T in C was 244 (134-390) ~mol/l.cells/hr. S i g n i f i c a n t l y e l e v a t e d levels of N a L i C T were found in all three p a t i e n t g r o u p s : D N P + 329 (162676) (p<0.02), DNP- 321 (189-627) (p<0.01) and RD 300 (142-655) (p<0.05). In C N a L i C T did not e x c e e d 390 pmo!/l.cells/hr. Values above this level w e r e found in 22.2% of DNP-, 29.7% Of DNP + and 21.4% of R D - p a t i e n t s (p=NS). In conclusion: in patients with diabetic n e p h r o p a t h y N a L i C T was not increased compared to patients w i t h o u t diabetic n e p h r o p a t h y or n o n - d i a b e t i c patients w i t h renal disease.

A13

5

6

ERYTHROCYTE SODIUM-LITHIUM COUNTERTRANSPORT ACTIVITY IS ELEVATED EARLY IN DIABETIC NEPHROPATHY. G.Zerbini, R.Mangili, F.Garbetta, D.Cusi, C.Barlassina, G.Bianchi and G.Pozza. Istituto Scientifico San Raffaele, Milan, Italy. The activity of erythrocyte sodium-lithium countertransport (Na/Li CT) is elevated in essential hypertension and in Type 1 (insulin-dependent) diabetic patients with clinical nephropathy, and is currently thought to be predominantly determined by genetic rather than environmental factors. We investigated whether elevated erythrocyte Na/Li CT activity may also associate with microalbuminuria - an early stage of diabetic kidney disease predicting clinical nephropathy. Na/Li CT activity was thus measured in erythrocytes from 16 patients (9 M, 7 F) with Type 1 (insulin-dependent) diabetes mellitus and urinary albumin excretion rate (AER) of 20-200 pg/min, and in 16 patients of similar age, sex and duration of diabetes, but normal AER. Na/Li CT activity was higher in the former group (median: 0.492 vs 0.345 mmol/I/hour; 2P=0.022). Glycated haemoglobin levels were similar in the two groups (9.2• vs 9.4 2.1%, mean• 2P=0.8), as well as intracellular sodium concentrations (7.0• vs 7.2• mmol/l). We suggest that elevated erythrocyte Na/Li CT activity may associate with early nephropathy in Type 1 (insulin-dependent) diabetic patients independently of glycaemic control, and might thus prospectively associate with overt nephropathy and renal failure. Whether elevated Na/Li CT activity may represent an independent risk factor for diabetic renal disease, remains to be established in longitudinal studies.

High sodium-lithium cotmtsrteassport activity in red blood cells (Na+/Li* CT)is associated with insulin resistance and ~ a c and renal hypertrophy in insulin dependent diabetes before the crset of nephropathy. Fiorettm P. ,Trevisan R. ,Doria A. ,Avogaro A. ,Se~plicini A., Dcc~adon V.,Abbruzzese E.,Crepaldi G.,Viberti G.C.,Nosadini R.Italy, Londcn, U.K. The rats of Na+/Li+ CT is on average elevated in insulin-dependent diabetic patients with abnormal alb~nin excr~tien rate. Hypertension, cardiac eo,plications and insulin r e s i s ~ are also frequently associated with microalbt[ainuria in diabetes. Little information is avsilable on the relatien afnon~t Na+/Li+ CT, hypertension, insulin resistance and renal and cardiac f~etion and morphology in diabetes before the onset of nephropathy. Two groups of hypertsnsive diabetic patients with Na+/LI+CT below (HID:II) and above (H2D:I3) the upper limit of normal subjects (0.41 mmal/l RBC/nr) were compared with a group of normotensive diabetic patients (Na+/Li+CT: 0.2_8i0.04)(CD). HID and H2D patients had similar age, se~ disthibutien, body mass index, dur~tien of hypertension (l.Z~_.2 vs 1.5+_0.3 yrs) and of diabetes (8._6i0.8 vs 8.1_+0.6 yrs) and actual levels of tmtreated diastolic blood pressmme (99&_i vs 101_+2 nnZdg). HZD c o ~ d to HAD patients showed a higher frequency of family history of hypertension (93% vs 50% p<0.Ol) elevated $1omerular filtration rate (130+_4 vs 122• ml/min/l.TS m2 p< 0.01) and albtmfin excretion rats (25!3 vs 9• p~/min p
(3.88+_0.27 m~I{g/rrin) than in HID (5.7~0.3 p<0.01). We conclude that an elevated Na+/Li~ CT identifies a subset of hypertensive diabetic patients with insulin resistance, cardiac and renal hypertrophy, hyperfiltration, microalbtmtinaria and positive family history for hypertensien. These patients ~my he particularly susceptible to develop diabetic ccf~olications.

OP 2 Hall A-2 Glucose Transport in Diabetics I 7

8

TRANSRACIAL STUDY OF INSULIN SENSITIVE GLUCOSE TRANSPORTER IN TYPE 2 (NON-INSULIN DEPENDENT) DIABETES MELLITUS.

POPULATION (NIDDM): GENE

J.U.Weaver, V. Mohan*, P. G.Kopelman, G.A. Hitman and M. Viswanathan. Medical Unit, The London Hospital, London,U.K. *Diabetes Research Centre, Madras, India.

Despite the strong genetic predisposition the inherited defects responsible for insulin resistance in Type 2 (non-insulin-dependent) diabetes mellitus remain unclear. Insulin facilitative glucose uptake is mediated by a family of glucose transporters with a tissue specific pattern of expression. Insulin sensitive glucose transporter (Glut 4) is expressed in the muscle and adipose tissue and therefore a likely candidate gene influencing post-receptor insulin resistance. We investigated whether an association exists between Kpn I polymorphism of Glut 4 with Type 2 (non-insulindependent) diabetes mellitus in 198 British Caucasoids (127 controls, 71 diabetic) and 93 South Indians (45 controls, 48 diabetics), clinically matched. DNA of studied subjects were extracted from the peripheral lymphocytes, digested using Kpn I and studied by Southern blot hybridisation using a [32p labelled Glut 4 probe. Two alleles were identified, 5.8 (G1) and 6.6 (G2) kb. The frequencies of genotypes G1G2, G1G1 and G2G2 in British Caucasoids were: controls 39%, 5 1 % , 10% and diabetic 48%, 49%, 3% (NS); Indian controls 49%, 27%, 24% and diabetics 67%, 21%, 12% (NS). We conclude that neither of these ethnic groups show an association between Type 2 (non-insulindependent) diabetes mellitus and the Kpn I polymorphism of Glut 4 gene.

GENETICS A STUDY OF

OF NON-INSULIN-DEPENDENT DIABETES THE GLUT1 (HepG2) GLUCOSAE TRANSPOETER

MG Baroni, P Pozzilli*, RS Oelbaum, SR Li, V Fiore*, JC Chamberlain, J Alcolado and DJ Galton. Department of Diabetes and Lipids, St Bartholomew's Hospital, London ECIA 7BE, UK, and *Cattedra di Endocrinologia (I), II Clinica Medica, Universita' di

Roma, I t a l y Genetic factors play a major role in the etiology of noninsulin-dependent diabetes mellitus (NIDDM), however, the inherited metabolic defects are still unknown. A primary defect may be present within the glucose transport system. To determine the possible contribution of the GLUT1 (HepG2) glucose transporter gene to the inheritance of NIDDM, two RFLP's and the related haplotypes at this locus were studied in 189 Italian diabetic and normal subjects. Genotype frequencies for the Xba-I and Stu-I RFLP's were significantly different between patients and controls (Xba-l: p <0.01; Stu-l: p <0.05). There was a significant difference for the Xl allele frequency between NIDDr~ patients and controls (Xl = 0.55 NIDD?[, X1 = 0.36 controls; p <0.025), whereas no difference was found for the Stu-I RFLP. Analysis of the haplotype frequencies revealed a significant difference between diabetics and controls (p <0.025). By comparing single haplotypes, a significant association was found between the XISI haplotype and diabetes (p <0.05), with a significant difference between the two groups for the X2S2 haplotype (p <0.005). These findings suggest this locus may contribute to the inherited susceptibility to the disease in this population.

A14

9

10

E X P R E S S I O N OF I N S U L I N R E S P O N S I V E G L U C O S E T R A N S P O R T E R S IN S K E L E T A L M U S C L E F R O M P A T I E N T S W I T H NON-INSULIN-DEPENDENT D I A B E T E S M E L L I T U S (NIDDM). O. P e d e r s e n , J. F. Bak, P. H. A n d e r s e n , S. L u n d , E. M o l l e r , J. F l i e r a n d B. K a h n . Steno Memorial and Hvidore Hospital, Copenhagen, Medical Department III, Aarhus Amtssygehus, Aarhus and Diabetes Unit, Beth Israel Hospital~ Harvard Medical School, Boston. Since a central feature of NIDDM is an i m p a i r e d a b i l i t y of i n s u l i n to e n h a n c e g l u c o s e disposal in s k e l e t a l m u s c l e w e h a v e e x a m i n e d the h y p o t h e s i s t h a t r e d u c e d e x p r e s s i o n of G l u t 4 is a characteristic finding in skeletal muscle of subjects with NIDDM. Biopsies of v a s t u s l a t e r a l i s m u s c l e w e r e o b t a i n e d f r o m 17 p a t i e n t s w i t h N I D D M a n d i0 l e a n a n d 9 o b e s e non-diabetics. Among the d i a b e t i c s u b j e c t s 7 were newly diagnosed and untreated. Compared with age-and body weight matched healthy control subjects there was no significant alteration in the level of Glut 4 mRNA d e m o n s t r a t e d by N o r t h e r n a n d slot b l o t or G l u t 4 protein determined by immunoblotting of muscle membranes. Glut 1 mRNA and protein levels were a l s o not s i g n i f i c a n t l y different between diabetics a n d m a t c h e d c o n t r o l s . Thus, unlike streptozotocin diabetes in rodents, t h e r e is no e v i d e n c e t h a t i m p a i r e d e x p r e s s i o n of G l u t i or 4 is r e s p o n s i b l e for i n s u l i n r e s i s t a n t g l u c o s e t r a n s p o r t in s k e l e t a l m u s c l e of NIDDM patients. We conclude that the impaired in v i v o insulin stimu]ated glucose u p t a k e in m u s c l e f r o m p a t i e n t s w i t h N I D D M is l i k e l y to be c a u s e d by d e f e c t s in t h e p a t h w a y s t h r o u g h w h i c h i n s u l i n e n h a n c e s t r a n s l o c a t i o n of g l u c o s e t r a n s p o r t e r s to the p l a s m a m e m b r a n e or by impaired intrinsic activation of g l u c o s e transporters.

EXPRESSION OF INSULIN REGULATABLE GLUCOSE TRANSPORTERS IN SKELETAL MUSCLE IS NOT AFFECTED BY TYPE II DIABETES.

Type II diabetes is characterized by a decreased insulin sensitive glucose uptake, predominantely in skeletal muscle. Since this decreased glucose uptake could be due to a reduced number of insulin regulatable glucose transporters (IRGT), we have developed a method for quantification of IRGT's in small tissue samples, readily obtainable by needle biopsy. Fifty mg of muscle was obtained by needle technique from the thigh of fasting Type II diabetics (N=II) and controls (N=I2). After homogenization, a vesicular fraction enriched in membrane proteins was isolated by a centrifugation procedure. IRGT was identified by Western Blotting technique using a specific polyclonal antibody. The antibody was produced by immunization of rabbits using a synthetic peptide (C13) identical with the 13 Cterminal amino acid residues of IRGT, and the resulting serum was affinity purified on C13 coupled to agarose. The amount of IRGT on blots was quantitated and normalized using a standard preparation. The content of IRGT in skeletal muscle was identical in Type II diabetics and controls (0.205• vs 0.208• arbitrary units, mean• On this basis we conclude that the decreased insulin responsiveness in muscle of Type II diabetics is not reflected in muscular IRGT content in the fasting state.

11

12

POLYMORPHISMS OF GLUCOSE TRANSPORTER GENES IN TYPE 2

GLUCOSE TRANSPORTER GENES AND TYPE INSULIN-DEPENDENT) DIABETES MELLITUS

DIABETES

Oelbaum RS, Li SR, Baroni MG, Stocks J, Alcolado JC and Galton DJ Diabetes and Lipid Research Pepartment, St Bartholomew's ~ospital, London ECIA 7BE, UK Both insulin resistance and defective insulin secretion have each been proposed for the primary defect in type 2 diabetes. Two possible candidate genes are the insulinsensitive glucose transporter (GLUT4) present in muscle and adipocytes, and the pancreatic B-cell glucose transporter (GLUT2). Restriction fragment length polymorphism (RF~P) studies at these two loci may help distinguish inherited from secondary defects. DNA was extracted f r o m 64 unrelated British Caucasian type 2 diabetics and 66 matched controls and digested with Kpn-i and Hind-III. The resulting fragments were separated by agarose gel e!ectrephoresis and blotted onto Hybond-N filters. These were hybridised with radiolabelled cDNA probes and the bands visualised by autoradiography. A Kpn-I GLUT4 polymorphism with 6.5 Kb (KI) and 5.8 Kb (K2) fragments and a Hind-III GLUT2 polymorphism with 4.2 Kb (HI) and 4 . 1 K b (H2) fragments were identified. There was no significant difference in allele frequencies between the diabetic (KI = 0.28, Hl = 0.12).and control (El = 0.32 Hi = 0.i0) groups. None of these four alleles appear to associate with the diabetic phenotype in this population. However there was a significant difference in the GLUT4 K1 allele frequency between Caucasian controls and 32 Japanese controls (KI = 0.66, p <0.001), suggesting different evolutionary histories of this R~LP marker between these racial groups.

Aa. Handberg, A. Vaag. P. Damsbo, H. Beck-Nielsen & J. Vinten. Inst. of Med. Physiol. B, Panum Inst.,Copenhagen; Hvid~re Hospital, Klampenborg; Dept. of Med. Endocrinol.,Univer. Hosp. of 0dense, Denmark.

A. Calderara, A.E. Pontiroli, Capra, G. Pozza, P.A. B o n i n i , and M. F e r r a r i . Istituto Scientifico San Raffaele ta' d i M i l a n o , M i l a n o , I t a l y .

2

(NON-

C. M a g n a n i , F. R. Taramelli

and Universi-

A n a s s o c i a t i o n b e t w e e n a l l e l e 1 of t h e glucose t r a n s p o r t e r g e n e (GLUT) a n d t y p e 2 d i a b e t e s h a s been claimed. We evaluated the GLUT probe and the m o r e s p e c i f i c a d u l t skeletal muscle glucose t r a n s p o r t e r g e n e (GLUT4, k i n d l y provided by G. Bell) in 68 p t s w i t h t y p e 2 d i a b e t e s [31 with Relative Body weight (RBW) 1 2 0 : l0 o n insulin, 9 on OHA] and i n 66 non diabetic c o n t r o l s m a t c h e d f o r age, sex and RBW, with no family history of diabetes, normal blood glucose levels throughout the day and normal HbAlc. All subjects were studied with the following probe/enzyme associations: GLUT/XbaI, GLUT4/KpnI. Statistical analysis was performed by x ~ test on alleles and genotypes. The study showed no significant differences between controls and diabetic patients for either G L U T or G L U T 4 p r o b e s ; the allele 1 of GLUT was not significantly more frequent in obese patients with type 2 diabetes than among obese controls. These data contradict an association of GLUT and GLUT4 with type 2 diabetes and suggest a n a s s o c i a t i o n of G L U T allele 1 with obese type 2 diabetes.

A15

OP 3 Hall A-3 Peripheral Neuropathy 13

14

DO NON-INVASIVE NERVE FUNCTION TESTS REFLECT MORPHOLOGICAL ABNORMALITIES IN DIABETIC NEUROPATHY ?

Somatosensory evoked potentials after tibial nerve stimulation in newly diagnosed Type 1 insulin-dependent) diabetic patients

RA Malik, A Veves, DJS Fernando, EA Masson, W Schady, AK Shsrma, RJ Lye and AIM Boulton Manchester Royal Infirmary and University of Aberdeen U.K.

D. Ziegler, H. Mtihlen, and F.A. Gries, Diabetes Research Institute, University of Dtisseldorf, F.R.Germany

There is currently no consensus as to which neurophysiological tests best reflect morphological abnormalities in peripheral nerve. In an early intervention study we compared morphological findings in sural nerve biopsies from 15 diabetic patients (mean age 47yrs) with control biopsies and related these changes to electrophysiological measurements including motor and sensory nerve studies and quantitative sensory tests for thermal and vibration perception. Myelinated fibre density (MFD)( mean t SEM) was reduced compared to control (4042 • 579 mm -:2 vs 6561 • 482 : p< 0.01), as was mean capillary density (53.3 • ~7 vs 67.9 • 5.6 p< 0.01). Endoneurial capillaries demonstrated widespread abnormalities including basement membrane thickening, endothelial cell hypertrophy and hyperplasia. Strong correlations existed between MFD and sural conduction velocity (NCV) (r = 0.69, p < 0.005) and amplitude (r = 0.66, p < 0.01) and peroneal NCV (r = 0.75, p < 0.0001), but no correlation was seen between MFD and any quantitative sensory test. MFD correlated with capillary density (r = 0.66, p < 0.01). These data have important implications for the selection of patients for clinical trials in diabetic neuropathy and suggest that i) conventional electrophysiology best reflects structural change and 2) widespread sural nerve morphological and microvascular changes may be present in patients with clinically early neuropathy.

Previous studies have demonstrated central nervous conduction deficits in diabetic patients with or without peripheral neuropathy, but the onset of these abnormalities is not known. We measured somatosensory evoked potentials after tibial nerve stimulation in 20 (12 male/8 female) newly diagnosed asymptomatic Type 1 (insulin-dependent) diabetic patients (mean -+ SEM: age: 29.0 • 1.6 years, height: 175 -+ 2 cm, HbA1 11.6 + 0.4 %) after correction of initial hyperglycaemia and ketonuria (duration of insulin treatment: 14.6 • 2.3 days; mean blood glucose 6.4 • 0.3 mmol/1) and in 23 control subjects (age: 29.6 • 0.9 years, height: 175 • 3 cm). The patients showed significantly prolonged latencies of the components N8 (popliteal fossa): 10.0 • 0.3 vs 9.3 • 0.2 ms; p<0.05; N22 (lumbal): 24.1 • 0.4 vs 23.4 • 0.3 ms; p<0.05, and N50 (cortical): 51.9 -+ 0.7 vs 49.9 -+ 0.7 ms; p < 0.01, as well as reduced amplitude of the first cortical positive peak N33/P40; 2.2 • 0.2 vs 2.9 • 0.3 V; p<0.05 as compared with the controls. No significant differences between the groups were observed for the cervical N30 and cortical N33, P40, P60, and N75 latencies. We conclude that dysfunction of the somatosensory afferent pathways occurs shortly after diagnosis of diabetes at lumbal and cortical levels, indicating an early onset of subclinical involvement of the central nervous system in Type 1 (insulin-dependent) diabetic patients.

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REPRODUCIBILITY OF THE PARAMETERS OF NERVE FUNCTION INVESTIGATIONS IN DIABETICS. J . R Attali, P. V a l e n s i and t h e F r e n c h Group f o r R e s e a r c h and S t u d y o f D i a b e t i c N e u r o p a t h y . F r a n c e . T h i s s t u d y was p e r f o r m e d d u r i n g a m u l t i c e n t e r t r i a l o f an aldose-reductase inhibitor (Statil) in 132 diabetic patients with moderate peripheral polyneuropathy assessed by m e d i a n m a l l e o l u s v i b r a m e t r y and by c o n d u c t i o n v e l o c i t y of peroneal nerve. The aim was to evaluate the reproducibility of nerve function parameters after a onemonth p l a c e b o t r e a t m e n t : q u a n t i t a t i v e visual scales of symptoms ( a c h e s , numbness and p a r e s t h e s i a ) , quantitative sensory assessment (vibration perception thresholds in m e d i a n m a l l e o l u s and b i g t o e , f o o t t h e r m a l p e r c e p t i o n threshold to hot and cold), electrophysiological investigations on the dominant side (conduction velocities and p o t e n t i a l a m p l i t u d e s o f s e n s o r y and m o t o r m e d i a n n e r v e , s u r a l and p e r o n e a l n e I ~ e s , a m p l i t u d e s o f F w a v e s o f m o t o r m e d i a n a n d p e r o n e a l n e r v e s ) and c a r d i a c autonomic tests (Yalsalva, deep-breathing, lying-tostanding). R e p r o d u c i b i l i t y was p o o r f o r ~#mptoms, t b e r m al sensitivity and potential amplitudes. It was satisfactory (total variation coefficient ~505) for all t h e o t h e r p a r a m e t e r s and e v e n v e r y good ( t o t a l v a r i a t i o n coefficient ~ 26~, within-subject variation factors corresponding to ~56~ of total variance) for velocities o f s e n s o r y and m o t o r m e d i a n and p e r o n e a l n e r v e s , t h e amplitudes of F waves and the 3 ~atonomic tests. This study shows which parameters are the most reproducible and thus appropriate for the assessment of diabetic neurol~thy and for therapeutic trials.

DIFFERENTIAL CHANGES IN A GENERAL NEURONAL MARKER AND NEUROPEPTIDES IN DIABETIC SKIN. DM LEVY, D SPRINGALL, G TERENGHI, G REYNOLDS, RR ABRAHAM and JM POLAK. Diabetic Neuropathy Research Group, Central Middlesex Hospital, London NW10 7NS, and Department of Histochemistry, Hammersmith Hospital, London W12 0NN, UK. We have previously demonstrated depletion of general neuronal marker PGPg.5 and ueuropeptides in cutaneous diabetic nerves. We have investigated these changes further in standardised mid-calf biopsies in 3 age-matched groups, mean 34y: 15 normals, 7 non-neuropathic diabetic patients (normal thermal thresholds and cardiovascular vagal tests), and 9 with neuropathy (abnormal thresholds and at least one abnormal autonomic test). All but two diabetic patients were insulin-dependent (type 1). Number and intensity of immuuofluorescent nerves were scored semi-quantitatively by two independent blinded observers. Epidermal PGP-immunoreaetive nerves were decreased in non-neuropathic subjects (p = 0.03), but nerves immunoreactive for vasoactive intestinal polypeptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP) were unchanged. Three neuropathic patients had nerves indistinguishable in number and intensity from normals, but the remainder had significantly decreased epidermal PGP- and CGRP-immunoreactivity (both p=0.03); VIP-immunoreactivity, but not SP or CGRP, was decreased around sweat-glands (number and intensity both p = 0.05). Epidermal nerves are depleted in diabetes, even in patients with normal neurological function. In established neuropathy there are inconsistent and variable regional changes in neuropeptide immunoreaetivity, but some patients have more nerves than expected. This may represent abnormal nerve proliferation.

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PATHOPHYSIOLOGICAL DISCRIMINATION BETWEEN PAINFUL AND PAINLESS DIABETIC NEUROPATHY. R.J. Young, A. White and C. Tsigos, Dept. of Medicine, University of Manchester, Manchester, U.K. Are the clinical syndromes of diabetic pelyneuropathy particularly painful and painless neuropathy - pathophysiologically distinct? We compared 17 diabetic patients with painful neuropathy to 15 with painless neuropathy (neuretrophic foot ulceration) and 19 without clinical neuropathy. In addition to conventional clinical and physiological criteria, we used plasma sympathetic activity, as measured by circulating (supine) nor-adrenaline (NA), to differentiate between them. Groups were matched for age, BMI, duration of diabetes and glycaemia. Neuropathy symptom and deficit scores, sensory thresholds, motor and sensory electrophysiology and cardiovascular autonomic function were grossly abnormal in both neuropathic groups compared to patients without neuropathy (p<0.001), but worse in painless neuropathy (p
EARLY IMPAIRMENT OF CENTRAL NERVOUS SYSTEM. A STUDY WITH MAGNETIC STIMULATION AND SOMATOSENSORY AVOKED POTENTIALS. G.Bax, S.Lelli, P.Negrin, V.Majellaro, L.Braghefto, A. Cospite, U.Grandis and D.Fedele.-Internal Medicine Dept. Padova University. Sensory and motor pathway damage at the cortical level are successfully studied today by recording both somatosensory evoked potentials (SEPs) and motor evoked potentials (MEPs). We examined 15 type i diabetic patients, mean age 30 yrs, mean disease duration 3 yrs, under good metabolic control (HbAlc 6.22+1.3%; fruetosamine 2.57~0.88/umol/i; plasma glucose evaluated before the tests 140+_30 mg/dl). Exclusion criteria were renal (microalbuminuria 10_+3 mg/dl), retinal or vascular complications and symptomatic neuropathy.lO healthy subjects, mean age 25 yrs, were studied as controls. Patient testing included: i) cardiovascular tests; 2) vibratory perception threshold (VPT) with biothesiometer; 3) motor conduction velocity (MCV) at the external sciatic and ulnar nerves and sensor conduction velocity (SCV) at the median and sural nerves; 4) SEPs and MEPs following magnetic stimulation. Our analyses resulted as follows: a) both the cardiovascular tests and VPT gave normal values; b) 50% of the patients showed distal symmetric neuropathy (p
OP 4 Hall A-15 Epidemiology I 19

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AN EPIDEMIOLOGIC EVALUATION OF HOST-ENVIRONMENTAL INTERACTIONS IN FAMILIAL TYPE 1 DIABETES RISK. J. S. Dorman, R. Lipton, J. Yoon, M. Trucco, J. Gavard and R. LaPorte, University of Pittsburgh, Pittsburgh, PA, USA and University of Calgary, Alberta, Calgary, Canada.

RISK OF DEVELOPING DIABETES MELLITUS BEFORE 35 YEARS OF AGE - INDICATIONS OF CLIMATOLOGICAL DETERMINANTS FOR AGE AT ONSET.

The genetic and environmental determinants of familial Type 1 diabetes risk can be investigated using population-based epidemiologic methods. To illustrate this approach, 31 Type I diabetic cases (excluding probands) and 99 non-diabetic controls from multiplex families identified from the Type 1 diabetes registries in Allegheny County, PA (age-adjusted familial incidence = 19O/1OO,O00/yr) were studied. Susceptibility was defined by homozygosity for an amino acid other than aspartate in position 57 of the HLA-DQ beta chain (nonAsp). Environmental exposure was characterized by CMV genome positivity. Among those susceptible, Ii of 27 cases and 5 of 48 controls were CMV+. Among those nonsusceptible, 2 of 4 cases and 9 of 51 controls were CMV+. Relative to Asp/CMV-, odds ratios for non-Asp/ CMV+, non-Asp/CMV- and Asp/CMV+ were 46, 8 and 5, respectively. Annual Type 1 diabetes incidence rates per 10O,O0O/yr were calculated (non-Asp/CMV+ and CMV-: 1349 and 228; Asp/CMV§ and CMV-: 137 and 29), revealing a multiplicative effect of CMV exposure among susceptible and non-susceptible individuals. This model is applicable for family and case-control studies and permits an evaluation of host and environmental interactions to Type 1 diabetes risk.

O.L. Nystr6m (1), G. Dahlquist (2), J. (Dstman (3), S. Wall (1), H, Arnqvist (4), G. Blohm@ (5), F. Lithner (6), B. Littorin (7). B. Scherst@n (7) and L. Wibell (8) From the dept of Epidemiology and Health Care Research, univ of Ume& (1), dept of Pediatrics, Sachs' Children's Hospital (2) and dept of Internal Medicine, Huddinge Hospital, Stockholm (3), dept of Internal Medicine, Regional Hospital, Link6ping (4), Sahlgrenska Hospital (5), Regional Hospital Ume~ (6), Akademiska Hospital (8) and dept of Community Health Sciences, University of Lund (7) Sweden. This study analyses data from two nationwide prospective diabetes registries now covering about 4000 cases from 19 million person years of follow-up in the age groups 0-34 years. The incidence rate showed a maximum for both boys and girls in early puberty. After puberta[ years a sharp increase in male to female incidence ratio in Type 1 (insulin-dependent) diabetes was notable at 10-14 years being 0.94 and at 20-24 years 2,08. The risk to develop Type 1 diabetes per 100 000 individuals before 15 years was 386 for boys and 391 for girls and by 35 years 701 for men and 562 for women. After 15 years of age a small but increasing number of cases were classified as Type 2 (noninsulin-dependent) diabetes (cumulative incidence (CI) by 35 years 63 for males and 50 for females) or secondary diabetes (CI by 35 years 31 for males and 35 for females). A Cox regression model was used to analyse the effects on time at onset for the cases of sex, populations density and climatological factors eg north-south living and season at onset. Thus the effect of sex was confirmed (p=0.000). A significant effect (p=0.004) of season was shown when classifying the four seasons according to a tour graded scale related to mean tempera[ure. When dividing Sweden into 11 regions according to north-south gradient a significant effect (p=0.038) was also found, However, no effect of population density or living in coastal-interior area was found. It is concluded the time at onset seems to be related to puberty but also to sex as well as to climatological factors as indicated by both the pattern of geographical and seasonal variations.

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I N C I D E N C E O F T Y P E 1 D I A B E T E S IN C H I L D H O O D IN ESTONIA AND FINLAND DURING 1980-88

GEOGRAPHICAL DISTRIBUTION OF CHILDHOOD TYPE 1 (INSULINDEPENDENT) DIABETES IN EUROPE: THE EURODIA8 SUBAREA A STUDY

T.Podar, J.Tuomilehto, A.Reunanen, I.Kalits, E . T u o m i l e h t o - W o l f and R.Lounamaa. N a t i o n a l P u b l i c H e a l t h Institute, E l i m @ e n k a t u 25A 00510 Helsinki Finland F i n l a n d has the h i g h e s t i n c i d e n c e of Type 1 diabetes. E s t o n i a n s are g e n e t i c a l l y and l i n g u i s t i c a l l y r e l a t e d to the ]Finns. The i n c i d e n c e in c h i l d r e n aged 0-14 y e a r s r e s i d e n t in E s t o n i a or F i n l a n d at the time of d i a g n o s i s d u r i n g 1980-88 w e r e i n c l u d e d in the c o m p a r a t i v e analysis. The c a s e a s c e r t a i n m e n t a p p r o a c h e d 100% in b o t h c o u n t r i e s . T h e r e w e r e 313 n e w l y d i a g n o s e d c h i l d r e n w i t h Type 1 d i a b e t e s (166 boys, 147 girls) in E s t o n i a and 2803 (1530 boys, 1273 girls) in Finland. The age s t a n d a r d i z e d a v e r a g e y e a r l y i n c i d e n c e of Type 1 d i a b e t e s in boys was 11.3/100 000 (95% CI 10.3-12.3) and 35.1 (33.4-36.9), and in girls I0.I (9.2-11.1) and 30.4 (28.8-32.1) in E s t o n i a and Finland, r e s p e c t i v e l y . In F i n l a n d the i n c i d e n c e i n c r e a s e d by 23% in boys and by 18% in g i r l s from 1980-82 to 1986-88, but in E s t o n i a it r e m a i n e d unchanged. T h e r e was a p e a k in boys a r o u n d the age 3-4 y e a r s and a s e c o n d one a r o u n d the age 12-13 y e a r s in b o t h c o u n t r i e s ; in girls the i n c i d e n c e r o s e e v e n l y up to the age 5-6 y e a r s to a level w h e r e it r e m a i n e d also for o l d e r age groups. This m a r k e d d i f f e r e n c e in the i n c i d e n c e of T y p e 1 d i a b e t e s b e t w e e n these two c o u n t r i e s w h i c h are g e o g r a p h i c a l l y and e t h n i c a l l y c l o s e r e m a i n s so far u n e x p l a i n e d .

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EURODIA8 is an EEC supported research programme addressing the epidemiological, public health and preventive aspects of diabetes in Europe. Subarea A of EURODIAB concerns a deseriptlon of the geographical distribution of childhood Type I (insulin-dependent) diabetes studied by complete enumeration of new cases diagnosed within geographically we/l-defined ~egions according to a standardized protocol. During 1989 24 study centres have registered 1552 new cases diagnosed in the age interval 0-14 years. The incidence of childhood onset diabetes shews statistically highly significant variation between centres ranging from 4.4 (Madeira Island, Portugal) to 49.0 (two counties in Finland) per 100,000 per year. No simple south-north gradient exists as in mld-Europe Sardinia and Luxembourg have particularly high rates. Further studies within the EURODIAB Subarea A collaborative research network will include continued epidemiologieal surveillance as well as extended studies of the genetic, clinical, immunological and pathogenetie aspects of childhood Type I (insulin-dependent) diabetes mellitus in Europe.

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C A U S E S O F DEATH, A N D A G E A T D E A T H A M O N G PATIENTS WITH YOUNG ONSET TYPE 1 DIABETES.

P. Lounamaa, A.Reunanen. ElimMenkatu

A. Green and the EURODIAB Subarea A Study Group, Institute of Clinical Genetics, Odense University, Odense, Denmark

R. Lounamaa, J. T u o m i l e h t o and N a t i o n a l P u b l i c H e a l t h Institute, 25A, 00510 Helsinki, F i n l a n d

D u r i n g 1965-79, 2839 m a l e and 2327 female T y p e 1 d i a b e t i c p a t i e n t s aged 0-17 y e a r s at d i a g n o s i s w e r e r e g i s t e r e d in t]he N a t i o n a l D r u g Registry. For e a c h c a s e two n o n - d i a b e t i c r e f e r e n t s m a t c h e d for age and sex (5617 males, 4619 females) w e r e chosen. By the end of 1987 139 d i a b e t i c s and 120 r e f e r e n t s had died; the s u r v i v a l a m o n g r e f e r e n t s was s i g n i f i c a n t l y b e t t e r t h a n a m o n g diabetics. D e a t h s due to d i a b e t e s c o m p r i s e d 36% of all d e a t h s in d i a b e t i c m a l e s (25% acute, 11% late c o m p l i c a t i o n s ) and 43% in d i a b e t i c f e m a l e s (26% acute, 17% late c o m p l i c a t i o n s ) . The r a t e r a t i o s ( d i a b e t i c / r e f e r e n t ) for o t h e r c a u s e s of d e a t h w e r e in males(M) and females(F): c a r d i o v a s c u l a r M5.6;F2.8, c a n c e r M0.8;FI.7, a c c i d e n t s MI.I;FI.2, s u i c i d e M 1 . 3 ; F O . 9 and others M2.3;FI.5. Compared with referents there was some e x c e s s m o r t a l i t y a m o n g d i a b e t i c s u n d e r 20 y e a r s of age, but at 20-29 y e a r s it was about 3-fold. In f e m a l e s the s u r v i v a l did not d e p e n d on the age-atd i a g n o s i s of diabetes, but in m a l e s who had b e e n d i a g n o s e d at 12-17 y e a r s of age m o r t a l i t y s t a r t e d to a c c e l e r a t e and at about i0 y e a r s after the d i a g n o s i s and to d i v e r g e from the t r e n d seen in p a t i e n t ~ d i a g n o s e d at 0-11 years.

THE M O R T A L I T Y OF CHILDREN WITH TYPE 1 (INSULINDEPENDENT) DIABETES M E L L I T ~ S IN NORWAY, 1973-1988 G . J o n e r - and Sarah Patrick- D e p a r t m e n t of Internal Medicine B, Aker U n i v e r s i t y Hospital, N o r w a y and D e p a r t m e n t of Epidemiology, Graduate School of Public Health, U n i v e r s i t y of Pittsburgh, Pittsburgh, PA, U.S.A. The ai~ of the study was to d e t e r m i n e the m o r t a l i t y among young d i a b e t i c s in Norway. The living status of all persons with Type I diabetes mellitus d i a g n o s e d from 1973 through 1982 and age at d i a g n o s i s b e l o w 15 years, was d e t e r m i n e d as of July ist, 1988. Of the 1908 cases included in the follow-up, 20 had died and i0 had emigrated. A two-fold increased risk for early m o r t a l i t y was found in this cohort compared to the b a c k g r o u n d p o p u l a t i o n with SMR= 207. L i f e - t a b l e analyses did not show sex or age at onset to be s t a t i s t i c a l l y s i g n i f i c a n t predictors of survival when c o n t r o l l i n g for diabetes duration. A review of death c e r t i f i c a t e s revealed that accidents and suicide a c c o u n t e d for the m a j o r i t y of the deaths (40%). Acute diabetes related c o m p l i c a t i o n s were the cause in 35% of deaths. Death by diabetic renal disease or c ~ r d i o v a s c u l a r disease was not found in this cohort. The cohort studied appears to be at a ten-fold d e c r e a s e d risk of early death when compared to a cohort of young diabetic patients from Oslo, Norway d i a g n o s e d 1925-1955 (mortablity rate 106 vs 1079 per i00~000 person-years). This result suggests vast i m p r o v e m e n t s in the survival of Type 1 diabetics in Norway.

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RELEASE OF INTERLEUKIN 1 AND INTERLEUKIN 6, AND HLA CLASS II EXPRESSION BY AN INSULINOMA CELL LINE M.G. Cavallo, M.G. Baroni, A. Toto, N. Visalli, D. Andreani and P. Pozzilli, Endocrinologia (I), University of Rome "La Sapienza", Italy; Nat. Inst. of Biol. Stand., South Mimms, UK. Viral infections may play a role in the pathogenesis of Type 1 diabetes. The aim of this study was to evaluate the effect of different viral infections (rubella, measles, mumps, polio) on an established human insulinoma cell line. The CM line was infected with above mentioned viruses; supernatants were collected every 24 hours for 3 consecutive days for the measurement of interleukin 1 (ILl) and interleukin 6 (IL-6), both using bioassays and immunoassays. At the end of the culture period, cells were analysed for HLA Class I and II expression using monoclonal antibodies and fluorescein a cell sorter analyser. IL-1 and IL-6 were undetectable in the supernatants from non-infected ceils. On the other hand, viral infection with all tested viruses induced the release of both IL-1 (range 30-100 pg/ml) and IL-6 (range 400pglng/ml). Infected cells showed hyperexpression of HLA Class I antigens and an "ex-novo" expression of Class II antigens. These data suggest that a viral infection of a human insulinoma cell line is able to induce the release of IL-1 and IL-6 which are known to be relevant cytokines triggering an autoimmune response. This phenomenon, together with the abnormal expression of HLA antigens, could be relevant in the pathogenesis of Type 1 diabetes.

STUDIES ON T H E MECHANISM OF M A C R O P H A G E MEDIATED ISLET CELL KILLING V. Burkart, B. Berschick and H. Kolb. Diabetes Research Institute, University of Dfisseldorf, Dfisseldorf, F R G We have shown previously that activated macrophages are able to lyse pancreatic islet cells in vitro and therefore may contribute to the decay of beta cells during the pathogenesis of diabetes in animal models of type 1 diabetes. In the present study we analysed the role of the cytoldnes IL-1 and TNF alpha and of reactive oxygen intermediates in macrophage-mediated islet celt killing in vitro. After 15h coincnhation activated macrophages lysed syngeneic 3H-leucine labeled islet cells at 87-+6% and the TNF sensitive L929 cells at 51-+4% (target: effector ratio 1:40). The lysis of L929 cells could be inhibited by anti-TNF antiserum (11-+5%) whereas islet cell lysis was unaffected by anti-TNF and anti-ILl. Separation of target- and effector-cells by a filter membrane (pore size 0.45um) resulted in a L929 lysis of 21+6% and a complete inhibition of islet cell lysis (1 -+4%). Superoxide-dismutase (SOD) and catalase (CAT) slightly reduced islet cell lysis to 77-+3% (SOD), 76-+2 (CAT) and 75-+6% (SOD and CAT). A strong dose-dependent inhibition of islet cell lysis could be achieved with inhibitors of poly(ADP-ribose)synthetase nicotinamide (12_+5%) and 3amino-benzamide (25-+2%). We conclude that macrophagemediated islet cell killing involves the activation of poly(ADPribose)synthetase. Lysis of islet ceils is neither caused by IL-1 nor by TNF but by a cytotoxic factor which does not act over a longer distance of diffusion.

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N I C O T I N A M I D E I M P R O V E S INSULIN S E C R E T I O N IN ISLETS U N R E S P O N S I V E A F T E R C H R O N I C E X P O S U R E TO IL-lfi. M.Buscema,C.Vinci,A.M.Rabuazzo,F.Forte,V.Caltabiano,S.Squatrito and F.Purrello.Cattedra di Endocrinologia, University of Catania, Italy.

INTERLEUKIN-1 ~ INHIBITS GLUCOKINASE ACTIVITY IN CLONAL HIT-T15 ~-CELLS P. Hammonds,J. E. Clarke, R. J. Mertz, J. Espinal and IV[.Beggs. Diabetes Section, Endocrinology Division, Giaxo Research Laboratories, Research Triangle Park, North Carolina, USA. The cytokine intefleukin-I g (IL-1 13)has been shown to have a bimodal effect on insulin secretion in isolated Islets of Langerhansand is involved in the onset of diabetesin animal models. We have recentlydemonstratedsimilar bimodal effects of IL-113on insulin secretionin HIT-T15 ceils which possess specific surface receptorsfor IL-I ft. In the present study, we have examined the effects of IL-1 g on glucosemetabolism. Short term (lh) exposureof HITT15 cells to IL-1 g (25 U/ml) evoked stimulation of both glucose utilisation (57%) and insulin secretion (22%; p<0.01 for both effects). In contrast, longer-term exposure (48h) to IL-1 g inhibited glucose utilisation (60%), glucose oxidation (64%) and insulin secretion (49%; p<0.01 for all effects). Accordingly,we examinedthe effects of IL-113on glycolyticflux. After 48h exposure of HIT-T15 cells to IL-1 13, the activities of glucokinase and hexokinaseWeredecreased39 and 54% respectively(p<0.05). We proposethat in HIT-TI5 cells exposedto IL-113,hexosephosphorylationmay becomeratelimiting for glycolysis.Physiologically,glucokinaseis the major contributor to hexosephosphorylationin the g-cell. Thereforethe present findingssuggest that the primary effect of IL-1 B on glucose utilisation may result from a decreasein glucokinaseactivity.

We studied the glucose-induced insulin release in rat pancreatic islets exposed to interleukin-lg (IL-lg) for 24 h in the presence or absence of nicotinamide. In static experiments, 5 islets were incubated for 24 h with or without 100 U/ml I L - l g in the presence or absence of nicotinamide (1-100 mM) and then exposed for 1 h at 1.4 or 19.4 mM glucose. In control islets the basal insulin secretion was 384_+76 pg/islet/h (m_+SE, n=6) and the glucose-stimulated insulin release was 2013_+427. In islets e x p o s e d to I L - l g (100 U / m l , 24 h) g l u c o s e - i n d u c e d i n s u l i n s e c r e t i o n w a s r e d u c e d (412.+_82). I n islets e x p o s e d to b o t h IL-IB and increasing concentrations of nicotinamide, a dose-dependent recovery of glucose-induced insulin secretion was observed. The maximum effect was obtained at 25 m M n i c o t i n a m i d e (960-2-_118, p < 0 . 0 1 ) . In g r o u p s o f 100 islets i n c u b a t e d for 24 h w i t h or w i t h o u t 100 U / m l ILl g in the p r e s e n c e o r a b s e n c e o f n i c o t i n a m i d e (25 m M ) , the k i n e t i c o f insulin release w a s studied in a p e r i f u s i o n s y s t e m . I n c o n t r o l islets, h i g h g l u c o s e s t i m u l a t e d the u s u a l b i p h a s i c i n s u l i n s e c r e t i o n (first peak=54.9_+9.7,second=70.8_+12.5 p g / i s l e t / m i n , n~+SE, n=7). In islets e x p o s e d to I L - l g b o t h p e a k s w e r e g r e a t l y r e d u c e d (3.9+1.1 a n d 7.8_+2.2). In the p r e s e n c e o f 25 m M n i c o t i n a m i d e i n s u l i n s e c r e t i o n w a s partially r e s t o r e d (first p e a k = 1 9 . 3 + 3 . 9 , p < 0 . 0 1 ) , a n d s e c o n d peak=28.1+8.7(p<0.01). The presence of nicotinamide t h e r e f o r e , is able to partially p r e v e n t t h e b l u n t i n g e f f e c t o f I L - I B o n g l u c o s e - i n d u c e d i n s u l i n secretion.

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INTERLEUKIN-IB I N C R E A S E S THE C Y T O S O L I C S O D I U M C O N C E N T R A T I O N IN I S O L A T E D P A N C R E A T I C ISLETS. J.Nerup, J.Johannesen, P.Bouchelouche and S.Helqvist. Steno M e m o r i a l H o s p i t a l and H e r l e v Hospital, Copenhagen, Denmark. I n t e r l e u k i n - i B (IL-I) is c y t o t o x i c to p a n c r e a t i c b e t a - c e l l s in vitro. All p r e v i o u s l y d e m o n s t r a t e d IL-I e f f e c t s on islet cells t a k e several h o u r s to develop. As IL-l was r e c e n t l y shown to i n c r e a s e the s o d i u m - c o n t e n t in a l y m p h o c y t e cell line, we s t u d i e d the e f f e c t of r e c o m b i n a n t h u m a n IL-I6 on free c y t o s o l i c s o d i u m (fNa+i) in i s o l a t e d rat p a n c r e a t i c islets. By use of the s o d i u m - s p e c i f i c f l u o r e s c e n t indicator, SBFI, we d e m o n s t r a t e d t h a t IL-I i n s t a n t l y and c o n t i n u o u s l y i n c r e a s e d fNa+i in a d o s e - d e p e n d e n t manner (IL-I 6x104 U/l, fNa+i: 21• v e r s u s 7• mmol/l, n=3) d u r i n g 1 h of exposure. The e f f e c t w a s a b o l i s h e d by e x t r a c e l lular d e p l e t i o n of sodium. The same IL-I conc e n t r a t i o n s t i m u l a t e d islet i n s u l i n - r e l e a s e by a p p r o x i m a t e l y 50% a f t e r i and 2 h of exposure, (16.2• v e r s u s 8.78• ng/10 islets, p < 0 . 0 5 a f t e r 2 h). A d d i t i o n a l l y , 12.5 ~mol/l of a m i l o ride, a b l o c k e r of Na§ + exchange, a b o l i s h e d the IL-I e f f e c t s on islet i n s u l i n - r e l e a s e . We h y p o t h e s i z e t h a t the IL-I e f f e c t on fNa+i leads to intracellular pH increase, superoxide anion f o r m a t i o n and b e t a - c e l l d e a t h due to t h e i r p o o r scavenger capacity (low c o n t e n t of M n S O D and glutathion peroxidase).

INTERLEUKIN-I INDUCED ACTIVATION OF POLY (ADP-RIBOSE) S Y N T H E A S E IN P A N C R E A T I C ISLETS. J.FernAndez ~ Alvarez and R.Gomis. Endocrinology and Diabetes Unit,Hospital Clinic. Barcelona. Spain. InterleuRin-i (IL-I) may play a key role in beta cell destruction ocurring during the coupse of insulin-dependent diabetes mellitus. However.the mechanisms by which IL-i exerts destructive actions on the beta cells remain elusive. A mechanism involving the formation of D N A - s t r a n d breaEs that lead to the overactivation of poly(ADP-ribose) synthetase enzyme and critical deplection of its substrate NAD could be proposed. Islet nuclear fractions display Mga+ dependent poly(ADP-ribose) synthetase activity catalyzing the incorporation of (U-CI~)NAD (Kin for Mg2+ and N A D amounts 0.86ram and 0.~2mM respectively). IL-i increased enzyme activity from 2.56+0.72 to 3.60+0.8i pmols/30 rain.islet (p<0.00a);nowever this effect was inhibited by nicotinamide though not m0di~ied by the presence o~ several concentrations of glucose. Preincubation o~ islets during 90 rain in presence of IL-i, interferon g a m m a and tumor necrosis factor,and 18 hours preculture with IL-i only,were also able to provoRe a slgni~icant increase o~ enzyme activity. Thus,it is conceivable that increase of poly(ADP-ribose) synthetase activity in conjuntion with D R A repair could be implieated in citotoxic e~ect of IL-i.

OP 6 Hall A-18 Insulin Receptors 31 I N S U L I N ANALOGUES: I N F L U E N C E OF R E C E P T O R A F F I N I T Y ON T H E I R E L I M I N A T I O N F R O M S E R U M A N D D E G R A D A T I O N IN PIGS U. Ribel, V. Kruse, U.D. L a r s e n and K. Drejer. N o v o R e s e a r c h Institute, DK-2880 Bagsvaerd, Denmark. The d i s a p p e a r a n c e from s e r u m of mono-125I( T y r A l 4 ) - l a b e l l e d h u m a n i n s u l i n (relative r e c e p t o r a f f i n i t y RA=I00%), B 2 5 A s p i n s u l i n (RA=0.05%), B 9 A s p + B 2 7 G l u i n s u l i n (RA=I8%), B l 0 A s p i n s u l i n (RA=327%), and A 8 H i s + B 4 H i s + B 1 0 G l u + B 2 7 H i s i n s u l i n (RA=687%) and the a p p e a r a n c e of t h e i r d e g r a d a t i o n p r o d u c t s w e r e s t u d i e d a f t e r i.v. i n j e c t i o n (18.5 MBq/animal) of each to two pigs (59-81 kg). S e r u m r a d i o a c t i v i t y was a n a l y z e d by a n i o n e x c h a n g e c h r o m a t o g r a p h y (Q-Sepharose-HP) for l a b e l l e d intact tracer, fragments, iodide and p r o t e i n b o u n d material. The d i s a p p e a r a n c e of intact l a b e l l e d m o l e c u l e s from s e r u m is f a s t e r the h i g h e r the affinity. L a b e l l e d f r a g m e n t s in s e r u m s h o w a n a d i r at 2-6 m i n f o l l o w e d by a m a x i m u m at 12-20 min, except for the "noa f f i n i t y " B 2 5 A s p insulin w h i c h shows a s t e a d y decline. The level of f r a g m e n t s from 4-8 m i n ranks in d e c l i n i n g order: B 2 5 A s p insulin, B 9 A s p + B 2 7 G l u insulin, h u m a n insulin, B l 0 A s p insulin, and A 8 H i s + B 4 H i s + B 1 0 G l u + B 2 7 H i s insulin. L a b e l l e d iodide a p p e a r s a f t e r a lag p h a s e of 2-6 minutes. I o d i d e from h u m a n i n s u l i n ranks highest, B 2 5 A s p i n s u l i n lowest and the low and the two h i g h - a f f i n i t y a n a l o g u e s inter-mediate. The slow a p p e a r a n c e of d e g r a d a t i o n p r o d u c t s from the h i g h a f f i n i t y a n a l o g u e s is m o s t l i k e l y c a u s e d by t h e i r slow d e g r a d a t i o n after r e c e p t o r - m e d i a t e d i n t e r n a l i z a t i o n as found in h u m a n h e p a t o c y t e s (Hep G2) and in rats.

32 IN VITRO POTENCY OF INSULIN ANALOGUES DEPENDS ON RATE OF RECEPTOR BINDING RATHER THAN ON AFFINITY L.Sch&ffer and A.R.Serensen, Novo Nordisk A/S, Bagsveerd, Denmark A number of insulin analogues have been examined with regard to in-vitro potency in the free fat cell assay (FFC), affinity to the soluble extracellular domain of the human insulin receptor, and rate of association to the receptor. The range of affinities of the analogues examined spans three orders of magnitude (7%6000%, human insulin=lOOt), whereas the corresponding FFC potencies are within a somewhat narrower range (11%-1200%). Association rates vary between 18% and 1650% and the relationship between affinity and association rate is not monotonous as one might expect. Most of the analogues have identical ranking in FFC and affinity but three analogues with affinities of 70%400% show FFC potencies of 11%-14%. These analogues exhibit association rates about five fold lower than human insulin in spite of their normal or increased affinity. Thus, the FFC potency is found to correlate better with the association rate than with the affinity. This suggests that the association step is rate determining for insulin action and implies that internalisation of the receptorinsulin complex is significantly faster than insulin dissociation thus rendering the dissociation rate, and thereby the affinity, of less importance for the potency than has previously been assumed.

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34

RECYCLING OF INSULIN RECEPTORS IN RENAL PROXIMAL TUBULES. S. Nielsen. Department of Cell Biology. Institute of Anatomy. University of Aarhus, Aarhus, Denmark.

RELATIONSHIP B E T W E E N D E F E C T I V E INSULIN RECEPTOR INTERNALIZATION AND T Y R O S I N E KINASE ACTIVITY IN MONOCYTES FROM NIDDM PATIENTS V. Trischitta, F. Giorgino, R. Scalisi, L. Frittitta and S. Squatrito. Endocrinology Department, University of Catania, Ospedale Garibaldi, Piazza S.M. Gesu', 95123 Catania, Italy

Insulin receptor-recycling and the sequence of involvement of dense apical tubules (DAT) in endocytosis and membrane recycling was investigated morphologically by developing a new method. Proximal tubules were perfused with insulin coupled to colloidal-gold (9~ 30 min), cross-linked to the receptor using DSS (9~ 30 rain), and fixed after functioning for 0-30 n'fin at 25~ or 37~ Controls were made without cross-linking. At 25~ endocytic vacuoles (EV) contained receptor cross-linked insulin-gold (R-Ins) after 4 min and EV showed tubular elongations with R-Ins. After 6 rain EV and few DAT contained R-Ins and after 8-30 rain both DAT and EV contained R-Ins, demonstrating that the endocytic uptake precedes from invaginations to EV and the receptors recycle via DAT. DAT are formed immediately after detach of invaginations from the surface. From 4-30 min R-Ins was located exclusively apically in DAT and in EV with inner-coat but not in EV without inner-coat demonstrating two types of EV. R-Ins was never delivered to lysosomes or other subcellular compartments nor basal located EV. In contrast controls demonstrated insulin-gold in LYS and basal located EV. In preliminary experiments at 37~ R-Ins was transferred back to the luminal membrane and was not re-internalized.

Insulin receptor (IR) tyrosine-kinase (TK) activity has been reported to modulate IR internalization. Since both IR-TK activity and internalization are reduced in NIDDM, our aim was to verify if the 2 defects described in these patients are related.Methods: Monocytes were obtained from 5 controls (C), 6 obese (Ob) and 7 obese NIDDM patients. IR internalization, induced by insulin, was studied by 125Iinsulin binding to cell surface tR (16 h, 4 C) after preincubation (37 C, 30 min) with 100 nM insulin, followed by acid wash (pH 4.5, 15 min).IR internalization was also induced by a monoclonal anti IR antibody (MA-10), a ligand that does not activate IR-TK activity. TK activity of WGA purified IR was studied by stimulating IR autophosphorylation and poly-glu4-tyrl (PGT) phosphorylation with 100 nM insulin. Results: a) IR internalization was reduced (p < 0.05) in NIDDM (5+_2% and 24+_5%, m+-SE, after exposure to insulin and MA10,) in comparison to Ob (20+_5 and 55 +8) and C (25+7,and 47+6). b) IR autophopshorylation was reduced (p<0.05) in Ob and NIDDM c) PGT phosphorylation was reduced in NIDDM but not in Ob (904+100 fmol ATP incorporated/ 100 pg PGT, vs 800+150, and 336+-70 in C, Ob, and NIDDM,). Conclusions:l) Although both Ob and NIDDM have defgctive IR-TK activity, IR internalization is reduced only in NIDDM. 2) This reduction is present also when IR internalization is induced by MA-10 that does not activate IR-TK activity. Our data suggest that in NIDDM the 2 defects are not related.

35

36

Different ~-subunit structure of type A and B human insulin receptor determines different tyrosine kinase a c t i v i t i e s

INSULIN BINDING TO ITS RECEPTOR INDUCES A CONFORMATIONAL CHANGEIN THE RECEPTOR C-TERMINUS.

M. Kellerer*, B. Ermel*, B. Vogt I , B. O b e r m a i e r Kusser I , A. U l l r i c h 2 and H.U. H~ring* IInstitUt f. Diabetesforschung, Munich FRG, 2 M a x - P l a n c k - I n s t i t u t , M a r t i n s r i e d FRG. The human i n s u l i n r e c e p t o r occurs in two isoforms which differ in the c - t e r m i n a l end of the s-subunit (aa 719-731 are d e l e t e d in type A). In order to study the impact of this d e l e t i o n on the sign a l i n g function of the i n s u l i n r e c e p t o r we chara c t e r i z e d the tyrosine kinase a c t i v i t y of HIR-A and HIR-B in vitro. A l t h o u g h equal amounts of wheat germ agglutinin-purified HIR-A and HIR-B were used, HIR-B showed a 3-fold (n=6) higher 32p-incorporation than HIR-A under basal and insulin stimulated conditions. The same d i f f e r ence was m e a s u r e d after further r e c e p t o r purification with insulin receptor a n t i b o d y B9. Kinetic analysis revealed almost same Km (HIR-A 14,3 s• HIR-B 20,2 s• n=4) whereas V m a x [~M/60 min Bg protein] was s i g n i f i c a n t l y d i f f e r ent (HIR-A 5,5 s• HIR-B 42,5 s• n=4). Tryptic peptide mapping of the ~ - s u b u n i t s rev e a l e d same p h o s p h o p e p t i d e patterns. In addition, phosphorylation occured only on tyrosine residues. The difference in 3 2 p - i n c o r p o r a t i o n of both r e c e p t o r types was a b o l i s h e d when p h o s p h o rylation was stimulated by tryptic c l e a v a g e of the ~ - s u b u n i t instead of insulin. In conclusion: HIR-B shows a 3-fold higher tyrosine kinase activity than HIR-A. The d i f f e r e n c e d i s a p p e a r s when the ~ - s u b u n i t s are c l e a v e d by trypsin. This suggests that d i f f e r e n t ~ - s u b u n i t s t r u c t u r e s affect tyrosine kinase a c t i v i t y of the ~-subunit.

J. Dolais-Kitabgi, V. Baron, N. Gautier, P. Hainaut, J.C. Scimeca and E. Van Obberghen, INSERM U145, Facult6 de M6decine, Avenue de Valombrose, 06034 Nice C6dex, France. Antibodies against peptides corresponding to sequences (peptide Ch amino acids 1309-1326; C2: amino acids 1294-1317) in the C-terminus of the insulin receptor B-subunit were used to approach the putative role of this domain in signal generation. The two antibodies produced distinct immunoprecipitationpatterns as a function of insulin receptor forms and recognized receptor changes induced by ligand binding and autophosphorylation.Thus, both antipeptideshad a decreasedrecognition capacity for receptors occupied by insulin compared to empty receptors. Further, anti-C1 had a lower affinity for phosphorylated receptors compared to unphosphorylated ones. Receptor immunoblotting showed that the phosphorylation-induced changes detected by anti-C1 are retained, suggestingthat they are likely not of a conformationalnature. In contrast, insulin-induced receptor changes disappear with denaturation pointing to their reversibility. In conclusion, antipeptides against the receptor C-terminus: (i) distinguish between phosphorylated and unphosphorylated receptors; (ii) detect insulin-induced changes in the receptor molecule. These data indicate that insulin binding to receptor leads to a reversible,phosphorylatinn-independentconformationalchange at the level of the receptor C-terminus. This could modify the receptor interaction with cellular structures, and as such, play a role in insulin action.

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OP 7 Hall A-1

Hypertension 37

38

ACUTE SODIUM RETENTION BY INSULIN: A POTASSIUM EFFECT?

NORMAL SODIUM RETAINING EFFECT OF INSULIN IN TYPE 2 (NON-INSULIN-DEPENDENT) DIABETES MELLITUS A STATE OF PERIPHERAL INSULIN RESISTANCE AND HYPERINSULINEMIA

C.E. Friedherg, H.A. Koomans, J.A. Bijlsma and E.J. Dorhout Mess. Department of Nephrology, University Hospital, Utrecht. Acute insulin administration causes sodium retention. We examined if this is an indirect effect. We studied the effect of insulin (i mE/kg/min for 3 hours) with the euglycemic clamp method on sodium, potassium and lithium excretion in 6 waterlnaded males. Each subject underwent 3 clearance studies: a time control followed by 2 clamp studies. During the last study potassiumchloride was also infused to clamp plasma potassium. During the second study plasma potassium fell: 3.90 .+ 0.06 to 3.2 _+ 0.06 (mmol/1, p < 0.01). Fractional sodium ~*d potassium excretion fell respectively 1.13 .+ 0.16 to 0.67 .+ 0.12 and 12.5 • 0.8 to 5.0 • 0.3 (% p < 0,01 and p < 0.05) (oneway ANOVA). During the last study neither plasma potassium nor fractional sodium and potassium excretion changed significantly. Fractional lithium excretion increased during both clamps (p < 0.01 and p < 0.05). Renin and aldosteron concentration didn't change during tests. Insulin reduced sodium and potassium excretion but not when plasma potassium remained constant. Insulin increased "diluting segment" reabsnrption and lithium clearance. Potassium infusion prevented this distal effect but not the increase in lithium clearance. We conclude that sodium retention after acute insulin administration is an indirect effect mediated by plasma potassium concentration. Probably this occurs in the diluting segment.

P. SkCtt, A. Vaag, N.E. Braun, O. Hother-Nielsen, M-A. Gall, H. Beck-Nielsen and H-H. Parring. (Hvid0re Hospital, Klampenborg, and Department of Clinical Physiology and Nuclear Medicine, Glostrup University Hospital, Copenhagen, Denmark). Insulin action on kidney function was evaluated in 10 non-insulin dependent diabetic patients (D, aged 56 (43-73) years), and 8 age matched control subjects (C). Body mass index was higher in D (29.5 vs 23.4 kg/m ~, p < 0.05). The renal clearances of ~mTc-DTPA, lithium, sodium were measured in a basal period of 90 min. Then 40 mU.min-l.l.73 m -2 insulin was infused. Blood glucose was clamped at the basal level (D: 9.9-+3.5, C: 5.3e0.5 mmol/1) and the clearance measurements were repeated. Insulin infusion resulted in similar increases in insulin concentrations (D: 0.12.+0.05 vs 0.56_+0.15, C: 0.05x0.02 vs 0.40_+0.07 pmol/ml). Although the steady state metabolic clearance rate of glucose during insulin infusion was lower in D (155_+62 vs 320.+69 ml.min~.1.73 m -~, p<0.01), the insulin induced decline in sodium clearance was similar (D: 1.8+1.1 vs 0.7-+0.4, p<0.01, C: 1.7_+0.3 vs 0.8.+0.3 ml.min~.l.73 m z, p <0.01). The glomerular filtration rate and lithium clearance was unchanged, consequently calculated distal tubular fractional sodium reabsorption increased (D: 92.9e4.1 vs 97.1.+1.5, p < 0.01, C: 93.1e 1.1 vs 96.5.+0.6%, p < 0.01). In conclusion, the sodium retaining effect of insulin is normal in D, despite peripheral insulin resistance and hyperinsulinemia. This may contribute to the extracellular volume expansion and the increased frequency of hypertension in this syndrome.

39

40

Atrial NatriureticFactor PossibleMediator Of ImpairedSodium Excre-

INSULIN RESISTANCE CORRELATES TO BLOOD PRESSURE IN PATIENTS WITH TYPE 2 DIABETES MELLITUS

tion In Response To Volume Expansion In DiabetesMellitos. J.Sgnchez, L.Fern~ndez,J.M. Lbpez Novoa, J. Gutkowska, Y. Diaz, A. Fernandez-Cruz. S.Car]osUniversityMespita],F~ndaci6nJim~nez Dfaz. Yedrid, Spain. C]inie2dResearch Instituteof NontreaT, Canada. The increasedexchangablebody sodium in diabetesme].]itus remains an umexp]ainedphenomenon. There is considerableevidence suggesting that AgY increases in response to volume expansion. We studied the responseof plasma ANF to an IV 2 h. 2 ]. saline infusion in 7 norrnotensivehealthy suOjects and 29 normotensivemetabolically stab]e diabetics (22 type I and 7 type II) aged 43.7+3.5 years. Na excretion in basal conditionswas: in controls 156.07+ 27.8; in type 1 196.4+_21.2and in type II: 172.~37.~ uEq/min. After saline infusionNa excretionwas increasedto 643.7_+67.2in control, to 372.8_+33.9in type I and to 344.4+3.'3.5in type It. ANF increased significantlyin all groups, p <0.01. However, the increment was blunted in both diabeticgroups. The basal plasma ANF was found to be in eontTD] group 7.1!1.5; in diabetics type I: 25.1~3.0; and tvfl3eII: ~.2~4.8 pg/m]. After saline infusion~NF was increased to 13.3+2.4 in control, to 35.9!_4.0in type I and to 47.5+ 7.7

pg/m] in type It. PRA and plasma a]dosteronewere suppressedin

all groups. These rssu]te demonstratethat the impairednat-riur~sis after volume expansion in diabetics is associatedwith bluntedANF response.

K I BIRKELAND, F CHATZIPANAGIOTOU, R GANZ*, K HANSSEN** AND S VAALER** HORMONE LABORATORY AND DEP OF MEDICINE** AKER HOSPITAL AND DEP OF MEDICINE ULLEVAAL HOSPITAL*, OSLO, NORWAY. Recently an association between insulin resist a n c e a n d h y p e r t e n s i o n in n o n - d i a b e t i c s w a s documented. The aim of the present study was to investigate the relationship between insulin r e s i s t a n c e a n d b l o o d p r e s s u r e in p a t i e n t s w i t h non-insulin dependent diabetes. We performed hyperinsulinemic e u g l y c e m i c g l u c o s e c l a m p in 9 w o m e n a n d 12 m e n a g e d 4 9 - 6 9 y e a r s ( m e a n 5 9 . 1 ) , diabetesduration 3 - 1 5 y e a r s ( m e a n 8.5) a n d b o d y mass index 20.9-33.2 kg/m2 (mean 26.9). Patients w e r e in s t a b l e m e t a b o l i c c o n t r o l t r e a t e d w i t h diet alone or combined with sulfonylureas. Glucose uptake was calculated from the amount g l u c o s e i n f u s e d t h e l a s t 20 m i n u t e s o f 2 h o u r s euglycemia. Measurements of weight, blood pressure, fasting blood glucose, HbAlc, and l i p i d s w e r e p e r f o r m e d e v e r y t h r e e m o n t h s in a 315 m o n t h s p e r i o d p r i o r t o t h e c l a m p s t u d y . Glucose uptake ranged from 2.01 to 9.13 mg/kg/min (mean 4.01). Mean blood pressure during the study period ranged from 125-207 systolic and 78-111 diastolic. There was a negative correlation between glucose uptake and d i a s t o l i c b l o o d p r e s s u r e (r= 0.60, 2 p < 0 . 0 1 ) . C o r r e l a t i o n b e t w e e n g l u c o s e u p t a k e a n d BMI, age, HbAlc, fasting insulin or C-peptide was not significant. We conclude that insulin resistance correlates strongly to diastolic blood pressure. This may s u g g e s t t h a t i n s u l i n r e s i s t a n c e is i n v o l v e d in t h e p a t h o g e n e s i s o f h y p e r t e n s i o n in p a t i e n t s with type 2 diabetes.

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THE NATUREOF INSULIN RESISTANCEIN ESSENTIALHYPERTENSION

INSULIN-STIMULATED GLUCOSE METABOLISM IN GENETIC MODELS OF HYPERTENSION S. Frontoni and L. Rossetti. University of Texas Health Science Center at San Antonio, U.S.A. In vivo insulin action was examined in four groups of 24h fasted, conscious unrestrained, rats: 1) Milan Hypertensive (MHS); 2) Milan Normotensive (MNS); 3) Spontaneously Hypertensive (SHR); 4) Wistar Kyoto (WKY). Mean arterial pressure and heart rate were increased in SHR vs WKY in fed (178+5 vs 119+2 mmHg and 399+8 vs 368_+11 bpm; p<0.01) and fasting (158_+4 vs 118+3 mmHg and 393+9 vs 368_+9 bpm; p<0.01) state. Fasting plasma glucose, insulin, hepatic glucose production (HGP) were similar in all groups. During high dose (129.3 pmol/kg'min) euglycemic clamps, glucose uptake (GU) was similar in MNS (159.4_+7.8 pmol/kg.min) vs MHS (186.7_+16.7 I~mol/kg.min) and in WKY (192.2+10.6 i~mol/kgmin) vs SHR (198.3_+13.3 ~mol/kg.min). Suppression of HGP, and stimulation of glycolytic and muscle glycogenic rates were also similar. During low dose (21.6 pmol/kg.min) euglycemic clamps suppression of HGP (19.6_+.3.5 vs 19.7_+3.6 ~mol/kg'min) and stimulation of glycolysi~ (91.1+11.7 vs 72.2+6.1 I~mol/kg'min) were similar in SHR vs WKY, while GU (136.7+10.6 vs 102.2_+6.7 ~mol/kg-min; p<0.05) and muscle glycogenic rate (32.8_+.2.8 vs 19.4_+2.2 t~mol/kg'min; p<0.05) were increased in SHR vs WKY. Conclusion: feeding augments blood pressure in hypertensive but not in normotensive rats; insulin sensitivity is normal or increased in two different rat models of genetic hypertension. Thus, hypertension is not invariably associated with insulin-resistance.

D.P. Rooney, R.D.G. Nee!y, K. Ennis, B. Sheriden~ A.B. ALkinson, E.R. Trimble and P.M. Bell. Sir George E. Clark Metabolic Unit and Regional Endocrine Laboratory, Royal Victoria Hospital and Department of Chemical Pathology, Queens University, Belfast. To investigate the nature of insulin resistance in essential hypertension, we measured peripheral and hepatic insulin action and the activity of the glueose/glucose-6phosphate (G/G6P) substrate cycle in 7 untreated nonobese hypertensive subjects and 7 matched controls. Euglycaemic glucose clamps were performed at sequential insulin infusion rates of 0.2 and 1.0 mU/kg/min. A simultaneous infusion of [2~H] and [6~H] glucose (2H and 6H) combined with s selective detritiation proeedure was used to determine glucose turnover, the differences being the activity of the G/G6P cycle. Endogenous hepatie glucose production (6H) was similar in hypertensives and controls in the posbsbsorptive state (lO.9• O• and at the 0.2mU infusion (5.0•177 At the l. OmU infusion glucose disappearance (6H) was lower in hypertensives than controls (20.3•177 umol/kg/min p~O.O05). Activity of the G/G6P cycle (2H-6H) was similar both in postabsorptive state (18.3•177 and during insulin infusion (0.2mU:21.1~2.2v24.2• 1.OmU:21.4•177 Following 3 months therapy with cyclopenthiazide 500ug daily in 7 patients postabsorptive endogenous glucose appearance increased (10.9• to 12.3+0.5umol/kg/min p
eyclopenthiazide therapy.

OP 8 Hall A-2 Glucose Transport in Diabetics II 43

44

EFFECT OF HYPERGLYCAEMIA ON GLUCOSE METABOLISM IN SKELETAL MUSCLES IN PATIENTS WITH TYPE 1 (INSULIN-DEPENDENT) DIABETES. A VAAG, O HOTHER-NIELSEN, P SKOTT, P ANDERSEN, P DAMSBO, EA RICHTER AND H BECK-NIELSEN. HVIDORE HOSPITAL, KLAMPENBORG, DENMARK. Patients with Type 1 (insulin-dependent) diabetes were used as a non-insulinaemic model to study the effect of hyperglycaemia per se on glucose metabolism in skeletal muscles (n = 11, C-pep. < 0.05 nmol/I, HbA,~ < 8.0%). After an over-night euglycaemic clamp, the i.v. insulin infusion rate was kept constant during the experiment. Using 33H-glucose and indirect calorimetry, glucose metabolism was assessed during a basal period (PG (plasma glucose) 5.0 mM) and during a hyperglycaemic period (4h i.v. glucose infusion, PG 12.1 mM). Biopsies were taken from the vastus lateralis muscle during both periods. Hyperglycaemia increased nonoxidative glucose disposal (50.9 _+ 9.1 vs 32.2 _+ 6.7 mg/m=/min, p<0.05), whereas glucose oxidation was unchanged (53.8 -+ 6.9 vs 54.9 _+ 6.0 mg/m=/min, NS). The activity of glycogen synthase (GS) in muscle biopsies (fractional velocity (0.1 and 10 mM G6P)) decreased slightly during hyperglycaemia (0.17 -+ 0.05 vs 0,14 _+ 0,06, p<0.05). The intracellular concentration of free glucose and glucose6-phosphate (G6P) increased during hyperglycaemia (0.56 _+ 0.11 vs 1.43 _+ 0.19 mM, p<0.01, and 0.14 _+ 0.04 vs 0.23 _+ 0.08 mM, p<0.02). In conclusion, in vivo hyperglycaemia may stimulate GS in skeletal muscles allosterically by increasing concentrations of G6P. The intracellular accumulation of free glucose and G6P indicates that glucose transport into the muscle cell may not be rate limiting for an increased glucose metabolism during hyperglycaemia.

LACK OF INSULIN RESPONSE OF MUSCLE GLUT4 mRNA AND PROTEIN IN INSUUN RESISTANT TYPE 1 DIABEfES H. YId-J~rvinen. H. Vuodnen-Markkola. L Koranyi. R.E. Bourey, M. Mueckler and V.A. Koivisto. Helsinki, Finland and St. Louis, MO. U.S.A.

We quantitated the effect of insulin on the expression of the muscle/adipose glucose transporter (GLUT4) mRNA and protein in 14 type 1 diabetic patients (DM) (age 29+2 yrs, BMI 22+1 kg/~) and 15 matched controls (CONT). MusCle biopsies w~e taken before and after a 240 min insulin infusion ('100" mU/I) under normoglycemic conditions (glucose 5.6+0.1 vs 5.3+0.1 mM, DM vs CONT). Total glucose uptake (M) v~as lower in-DM (6.0+0.3) than in CONT (7.6+0.4 mg/kg.min, p<0.01). The basal GLLTI'4 content was 43 % 15wer in DM than in CONT (p
A23

46

45 GLUCOSE TRANSPORT IN SKELETAL MUSCLE IS INSULIN-RESISTANT IN TYPE 2 DIABETES. R.C. Bonadonna, S. Del Prato, C. Cobelli, M.P. Saccomani, A. Stocco, D. Bier, E. Ferrannini and R.A. DeFronzo. San Antonio, U.S.A.; Saint Louis, U.S.A.; Padova, Italy; Pisa, Italy. We tested the hypothesis that, in type 2 diabetics, glucose transport in skeletal muscle is insulin resistant. We studied 5 diabetics (age=49+6 years, BMI=25.9+1 k g / m 2, FPG=I4.7+I.I retool/I) and 5 controls (age=50+3 years, BM1=23.9• kg/m2), by the euglycaemic insulin (1 mU/min/kg, ~70 mUff) clamp technique, in combination with the forearm balance technique (brachial artery and deep vein catheterisation) and with the intra-arterial pulse injection (basal and hyperinsuli~aemic state) of 1 2 C-mannitol (not transportable) and 3-O-1 4 C methyl-D-glucose (transportable, but not metabolizable). The wash-out curves, measured in the deep vein, were analyzed by an, "ad hoc" multicompartmental model to quantitate transmembrane inward (kin) and outward ( k o u t ) 3 . O - 1 4 C - m e t h y l - D glucose transport. Basal plasma glucose was normalized (5.1+0.1 retool/I) in Ihe diabetics by an overnight intravenous insulin infusion. The exogenous glucose (M) metabolised during the clamp was decreased in the diabetics (9.4+2.1 vs 23.4+2 p.mol/min/kg, p<0.01) as was forearm glucose uptake (4.3=1:1.2 vs 18.8+4.7 i.tmol/min/kg, p<0.01). In the basal state, kin and kout were comparable in ~ontrols and diabetics (0.07+0.01 vs 0 . 0 6 2 + 0 . 0 1 . r a i n - l*a n d 0.04:1:0.01 vs 0.03+0.01 min" i , respectively). In contrast, during the clamp, kin was significantly stimulated in controls, but not in diabetics (0.074-0.01 vs 0 . 1 4 + 0 . 0 2 . r e i n ' l , p<0.05). We conclude that transmembrane inward glucose transport in human skeletal muscle is defective in type 2 diabetes and may account for the insulin resistance of this condition.

FASTING-INDUCED CHANGES IN GLUCOSE TRANSPORTER EXPRESSION" PARALLEL BASAL MUSCLE GLUCOSE UPTAKE E.W. Kraegen, J.A. Sowden, P.W. Clark, M.B. Halstead and D.J. Chisholm. Garvan Institute of Medical Research, St. Vincents Hospital, Sydney, NSW 2010, Australia. Fasting reduces glucose transporter mRNA in adipose tissue. We aimed to examine the heterogeneity and relationship to glucose uptake of the glucose transporter mRNA response in muscle with 48h fasting (Wistar rats; n=4-6; results expressed as percent of 5h fasted values). Basal and insulin-stimulated glucose uptake (Rg', 2,6[3H]deoxy glucose plus conscious euglycemic clamp) were compared to insulinregulatable (IRGT) and HepG2-type glucose transporter mRNA levels in heart, diaphragm and red and white hindlimb muscle, (Northern Blots, corrected for total mRNA). Fasting-induced changes in IRGT mRNA paralleled basal Rg' (r=0.99, p<0.01; Heart Rg' 2.9_+0.3%, [RGT 48+10%; diaphragm Rg' 28.5+7.8%, IRGT 76+10%, red muscle Rg' 40L-_12%,IRGT 95+_19%, white muscle Rg' 67.5_+9.2%, IRGT 131+18%). Neither incremental Rg' to insulin elevation (100 mU/L) nor IRGT mRNA declined significantly with 48h fasting in red or white muscle. HepG2 glucose transporter mRNA, reliably detected only in heart, was 11+10% of 5h fasted values. Thus, heterogeneity of IRGT mRNA response to fasting among muscle types corresponds to effects on basal glucose uptake. The large decline in HepG2 and IRGT expression in heart may contribute to observed fasting-induced glucose sparing. Maintenance of IRGT expression in skeletal muscle may be important in retaining normal insulin sensitivity with fasting.

47

48

E X P R E S S I O N OF I N S U L I N - R E S P O N S I V E G L U C O S E T R A N S P O R T E R m R N A IN S K E L E T A L M U S C L E OF P A T I E N T S W I T H T Y P E 2 DIABETES. L.C.Groop, L.Koranyi, J.Eriksson, C . S c h a l i n and A. Permutt. Helsinki University Hospital, Helsinki Finland, and Washington University School of Medicine, St. Louis, Missouri.

~UCOSE UPTAKEIN MYOCARDIALAND SKELETAL MUSCLE ESTIMATED WrlH POSIn:K)N EMISSION TOMOGRAPHY IN TYPE 2 DIABEI'ES

To study w h e t h e r i n s u l i n r e s i s t a n c e in T y p e 2 diabetes is a s s o c i a t e d w i t h a d e f e c t in the e x p r e s s i o n of the GLUT4 gene in s k e l e t a l muscle, we p e r f o r m e d N o r t h e r n b l o t a n a l y s i s of G L U T 4 m R N A in m u s c l e b i o p s i e s from v a s t u s l a t e r a l i s of 9 i n s u l i n - r e s i s t a n t T y p e 2 d i a b e t i c s (age = 60 • 2 yrs; BMI 28.7 • 1.5) and of 6 i n s u l i n s e n s i t i v e control s u b j e c t s (age = 31 • 9 yrs; BMI 22.6 • 0.3). Insulin sensitivity was measured with a euglycemic insulin clamp in combination with indirect calorimetry and i n f u s i o n of 33-glucose. C o m p a r e d w i t h controls, Type 2 d i a b e t i c s had i m p a i r e d i n s u l i n - s t i m u l a t e d glucose metabolism (3.3 • 0.4 vs 7.7 • 0.3 m g / k g , rain ; p<0. 001) , w h i c h was due to an i m p a i r m e n t in b o t h the o x i d a t i v e (1.7 • 0.2 vs 2.8 • 0.I mg/kg.min) and n o n - o x i d a t i v e (1.6 • vs 4.8 • 0.3 mg/kg.min) (both p<0.01) p a t h w a y of g l u c o s e m e t a b o l i s m . The levels of G L U T 4 m R N A e x p r e s s e d p e r ~g RNA (167 • 36 vs 50 • 17 pg;p<0.05) or per mg t i s s u e (115 • 22 vs 56 • 17pg; p=0.07) w e r e h i g h e r in c o n b t r o l s t h a n in Type 2 diabetics. In addition, the level of m u s c l e GLUT4 m R N A c o r r e l a t e d i n v e r s e l y w i t h the rate of total body glucose metabolism (R=0.55;p<0.05). C o n c l u s i o n s : I n s u l i n r e s i s t a n c e in T y p e 2 d i a b e t e s is not a s s o c i a t e d w i t h a d e f e c t in the e x p r e s s i o n of GLUT4 m R N A in s k e l e t a l muscle.

P. Nuufila, J. Knuuti, U. Ruotsalainen, M. Teras, O. Solin, V. Koivisto, U. Wegelius and L-M. Voipio-PulkkJ, Turku and Helsinki. Rnland The rate of glucose uptake (GU) was examined in myocardium, forearm muscle and total body by insulin clamp technique and positron emission tomography (PET). 'SF-Fluoro-2Deoxy-Glucose ('SFDG) was administrated to 9 type 2 diabetic patients (age 58+2.3 yrs; BMI 27.3-+0.8 kg/m 2, mean-+SEM) and 13 matched controls in a steady state of hyperinsulinemia (167+14 vs. 1754-32 mU/I, respectively) and glycemia (7.3-+0.8 vs. 5.44-0.7 mmol/I, respectively). The whole body GU was similar in diabetics (5.4+0.6 mg/kg/min) and controls (6.04-0.9 mg/kg/min). The GU in myocardium and forearm muscle was estimated using the Patlak graphical analysis of 'SFDG. Myocardial GU was not significantly different in the diabetics (14.8+2.0 mg/min/100 g) and controls (22.5+3.3 mg/min/100 g), although influx constants for 'SFDG in myocardium were different (0.08-+0.01 vs. 0.18-+0.02, p<0.01, respectively). The GU rates of forearm were similar in type 2 diabetic and control subjects (2.44-0.3 vs. 3.1-+0.8 mg/min/100 g, respectively). In conclusion, during hyperinsulinemia with similar whole body glucose uptake in type 2 diabetic and control subjects. 1) Myocardial and skeletal muscle glucose uptake are similar in the two groups, and 2) glucose uptake in forearm is 11-14% of that in the myocardium.

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OP 9 Hall A-3 Diabetic Foot 49

50

SKIN BLOOD FLOW REACTIVITY TO DISTANT COOLING, A TEST TO DETECT EARLY SYMPATHETIC DYSFUNCTION.

DISSOCIATION OF SKIN OXYGENATION FROM ARTERIAL BLOOD PRESSURE IN DIABETIC LIMB AMPUTATION R.E. Pecoraro and G.E. Reiber, University o f Washington and Seattle V A Medical Center, Seattle, W A , U.S.A. W e compared the preoperative dorsalis pedis Doppler blood pressure and the transcutaneous oxygen tension at the dorsal foot, a physiologic measure of capillary perfusion, in 66 diabetic men who required initial lower limb amputations, 27 dysvascular nondiabetic amputees, and 40 diabetic outpatients with nonhealing foot ulcers. Controls included 236 hospitalized diabetics without limb lesions, 20 hospitalized nondiabetics, and 23 healthy, physically active nonsmokers recruited by advertising in the newspaper. All groups were m e n and were matched for age. Mean values (+SD) (p by t-test vs healthy controls) for dorsalis pedis blood pressure (ram Hg) were: 111+50 (p<0.001), 75+48 (p<0.001), 119+46 (p<0.001), 141+34 (p<0.05), 145+30 (p=NS), and 155+22, respectively. Dorsal foot transcutaneous oxygen tension (mm Hg) at 44~ was 25+18 (p<0.001), 21_+22 (p<0.00l), 38+15 (p<0.001), 49+12 (p=NS), 52+12 (p=NS), and 53+13, respectively. Although both the dorsalis pedis Doppler blood pressure and the dorsal foot transcutaneous oxygen tension were significantly decreased among diabetic and nondiabetic amputees and those with foot ulcers, the two parameters correlated poorly w h e n compared within subjects among all groups (R2 = 0.141, 0.088, 0.004, 0.038, 0.135, and 0.017, respectively). W e conclude that cutaneous oxygenation is an important determinant o f limb viability and tissue repair, but it is poorly predicted by traditional measurements o f distal arterial pressure.

PM Netten, H Wollersheim, Th Thien and JA Lutterman. Dept. of Medicine, Division of General Internal Medicine, University Hospital N i j m e g e n , The N e t h e r l a n d s . The increased local skin blood flow in a d i a b e t i c n e u r o p a t h i c foot is the result of an abnormal high degree of shunting through arteriovenous anastomoses who are richly s u p p l i e d by s y m p a t h e t i c nerve endings. To study sympathetic skin vasomotor responses, we u s e d Laser Doppler f l u x m e t r y (LDF; Periflux) d u r i n g distant cooling, after 20 minutes a c c l i m a t i s a t i o n in a c l i m a t e room (24.7 + 0.3 ~ In 36 c o n t r o l s i m m e r s i o n of the h a n d or foot in a waterbath of 10~ d u r i n g 1 minute, was f o l l o w e d by a LDF decrease Of the noncooled extremity of 49.5 • 12.5% (range 2786). In 14 patients who underwent a s y m p a t h e c t o m y the LDF d e c r e a s e was 13.2 • 9.8% (range 0-30). F r o m these results a d e c r e a s e of less then 25% was c o n s i d e r e d to be abnormal, i n d i c a t i n g a s y m p a t h e t i c dysfunction. Out of 275 d i a b e t i c patients, 42 w e r e c l i n i c a l free of late c o m p l i c a t i o n s (vibration sense, ankle and knee jerks, a n k l e - b r a c h i a l index, k i d n e y f u n c t i o n and a l b u m i n e x c r e t i o n rate w e r e normal, no retinopathy) and in g o o d m e t a b o l i c c o n t r o l (HbAI < 10%), 26 gave i n f o r m e d c o n s e n t to participate in this study. Of these 26 patients Ii showed a p e r c e n t a g e LDF d e c r e a s e of less t h e n 25%. The c o e f f i c i e n t of v a r i a t i o n was 23%. In conclusion: skin blood flow r e a c t i v i t y to d i s t a n t c o o l i n g is a simple test to detect early sympathetic dysfunction in diabetic patients without clinical signs of diabetic foot abnormalities, but the r e p r o d u c i b i l i t y w a r r a n t s improvement.

51

52

IMMUNOSCINTIGRAPHY FOR DETECTION OF F O O T INFECTIONS IN DIABETIC PATIRNTS

EFFECTIVENESS OF THE ACTIVITIES OF CHIROPODIST IN THE OUTPATIENT FOOT CARE OF DIABETIC PATIENTS

G.Weidlieh, A.Kroiss, Ch.Auinger and G.Schernthaner Department of Medicine I & Institute of Nuclear Medicine, Rudolfstiftung Hospital, Vienna Foot infections is one of the central problems in patients with diabetes mellitus. It is clinically important to distinguish between soft tissue diseases as cellulitis and ulceration from osteomyelitis. The aim of this study was to evaluate the clinical relevance of immunoscintigraphy (IgG isotype, directed against on an epitope of NCA-95) and three phase bone scans. Immunoseintigraphy was performed with 10 mCi (370 MBg) Tc99m labeled antibodies (BW 250/183); Behringwerke, FRG). The images were done 4 hrs planar. Three phase skeletal scintigraphy consisted of a radionuclide angiogramm, an immediate pestinjection "blood-pool image, and 3 hrs delayed image. We investigated 20 diabetic patients with clinical foot infections (11 male, 9 female; mean age 65.3 + 13 yrs). The final diagnosis was based on clinical course, f~llow-up (i.g. Xray) examinations, biopsy materials and surgical specimen. By immunoseintigraphy a correct positive diagnosis was made in 13 and a correct negative assessment in 6 out of the 20 patients. No false positive diagnosis was made in any of the patients, whereas a wrong negative assessment was made in only one of the patients. We found a sensitivity of 90% and a specificity of 90% for the immunoseintigraphy. In conclusion, the combined investigation of immunosointigraphy and three-phase skeletal sointigraphy seems to be a helpful diagnostic tool for detecting foot infections and osteomyelitis in diabetic patients.

T. R6nnemaa, I. Liukkonen, L.-R. Knuts, P. Sepp~il~l and V. Kallio, Rehabilitation Research Centre of the Social Insurance Institution, Turku, Helsinki IV College for Health Care Personnel, and Turku University Central Hospital, Finland Our aim was to evaluate the activities of chiropodist in diabetic outpatient foot care. We studied 623 patients, aged 10-80 years, who had not visited chiropodist during previous 6 months. 93 patients (group A) had obvious needs for foot care and were referred to chiropodist. The rest were randomized into chiropodist (B) and control (C; written instructions} groups. An independent chiropodist examined the patients at baseline and one-year follow-up. The knowledge about foot care increased (p<0.001) in all groups. The self-care score (0-12) also increased significantly (p<0.001; A: 5.5 to 6.9, B: 5.4 to 7.0, C: 5.3 to 6.0). The change in knowledge and self-care scores was greater (p<0.01) in B than in C. The prevalence of callosities decreased significantly (p<0.01) only in B: in calcaneal region from 18.5% to 12.0% and other regions from 54.5% to 39.5%. The size of callosities decreased in A and B. The prevalence of corns decreased in A (p<0.05) and B (p<0.01) but not in C. The prevalences of nail disorders and abnormal foot or toe posture were unchanged. The activities of chiropodist in diabetic out-patient care can significantly improve self-care habits and knowledge about foot care and reduce the occurrence of hyperkeratoses.

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54

TREATMENT OF ISCHA[~IC ULCERATION OR REST PAIN WITH INTRAVENOUS ILOPROST: A DOUBLE-BLIND PLACEBO CONTROLLED STUDY. V. Fonseca, P. Dandona, C. Belcher and U.K. severe limb ischaemia study group, The Royal Free Hospital, London and Schering Limited, Burgess Hill., U.K.

LOCAL LOW-DOSE UROKINASE IN DIABETIC PATIENTS WITH PERIPHERAL ARTERIAL OCCLUSION AND RAPIDLY PROGRESSIVE FOOT LESIONS. P. VANNINI, A. CIAVARELLA, A. MUSTACCHIO, C. ROSS I *, G. FORLANI, S. GIANGIULIO and V.GALUPPI. Dept. of Metabolic Disease and ~Inst. of Radiology, St. Orsola University Hospital, Bologna, Italy. To improve blood flow in small distal arteries of the foot, 7 type 2(non-insulin-dependent)diabetic patients, 6M/IF, age 60+7 yr,with angiographically demonstrated occlusion of infrapopliteal arteries and rapidly progressive ischaemic foot lesions were treated by local Urokinase infusion. Using a peristaltic pump,Urokinase (70000 Ul/h for 48-h and 35000 Ul/h for the successive aS-h)was infused by a 5/8 F catheter introduced through the femoral artery and placed at the occlusion site.Heparin was also administered subcutaneously at the dose of 7000 U at 8-h intervals. After baseline, arteriography was repeated after 48-h therapy and at the end of infusion period. Five patients had substained angiographic improvement as judged by recanalization of collateral vessels of trifurcation arteries and by improvement of blood flow in small arteries of forefoot and toes; in any case primary recanalization of larger arteries was achieved. Clinical improvement was evident in all but one patient as confirmed by hyperaemia and reduction of pain in the extremity,by reappearance of pedidial pulse(one case)and ameloration of foot lesions. None experienced hemorrhagic complications,catheter-related thrombosis or infections.Although primary recanalization of the large arteries of the trifurcation was not achieved,local Urokinase, opening collateral vessels, may be effective to improve blood flow in the leg and foot in diabetic patients with ischaemic foot lesions.

In a multi-centre study 151 patients with lower limb ischaemic presenting as ulcers and/or rest pain were randomised to receive either Iloprost (a stable analogue of Prostacyclin, Group I, n = 80 diabetics = 25) or placebo (Group II, n = 71 diabetics = 35). Iloprost up to 2ng/kg/in, or placebo were administed double-blind as an intravenous infusion for six hours daily over fourteen days in patients with rest: pain and twentg~eight days in patients with ulcers. Rest pain was relieved and ulcer healing improved in 45% of patients in Group I and 29% of patients in Group II (p < 0.05). During six months follow up, in Group I 38% of patients underwent amputation and 6% died, whereas in Group II 52% underwent amputation and 9% died (p < 0.05). In the subgroup of patientswith diabetes 50% in Group I and 28% in Group II survived without amputation (0.i> p > 0 . 0 5 ) . We conclude that Iloprost improves ulcer healing and relieves rest pain due to severe limb ischaemia. This improvement is maintained over sixmonths. Further trials are required in larger nt~nbers of diabetic patients.

OP 10 Hall A-15 Immunology in Man I 55

56

CIRCULATING LEVELS OF INTERLEUKIN-Ia, TUMOUR NECROSIS FACTOR-a AND INTERFERON-u ARE HIGH IN NEWLY-DIAGNOSED TYPE 1 DIABETES.

INTERLEUKIN-2, SOLUBLE 1NTERLEUKIN-2 RECEPTORS AND INSULIN AUTOANTIBODIES IN NEWLY DIAGNOSED TYPE I DIABETIC PATIENTS 1. Testa, A. Cavarape, A. Bottaccio, *E. B a r t o l o t t a , **A. Recchioni, **G. De Sio and op. Fumelli. I n s t i t u t e of I n t e r n a l Medicin and * I n s t i t u t e of P e d i a t r i c s , U n iv e r s i ty of Ancona, **Department of C l i n i c a l Pathology , Ancona Department of Diabetology 1NRCA, Ancona ( ITALY ) As in many immune diseases, I n t e r l e u k i n - 2 ( IL-2 ) and its soluble receptors ( slL-2R ) would have a ro le in pathogenesis of Type I diabetes m e l l i t u s . Aim of our study was to v e r i f y modifications of plasma l e v e l s of I n t e r l e u = kin-2 , l n t e r l e u k i n - 2 soluble receptors and i n s u l i n autoan= t i b o d l e s in young subjects with insulin-dependent diabetes m e l l i t u s observed a t the onset of disease . We studied 42 d i a b e t i c p a t i e n t s , 8 - 16 aged, with recent disease onset ( 5-30 days before ). IL-2 and slL-2R plasmatic le ve ls were determinated using immunoenzymatic methods, and i n s u l i n autoantibodies by radioimmunoassay. 24 of 30 c o n t r o l subjects showed e• low IL-2 values ( < 2 U/ml ), and a mean slL-2R l e v e l of 483 • 56 U/ml . 19 of 42 d i a b e t i c p a t i e n t s showed IL-2 le ve ls lower than 2 U/ml, 12 p a t i e n t s had values between 2 and 10 U/ml and 11 p a t ie n t s l e v e l s higher than 30 U/m1. S i g n i f i c a n t l y increased plasmatic l e v e l s were found in 32 d i a b e t i c subjects ( 896 • 162 U/ml ) . 26 of them showed 1L-2 le ve ls lower than 10 U/ml. I n s u l i n autoantibodies were found in 12 p a t ie n t s ; in the same group of subjects, slL-2R plasmatic l e v e l s were higher than in co n t ro l subjects. Abnormality of these parameters may be the expression of immunological disorders observed in Type I diabetes m e l l i t u s ; they would be useful t o value the e vo lu t io n of disease and the p o s s i b i l i t y of immunotherapy.

Hussain Watkins

MJ, P e a k m a n M, L e s l i e P J a n d V e r g a n i D.

RDG,

Viberti

Depts Immunology, Diabetes and Medicine, C o l l e g e S c h o o l o f M e d i c i n e , L o n d o n U.K.

GC,

King's

IL-I a a n d TNF-a have been reported to be c y t o t o x i c to i s l e t B c e l l s i n v i t r o , a n d T N F - a a n d i n t e r f e r o n - u a r e k n o w n to i n d u c e e x p r e s s i o n of MHC products on islet cells. Using sensitive enzyme-linked techniques, we measured plasma l e v e l s o f IL-la, T N F - a a n d I F N - y in p a t i e n t s with newly-diagnosed (all w i t h i n 6 m o n t h s o f diagnosis, n=34) and long-standing (more than 15 y e a r s s i n c e d i a g n o s i s , n = 4 5 ) t y p e 1 ( i n s u l i n dependent) diabetes and type 2 (non-insulind e p e n d e n t ) d i a b e t e s (n=29) c o m p a r i n g t h e m w i t h controls (NCs, n = 3 3 ) . L e v e l s o f IL-la, T N F - a and IFN-u were all significantly higher in newly-diagnosed type I diabetics compared to NCs (p<0.005) and compared to long-standing type I and type 2 diabetics (p<0.03). In newlydiagnosed patients, levels of TNF-~ were higher in those tested before starting insulin t r e a t m e n t ( p < 0 . 0 5 ) . In a d d i t i o n , l e v e l s o f T N F a w e r e h i g h e r in l o n g - s t a n d i n g t y p e 1 d i a b e t i c s and type 2 diabetics t h a n in N C s (p<0.001, p<0.01). In c o n c l u s i o n , high levels of the c y t o k i n e s IL-la, T N F - a a n d I F N - y c h a r a c t e r i s e newly-diagnosed patients with type I diabetes, supporting the hypothesis that they are i n v o l v e d in t h e p a t h o g e n e s i s of islet B cell destruction.

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SOLUBLE I N T E R L E U K I N - 2 R E C E P T O R S IN S I B L I N G S OF TYPE 1 (INSULIN-DEPENDENT) DIABETIC PATIENTS. G. I m p e r i a l e , M. C a s s a d e r a n d G. P a g a n o . Istituto di M e d i c i n a I n t e r n a deil'Universita'. C o r s o P o l o n i a 14 - 1 0 1 2 6 T o r i n o , Italy. We evaluated the serum levels of soluble inter leukin-2 receptors ( s I L - 2 R J as an early ma~ker iu the development of overt type 1 (insulin-dependent) diabetes mellitus. A group of 61 healthy s i b l i n g s of type 1 (insulindependent] d i a b e t i c p a t i e n t s a n d a g r o u p of i0 healthy subjects, whose family history were negative for diabetic and for immunological d i s e a s e , w e r e e v a l u a t e d for H L A s y s t e m a n d e v e r y s u b j e c t ~as s u b m i t t e d to an i.v G . T . T (glucose 0.33 g / k g b.w. J a n d t e s t e d for s e r u m level of sIL-2R. The insulin response, expressed as immunoreactive insu] in (IRI) at 2" during i . v . G . T . T . , a n d s e r u m l e v e l of s I L - 2 R for each c l u s t e r h a v e g i v e n , r e s p e c t i v e l y , the following r e s u l t s ( m e a n + S E M 7: c o n t r o l s (n=10) 1 8 3 . 2 0 + 5.40 ~ U / m l & 2 9 0 . 0 0 + 3 3 . 8 0 U/mi; D R 3 - D R 4 ( n = 2 4 7 58.86 . 5.93 p U / m l & 6 6 9 . 5 8 + 4 7 . 8 8 U/m!; HLAidentic~l (n=23) ~ 5 . 0 9 + 7 . 3 ~ }aU/ml & 4 6 5 . 8 7 _+ 2 7 . 9 9 Ulml; H L A - h a p l o - i d e n n i c a l
INDUCTION OF ANTI-ISLET CYTOTOXICITY OF MNC FROM RISK PERSONS, NEWLY DIAGNOSED AND LONG TERM TYPE-I DIABETICS BY I S L E T ANTIGEN IN VITRO H. Wanka, E. K ~ h l e r , S. Knospe, D. M i o h a e l i s C e n t r a l I n s t i t u t e o f D i a b e t e s , K a r l s b u r g , SDR The aim of the study was t o compare the inducability of anti-islet cytotoxicity of mononuclear c e i l s (MNC) f r o m patients with d i f f e r e n t immunological and m e t a b o l i c s t a t e s of d i a b e t e s m a n i f e s t a t i o n by i s l e t a n t i g e n . F r e s h l y isolated MNC w i t h o u t spontaneous a n t i - i s l e t cytotoxicity o b t a i n e d from h e a l t h y c o n t r o l s , risk persons f o r d e v e l o p i n g T y p e - I diabetes, newly diagnosed and long term d i a b e t i c patients ( d u r a t i o n > 5 y e a r s ) w i t h o r w i t h o u t r e s i d u a l Cpeptide were cultured with antigen (250 homogenized r a t i s l e t s per ml medium) f o r 2, 3 and 7 days. Thereafter the anti-islet cytotoxicity was estimated in a 6 h incubation (mXCr-release assay using neonatal rat islets as target). Induction of anti-islet cytotoxicity was achieved in all newly diagnosed Type-I diabetics, in 50 % o f r i s k persons but n o t in any o f healthy controls. MNC from long term diabetic p a t i e n t s c o u l d o n l y be r e a c t i v a t e d t o anti-islet c y t o t o x i c i t y i f t h e p a t i e n t s were Cp e p t i d e p o s i t i v e . I t i s concluded t h a t (1) i t i s possible to r e c o g n i z e probands w i t h immune pathogenesis of i s l e t d e s t r u c t i o n a l s o during immunologically inactive phases, (2) the presence o f c y t o t o x i c c e l l s i n r i s k p e r s o n s does not a l l o w c o n c l u s i o n s on a p o s s i b l e d i a b e t e s manifestation, (3) i n long term d i a b e t i c s w i t h residual C-peptide c y t o t o x i c memory cells survive.

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60

CD5 p o s i t i v e B cells in type I (insulindependent) d i a b e t i c p a t i e n t s . A. Mu~oz, T. G a l l a r ~ , 0. V i ~ a ~ , Y. S a r r i and R. Gomis. Endocrinology and Diabetes U n i t . * Immunology Unit. Hospital Clinic. Barcelona.Spain CD5 p o s i t i v e B c e l l s , which n o r m a l l y r e p r e s e n t a minor subset i n v i v o , have a t t r a c t e d much attention because of their involvement in autoimmune responses. We have investigated B CD5 c e l l s in the peripheral blood of 28 patients with t y p e 1 d i a b e t e s , aged between 730 y e a r s (mean~SD) 19.2~5.93. P a t i e n t s were divided in two groups according to disease duration: group I (< 2 months, with n=12 p a t i e n t s ) and group I I (2 months t o 2 years, with n=i6 patients). Eighteen age matched h e a l t h y s u b j e c t s were used as c o n t r o l s . B CD5 was i d e n t i f i e d by means o f double i m m u n o f l u o r escence s t a i n i n g w i t h CD5 ( B e c t o n ) , a n t i h u m a n Immunoglobulin (Tago) and analyzed by c y t o f l u o r i m e t r y (Fats Star P l u s ) . In group I , 5 patients out of 12 (41.6%) showed an increase of CD5+ B c e l l s compared to c o n t r o l subjects, in t h e same way, in group I I , 6 patients out of 16 (37.5%) showed this increase. We d i d n ' t observed any relation between the i n c r e a s e d numbers o f CD5 B c e l l s and t h e presence o f c i r c u l a t i n g a u t o a n t i b o d i e s (antiislet, a n t i i n s u l i n ) i n our p a t i e n t s . T h i s f i n d i n g c o u l d suggest t h a t CD5 B c e l l s may be involved in the pathogenesis of type 1 diabetes.

BETA-CELL CYTOADHERENT LYMPHOCYTES IN VITRO : A M A R K E R OF C E L L U L A R I M M U N I T Y IN SOME SUBJECTS AT RISK FOR TYPE 1 (INSULIN-DEPENDENT) DIABETES. V. Rohmer, S. Bardet, D. Maugendre, Y.Gallois, H. Stetieh, M. Marre, H. Allannic, B. Charbonnel and P. SaL Nantes, A n g e r s , R e n n e s - F R A N C E .

The increased binding of T-lymphocytes from type 1 diabetic patients to B-cell m e m b r a n e a n t i g e n s ("Diabetic rosettes") was previously described as a marker of cell-mediated immunity. "Diabetic rosettes" were here investigated in 247 subjects at risk for type 1 diabetes (186 first degree relatives and 61 nondiabetic subjects with transient hyperglycaemia). The number of B(RINm5F)cell-adhering lymphocytes (B-L) was higher in subjects at risk than in 228 age- and sex-matched control blood bank donors (p<10-6), and higher in 76 type 1 diabetics than in subjects at risk ( p < 1 0 - 6 ) ; the 3 populations being different for 6-L values (Kruskal-Wallis : p <0.001). T w e n t y % of subjects at risk gave "positive" tests (defined as B-L values>95th percentile of controls. The two subgroups of subjects at risk were not different for mean values +SEM of B-L or for percentages of individuals with "positive" tests. "Diabetics rosettes" were slightly a s s o c i a t e d with i n s u l i n r e s p o n s e to IV g l u c o s e (AIRG)<5th percentile of controls (IRI 1+3 rain = 35 ~tU/ml) (X2: p =0.04) and tended to be associated with HLA DR 3/4 (X2: p=0.05), but not with ICA, IAA, or with HLA DR +) T- lymphocytes. "Diabetic rosettes", a marker of cell-mediated immunity, could be detected in some subjects at risk for diabetes.

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OP 11 Hall B-3 Neural Control of B-cell Function 61

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REDUCED PANCREATICCONTENTOF THE ]iNHIBITORYNEUROPEPTIDE, GALANIN,IN GENETICALLYOBESE,HYPER]NSULINEMICMICE. B.E. Dunning and B. Ahrtn. University ef Washington, Seattle, WA, USA, and University of Lund, Lund, SWEDEN. The neuropeptide, galanin, is present in sympathetic nerves of pancreatic islets and has been implicated as a physiologic inhibitor of insulin secretion. Since abnormal autonomic neural activity occurs in

WHICH G-PROTEINS MEDIATE THE ACTIONS OF GALANIN TO INHIBIT INSULIN SECRETION? G.W.G. Sharp, M. Cormont, Y. LeMarchand-Brustel, E. VanObberghen and A.M. Spiegel, INSERM U 145, Nice, France, the Molecular Pathophysiology Branch, NIDDKD, NIH, Bethesda, USA and the Deparanent of Pharmacology, College of Veterinary Medicine, Comell University, Ithaca, USA. Galanin has multiple actions in 13-cells, including changes in ion channel activity, inhibition of adenylyl cyclase and a "distal" effect of unknown mechanism, all of which inhibit stimulated insulin secretion. As the effects of galanin are sensitive to pertussis toxin, we are studying which G protein(s) mediate these multiple effects. (1) By ADP-ribosylation and immunoblot studies, we identified Gil, Gi2, Gi3 and two forms of Go in the RINm5F cell. (2) In RINm5F cell membranes 125I-galanin binding was decreased by GTP and GTTS by up to 60% because of reduced galanin-receptor affinity (shown by Scatchard analysis and dissociation rate studies) which, in turn, is caused by dissociation of the G protein(s) from the receptor. Therefore, with specific antipeptides against the C-terminal decapeptides of the c~-subunitsof the G-proteins, it should be possible to block the G protein-receptor interaction and identify the G protein(s) specific for the galanin receptors. Preincubation of membranes with antipeptide reacting with GiM and Gi~.2decreased the binding of 125Igalanin under conditions in which preincubation with antipeptides against Gia3, Go and control IgG had no effect. These data implicate Gil and Gi2 as potential mediators of the multiple effects of galanin in ~-ceUs.

several animal models of obesity, we hypothesized that the hyperinsulinemia of genetically obese (ob/ob) mice may result from reduced pancreatic "galaninergic" input. As an index of this input, we measured pancreatic content of galanin-like immunoreactivity (GLIR). We compared pancreatic GLIR and plasma insulin in 14 female ob/ob mice (aged 2-5 months) with 16 female age-matched lean littermates. In lean mice, pancreatic GLIR was inversely correlated with plasma insulin (r=-0.601, p<0.01), supporting a role for pancreatic galanin in regulating insulin secretion. Pancreatic GLIR was 13-fold lower in ob/ob v s lean (0.38+0.03 v s 5.15+0.59 fmol/gg protein, p<0.001), reflecting both higher protein (1390-/--70 v s 730-/--30 gg/pancreas) and lower GLIR (520-/-_50v s 3760i-_470 fmol/pancreas). Correspondingly, plasma insulin was 12-fold higher (1250!-_30v s 108+18 pM, p<0.001 ). The reduced pancreatic GLIR in ob/ob mice appeared organ-specific, since adrenal GLIR was not reduced in ob/ob v s lean (5.20i-_0.20 v s 3.32+0.25 fmol/gg protein). We conclude that pancreatic galanin may be an important factor in determining plasma insulin in lean mice and a specific reduction of pancreatic galaninergic input may contribute to hypefinsulinemia in ob/ob mice.

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CHOLINERGIC POTENTIATION OF THE IM/~DIATE INSULIN RESPONSE TO GLUCOSE AND FOOD INTAKE

ADRENERGIC INHIBITION OF INSULIN SECRETION FROM MOUSE B-CELLS DOES NOT INVOLVE REDUCED CALCIUM CURRENT

J.H.Strubbe, J.E.Bruggink and A.B.Steffens, of Animal Physiology, Haren, The Netherlands.

K. W&hla~der, C. Amm~l~, K. Bokvist, P. Arkhammar*, P.O. Berggren and P. Rorsman. Department of MEdical Physics, GSteborgs Universitet, GSteborg and Department of Endocrinology, Karolinska Institute, Stockholm, Sweden.

Department

The aim of the present study was to investigate the in vivo influence of the parasympathetic nervous system on the dynamics of insulin secretion during intravenous glucose infusion and food intake in rats. This was performed in 7 male rats by rapid blood sampling every i0 seconds via permanently implanted cannulas in the hepatic portal vein without disturbing the animals. Already i0 seconds after the start of glucose infusion (i0 mg/kg/ min) via a chronic cardiac cannula, plasma insulin increased in the portal vein 47 • ii mU/l to a maximum increment of 168 • 21 mU/l (mean • SEM) at 40 seconds. In con trast after blockade of peripheral cholinergic muscarinic receptors with methylatropine (0.5 mg/kg), =he insulin response was delayed and rose from 30 seconds onward to a maximum increment of i01 • 17 mU/l at 60 seconds which was lower than without blockade (p < 0.05). After food intake plasma insulin already rose 38 • 19 mU/l at i0 seconds. This was independent of a rise in blood glucose levels which occurs only after 2 minutes. The maximum increment of 105 • ii mU/l was attained after 50 seconds. After treatment with methylatropine this response was abolished. This study demonstrates that after glucose infusion and food intake insulin is secreted with an extraordinary rapidity which is dependent on a strong potentiating influence of the parasympathetic nervous system.

We have investigated whether ~2-adrenergic inhibition of insulin secretion from mouse ~-cells involves reduction of the Ca-current. Insulin secretion from individual E-cells was determined using the haemolytic plaque assay. In the presence of a low glucose concentration (2.8 mM), 4% of the cells formed plaques. The figure increased to >70% in the presence of 20 mM glucose. Addition of 2 ~M clonidine abolished glucose-stimulated insulin release and only 5% of the cells formed plaques. Clonidine (5 gM) inhibited electrical activity evoked by glucose (20 mH) and tolbutamide (200 ~M). This action was antagonized by yohimbine (20 ~M). Whole-cell Ca-currents were measured using standard patch-clamp techniques. Addition of 5 NM clonidine increased the Ca-current density from 24 pA/pF to 32 pA/pF (P<0.05). This effect was not observed after inclusion of GTP (i mM] or the non-hydrolyzable analogue GTPzS (0.2 mM) in the intracellular solution. When Ca-currents were measured in intact E-cells using the perforated patch whole-cell configuration, clonidlne (1-5 ~M) also failed to reduce the Ca-current amplitude. It is concluded that inhibition of ~-cel] Ca-currents is not involved in ae-adrenergic suppression of insulin secretion in mouse E-cells.

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CHARACTERIZATION OF THE me+RECEPTOR SUBTYPE RESPONSIBLE FOR INHIBITION OF INSULIN SECRETION IN RAT ISLETS.

BLOCKERS OF o~-ADRENOCEPTORS HAVE MORE THAN ONE SITE OF ACTION IN PANCREATIC B CELLS. T.D. Plant and J.C. Henquln. I. Physiologisches Institut, University of Saarland, Homburg/Saar, Germany and Unit6 de Diabttologie et Nutrition, University of Louvain, Brussels, Belgium.

S.L.F. Chan and N.G. Morgan, Department of Biological Sciences, University of Keele, Staffs, ST5 5BG, U.K. Recent evidence has demonstrated that ~2-adrenergic receptors do not comprise a homogeneous population but that receptor subtypes exist. A subclassification scheme of ~2A and ~2B has been proposed based on differences in the relative binding affinity of the antagonists, oxymetazoline (~2A-selective) and prazosin (~2B~-selective). To investigate which subtype of ~2-adrenoceptor is responsible for inhibition of insulin secretion in rat pancreatic islets, we have employed oxymetazoline and prazosin, together with two novel ~2A- and ~2B-S~lective antagonists, BRL 44408 and BRL 41992. BRL 44408 (~2Aspecific) significantly reversed the inhibition of i n s u l ~ secretion induced by IBM UK, 14-304. This effect was dose-dependent (0.1-100DM), with an ECs0 of approximately 5BM and complete reversal at 1OONM. In contrast, BRL 41992 (~2R-specifie) caused only slight relief of inhibition (10% at 100~M). These results suggest that the functional ~ - r e c e p t o r in islets is of the ~2A subtype. Data from similar studies with oxymetazoline and prazosin confirm this, since oxymetazoline exhibited a greater capacity to relieve the ~-adrenergic inhibition of i n s u l ~ secretion. In further support of these conclusions, islet ~z-receptors could be labelled with 3H-RX821002, a newly available ligand which labels ~2A-receptors. The KD for binding was 5.90 • 0.79nM and the total number of receptor sites (Bma• averaged 589 • 78 fmol/mg protein

(n=9).

Circumstantial evidence suggests that phentolamine might increase insulin release by mechanisms unrelated to the blockade of B cell adrenoceptors. Normal mouse islets were used to investigate whether ATP-sensitive K§ channels (K§ channels) could be a target for phentolamine. In 3 mmol/l glucose, phentolamine irreversibly inhibited ~Rb efflux from islet cells. The rate of this inhibition, but not its steady state magnitude was dose-dependent (20-100 ~tmol/1 phentolamine). The large acceleration of 8~Rb effiux caused by 100 ~tmol/l diazoxide in 6 mmol/1 glucose was almost completely prevented by 20 ~tmol/1 phentolamine. ATPsensitive K § currents in B cells were measured by the patchclamp technique in the whole cell mode. They were progressively but nearly completely inhibited by phentolamine which also prevented their activation by diazoxide. In contrast, voltagedependent K+ currents were only 30 % inhibited by phentolamine. Yohimbine, a blocker of oh-adrenoceptors, which is structurally unrelated to phentolamine, mimicked the effects of the latter on StRb efflux and K+-ATP channels. However, its potency was much less than that of phentolamine and its effects were reversible. In conclusion, phentolamine exerts a sulphonylurealike action in B cells. Blockade of K+-ATP channels rather than of r may account for the increase in insulin release that phentolamine causes at high concentrations.

OP 12 Hall A-18 Metabolism in vitro 67

68

INSULIN STIMULATES RAT SCIATIC NERVE Na+/K § ATPASE VIA PROTEIN KINASE C C.B. Etflinger, S. Parton and D.R. Tomlinson, Department of Pharmacology, Medical College of St. Bartholomew's Hospital, London, U.K. This study examined the effect of insulin on Na+/K+-ATPase activity in peripheral nerves in vitro. Sciatic nerves were removed from freshly-killed rats, desheathed and the perineurium of the major fascicle fenestrated by microdissection. These preparations were incubated in Krebs-Henseleit bicarbonate buffered saline containing 0.5 mM myo-inositol, 5.0 mM glucose and 3% bovine serum albumin, gassed with 95% Oz/5% CO z. Na+/K+-ATPase activity was measured as [86Rb§ during a 30 rain incubation -+ 2 mM ouabaln. Insulin (10 nM) increased basal Rb uptake by 21% (p<0.01) in the absence of ouabain, but had no effect when ouabain was present, indicating a selective stimulation of Na+/K+-ATPase. The effect of insulin was completely prevented by the protein kinase C antagonist H-7 (1,5 (isoquinolinylsulfonyl)-2-methylpiperazine) at 50/~M, but H-7 was without effect on basal Rb pumping. Basal Rb pumping was also increased by 30% (p<0.05) by 20 /.tM phorbol dibutyrate, demonstrating that a protein kinase C activator has an effect similar to that of insulin. Again ouabain-resistant Rb pumping was unaffected by phorbol dibutyrate. These findings indicate that insulin stimulates Na+/K+-ATPase via activation of protein ldnase C.

EFFECTS OF INSULIN ON LACTATE METABOLISM IN ISOLATED HEPATOCYTES. T.M. Greenaway, G.J. Cooney, I.D. Caterson and J.R. Turtle, Dept.of Medicine, University of Sydney, N.S.W. 2006, Auslralia. Hepatic insulin resistance is a major feature of non-insulindependent diabetes and obesity. However little is known about the subcellular mechanisms that result in increased hepatic glucose and triacylglycerol production in the presence of normal or elevated insulin concentrations. This study was aimed at identifying insulin sensitive pathways of lactate metabolism in hepatocytes that could help determine the intracellular changes occurring in liver in insulin resistance. Hepatocytes were isolated from fasted rats by a collagenase perfusion technique. Hepatocytes were incubated with 5mM L-[u-lac] lactate with or without insulin (10-tM), and lactate conversion to CO2, glucose and triacylglycerol determined. Insulin had no effect on the conversion of lactate to CO2 but lactate conversion to glucose was decreased 15% by insulin (42.5+1.3 vs 34.8+l.7nmol/30mird106cells; P<0.05). The major effect of insulin was to decrease lactate conversion to triacylglycerol by 37% (6.1_+0.5 vs 3.9+0.7 nmol/30min/106cells; P<0.01). This effect was entirely due to a decrease in lactate incorporation into the glycerol moiety of triacylglycerol and represents an insulin-induced inhibition of the gluconeogenic conversion of lactate to glycerol-3phosphate. Absence of this insulin effect may partly explain the increased hepatic glnconeogenesis and lipogenesis observed in insulin resistance.

A29

69 OVERTRANSCR}PTION OF GENES INVOLVED IN FAT STORAGE IN A MODEL OF GENETIC OBESITY, A M P L I F I C A T I O N BY H Y P E R I N S B L I N E M I A . I. D U G A I L , A. Q U I G N A R D - B O U L A N G E , X. Le L I E P V R E , C. G U I C H A R D , and M. LAVAO. I N S E R M U177, 15 rue de l ' @ c e l e de m ~ d e c i n e Paris, F R A N C E . We h a v e shown previously that adipose tissue from young genetically obese Zucker rats was c h a r a c t e r i z e d by a higher content of mRNA e n c o d i n g for F a t t y a c i d s y n t h e t a s e (FAS), M a l i c enzyme (ME) and Glyceraldehyde-3-phesphate d e h y d r o g e n a s e (GAPDH). The aim of t h i s s t u d y was to examine whether increased rates of gene transcription could explain these observations. N u c l e i w e r e i s o l a t e d f r o m a d i p o s e t i s s u e of lean (Fa/fa) and obese (fa/fa) Zucker rats aged e i t h e r 16 or 30 days. N u c l e a r r u n - o n a s s a y was p e r f o r m e d by incubating nuclei w i t h lOOuCi of ~P CTP, and labelled transcripts were h y b r i d i z e d to recombinant plasmids. In 3B-day old hyperinsulinemie o b e s e rats, both m R N A and t r a n s c r i p t i o n rate w e r e markedly stimulated : ME x 8, FAS x 15, G D P H x I0 and G A P D H x 6, over control values. At 16 days of age, w h e n m u t a n t pups are still normoinsulinemic, the h i g h e r level of mRNAs in a d i p o s e tissue could be explained by a parrallel increase in t r a n s c r i p t i o n rates : ME x 2, G A P D H x 2, and FAS x 3, suggesting a genotype effect i n d e p e n d a n t of h y p e r i n s u l i n e m i a . At this age, G P D H mRNA levels and t r a n s c r i p t i o n rates were u n a f f e c t e d in o b e s e rats. In conclusion, the fatty genotype exerts a control on the e x p r e s s i o n of genes encoding for lipid s t o r a g e enzymes at the transcriptionnal level. This genotype effect is greatly amplified by the d e v e l o p m e n t of hyperinsulinemia.

71 EFFECTS OF GROWTH HORMONE ON INSULIN RECEPTOR AND GLYCOGEN SYNTHASE ACTIVITY IN HUMAN MUSCLE J.F. Bak 1, N. Moller2~ and O. Schmitz 1. Medical Endocrinological Dept III, Aarhus Amtssygehus 1, and 2nd Univerity Clinic of Internal Medicin, Aarhus Kemmunehospital 2, DK-8000 Aarhus C, Denmark Elevated serum growth hormone (GH) levels in malregulated insulindependent diabetes may impair insulin sensitivity. To elucidate this effect of GH seven healthy male subjects were examined twice with a 5 h euglycemic clamp. Insulin and GH (Velosulin and Norditropin, Novo-Nordisk, Copenhagen) were infused at rates of 0.5 m U / k g / m i n and 45 n g / k g / m i n , respectively. In the control experiment no GH was given. GH infusion inhibited the glucose disposal by 27 % from 4.44 + 0.74 to 3.25 • 0.39 m g / k g / m i n (p<0.02). In muscle biopsies obtained 15 minutes before termination of the clamp experiment insulin receptor binding as well as basal and insulin-stimulated kinase activity of the solubilized receptors were similar in the control and the GH clamp. However, the GH infusion decreased the fractional velocity of the muscle glycogen synthase during moderate hyper-insulinemia by 41 from 14.1 • 2.5 ~ to 8.3 + 1.4 % (p<0.03). The total activities of the glycogen synthase were identical: 40.6 • 3.2 vs. 38.8 • 1.6 U/rag protein. In conclusion, the diabetogenic effect of GH involves a post-insulin-receptor antagonism of insulin action on the glycogen synthase in skeletal muscle.

70 INTERACTION BETWEEN GLUCOSE AND INSULIN ON D E V E L O P M E N T OF I N S U L I N R E S I S T A N C E IN M U S C L E B.F.Hansen, T.Ploug, J.Bak, S.A.Hansen and E.A.Richter, August Krogh Institute; Panum Institute, University of Copenhagen, Arhus A m t s s y g e h u s , Arhus, Denmark.

R a t h i n d q u a r t e r s w e r e p e r f u s e d for 2h w i t h O, 5 or 25 m M g l u c o s e and O, 50 or 2 0 , 0 0 0 H U / m l insulin whereupon responsiveness to 20,000 gU/ml i n s u l i n w a s tested. P e r f u s i o n w i t h 25 m M g l u c o s e and m a x i m a l i n s u l i n i n i t i a l l y r e s u l t e d in g l u c o s e u p t a k e of 43.6 • 3.9 B m o l / g / h w h i c h d e c r e a s e d to 28.7 • 1.6 B m o l / g / h a f t e r 2h (p
72 DIFFERENTIAL SUPPRESSION OF GLUCOSE UTILIZATION AND PYRUVATE OXIDATION IN MUSCLE BY LIPID FUELS M.J. Hoiness, Y.-L. Lui and M.C. Sugden, Department of Biochemistry, London Hospital Medical College (University of London), Turner Street, London E1 2AD, U.K. Several insulin-resistant states are associated with increased lipid oxidation, but the tissues and loci at which insulin resistance is manifested are not precisely defined. The aim was to delineate the effects of an increased lipid supply on glucose phosphorylation and oxidation in cardiac and representative working or non-working skeletal muscles. Glucose phosphorylation was estimated in conscious, resting rats using 2-[~H]deoxyglucose. Oxidative capacity was assessed by measurement of active pyruvate dehydrogenase complex (PDHa). Acute elevation of fatty acid supply was achieved by intragastric administration of lipid followed by heparin injection. After 3h, glucose phosphorylation was decreased in heart, but not in either working or non-working skeletal muscles. PDHa was uniformly decreased. After 1-2h, cardiac glucose phosphorylation was not decreased, but cardiac PDHa was reduced. After 4-6h, modest suppression of glucose utilization was observed both in working skeletal muscles and heart. The results demonstrate: (i) a variable, time-dependent effect of fatty acids on glucose utilization in individual muscles; (ii) differential sensitivity of glucose phosphorylation and pyruvate oxidation to lipid, with greater impairment of pyruvate oxidation than glucose phosphorylation. Therefore, both tissue and pathway specificity exists with respect to the extent to which fat oxidation affects in r i v e glucose disposal.

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OP 13 Hall A-1 Microalbuminuria and

Nephropathy

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Diabetic n e p h r o p a t h y - an inherited complication ? B o r c h - J o h n s e n K., Norgaard K., Hommel E., Mathiesen, E.R., Jensen J.S., Parving H.-H. and Deckert T.. Steno Memorial Hospital, Gentofte and Hvid~re Hospital, Klampenborg, Denmark.

PREVALENCE OF M I C R O A L B U M I N U R I A WITH TYPE 1 DIABETES.

Only 30-45% of I D D M - p a t i e n t s d e v e l o p diabetic n e p h r o p a t h y (DN). Poor glycemic control in itself does not explain this phenomenon, and genetic p r e d i s p o s i t i o n to DN has been suggested. If so, familial c l u s t e r i n g of DN should be expected. Among a r e p r e s e n t a t i v e p o p u l a t i o n of patients with IDDM, we i d e n t i f i e d all patients with DN ( U r i n a r y A l b u m i n E x c r e t i o n (UAE) > 300mg/24h ) who had a sibling w i t h IDDM (N=20) and a c o m p a r a b l e group of n o r m o a l b u m i n u r i c patients ( UAE < 30mg/24h ), also h a v i n g a sibling with IDDM (N=29). Diabetic n e p h r o p a t h y was found in 7 of 21 siblings to patients with DN and in 3 out of 30 siblings to n o r m o a l b u m i n u r i c patients ( p = 0 . 0 3 5 ) . Incipient or overt n e p h r o pathy (UAE > lOOmg/24h) was found in 43% and 13% of the siblings r e s p e c t i v e l y (Odds ratio 4.9). Age at diagnosis, diabetes duration, blood pressure and HbAlc did not differ b e t w e e n the two groups of siblings. HbAlc showed a significan positive c o r r e l a t i o n w i t h i n sib-pairs (R=0.47, p< 0.001). In conclusion: Familial c l u s t e r i n g of DN does occur, either due to genetic s u s c e p t i b i l i t y or due to "environmental inheritance" (e.g. sibs i m i l a r i t i e s due to shared environment).

A nation-wide screening for m i c r o a l b u m i n uria in D e n m a r k was p e r f o r m e d in 1020 children with Type 1 diabetes (approximately 80% of total). In 209 n o n - d i a b e t i c subjects upper 95% limit for albumin excretion rate (AER) was 20 ~g/min. Mean AER was s i g n i f i c a n t l y elevated in diabetic (3.0 vs. 1.7 ~g/min, p<0.001) and non-diabetic (2.5 vs. 1.3 ~g/min, p<0.001) adolescents (>12-19 years) compared with preadolescents (<12 years). In the diabetic group AER was p o s i t i v e l y c o r r e l a t e d to body surface area and age. Among the diabetic patients 5% had AER above 20 ~g/min (persistent microalbuminuria) and seven had overt proteinuria (>150 ~g/min) in at least two timed overnight urine collections. Females with AER >20 ~g/min had s i g n i f i c a n t l y higher mean HbAlc level (10.8%) than those with AER <20 ~g/mln (9.8%, p<0.03) and impaired linear growth (SDS score: -0.25 vs. +0.16, p<0.003). These a s s o c i a t i o n s were not found in males. Mean blood pressure was s i g n i f i c a n t l y higher (94 mmHg) in the group of diabetic patients w i t h AER >20 ~g/min than in the group w i t h AER <20 ~g/min (87 mmHg, p<0.0001). The "analyses suggest that hormonal changes in puberty, poor glycemic control (particularly in females) and elevated arterial blood pressure may be related to the i n c r e a s e d prevalence of microa l b u m i n u r i a o b s e r v e d in adolescents with Type 1 diabetes.

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Progression to microalbuminuria in normealbuminuric Typel (insulin-dependent)diabetic patients.

ASSOCIATION OF MICROALBUMINURIAWITH ACETYLATORPHENOTYPE IN TYPE I (INSULIN-DEPENDENT) DIABETIC CHILDREN L.Maddcsy,I.SzGrddy,A.Sdnta,L.Barkai and I.Vdmosi ( I I Dept

J. Messent, Guy's Hospital, London. U.K. One hundred and forty-nine normoalbuminuric (albumin excretion rate AER<30mcg/min) and non-hypertensive (blood pressure SP<160/95mmHg without medications) Type 1 (insulin-dependent) diabetic patients (age 18-60yrs,duration of diabetes<35yrs) were recruited into a prospective study of the natural history of AER. They were seen every 6 months when sitting BP was recorded to the nearest 2mmHg in the right arm using a random zero sphygnomanometer and a timed overnight urine specimen and blood were collected for the measurements of AER and glycosylated haemoglobin (HbAI). We report on 133 patients (73M,60F] who have completed at least 24 months follow-up. During this period 6 patients (4M,2F, age 22-62yrs, duration of diabetes ll-35yrs) developed microalbuminuria giving a cumulative incidence of 4.6%. The progressors had a significantly higher HbAI at baseline and over the study period (mean 9.9 vs 8.8%, p<0.01). Mean BP was also higher in the progressors at baseline (mean 98.4 vs 89.1mmHg, p<0.05) and throughout the study. At recruitment mean AER was 16.5mcg/min (CI 7.3-25.Smcg/min) in the progressors and 8.3mcg/min (CI 6.9-9.8mcg/min)(p<0.05]. In conclusion the progressors had poorer glycaemic control, higher baseline mean BP and pre-study AER than the non-progressors.

of Ped.,Postgrad.Med.Univ.,Miskolc and Children's Hospita~ Szeged,Hungary) The aim of the study was to investigate the genetically determined acetylator phenotype in diabetic children withand without increased urinary albumin excretion.Acetylator phenotype was determined according to Evans and 24 h albumin excretion rate (AER) was measured using a RIAmethod in 86 children and @dolescents with Type l (insulin-dependent) diabetes mellitus.In all subjects, the fast acetylator phenotypa was found in 36 (41.9%) cases and the slow one in 50 (58.1%).Ameng patients w~th

IN

CHILDREN

H.B. Mortensen, K. Marinelli, K. Nergaard, K. Main, K.W. Kastrup, K.K. Ibsen, J. Villumsen, H.-H. P a r v i n g and The Danish Study Group of Diabetes in Childhood. Hvidore Hospital, Klampenborg, Denmark.

normal albumin excretion (Group ~: n=70,mean age:ll.9-).5 years,mean diabetes duration:5.)-3.8 years,AER<20 pg/min) ]5 (50%) fast acetylators and 35 (50%) slow acetylators

were found.In patients with ~levated albumin excretion (Group B: n=~6,mean age:14.0-).2 years,mean diabetes duration:4.9-}.O years,AER>20 ~g/min) 1 (6.]%) patient was fast acetylator and 15 (93.7%) were slow acetylators. A significant difference has been found between Groups A and B in the rate of slow/fast acetylators,and in the age of patients (p
detect patients

"at risk" of

nephropathy.

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INCREASED SPERMIDINE CONTENT IN ERYTHROCYTES OF TYPE 1 (INSULIN-DEPENDENT) DIABETIC PATIENTS L~ITH NEPHROPATHY G.Seghieri, A.Gironi,L.Alviggi,P.Caselli,L.A.De Giorgio 7 and G.C.Bartolomei. Diabetes Unit, Spedali Riuniti,Pistaia, Italy. Altered cell growth processes have been described as an hallmark of Type I (insulin-dependent) diabetes,especially when associated with its microsngiopatic complications. Since polyamines spermidins (Sd) and spermine (Sm) are involved in the pathways of cell growth ,this study is aimed at evaluating whether the erythrocyte content of polyamines, assayed by HPLC and expressed as nmol/lOOmlRBPC~ is modified in patients

GROWTH DISTURBANCES AND LONG TERM GLYCEMIC CONTROL IN CHILDREN AND ADOLESCENTS WITH MICROALBUMINURIA. II.Glosli, H.J.Bangstad, K.Dahl-J~rgensen and ~.Aagenms. Aker Diabetes Research Center, Aker University Hospital, Oslo, Norway. The aim of the study was to compare growth and long term (5 years) glycemic control in diabetic children and adolescents with and without mieroalbuminuria. Twenty children with urinary albumin excretion rate (UAE) >15ug/min in at least 2 out of 3 overnight urine collections and at least four HbAl-measurements annually for five years were compared with a group of normoalbuminuric patients (n=20) matched by an independent investigator for sex (9 girls, Ii boys), age (mean 18.4 vs 18.5 years) and duration of diabetes ~I0.8 vs i0.0 years). UAE in the microalbuminuric group was 51.7+37.0 ug/min (range 15.9 to 140.2 ug/min) and 6.8+3.2 ug~min (2.6-13.3 ug/min) in controls (p<0.001), an~ mean 5-years HbAI: 12.3% (95% confidence intervall ii.5-12.9%) vs 10.3% (95% confidence intervall 9.7-11.1%) in controls (p<0.01). Considered as a group the boys had a reduced growth spurt, and the girls nearly no pubertal growth spurt at all. The growth spurt was delayed mean 6 months in boys. Menarche appeared at 14.1+1.7(SD) years of age. In boys height velocity (cm/year) was reduced in microalbuminuric patients compared to controls (p<0.05). The growth spurt in girls and boys was mostly reduced in the patients with the 50% highest HbAI (HbAI>II.4%) (normal <7.6%). In conclusion: Diabetic teenagers with microalbuminuria and high HbAI are not only at risk for development of late complications~ but also for growth disturbances.

affected by Type 1 (insulin-dependent) diabetes (n=38) with or without retinopathy~ persistent microalbuminurie (HA; urinary albumin excretion between 20 and 200 tJg/min, UAE) or overt diabetic nephropathy (ON; UAE persistently> 200/~g/min), as compared with 78 sex and age matched controls. ~hile mean Sm was similar in both diabetics (19.51+13.3(SD)nmoI/IDOmlRBPC) and controls (16.95+6.56nmo~/100mlRBPC), Sd was higher in the former group (30.62+11.53nmol/100mlRBPC) then in controls (23.65+6.44nmol/100mlRBPC; p=O.O01). Only Sd was significantly higher in either HA (n=11; 36.07+14,73 nmoI/IOOmlRBPC) or ON patients (n=~; 35.6+g.22nmol/IO0 mlRBPC) than in both normoslbuminurics (n--'23; 27.14+ 9.04nmol/1OOmiRBPC) and controls (F=9.78; p=O.O001), being correlated with IogUAE (r=O.~1; p=O.O09). Similarly proliferative rstinopathy we~3 associated with a significant increase in erythrocyts Sd content (n=5; 32.08+11.21nmol/1OOmlRBPC), as compared to controls (p=O.O009)~ In conclusion a raised erythrocyte Sd content seems to be a selective marker of both nephropethy and advanced retinal damage in Type I (insulin-dependent) diabetes.

OP 14 Hall A-2 Glucose Transporters in Animals 79

8O

TISSUE SPECIFIC UP OR DOWN REGUI .&TIONOF GLUT-4 GLUCOSE TRANSPORTERS mRNA BY HYPERINSULINEMIA.

UBIQUITOUS GLUCOSE TRANSPORTER GLUT- 1 IS A PUTATIVE GLUCOSE-REGULATED PROTEIN (GRP). E. Wertheimer, S. Sasson, E. Cerasi and Y. Ben-Neriah. Departments of Endocrinology, Pharmacology and Immunology, Hebrew University Hadassah Medical Center, Jerusalem, Israel.

I. Cusin, J. Terrettaz, F. Rohner-Jeanrenaud, N. Zarjevski and B. Jeanrenaud. Laboratoires de Recherches M6taboliques, Universit6 de Gen6ve, Switzerland. The role of hyperinsulinemia induced by insulin infusion (via minipumps) for 4 days to normal rats has been investigated on the relative abundance of insulin-responsive glucose transporter mRNA (GLUT-4) of white adipose tissue and muscles. Three groups were included : 1) control rats infused with saline; 2) hyperinsulinemic rats with resulting hypoglycemia; 3) hyperinsulinemia with normoglycemia obtained by superimposed glucose infusion. Hyperinsulinemia was in the range of 300 #U/ml. At day 4, all rats were sacrificed, the inguinal adipose tissue, the tibialis and the diaphragm were removed. The relative abundance of GLUT-4 mRNA (Northern blots) in adipose tissue was increased 17-fold and 5-fold in hyperinsulinemichypoglycemic and hyper-insulinemic-normoglycemic rats respectively, compared to controls. In contrast such relative mRNA abundance was decreased in tibialis and diaphragm by 3-fold and 2-fold in hyperinsulinemic-hypoglycemic and hyperinsulinemic-normoglycemic rats, respectively, compared to controls. These changes were corroborated with an increase in the glucose utilisation index in adipose tissue and a decrease in that of the muscles during clamps associated with the labelled 2-deoxyglucose method. Conclusion : chronic hyper-insulinemia produces a tissue specific up-, or down-regulation of the expression of GLUT4 mRNA which may be of relevance for the understanding of type 2 diabetes.

GLUT-1, the brain/erythrocyte glucose transporter, is expressed in most cells, in contrast to GLUT-4 which is restricted to insulinresponsive tissues. It is not clear whether GLUT-1 has a function distinct from that of tissue-specific transporters. Previous work has shown that GLUT-1 mRNA and protein are regulated by glucose, its withdrawal being a strong stimulus. Since ceils respond to glucose starvation by increasing the stress protein family of glucose-regulated proteins (GRP), we investigated whether GLUT-1 is regulated similarly. We studied the mRNA levels of GLUT-1 and GLUT-4, and of GRP 78 and GRP 94 by Northern and slot-blot analyses in L8 myocytes and other cell lines, subjected to stimuli known to increase GRPs (glucose deprivation, calcium ionophore A23187,tunicamycin and 13-mercaptoethanol). All conditions strongly stimulated timedependently the mRNA levels for GLUT-1 and GRPs in parallel, while GLUT-4 and g-actin mRNAs were not increased. Total myocyte GLUT-1 protein, studied by Western blot with GLUT-1 specific antiserum, increased several fold after exposure to the stimuli. We suggest that GLUT-1 belongs to the family of GRPs and plays an important role for cell survival under stress conditions.

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EVIDENCE FOR THE EXISTENCE OF A SPECIFIC CONTRACTION REGULATABLE GLUCOSE TRANSPORTER IN SKELETAL MUSCLE. T. Ploug, H. Galbo and J. Vinten. Institute of Medical Physiology B, The Panum Institute, University of Copenhagen, Denmark. In skeletal muscle contractions and insulin stimulate glucose transport in an additive manner, suggesting that stimulated transport is mediated by different glucose transporters (GT). To investigate this we prepared a plasma membrane enriched vesicular fraction from rat hindleg muscle. Glucose transport into vesicles prepared from muscle stimulated with both insulin and contractions was increased 20 fold compared to control. Contractions alone accounted for about 60% of the maximum increase in transport whereas insulin alone accounted for about 35%. Cytochalasin B binding sites were identical in membranes from control and stimulated muscle (15.5 pmol/mg). Following photolabelling with 3H-cytochalasin B membranes were solubilized and GT precipitated with an antibody against the muscle/adipocyte GT (GLUT4) or an antibody against the HepG2/erythrocyte GT (GLUT1). Approximately 30% of total photolabelled GT could be removed by GLUT4 antibodies whereas only I0 % could be precipitated by GLUTI antibodies. Immunoprecipitation efficiency was better than 90% as verified by Western blotting. Thus, approximately 60% of the GT in our membrane fraction is neither GLUT4 nor GLUTI. Our findings indicate that contraction stimulated glucose transport is mediated by an unique, yet unsequenced protein. This new transporter may account for the major part of glucose transport in adult skeletal muscle.

DEFECTS OF INSULIN-RESPONSIVE GLUCOSE TRANSPORTER mRNAs IN PRE-OBESE PUPS. N. Zarjevski, J. Terrettaz, I. Cusin, F. Rohner-Jeanrenaud and B. Jeanrenaud. Laboratoires de Recherches M6taboliques, Universit6 de Gen~ve, Switzerland. It is not clear whether insulin resistance of type 2 diabetes is an early event possibly preceding other metabolic-hormonar changes. To attempt answering this question, young lean and pre-obese pups of the fa/fa strain were investigated. As pups are small, metabolic studies are difficult. Thus, the measurement selected was that of the relative abundance of insulinresponsive glucose transporter mRNA (GLUT-4) of white adipose tissue and muscles in 21-day (before weaning) and 31-day (after weaninz) old lean and oreobese pups. The relative abun/tance of GLUT-4 mRNA m adipose tissue was increased by 2-fold in 31day-old pre-obese pups, compared to controls. More strmkingly, the relaiive abundance of mRNA was also increasei2 in the extensor digitorum lon~us-tibialis muscles of 31-day-old pre-obese compared fo controls (arbitrary units: 4862+458 versus 2784_+301, respectively) and. in the diaphragm of pre-obese ~roups compared to controls (arbitrary units: 2603-+357 vs 510___115, respectively for 21-day-old pups; 2921-+627 vs 1460-+141, respectively for 31-dayold pups ). All differences were: p<0.05 or smaller, n=5-6. Although mRNA levels may not always reflect the actual amount of protein formed (currently investigated), these results favor the view that insulin resistance a f the level of glucose transporter mRNAs is not an early event ot the pre-obese state.

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84

SPECIFIC OVER-EXPRESSION OF ADIPOSE CELL~MUSCLE GLUCOSE TRANSPORTER, IN YOUNG, HYPERINSULINEMIC, OBESE ZUCKER RATS. I . H a i n a u l t , M . G u e r r e - M i l l o , C.Guichard and M.Lavau. INSERM U 177, 15 rue de l ' E c o l e de M~decine, PARIS, FRANCE.

Substitution of leucine for tryptophan 412 of GLUT1 glucose transporter decreases transport activity. Y. OKA, T. ASANO, H. KATAGIRI Y. Akanuma* and F. Takaku. Third Department of Internal Medicine, University of Tokyo, *Institute for Diabetes Care and Research, Asahi Life Foundation,Tokyo, Japan To investigate the structure-function relationship, rabbit HepG2-type (GLUT1) glucose transporter cDNA was engineered such that tryptophan 412, putative cytochalasin B photoaffinity labeling site, of glucose transporter was mutated to leucine. The mutated cDNA was subcloned into the expression vector and transfected into Chinese hamster ovary (CHO) cells. Immunoblotting with glucose transporter antibody demonstrated a stable over-expression of the mutated glucose transporter. In addition, incorporation of the mutated glucose transporter into plasma membranes was demonstrated by cell surface labeling of glycoproteins and subsequent immunoprecipitation of glucose transporter. However, the uptake of 2deoxyglucose (0.1 mM) was not significantly increased compared with control CHO cells, whereas 5-fold increase in 2-deoxyglucose uptake was observed in CHO cells with a similar amount of expressed wild-type glucose transporter. Although cytochalasin B labeling was observed in the mutated glucose transporter, inhibition of the labeling by D-glucose was markedly decreased compared with wild-type glucose transporter. These results suggest that 1. cytochalasin B photoaffinity labeling site is not tryptophan 412, 2. substitution of leucine for tryptophan 412 markedly decreases the glucose transport activity due to the decreased affinity for D-glucose.

At l e a s t two glucose t r a n s p o r t e r s c o e x i s t i n a d i p o c y t e s : t h e e r y t h r o i d (E-GT) and t h e a d i p o c y t e / m u s c l e (AM-GT) t y p e . These are differentially r e g u l a t e d by i n s u l i n i n c u l t u r e d a d i p o c y t e s or by i n s u l i n e m i a in r a t adipose t i s s u e . Here, we examine E-GT and AM-GT e x p r e s s i o n i n young g e n e t i c a l l y obese Zucker r a t s , c h a r a c t e r i z e d by a l a r g e pool of t r a n s p o r t e r s ( C y t o c h a l a s i n B) i n a d i p o c y t e s . E-GT and AM-GT s p e c i f i c probes were used f o r Western and Northern a n a l y s i s of adipose t i s s u e , from obese ( f a / f a ) versus lean ( F a / f a ) r a t s . In c o n t r a s t w i t h v i r t u a l l y unchanged l e v e l s o f E-GT mRNA and p r o t e i n , AM-GT mRNA and p r o t e i n amounts were markedly i n c r e a s e d , i n 30-day o l d , h y p e r i n s u l i n e m i c , obese r a t s . The o v e r - e x p r e s s i o n of AM-GT mRNA was a l r e a d y p r e s e n t , a l t h o u g h l e s s marked, i n adipose t i s s u e from s u c k l i n g normoinsulinemic mutant r a t s (16-day o l d ) , and was not observed i n brown adipose t i s s u e or muscles. These d a t a d i s c l o s e t h a t t h e f a / f a genotype s t i m u l a t o r y e f f e c t on glucose t r a n s p o r t i n a d i p o c y t e s , i s mediated t h r o u g h a s e l e c t i v e i n c r e a s e i n t h e e x p r e s s i o n of AM-GTy a t a p r e - t r a n s l a t i o n a l level.

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OP 15 Hall A-3 Autonomic Neuropathy 85

86

THE AVONCHILDHOODDIABETES STUDY: PREVALENCEOFAUTONOMIC AND PERIPHERALSENSATION ABNORMALITIES. K.Karavanaki, A.G.Davies and J.D.Baum The Institute of Child Health,Bristol, England.

A U T O N O M I C N E U R O P A T H Y A N D CIRCADIAN B L O O D PRESSURE: POSSIBLE PROGNOSTIC IMPLICATIONS

129(87%) of the identified diabetic children in Avon Country,UK,and 144 age and sex matched controls,participated in a longitudinal study of microvascular disease.Diabetic children had a mean age 11.9 years (range: 3.6-16.8), mean diabetes duration 3.9 years (range: 0.1- 13.4),mean glycosylated haemoglobin 11.2% (range: 5.8-17.9).Pupillary dialation in darkness was estimated by a portable Polaroid Pupillometer, heart rate by a monitor and personal computer, and Vibration sensation threshold by a biothesiometer. 14 diabetic children (13.8%) had pupillary dialation in darkness narrower than the confidence intervals. These children in comparison with the rest of the diabetic population had: similar age, longer diabetes duration (7.0 years vs 4.2, p• d i a s t o l i c BP phase IV. Heart rate abnormalities were present in 7 (5.4%) diabetic children. There was no difference in mean age, diabetes duration and mean HbA1 between these children and the rest of the diabetic population. However they had an increased diastolic BP phase IV >+2SD above the mean. There was no difference in vibration sensation between diabetic and control children. In conclusion, prevalence of sympathetic dysfunction was higher than the parasympathetic dysfunction, and depended upon diabetes duration and glycaemic control.

87 QT INTERVAL, DIABETIC UNEXPECTED DEATH

C. Liniger, L. Favre and J.-Ph. Assal, Diabetes Treatment and Teaching Unit and Division of Endocrinology, University Hospital, Geneva, Switzerland. To assess the effect of autonomic neuropathy on circadian blood pressure (BP) profiles, 24 h ambulatory BP recordings were performed on 24 diabetic patients, 18 of whom had abnormal cardiovascular reflexes (CVR). Mean arteriai pressure (MAP) rose at night in 6 of the patients with abnormal CVR (Group A: median: +5 mm Hg; range: +3 to +10 mm Hg) and fell by <_5 mm Hg in 7 (Group B: -3[-1 to -5] mmHg). In the remaining 5 patients with abnormal CVR (Group C), and in the 6 with normal CVR (Group D), nocturnal MAP fell comparably to that of 11 non-diabetic hyperte nsives (C: - 18 [- 14 to -24] mm Hg; D: -20[- 13 to -23] mmHg; non-diabetics: - 12 [+4 to -23] mm Fig). The three neuropathic groups were comparable for age, known diabetes duration, autonomic symptom prevalence(A: 5/6 patients; B: 7/7; C: 4/5) and 24h MAP (,4:103 [92-117] mm Hg; B: 99 [92I09] mm Hg; C: 99 [96-103] mm Hg). Twenty-seven (14-52) months after monitoring, 4/6 patients with raised nocturnal MAP (Group A) had sustained 5 severe non-fatal and 3 fatal cardiovascular or renal events, compared with one non-fatal event in Group B and none in Group C (A vs B + C:p=O.02). These findings show that differing circadian BP patterns occur in diabetic patients with abnormal CVR and suggest an association between nocturnal BP rise and poor prognosis.

88 AUTONOMIC

NEUROPATHY

AND

D J EWING, 0 M IN)LAND, C G CHO, J M M NEILSON AND B F CIARKE, University Departments of Medicine, and Medical Physics and Bioengineering, and Diabetic Department, Royal Infirmary, Edinburgh Q T i n t e r v a l d y n a m i c s m a y b e a l t e r e d in d i a b e t i c autonomic neuropathy. We examined Q T / R R interval relationships during 24 hour ambulatory ECG recordings in age-matched males (12 normal (N), 13 diabetics without (DAN -ve) and 13 w i t h (DAN +ve) autonomic neuropathy) . In each subject QT was measured at six to eight different RR intervals ranging frcrn 500 to I000 ms. There was a very close linear relationship between QT and RR in all subjects (r = > 0.9). QT/RR slopes w~re significantly greater in the DAN +ve group conioared with normal (p = 0.017) (N, 0.205 + 0.03, DAN -ve 0.212 + 0.05, DAN +ve 0.243 + 0.04). We also undertook a retrospective analysis ~f 71 diabetics (39 DAN -ve, 32 DAN +ve) under 60 years, whose C!P was measured frcra a 24 hour ECG at an RR interval around 750 ms. 13 (3 DAN ve, 10 D A N +ve) h a d d i e d w i t h i n 3 y e a r s o f the recording, 7 unexpectedly, 2 frc~ unrelated causes, and 4 unknown. Of those with definite autonomic neuropathy, QT and corrected QT (_OTC) were significantly longer in the group who subsequently died ( Q T : alive 364 + 17, dead 391 + 35 ms, p < 0 . 0 1 ; QTC: alive 422 + 20, dead 452 + 37 ms, p<0.01). We conclude that these results are fl--lrtherevidence of disturbed QT dynamics in diabetic autonomic neuropathy. There m a y be an association b e t w e e n QT p r o l o n g a t i o n and the risk of dying unexpectedly in this group.

ANTI-SYMPATHETIC AND ANTI-PARASYMPATHETIC NERVE ANTIBODIES IN AUTONOMIC NEUROPATHY G. Sundkvist, P. Lind, B. Bergstr6m, B. Lilja, and S.L. Rabinowe. Departments of Medicine and Clinical Physiology, Malmo General Hospital, University of Lurid, Malta0, Sweden and the Immunology Section, Joslin Diabetes Center, Brigham and W o m e n ' s Hospital, Department of Medicine, Harvard Medical School, Boston, USA To evaluate possible associations between autoimmunity and autonomic neuropathy complement fixing anti-adrenal medulla (CF-ADM), anti-sympathetic ganglion (CF-SG), and anti-vagal (CF-V) nerve autoantibodies were determined in 74 patients with Type 1 diabetes (age 20-74 years, mean 42: duration 2-62 years, mean 23) and in 38 patients with Type 2 diabetes (age 42-59 years, mean 50; duration 3-17 years, mean 9). Autonomic neuropathy, ie abnormal heart rate reactions to deep breathing (E/I ratio) and to tilt (acceleration and brake indices), occurred in 44 Type 1 and 24 Type 2 diabetic patients. CF-ADM and/or CF-SG were less prevalent in Type 1 diabetic patients with autonomic neuropathy than in those without (5/44 [11%] versus 14/30 [47 %]; p<0.002). In agreement with this, the brake index, a sign of sympathetic nerve function, was significantly higher in Type 1 diabetic patients with CF-ADM and/or CF-SG than in those without (-0.56 • versus -1.04 • In contrast, Type 2 diabetic patients with CF-V had a significantly lower mean E/I ratio, signifying more advanced parasympathetic dysfunction, than those without (-1.81 • versus -1.20 • p<0.03). In conclusion: 1. CF-V were associated with advanced parasympathetic neuropathy, 2. CF-ADM and/or CF-SG were increased in patients without established autonomic neuropathy; CF-ADM and/or CF-SG may antedate and predict sympathetic neuropathy.

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89

90

V I S C E R A L A F F E R E N T N E U R O P A T H Y IN D I A B E T I C GASTROPARE S IS W. R a t h m a n n , P. E n c k * , T. F r i e l i n g * a n d F.A. Gries, Diabetes Research Institute, *Department o f G a s t r o e n t e r o l o g y U n i v e r s i t y D~lsseldorf A lack of symptoms was reported in d i a b e t i c patients with gastrointestinal motility disorders. We investigated cerebral evoked potentials (EPs) following esophageal stimulation in 6 patients with motor dysfunction of the GI-tract and in 6 healthy controls. All patients had "type 1 (insulind e p e n d e n t ) " d i a b e t e s m e l l i t u s (4:2 m a l e : f e m a l e , age range: 32-60 years, diabetes duration: 8-31 years ; 6/6 polyneuropathy, 4/6 cardiac autonomic neuropathy). Their esophageal and gastric motor disorders had been diagnosed by scintigraphy or X-ray, and GI-stenosis had been excluded by gastroscopy. Only 1 patient had severe symptoms while 4 patients complained only minor discomfort (distension, bloating) and 1 patient was symptom free. EPs were recorded after electrical stimulation of t h e esophagus (32 c m inc.) a t i n t e n s i t y j u s t a b o v e the perception threshold. All controls exh i b i t e d r e g u l a r E P s a t 0.i m s / 3 0 m A s t i m u l a t i o n intensity. In 5 d i a b e t i c p a t i e n t s n o E P s w e r e d e t e c t e d a t 0.I a n d 0.3 m s / 3 0 mA, a n d t h e p e r ception thresholds were significantly elevated. In one patient with normal perception threshold, EPs of regular shape but decreased amplitude were recorded. Only this patient had severe gastroparetic complaints. These data show for the first time that the lack of s y m p t o m s in d i a b e t i c g a s t r o i n t e s t i n a l motility disorders may be due to visceral afferent neuropathy.

Fogow-up of a u t o n o m i c neuropalthy a l t e r pancreas andltor Iddeev tmnsplmmation 91. Nusser, M. Kehrle, R. Scheuer, W. Iilne6 D. Abendroth, 14/. Land, R. Landgraf Medizinische Klinik Innenstadt and Transplantation Center. University of Munich, FFIG

The fate of diabetic complications after successful simultaneous pancreas/kidney or single kidney transplantation is still under debate. In a prospective study we tested 26 b)pe-Idiabetics (group 1, mean age 32_+1.6 years, duration of diabetes 23+1 years), who underwent simultaneous pancreas/kidney transplantation and 14. tMoe-l-diabetics after kidney grafting (group 2, mean age 33,2 years, duration of diabetes 21,2 years). HbA1 at the latest follow-up was 6.6,0.2% vs 8.5,0.3%. Serum creatinine was comparable 1.91-0.4 vs. 1.5,0.2. All patients underwent autonomic testing by deep breathing, lying/standing, Valsalva-manouvre and filled in a standardized questionnaire concerning autonomic symptoms in six months intervals. Mean observation pedod was 35,2 months for group 1 and 2 5 , 3 months for group 2. The mean RR-parameters (E/I-, 30/15-, Valsalva-ratio and score) in both groups were very similar and did not improve, although the mean heart rate in group 1 was significantly lower. Orthostatic dysreguiation ameliorated in group 1 and worsened in group 2. The symptom scores were comparable but the symptoms tended to Improve in group 1. After pancreatic graft loss reappearance of diabetes showed worsening of RR-parameters within one year after graft loss. It is concluded that Ion~erm normalization of blood glucose is necessary for amelioration of autonomic neuropathy.

OP 16 Hall A-15 Technical Devices 91

92

~UT.TTCENTBR FEASIBILITY AND SAFETY STUDY OF ~N IMPLANTABLB PROGRAM94ABLE INSULIN PUMP IN TYPE 1 DIABETES. P. Micossi, J.L. Selam, F. Dunn and D.M. Nathan for the Study Group. Milano - Italy, Irvine - CA, Durham - NC, Boston - MA, USA Sixty-five Type 1 C-peptide negative diabetics were admitted to a feasibility and safety multicenter trial of an implantable programmable insulin pump (INFUSAID Model i000). After a 3 month run-in period of intensive subcutaneous (SC) treatment, pumps were implanted either in the abdominal wall (n=60) or in the infraelavicular fossa (n=~) and catheters placed in the peritoneal space (IP n=48) or in the superior vena cava (IV n=17). After an overall experience of 845 patient months with 35 patients treated for more than 6 months and 14 treated for more than 1 year, there have been 531 pump refills and 123,420 progr~muning sequences with no errors. Mean normalized pump flow calculated as ratio actual/programmed is 1.01• Nine pumps showed a progressive flow rate decrease. In 5 cases flow was restored by alkaline pump flushing procedure, 3 are pending, i pump was replaced. Eleven catheter obstructions were resolved by buffer flushing through the sideport. One IP catheter was replaced. Six IV catheters migrated: 3 were repositioned, 2 explanted with the pump and I resutured. Patients performed 4-5 blood glucose (BG) measures daily, directly loaded on a memory system. Severe hypoglycaemic events decreased from 0.29 SC to 0.02 IP patient-yrs. ENTRY SC INFUSAID INFUSAID 3 months 3 months 6 months mean BG(mg/dl) 182• 173• 146• 151• median BG(mg/dl) 174 167 148 139 mean HbAlc(%) 8.12• 7.55• 6.79• 6.85•

INTRAPERITONEAL IMPLANTABLE PUMPS VS INTENSIVE SUBCUTANEOUS INSULIN: A RANDOMIZED COMPARISON

It is concluded that the programmable implantable pump offers a safe and effective option for intensive insulin treatment of diabetic patients.

J.L. Selam, K. Wucetich, J.L. Lozano, L. White, D. Raccah, N. Jean-Didier, and M.A. Charles. University of California Irvine, Irvine, California. Efficacy of insulin with implantable pumps to control diabetes has only been assessed during uncontrolled safety trials. To compare intraperitoneal implantable (IP) to subcutaneous (SC) intensive insulin, 21 Type 1 adult diabetic patients underwent a 3-month treatment optimization using multiple injections or external pumps. They were then randomized to IP infusion using Infusaid programmable pumps or continuation on SC intensive insulin for 6 months. HbA1C improved in both groups: IP: 9.0 + 0.5% ~ 7.8 + 0.6% (p < 0.01), SC: 8,4 • 0.5% x~ 7.5 + 0.3% (p < 0.01) at 0 and 6 months respectively (normal: < 6.9%). The percentage of glycemic tests above 200 mg/dl at 0 and 6 months was 28 + 5% vs 13 + 5% in the IP (p < 0.01) and 22 +__5 x,s 24 + 7% in the SC group (p = NS). At 0 and 6 months, the standard deviation of blood glucose values was 78 + 7 vs 59 + 9 mg/dl in the IP group (p < 0.01) and 68 + 5 x,s 70 + 7 mg/dl in the SC group (NS). Weight, insulin doses, circulating insulin and lipids, and the frequency of hypoglycemic reactions were similar and did not change in the two groups. Thus, IP implantable pumps, when compared to SC intensive insulin therapy are equally effective at achieving near-normal glycemia, but are more effective at limiting glyeemie fluctuations.

A35

94

93 PULSATILE INSULIN ADMINISTRATION DEPENDENT (TYPE I) DIABETES.

IN INSULIN-

G.M. Ward, J.M. Wahers, P.M. Aitken, A. Kalfas, and F.P. Alford. Endocrinology Unit and University Department of Medicine, St. Vincent's Hospital, Melbourne, VIC 3065, Auslxaiia. Intravenous pulsatile insulin (PI) infusion at near therapeutic doses in Type I diabetes was compared with an equivalent continuous infusion (CI). The 9 young nonobese Type I diabetic w)lunteers included only 4 with detectable fasting plasma C-peptide (0.05 _+ 0.01 pmol/ml SEM) . Insulin was infused intravenously at 12 mU/kg/h overnight for 18h either continuously (CI) or as 40-second pulses every 13 minutes (PI), with glucose infused to maintain euglycaemia. An intravenous glucose tolerance test was then performed with insulin infused to simulate the non-diabetic pattern of endogenous insulin and analysed by the minimal model of Bergman et al. In the 4 C-peptide positive subjects, the hypoglycaemic effect of PI (measured by the glucose infused to maintain euglycaemia) was uniformly greater than with CI (PI vs CI, 8.5 + 4.1 vs 3.0 + 0.8 umol/kg/min) but not in the 5 C-peptide negative subjects (PI vs CI, 6.3 +_ 2.4 vs 6.3 + 2.5 umol/kg/min). The 4 C-peptide positive subjects showed insulin sensitivity uniformly greater after PI than CI (PI vs CI, 6.0 +_ 1.8 vs 2.1 + 1.5 min -1 per mU]l xl04) In contrast, the five C-peptide negative subjects showed uniformly greater insulin sensitivity with CI than with PI (PI vs CI, 2.5 + 2.2 vs 10.9 + 5.2 min -1 per mU/l xl04) - significantly different to the C-peptide positive group (P < 0.02). Therefore, in Type I diabetics with measurable endogenous insulin secretion, PI at near therapeutic levels produced greater hypoglycaemic effect and insulin sensitivity,, when compared with equivalent continuous infusions.

HIGHERPLASMAC-PEPTIDELEVELSDURINGPULSATILETHANCONTINUOUS INTRA-VEtNOUSINSULINDELIVERYIN TYPE2 DIABETESMELLITUS. Ph. Giraud, Y. Gallois, D. Six, V. Leforestier a n d V. Rohmer. CHRU. 49033 Angers Cedex. France."

A glucose clamp study showed that pulsatile insulin administration could modulate 8-cell function. In type 2 diabetes, irregular oscillations have been noted. The aim of this study consisted in investigating wether plasma CPeptide levels could be modified by intra-venous (IV) pulsatile insulin treatment. Seven type 2 diabetic male, aged 69+2 years (mean_+SE), HbAlc 6.7+1.8%, were treated, in a random order, during two successive periods of four days by pulsatile or continuous administration with an Ulis pump (Medicorep) or a 304S-pulsatile Minimed pump (Pacesetter). At 8h a.m. on the fifth day, blood samples were taken every 2 minutes during 40 minutes for plasma free insulin and C-Peptide determination. Comparisons were made between mean values of insulin (or C-Peptide) levels obtained during the 40 min periods. A Wilcoxon test for paired data was performed. Fasting glycemia were strictly similar for the two treatments. Basal insulin rate was reduced by 19+4% (p<0.02) during pulsatile administration. Plasma free insulin levels were strictly comparable within the two treatment (16.14+3,97 vs 15.99+4.21 mU/L), whereas C-Peptide levels w e r e increased during pulsatile insulin administration (2.34+0.87 vs 1.84+0.96 pg/L; p=0.003). We conclude that C-Peptide levels were increased during IV pulsatile insulin administration ; it could explain the similar insulin levels noted during the two treatments with different insulin dosages.

95

96

THE GLUCO-DAY: A NEW WEARABLE 24 HOURS C O N T I N U O U S BLOOD G L U C O S E M O N I T O R L. Bernardi*+, P. Bernardi+, A~M. De Luca* and G. B o m b a r d i e r i * , +Ampliscientifica, V. Svevo 85, Rome, *Catholic University, Rome, Italy. In our l a b o r a t o r i e s we d e v e l o p e d the Gluco-Day, a wearable device which permits the quantitative determination of b l o o d g l u c o s e over a p e r i o d of 24 hours w i t h o u t r e m o v i n g blood from the patient while allowing the p a t i e n t to live an almost normal daylife. By m e a n s of a s p e c i a l l y d e v e l o p e d m i c r o d i a l y z i n g n e e d l e i n s e r t e d into a vein of the p a t i e n t a fluid is obtained in which the glucose concentration is p r o p o r t i o n a l to that of the blood, w i t h o u t the need of blood drawing. A G l u c o s e O x i d a s e - H 2 0 2 sensor is used to m e a s u r e glucose concentration in t h i s f l u i d . Every m i n u t e g l y c e m i a s are d i s p l a y e d and s t o r e d in an internal memory; they are trasmitted to a computer a n d / o r to a p u m p to o b t a i n blood g l u c o s e c o n t r o l or to p e r f o r m g l u c o s e clamping. D u r i n g c l i n i c a l t r i a l s on d i a b e t i c subjects b l o o d s a m p l e s w e r e t a k e n e v e r y 2 h o u r s to e v a l u a t e the c o r r e s p o n d e n c e (R2=0.97) b e t w e e n our monitor and a reference instrument (Beckman). Clinical trials c o n f i r m that, due to its low costs, small size and easy use, this d e v i c e can be e x t e n s i v e l y used for r o u t i n e and r e s e a r c h blood g l u c o s e p r o f i l e evaluations~

KINETICS OF SUBCUTANEOUS GLUCOSE CONCENTRATION IN NORMAL AND DIABETIC DOGS K. Rebrin, U. Fischer, Th. v . Woedtke and E. Brunstein. Central Institute of Diabetes " G e r h a r d t K a t s c h " K a r l s b u r g , GDR According t o our p r e v i o u s i n v e s t i g a t i o n s , steady state g l y c a e m i a i s r e p r e s e n t e d by t h e subcutaneous g l u c o s e c o n c e n t r a t i o n (SCG) and SCG may be reliably feedback-controlled. This study characterizes kinetic properties of SC8 with respect to therapeutic decisions. Normal and diabetic dogs on glucose-controlled insulin infusion were instrumented with subcutaneous a m p e r o m e t r i c H=O=/GOD e l e c t r o d e s f o r SCG t e l e m e try. For reference, g l u c o s e was m o n i t o r e d in b l o o d and plasma. - Results: (1) S t a b l e sensor f u n c t i o n was a c h i e v e d o v e r between 10 and 48 h r (n = 12). At s t e a d y s t a t e g l y c a e m i a ( 5 m m o l / 1 ) , the coefficient of v a r i a t i o n o f SCG ( ~ t = 1 min) was between 5 and 10 % w i t h different sensors (in v i t r o v= < 3 %) i n d i f f e r e n t animals. (2) First-order non-linear regression analysis o f responses t o square a l t e r a t i o n s in intravenous glucose input (12 mg.kg-*.min-~) revealed the following time constants T (n=9): SCG ~ = 3 0 . 8 min ~range 1 2 . 0 - 1 0 8 . 7 min), plasma glucose x = 17.5 s i n ( 1 0 . 0 - 3 3 . 8 min>, blood g l u c o s e ~ = 2 5 . 0 min ( 1 0 . 2 - 4 3 . 2 min) w i t h no a p p r e c i a b l e d i f § n e i t h e r between normal and d i a b e t i c a n i m a l s n o r between i n c r e a s i n g and d e c r e a s i n g c o n c e n t r a t i o n s . The i n v i t r o T - v a l u e s o f t h e same e l e c t r o d e s were ~ = 2.2 min ( r a n g e 0 . 5 - 3.0 min). - Conclusion: Functional p r o p e r t i e s of a v a i l a b l e glucose sensors are s u i t a b l e t o r e p r e s e n t any alteration i n S C 8 . But t h e k i n e t i c p a r a m e t e r s o f SCG must explicitely be c o n s i d e r e d i n g l u c o s e m o n i t o r i n g or feedback c o n t r o l o f d i a b e t i c p a t i e n t s .

A36

OP 17 Hall B-3 Biosynthesis in B-cells 97 INTERLEUKIN-IB ISLETS WITHOUT

98 INHIBITS IMPAIRING

INSULIN GLUCOSE

PRODUCTION METABOLISM

IN MOUSE

D.L. Eizirik, E. Strandell, N. Welsh and S. Sandler. Department of Medical Cell Biology, Uppsala University, BMC, P.O. Box 571, S-751 23 Uppsala, Sweden. In order to characterize the mechanisms behind interleukin-lB (rlL-l[~) induced impairment in islet function, mouse pancreatic islets obtained from NMRI, NOD, C57BL/6 and C57BL/Ks mice were exposed to rlL-1[~ (25, 50 or I00 U/ml) for a 48 h-period. In all groups and at the various rlL-IB concentrations tested, there was a similar 30-50% inhibition in glucose-induced insulin release (P<0.01), a 70-80% decrease in islet insulin content (P<0.001) and no significant differences in DNA content or insulin accumulation in the culture medium. Further experiments showed that exposure to 50 U/ml rIL-l[3 reduced the insulin biosynthesis in NMRI and C57BL/6 islets by 40-50% (P<0.05 or less) and the insulin mRNA content by 80-90% (P<0.01). However, rlL-1B did not decrease islet total protein biosynthesis, glucose oxidation, ATP content, ATP/ADP ratio, cAMP content and polyamine content. In conclusion, these data suggest that rIL-1B preferentially reduces insulin mRNA levels and insulin biosynthesis in mouse pancreatic islets, without equally impairing other cell functions. The mechanism behind this reduction seems thus to be different from that observed in rat islets, and can not be explained by an altered generation of any of the known regulators of the insulin gene expression.

GLUCOSE REGULATES ITS OWN TRANSPORT AND THE GLUCOSE CARRIER GENE EXPRESSION IN CULTURED PANCREATIC CELLS. F.Purrello,M.Vetri,M.Buscema,C.Vinci,C.Gatta and R.Vigneri Cattedradi Endocrinologia,Universityof Catania,Italy To study the effect of chronic high glucose on the regulation of glucose transport in B-cells we used an in vitro model where rat pancreatic cells in permanent culture (RIN) were exposed for 48 h to glucose concentrations of 5.5 mmol/1 (G5.5) or 16.7 mmol/1 (G16.7). After this period the glucose transport was studied by measuring the uptake of (3H)-2-deoxyglucose (2DG) in KRP buffer (5 and 10 rain at 24 C). 2DG uptake in cells pre-exposed to G16.7 was lower in respect to cells pre-exposed to G5.5 (respectively 16 + 2.6 nmol/mg vs 24 + 2.5 at 5 min, and 29 _+4.8 vs 51 + 5.9 at 10 min, n~+SE, n=5, p<0.01). The mechanism for high glucose impairment of glucose transport was studied in the same cells by evaluating the glucose carrier gene expression (Northern Blot analysis). Total RNA (20 gg) was electrophoresed, fixed onto a nylon filter, and then hybridized to a 32P-labelled cDNA probe obtained from HEPG2 cells. After high stringency washes cells exposed to high glucose (G16.7) have less (-48.9 %) glucose-carrier mRNA levels than control cells (G5.5).Therefore, in pancreatic RIN cells, chronic exposure to high glucose concentrations impairs both glucose transport and glucose-carrier gene expression. These effects may play a role in the altered glucose sensitivity of B-cells in type II diabetic patients.

99

100

Human and mouse insulin gene expression in transgenic mice

ISLET CELL STIMULATING ANTIBODIES INCREASE PREPROINSULIN M E S S E N G E R RNA IN C U L T U R E D ISLETS T.J. Wi]kin, L. F o g g e n s t e i n e r , K. W e b s t e r and A.J. Bone Endocrine Section, Medicine II, Southampton Genera] Hospital, S o u t h a m p t o n , UK We have r e c e n t l y d e s c r i b e d novel islet cell stimulating antibodies (ICSTA) which are distinct from insulin receptor antibodies, occur spontaneously and stimulate insulin r e l e a s e in v i v o and in v i t r o over ]ong p e r i o d s of time. To i n v e s t i g a t e w h e t h e r ICSTA cause the synthesis of insulin, and not m e r e l y its release, we used d o t - b l o t s to make s e m i q u a n t f t a t i v e m e a s u r e m e n t s of insu]in mRNA. Collagenase-isolated rat islets w e r e i n c u b a t e d in m u l t i w e l l s w i t h the i n s u l i n a n t i b o d y - , i n s u l i n - and glucosefree globulin (G) a n d n o n - g l o b u l i n (non-G) f r a c t i o n s of h e a t - t r e a t e d sera f r o m patients with insulin-dependent diabetes (IDDM), the i n s u l i n a u t o i m m u n e s y n d r o m e (IAS) and controls. The G, b u t not the n o n - G s e r u m f r a c t i o n s from the 3/4 IDDM and i/i IAS p a t i e n t s , and n e i t h e r f r a c t i o n from controls, s t i m u l a t e d i n s u l i n release, from 5 to 8 - f o l d b a s a l at a d i l u t i o n of 1/20. S i m i l a r l y , the G f r a c t i o n s of sera f r o m the ICSTA p o s i t i v e patients, but not from the controls, i n c r e a s e d p r e p r o i n s u l i n m e s s a g e in rat islets in a d o s e - d e p e n d e n t f a s h i o n (see table of dotblot d e n s i t i e s ) .

B. Sctmetzler, R.F. Selden and P.A. Halban Laboratoires Jeantet, Geneva University School of Medicine, Switzerland, and Dpt. Molecular Biology, Massachusetts General Hospital, Boston, USA Human insulin expression in transgenic mice has been described, but the regulation of the trans-gene and the impact of its expression on endogenous mouse insulin production has not. We have developed an HPLC method for quantification of pancreatic human, mouse I and mouse II insulins and studied the effects of fasting/refeeding on insufin ratios in controls and transgenie mice expressing the intact human insulin gene. Plasma glucose and immunoreactive insulin (respectively) decreased from 153 mg/dl; 31 uU/ml (controls) and 142 mg/dl; 30 uU/ml (transgenies) in fed mice, to 60 mg/dl; 13.5 nU/ml (controls) and 60 mg/dl; 11.5 uU/ml (transgenics) during a 48 h fast. In controls, insulin I was 30.9 + 4.8 % of total (I + U). This value was not significantly affected by 48 h fasting (29.2 + 3.7 %) or by 24 h fast + 24 h refeed (28.4 + 3.7 %). In transgenics, there was 28.2 + 3.1% human, 21.8 + 1.8 % mouse I and 50.0 + 4 . 1 % mouse II (the amount of mouse I relative to mouse n thus being similar to controls). These values were unchanged throughout the fasting and fast/refeed protocols. These results indicate coordinate regulation of pancreatic content of human and mouse insulins in the trausgenic mice, with no effect of trans-gene expression on the relative mouse I and II insufin levels, under conditions of decreased (fasting) or restored (fast + refeed) pancreatic insulin turnover.

Diln. mRNA i:I 1:2 1:4 1:8

Glucose 2.7mM 0.12 0.14 0.01 0.00

Glucose 20~M 1.00 0.78 0.36 0.26

G1 0.52 0.38 0.17 0.00

G2 0.75 0.44 0.ii 0.00

G3 0.59 0.52 0.00 0.00

G4 0.08 0.25 0.17 0.00

Mean G control 0.07 0.00 0.00 0.00

We c o n c l u d e that I C S T A s t i m u l a t e i n s u l i n synthesis, not on]y its r e l e a s e and may i n f l u e n c e g l u c o s e h o m e o s t a s i s in prediabetes.

A37

101

102

PROINSOLIN PROCESSING IN ELECTRICALLY p~M~ARILISED RAT ISLETS OF L/~C~RHANS. D . J . S l e e , P.M. Jones and S.L. H o w e l l . Biomedical S c i e n c e s Division, King's College London, Campden Hill Rd, London W8 7AH, O K .

INDUCTION OF INSULIN ENHANCER BINDING FACTORS DURING 13CELL DIFFERENTIATION

Pancreatic

B-granules

have

an acidic interior (pH 5-6),

thought to be formed by a HgATPase p r o t o n pump. T h i s may c o n t r o l the a c t i v i t y o f p r o t e o l y t i c enzymes which c l e a v e p r o i n s u l i n ( P I ) . We have used e l e c t r i c a l l y permeabilised islets t o study the r e g u l a t i o n o f i n t r a g r a n u l a r P I c version in situ. Islets preincubated with [~S]-cysteine-- (45mins) were permeabilised and further i~ubated for 60 mins under various conditions. [ S]-incorporation i n t o P I and I n s u l i n ( I ) , s e p a r a t e d by h p l c , was m e a s u r e d a t t - 0 and 60min and r e s u l t s expressed as g c o n v e r s i o n . Conversion r a t e s were s i m i l a r in p e r m e a b i l i s e d and i n t a c t islets. P I c o n v e r s i o n was pH sensitive i n p e r = e a b i l i s e d i s l e t s ( c o n v e r s i o n 4 0 . 8 + 1.7% a t pHS.5, 31.8~1.7% pHT.4, p < 0 . 0 5 , mean~SEH, nffiS).The effect

of

intragranular

pH

was

furrier

studied

by

collapsing the pH gradient with 40mM ammonium acetate, conversion was: pH 7.4: 28.3~1.8Z, pH 5.5: 50.3+5.0%, pH 4.5: 49.3~7.6%, pH 3.5: |0~0%, n=3-7, ATP (5,@I) stimulated conversion above a basal rate at pE 7.4 (3].8+1.7% -ATP, 43.3+3.8% +ATP, p
P. Serup, B. Michelsen, K. Lund and 0.D.Madsen. Hagedorn Research Laboratory, Gentofte, Denmark The glucagon producing pancreatic islet cell line, NHI6F, undergoes differentiation by in vivo culture to express the endogenous insulin genes as well as a stably transfected human insulin gene. Expression of the rat insulin I gene is regulated by cis-acting enhancer elements containing discrete protein binding domains (El to E5). By electrophoretic mobility shift assay we detect induction of proteins, able to bind this enhancer, concomitantly with induction of the insulin gene in NHI-6F cells. By DNase I footprinting we show that the proteins primarily interact with the E1 and the E4 binding domains and a novel DNase I hypersensitive site appears at the 5'-border of the E4 region. The E1 footprint and the DNase I hypersensitive site are present in control insulin producing cell lines. De-differentiatlon by prolonged in vitro culture of NHI-6F tumor isolates results in loss of E1 binding and disappearance of the DNase I hypersensitive site. The E1 and E4 regions have previously been implicated in the tissue-specific regulation of the rat insulin I gene and we propose that the induction of binding activities to these regions reflects islet cell differentiation during pancreas ontogeny.

OP 18 Hall A-18 Clinical Transplantation 103

104

THE EFFECTS OF PANCREAS T R A N S P L A N T A T I O N ON DIABETIC NEPHROPATHY IN MAN RW Bilous, SM Maner, DER Sutherland, and M W Steffes. Depts Lab Med and Pathol, Pediatrics and Surgery. University of Minnesota, Minneapolis, USA.

QUALITY OF LIFE IN DIABETICS PRIOR TO OR AFTER TRANSPLANTATION IN RELATION TO ORGAN FUNCTION

No studies of the effect of improved glycaemic control on diabetic nephropathy have included renal structural analysis. We have studied the effects of up to 5 years normoglycaemia induced by p a n c r e a s t r a n s p l a n t a t i o n (PTx) on g l o m e r u l a r structure and function in the native kidneys of 14 (6M, 8F) type 1 (insulin dependent) diabetics. Patients were studied before, 2 and 5 years (6 subjects) after PTx. Mean duration of diabetes before PTx was 22_+5 years and 7 had dipstick positive proteinuria. Baseline HbA 1 was 10.2+i.3% normalizing to 7.0+1.0% at 2 years and 6.8_+0.8% at 5 years. Creatinine clearances initially fell from 103+25 to 63+12 ml/mirdl.73 m z (p<0.001) at 2 years and stabilised at 69+_27 (pNS) at 5 years. Most clinically proteinuric patients showed an initial decline in albumin excretion rate. Blood pressure levels did not change. Glomerular structural analysis revealed no significant changes in mesangial volume fractions, mean glomerular volume or g l o m e r u l a r b a s e m e n t m e m b r a n e widths but s o m e patients showed an increase in interstitial tissue and global glomerular sclerosis. These data suggest that diabetic glomerulopathy remains stable after PTx in m a n despite a significant initial reduction in creatinine clearance. Carefully controlled studies of the natural h i s t o r y o f g l o m e r u l a r l e s i o n s in d i a b e t e s are n e c e s s a r y to determine if PTx confers any benefit in patients with nephropathy.

W. Piehlmeier, J. Nusser, A. K,~nig, F. A. Muthny, W. D. /#net', D. Abendroth, W. Land and R Landgraf. Dept. of Internal Medicine ~/nnenstadt ~, Transptantation Center, University of Munich, Munich, and Dept. of Rehabilitation Psychology, University of Fre~urg, Freburg, FRG Improvement of the quality of life is one of the main goals in organ transplantation. We therefore evaluated a broad spectrum of rehabilitation criteria using a self-administrated questionnaire ( scores from 1-5 ) in 157 type I diabetics divided in ,5 groups: A: n = 29 pretransplant without endstage renal insufficiency. B: n=,H pretransplant on chronic dialysis.C: n=31 after successful pancreas and kidney grafting (27+4 months).D: n=29 after grafting with diabetes posttransplant,but good renal function.E: n=24 after grafting with diabetes and on chronic dialysis.The age of the patients (36+_1 y) and the duration of diabetes (24-!-_1 y) were very similar in all groups.The results demonstrate that the satisfaction with the physical activity (mean scores:3,6 _+0,2 vs 2,4__.0,3)and mental status (3,9+-0,1 vs 3,1+0,3) is best in C and worst in E.Sexual life dissatisfaction is high in B and E and better in A,C and D.Quality of familiy life is best in successfully kidney/pancreatic graft recipients and much less in all other groups.Fears for drug side effects are very similar in all patients.General life satisfaction is superior in C (4,0_+0,2) and lowest in B (2,7+-0,2),but comparable to D (3,8+_0,2).Vocational rehabilitation however is discrepant to expectation with no occupation in:A 38%,B 64%,C 74%,D 66%,E 83%.1t is concluded that despite a very good medical result after successful combined grafting improvements of psychosocial rehabilitation are less impressive probably due to severe and complex diabetic problems pretransplant and the rather short observation time.

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PRESERVED INCRETINEFFECTAFTER HETEROPTOPICPANCREAS TRANSPLANTATION IN TYPE I-DIABETIC PATIENTS M. Nauck, M. BOsing, E.G. Siegel, J. Talartschik, Th. Baartz, A. K6rner, U.T. Hopt, H.D. Becket and W. Creutzfeldt, Dept. of Medicine, Univ. G6ttingen, and Dept. of Surgery, Univ. TObingen, FRG I t is not clear whether the incretin effect is reduced in patients who received a pancreas transplant with insulin drainage into the systemic circulation. I t was the aim of the present study to quantify incretin effects after oral glucose in such patients with a t o t a l l y denervated pancreas. 9 patients after combined heterotopic pancreas (plus kidney) transplantation, and 7 control patients who received a kidney transplant only, matched for kidney function and immunosuppressive medication, were studied. The B cell secretory response (integrated incremental IR-insulin and IR-C-peptide area) after 50 g oral glucose and during a matched ("isoglycaemic") intravenous glucose infusion were determined in blood drawn from arteriovenous shunts. Glucose tolerance was normal or mildly impaired in both groups of patients. Oral glucose e l i c i t e d a s i g n i f i c a n t l y greater B cell secretory response in both groups (p~O.02) than did isoglycaemic intravenous glucose. The i n c r e t i n e f f e c t ( A ) contributed 57.2• % to the C-peptide response a f t e r oral glucose in pancreas-kidney-transplanted p a t i e n t s , as compared to 48.2• % in kidneytransplanted patients (58.4• % in p r e v i o u s l y studied normal subjects). In conclusion, i n c r e t i n factors remain an important determinant of i n s u l i n secretion a f t e r oral glucose even a f t e r denervation of the pancreas, and in spite of systemic i n s u l i n delivery.

INSULIN ACTION AFTER PANCREAS TRANSPLANTATION F. Saudek, T. Pelik~nov6, V. Barto~: I.Reneltov~, L. Kazdov~ and J. Kov6~. Institute for Clinical and Experimental Medicine, Prague, Czechoslovakia Insulinoresistance may contribute to hyperinsulinemia after successful pancreas transplantation. Using the euglycemic clamp technique, insulin action was examined in lO diabetics 1 year after simultaneous pancreas and kidney transplantation (group i), in 9 non-diabetic kidney recipients (group 2) and in i0 age- and weight-matched healthy subjects (group 3). Insulin action was expressed as the metabolic clearance of glucose at insulin concentration of approx, i00 ~U/ml (MCRsubmax) and approx. 2000 )JU/ml (MCRmax). MCRmax reflects the postreceptor insulin effect. Further, insulin binding to specific receptors of erythrocytes (SB) was determined. All transplant recipients were normoglycemic. In groups 1 and 2 fasting insulinemia was significantly higher than in controls (mean + SO 32+17, 21+7 and 14+6 ~JU/ml, respective-ly). M~Rsubma~ and MCRmax were decreased in groups i and 2 (p~:O.01). In groups 1, 2 and 3 MCRsubmax were 6.4+1.8: 6.1+2.2 and 10.4+3.5 ml/min.kg and MCRmax--12.7+2.5T 13.1+2.9 and19.3+4.7 ml/min.kg, respeEtively. SB-was decreased only in group 1 ( p < O . O 1 ) due to a 31% decrease of receptor number vs controls. It is concluded that the postreceptor defect in insulin action is common to all immunosuppressed organ recipients and not related to the pancreas graft. In addition, a receptor defect was found in pancreas transplantation.

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CYCLOSPORINEEFFECT ON BETA-CELL FUNCTIONIN PATIENTSWITH KIDNEY TRANSPLANT Esmatjes E., Ricart M.J., Ferrer J., Conget I, Oppenhaimer F., Vilardell J, Casamitjana R. Endocrinology and Diabetes Unit, Renal Transplant Unit, Hormonal Laboratorie. Hospital Clinic i Provincial. Barcelona Adverse e f f e c t s of cycl osporine (CsA) on glucose metabolism have been reported in patients with kidney t r a n s p l a n t (KTx), but steroids were present in a l l the immunossupressive schedules evaluated. We have studied endocrine pancreatic function (OGTT measuring plasma i n s u l i n (IRI) and C peptide (CP)) in 3 groups of 6 p a t i e n t s , matched for age and body mass index, 16 to 66 months a f t e r a functioning ~
T R A N S P L A N T A T I O N OF FRESH H U M A N ISLETS IN TYPE I D I A B E T I C PATIENTS: E V I D E N C E OF FUNCTION. C. SOCCi, L. Falqui, A.M. Davalli, P. M a f f i , M. Cristallo, A. Secchi, G. Ferrari, S. Braghi, M. Carlucci, M. Freschi, P. Magistretti, V. Di Carlo and G. Pozza. S c i e n t i f i c I n s t i t u t e S. Raffaele, university of Milano, Milan, I t a l y The recent development of the automathed method for h u m a n islet i s o l a t i o n allows to obtain p r e p a r a t i o n s of islets suitable for t r a n s p l a n t a t i o n in h u m a n patients. Two Type I diabetic patients, C-PEP negative (<0.15 ng/ml), were r e c e n t l y t r a n s p l a n t e d via the portal vein. T h e y h a d r e c e i v e d a k i d n e y and pancreas transplant with early pancreas failure and were on triple immunosuppressive therapy (CSA, AZA, PRED). The first patient r e c e i v e d 270,000 fresh h u m a n islets (insulin content: 69 units). P r e d n i s o n e was increased to 60 m g / d i e for 15 days. The s e c o n d p a t i e n t r e c e i v e d 350,000 fresh h u m a n islets (insulin content 90 Units). A 7 day course of antilymphocyte g l o b u l i n was also administrated. I.v. insulin was given to b o t h p a t i e n t s for the first 15 days to keep the islets in a near resting state. C-Pep values raised progressively in b o t h patients. The first p a t i e n t went up to 2.4 ng/ml at 8 days. Then the C-pep value s u d d e n l y d r o p p e d back to <0.15 ng/ml at 12 days p r o b a b l y due to a r e j e c t i o n episode. The s e c o n d p a t i e n t s h o w e d a p r o g r e s s i v e C-PEP increase to 3.16 ng/ml still p r e s e n t at 19 days . In c o n c l u s i o n the automathed m e t h o d yields a preparation of viable islets, as d e m o n s t r a t e d b y the C-Pep values that can be used in clinical transplantation.

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OP 19 Hall A-1

Amylin (lAPP) 109

110

BASAL AND S T I M U L A T E D P L A S M A A M Y L I N LEVELS IN DIABETES MELLITUS B.Ludvik, T.Swoboda, E.Hartter, E.KHenburg, M. Brunnbauer, W. W o l o s z c z u k and R. Prager, 2 "d M e d i c a l Department, U n i v e r s i t y of Vienna, Austria Amylin, a new peptide isolated from amyloid deposits of type II d i a b e t i c islets, is t h o u g h t to p l a y a p o t e n t i a l role in the p a t h o g e n e s i s of Type 2 diabetes mellitus. We therefore developed a r a d i o i m m u n o a s s a y (RIA) to m e a s u r e basal amylin levels in h e a l t h y controls (n=23) and in obese patients w i t h impaired g l u c o s e t o l e r a n c e (IGT) or type 2 d i a b e t e s m e l l i t u s (n=20) and in patients w i t h type 1 diabetes m e l l i t u s (n=14). The lowest level of d e t e c t i o n of the a m y l i n RIA was 6 pg/ml. The r e c o v e r y of the assay was 80%. In healthy controls amylin levels were significantly lower than in obese patients (10.1• vs.18.7• p<0.05). In type 1 diabetic patients basal a m y l i n levels were not detectable. D u r i n g an oral g l u c o s e t o l e r a n c e test (75 g glucose) s t i m u l a t e d a m y l i n levels were h i g h e r in obese (n=7) c o m p a r e d to controls (n=7; 48.7• vs. 30.3• pg/ml at 60 minutes, p=0.09 and 36.1 vs. 19.3• pg/ml at 180 minutes, p<0.05). Basal and s t i m u l a t e d a m y l i n levels correlated closely to plasma insulin values (r=0.92, p<0o01). In c o n c l u s i o n a m y l i n levels are e l e v a t e d in obese p a t i e n t s with IGT or type 2 diabetes mellitus and are not detectable in patients w i t h type 1 diabetes mellitus. Amylin values c o r r e l a t e c l o s e l y to p l a s m a insulin levels and are p o s s i b l y c o s e c r e t e d by pancreatic B-cells.

MEASUREMENT OF AMYLIN IN PANCREATIC TISSUE AND PLASMA IN TYPE 2 DIABETES A.E. Butler, P.C. Roche, W.B. Carter, .1. Chou, R.A. Rizza, and P.C. Butler, Endocrine Research Unit and Department of Pathology, Mayo Clinic, Rochester, MN 55905, USA Amylia has recently been implicated in the pathogenesis of type 2 diabetes. By use of a specific pelyclonal rabbit antiserum to human amylin and peroxidase antiperoxidase immunostaining we sought to confirm the localization of amylin to beta cells in normal man (6), and extracellular amyloid in insulinoma (13) and type 2 diabetic (9) pancreases as well as measuring circulating concentrations by radioimmunoassay with the same antibody in extracted plasma. Extracellular deposits of amylin were present in 100% and 56% of insulinoma and diabetic pancreases, respectively. Amylin colocalized with insulin in 100% normal pancreases. Plasma amylin was measured before and following meal ingestion in lean controls (LC, n=10), patients with type 2 diabetes (D, n=10) and matched controls (MC, n=5). Both amylin (2.03 _+ 0.22 to 3.78 + 0.39 pmol/l) and insulin (48.3 + 3.1 to 265 + 44 pmol/1) increased (p < 0.001) following meal ingestion (LC) implying co-secretion. Neither fasting (6.7 + 0.7 vs 4.8 _+ 2.0 pmol/l, p=0.3) or postprandial (1.6 _+ 0.2 vs 1.4 + 0.5 nmoI/1/1/3 hours, p=0.7) amylin was different in D versus MC. In conclusion, amylin is co-localized with insulin in beta cells of healthy man, and is co-secreted with insulin following meal ingestion. Extracellular amylin deposits are often but not invariably present, and circulating concentrations are not increased in patients with type 2 diabetes.

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112

RAT AMYLIN: DISTRIBUTION, SECRETION AND BINDING SITES. J. Chou, Y. X. Zhu, Y. N. Wang, P. Butler, D. Chang and J. K. Chang. Peninsula Laboratories, Inc. Belmont, CA 94002 Mayo Clinic, Rochester, MN 55905 Amylin, a 37 amino acid peptide isolated from amyloid deposits from islet deposits of patients with type 2 diabetes has been implicated in the pathogenesis of this disease. By use of specific rat amylin antiserum we sought to determine: 1 ) the localization and quantity of amylin in rat pancreas and liver; 2) the circulating concentration of amylin and its response to intravenous glucose. Immunofluorescence indicated that amylin was co-localized with insulin in the beta cells. Radioimmunoassay demonstrated amylin content was 105 + 9.9 pg/mg wet weight in pancreas, but none detectable in liver. Radioimmunoassay of extracted plasma indicated lower amylin concentration in fasted versus fed rats (6.8 _+0.7 versus 9.8 + 0.9 pg/ml, p<0.05). Intravenous glucose injection (30 mg) led to an increase in both insulin and amylin (correlation r=0.7, p<0.001 ). Use of I I~-rat amylin (0.5 nM) in the presence and absence of 1 ~M amylin in crude membrane extracts indicated most displaceable binding in liver, and 45%, 5%, 1% relative to liver in muscle, brain and pancreas. In conclusion, amylin is co-localized with insulin to beta cells and is co-secreted following intravenous glucose injection. Our preliminary data indicates that while amylin is predominantly localized in pancreas, displaceable binding is most abundant in liver.

FAILURE OF AMYLIN TO AFFECT INSULIN SENSITIVITY IN THE ISOLATED, PERFUSED RAT LIVER M. Roden, I. Kothbauer, H. Vierhapber and W. Waldh~usl Dept. of Endocrinol., I.Med.Univ.Klinik, Vienna, Austria The recently purified polypeptide, ~mylin (islet ~nyloid polypeptide, diabetes-associated pept~de), is synthetized by pancreatic beta-cells and was shown to be increased in diabetic humans. In vivo- and in vitrostudies in mammalian sceleta] muscle and isolated adipocytes indicate that amylin induces insulin resistance. To elucidate effects of ~ylin on hepatic insulin sensitivity we measured glucose output of isolated rat livers, perfused with an oxygenated Krebs-RiDger buffer and dialyzed human erythrocytes in a non-recirculating system. Amylin (10E-7 M) alone had no influence on hepatic glucose release. Infusion of 10E-If M glucagon alone (control) resulted in an expected elevation of the glucose production rate ( ~ ] / ( m i n x 100g b.w.)) reaching a maximum of 12.69+3.95 after 30rain. Insulin (50mU/l) submaximally decreas-ed the glucagon-stimulated glucose production (2.7]+1.65 at 30min). A simultaneous infusion of 10E-7 M amylin was not able to reverse the insul in-dependent inhibition of g] ucagon-st imulated glucose production (1.77_+0.51 at 30rain). Glucose release within 86min, estimated as AUC (umol/100g b.w.; control: 951.3+272.7), was similar in the presence of amylin (128.1+_-29.6) and in its absence (166.2+95.4). At a concentration that induces insul~n-resistance in sceletal muscle and adipocytes amylin has no impact on hepatic insulin sensitivity in vitro. This does not exclude an effect of amy]in on hepatic gluconeo~enesis by provision of glucogenic metabolites due to ~ i p h e ral insulin resistance in vivo.

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ISLET AMYLOID POLYPEPTIDE INHIBITS INSULINS T I M U L A T E D G L U C O S E T R A N S P O R T IN S K E L E T A L M U S C L E I N D E P E N D E N T OF C H A N G E S IN G L Y C O G E N LEVELS H W a l l b e r g - H e n r i k s s o n , J R Zierath, K A n d r 4 a s s o n , D Galuska, U Engstrom, C Betsholtz, P W e s t e r m a r k . Dept of Clin Physiol, K a r o l i n s k a Hospital, Stockholm, S w e d e n

RAT AMYLIN INHIBITS INSULIN RELEASE WITHOUT AFFECTING GLUCAGON AND SOMATOSTATIN OUTPUT IN THE RAT PANCREAS

D e p o s i t i o n of islet a m y l o i d p o l y p e p t i d e (IAPP) in the islets of L a n g e r h a n s characterizes noni n s u l i n - d e p e n d e n t d i a b e t e s mellitus. This p e p t i d e has b e e n s u g g e s t e d to d e c r e a s e g l y c o g e n s y n t h e s i s in skeletal muscle. We i n v e s t i g a t e d the e f f e c t of rat (r) and human (h) IAPP on g l u c o s e t r a n s p o r t and g l y c o g e n c o n t e n t in the i s o l a t e d rat epit r o c h l e a r i s m u s c l e and in h u m a n s k e l e t a l muscle. M u s c l e s p e c i m e n s from the q u a d r i c e p s m u s c l e w e r e o b t a i n e d from 6 h e a l t h y subjects, by u s i n g a newly d e v e l o p e d open m u s c l e b i o p s y technique. 30-Methylglucose uptake measurements were performed on in v i t r o i n c u b a t e d e p i t r o c h l e a r i s m u s c l e s and on h u m a n m u s c l e strips. IAPP (i-i00 nmol/l) did not a f f e c t basal g l u c o s e transport. I n s u l i n s t i m u l a t e d (i00 ~U/ml) g l u c o s e t r a n s p o r t in rat m u s c l e s was d e c r e a s e d by rIAPP (i00 nmol/l); 6.57• v e r s u s 5.50• ~ m o l / m l / h (p<0.01). In h u m a n skeletal m u s c l e strips, the p r e s e n c e of h I A P P at i00, i0 and 1 nmol/l d e c r e a s e d i n s u l i n s t i m u l a t e d (i00 ~U/ml) g l u c o s e t r a n s p o r t by 40% (p<0.01), 39% (p<0.01) and 24% (p<0.05), r e s p e c tively. M u s c l e g l y c o g e n levels w e r e u n a f f e c t e d by the p r e s e n c e of i00 nmol/l of IAPP. In c o n c l u sion, IAPP inhibits i n s u l i n - s t i m u l a t e d g l u c o s e t r a n s p o r t in b o t h human and rat skeletal muscle, c o n c o m i t a n t w i t h u n c h a n g e d g l y c o g e n levels. Thus, IAPP m a y cause p e r i p h e r a l insulin r e s i s t a n c e by p r i m a r i l y a f f e c t i n g the t r a n s p o r t of g l u c o s e across the cellmembrane.

R.A. Silvestre, E. Peir6, P. D4:gano, A. Iglesias, P. Mirailes and J. Marco. Hospital Puerta de Hierro, Universidad Aut6noma de Madrid, Madrid, Spain. Amylin, a 37-amino acid polypeptide, is the main component of amyloid deposits in the pancretic islets and has been identified in the B-cell secretory granules. We have investigated the effect of synthetic rat amylin (Peninsula Labs.) on insulin, glucagon and somatostatin release in the perfused rat pancreas. Perfusate consisted of Krebs-Henseleit buffer supplemented with albumin (0.5%), dextran T-70 (4%) and glucose (5,5 mmol/I). Hormones were analised by RIA. Amylin infusion (750 nmol/I) inhibited unstimulated insulin release (by 50%, p<0.01) as well as the insulin response to 9 mmol/I glucose (by 80%, p<0.05). At a lower concentration (500 nmol/I), amylin also reduced the insulin responses to 11 mmol/I glucose (by 70%, p<0.01) and to 3.5 mmol/I arginine (by 40%, p<0.025). Amylin failed to significantly modify glucagon and somatostatin release under any of the above experimental conditions, in summary, in the rat pancreas, homologous amylin: 1) reduces unstimulated insulin release as well as the insulin responses to metabolic substrates (glucose and arginine); 2) does not affect glucagon or somatostatin release. These observations suggest that amylin influences the B-cell directly, and not through an A-cell or D-cell paracrine effect. The inhibitory effect of amylin on insulin secretion favours the concept of this novel peptide as a diabetogenic agent.

OP 20 Hall A-2 Education 115

116

THE STOCKOLM DIABETES CONTROL PROGRAM - ORGANIZATIONAL CHANGE AND CONTINUING MEDICAL EDUCATION

A M O D U L A R O U T P A T I E N T E D U C A T I O N P R O G R A M FOR TYPE 2 D I A B E T I C PATIENTS K. Nelson, K.Howorka, H.Grillmayr, D.Heydarian, H.Thoma, P . S c h e n k and Ch. Schlusehe, Institute for B i o m e d i c a l Engineering, U n i v e r s i t y of V i e n n a The p r e s e n t study was a pilot p r o j e c t to test a p a t i e n t e d u c a t i o n p r o g r a m d e v e l o p e d to meet the varied requirements of Type 2 (non-insulindependent) diabetic patients. The p r o g r a m consists of s t r u c t u r e d teaching m o d u l e s w h i c h can be used in group instruction by paramedical p e r s o n n e l on an o u t p a t i e n t basis, including: (i) review of t h e r a p y and rehabilitation; (2) basic information on diabetes; (3) behavior modification techniques for weight loss; (4)instruction for p e r i o d i c subtotal fasting; (5) i n f o r m a t i o n and d i e t a r y g u i d e l i n e s regarding blood lipids; (6) training in functional insulin treatment (7) a n t • training; (8) rehabilitation of d i a b e t i c late complications (e.g., visual -impairment); (9) psychological counseling. Subjects were 36 Type 2 diabetic patients (age: 61• d u r a t i o n of diabetes: 12• yrs/x+SD~. 78% of p a r t i c i p a n t s were overweight. 62% were insulin treated. P a r t i c i p a t i o n in the various modules (according to individual need) was as follows: Module (i): 97%; (2): 69%; (3): 44%; (4): 19%; (5): 56%; (6): 54%; (7): 11%; (8): 11%; (9): 14%. A follow-up examination c o n d u c t e d 8• months after program c o m p l e t i o n showed changes in HbAlc(%): (before) 7.4• 6.6• (p<0.0001), body weight(kg): 76• 75+11 (p<0.03),

A Carlson and U Rosenqvist, LUCD, Karo]inska Hospital 104 Ol Stockholm, sweden The aim was to evaluate a diabetes training program which included organizational development measures and @ CME course. Thirty-Four primary health care centers had volunteered For the program - 17 centers were randomized to the training program, which lasted For 18 months, and 17 centers comprised the control group. In total 4538 diabetic patients, 127 general practitioners and 249 registered nurses were involved in the study. The study of organizational change included content analysis of problems identified and solutions designed by staff and patients in the intervention group. Organizational effects of the program was measured by the extent to which the solutions had been implemented after 18 months. Process of care was studied through measures of staff competence, involvement in diabetes care, resources, cooperation and quality of service. Patient outcomes were studied by means of patients" dietary knowledge, self-care behaviors and metabolic control. The results showed that the program had been highly successful as a means for organizational change and for improvements in process of care. The patients" metabolic control, however, had not been affected. Reasons for this are discussed, including short time of exposure to the changed organization, and inadequate measures to detect beneficial effects of the program.

cholesterol(mg/dl):

243•

(p
blood pressure(mmHg): systolic 163•177 (p<0.02), diastolic 87•177 (p<0.02), metabolic self-monitoring (% of patients): 26%/97% (p<0.0001). A modular outpatient education program provides the necessary flexibility for the v a r i e d problems of Type 2 diabetes.

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117 DIET EDUCATION IMPROVES METBBOLIC CONTROL PATIENTS WITH NON-INSULIN-DEPENDENT DIABETES

OF

J.Jormanainen, M.Uusitupa, O.Siitonen Departments of C l i n i c a l N u t r i t i o n and Medicine, U n i v e r s i t y of K u o p i o The aim of the s t u d y was to find out w h e t h e r an intensive diet education p r o g r a m for r e c e n t l y diagnosed patients with non-insulin-dependent diabetes, aged 40-65 years, w o u l d r e s u l t in a more s a t i s f a c t o r y c o n t r o l of b l o o d g l u c o s e and cardiovascular risk factors than conventional diabetes management. Altogether 49 recently d i a g n o s e d obese patients w e r e r a n d o m i z e d after three m o n t h s b a s i c diet e d u c a t i o n into i n t e n s i v e diet e d u c a t i o n (ID) and control g r o u p s (CD). After 15 m o n t h s b o t h g r o u p s s h o w e d significant i m p r o v e m e n t s in w e i g h t (93,8+].4,2 kg (mesn~sd) vs. 8 6 , 8 ~ 1 3 , 0 kg in ID, p < 0 , 0 O I and 9 0 , 5 + 1 4 , 1 kg vs. 8 6 , 8 + 1 4 , 1 kg in CD, p<0,001); in fasting blood glucose (Ii,3~5,3 mmol/l vs. 5,9~i,3 mmol/l in ID, p < O , 0 0 1 and 11,0+_3,5 mmol/l vs. 7 , 9 + 2 , 4 m m o l / l in CD, p<0,001); in g l y c o s y l a t e d HBAIc (8,1+2,3 % vs. 6 , 4 ~ i , 0 %, in ID, p<0,01 and 8 , 9 ~ 2 , 6 % vs. 7 , 7 + 1 , 6 % in CD, p<0,01); in serum triglycerides --(3,88+2,32 mmol/l vs. 2,29~i,04 mmol/l in ID, p
Cognitive function deficits in Type-2 (non-insulin-dependent) diabetes patients in the general population. H. de Vrias, L.R. van Houtc, s van Eijk and * s Lindeboom, Department of General Practice / Institute of F.xtramural Medicine and * Department of Medical Psychology, Faculty of Medicine,Free University,Van der Bocchorststraat7, 1081 BT Amsterdam, The Netherlands In order to evaluate cognitive functioning in Type 2 (noninsulin-dependent) diabetic patients in the general population 29 of these patients (criteria: WHO-study group 1985; age 45 75 years) from general practice lists and 29 non-diabetic controls, matched for gender, age and education, were submitted to neuropsychological testing by a trained psychologist blinded for N1DDM/control. Excluded were persons with organic brain disorder, renal or hepatic insufficiency, centrally acting medication or alcohol abuse. The neuropsychological battery comprised tests for memory, concentration and reaction time. A questionnaire focussed on cognitive symptoms. The mean total-score of a serial learning test of 15 words in 5 trials was in diabetic patients 37.1 +_ 8.3(SD), in controls 42.2 +_ 8.3(SD) ( p=0.008, Students paired-test). The mean score for Digit Span backwards was in dlabetiepatients 5.1 + 1.8(SD), in controls 6.2 _+ 1.6(SD) (p=0.025, Students paired-test). No differences in reported cognitive symptoms were found. The outcome supports the hypothesis of a mild organic brain syndrome as a complication of Type 2 (non-insulin-dependent) diabetes mellitus in the general population. This may have implications for patient compliance and diabetes-education.

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A NEW S T R A T E G Y FOR C O N T R O L L E D T R A N S F E R OF "NEARNORMOGLYCEMIC INSULIN SUBSTITUTION" H.Thoma, K . H o w o r k a and H.Grillmayr, Institute of B i o m e d i c a l Engineering. U n i v e r s i t y of Vienna, S e n s e n g a s s e 8, 1090 Vienna, Austria. Numerous methods for education of Type i (insulin-dependent) p a t i e n t s exist, but only a few groups - e.g. M.Berger, D U s s e l d o r f - are concerned with q u a l i t y control in t r a n s f e r to other centers. Our m e t h o d of t r a n s f e r for the training p r o g r a m in n e a r - n o r m o g l y c a e m i c insulin s u b s t i t u t i o n (NIS) includes: (I) s t a n d a r d i s a t i o n of the educational programme using different media; (2) o r g a n i s a t i o n of annual w o r k s h o p s for physicians and diabetes counselors; (3) evaluation of results one year after the workshop; and (4) organisation of the "International W o r k i n g G r o u p NIS". So far 147 persons (52% from Austria, 42% BRD and 6% from other countries) have taken part in 3 annual workshops. 64% of p a r t i c i p a n t s were physicians, 27Z d i a b e t e s c o u n s e l o r s and 9% representatives of l a y - o r g a n i s a t i o n s . A s i g n i f i c a n t increase in p a r t i c i p a n t s ' d i a b e t e s k n o w l e d g e was o b s e r v e d in a q u e s t i o n n a i r e a d m i n i s t e r e d before and after each w o r k s h o p - mean increase from 71% to 94% correct answers (p<0.0001). One year after the workshop, p a t i e n t s e d u c a t e d in the transferred programmes showed a decrease in HbAlc from I@0Z+20 of the normal c u t - o f f value to 125%• (x• This project shows that a health education program can be standardized and transferred. The skills and organisational talents of bioengineers can be particularly useful in the t r a n s f e r of such medical methods.

Determinants of the behavlour 'in3ecting insulin' in patients with type 2 diabetes mellitus. B.H.R. Wolffenbuttel, C.H.C. Drossaert, A.Ph. Visser. State University Limburg, The Netherlands. In type 2 diabetic patients metabolic control may worsen despite diet and hypoglycaemic drugs. We investigated which factors determine the choice for elderly type 2 diabetics to start insulin therapy. According to Fishbein and A]zen a behaviour-model was constructed which included the determinants intention, attitude, subjective norm and personal efficacy. Fiftythree type 2 diabetic patients (age 68• years) participated: 26 already used insulin for 7 (I-12) months, 27 were tablet-treated but poorly-controlled (fasting blood glucose >8.0 mmol/l). A high correlation (r=0.81, p<0.001) existed between intention and the desired behaviour. The main factor (70%) explaining intention was the subjective norm: patients are guided by the opinlon of important other persons, especially the treating internist and the family physician. Tablet-treated patlents reported a negative opinion of their physician towards insulin therapy. Intention was also determined by personal efficacy, e.g. presence of skills concerning injecting insulin. Attitude correlated with intention (r=0.60, p<0.001), but did only indirectly influence behaviour. Selfmonitoring of blood glucose and knowledge about diabetes positively influenced personal efficacy and therefore intention. In conclusion, for the patient's choice of starting insulin therapy the opinion of the physician is most important. Education needs to improve the patient's personal efficacy and attitude, but a stimulating attitude of the treating physician is essential.

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OP 21 Hall A-3 Experimental Transplantation 121

122

MODIFICATION~ IN VITRO AND IN VIVO EVALUATION OF MICROENCAPSULATED RAT ISLETS: (TRANSPLANTATION INTO NOD MICE AND BB RATS), A.M. Sun, L. Levesque, Z. LumjM. Fan and J. Norton. University of Toronto, Toronto, Canada

INTRAVASCULAR TRANSPLANTATION OF NICROENCAPSULATED ISLETS IN DIABETIC DOGS. R.Calafiore, G.Basta, A.Falorni jr., M.L.Picchio, *G.Brotzu and P.Brunetti. Istituto di Patologia Soeciale Medica, University of Perugia, Perugia and ~Istituto di Patologia e Chirurgia, Universlty of Cagliari, Cagliari, Italy. We had shown that microcapsules comprised of alginic acid and aminoacidic polycations may immunoprotect islets of Langerhans in vitro and in rive after intraperitoneal transplant,in rodents with either spontaneous (NOD mice) or Streptozotocin induced (Balb/C mice) diabetes, as well as to provide islets with a favorable microenvironment for long-term survival and function (Calafiore et al., Novel drug delivery and its therapeutic application, John Wiley & Ltd., 305-312, 1989). To improve the life-span of islet grafts and to achieve success of this approach also in high mammalian with diabetes, we have developed a coaxial, artificial vascular prosthesis (PR), which is directly anastomized to blood vessels, with the microencapsulated islets being placed between the inner (permeable) and the outer (impermeable) layer of the chamber. This approach is seemingly associated with better oxygen and nutrient supply, compared to the peritoneal site of implant, to the encapsulated islets. 6 out of 6 non immunesuppressed dogs with spontaneous diabetes (daily regular + NPH insulin: 25 U) receiving 150,0OO microencapsulated porcine islets within the vascular aortic-femoral vein PR, showed normalization of blood glucose along with either suspension (1/6 x 7 d) or dramatic dropping of exogenous insulin (6/6 x at least 90 d). Microcapsules retrieved from the PR were free of fibrin overgrowth and contained well preserved porcine islets. Vascular prostheses may represent a potential strategy for the large scale transplantation of microencapsulated islets in high mammalians with diabetes.

Rat islets were transplanted into spontaneously diabetic NOD mice and BB rats 9 using a modified encapsulation procedure and reducing the capsule diameter to 0.2-0.3 mm. In 24-hour culture studies, islets enclosed in the modified capsules responded to glucose challenges and glucose plus IBMX (3-isobutyl-l-methyl-xanthine) or PMA (phorbol 12-myristate 13-acetate) challenges with nearnormal increases in insulin secretion. Single transplants of 1,000 encapsulated rat islets into 16 NOD mice reversed hyperglycemia for an average of 176+60 days without immunosuppression. Removal of the implanted capsules at 4 and 6 months post-~ransplant resulted in a quick return to the diabetic state, but hyperglycemia was again reversed ,by second transplants. More than 80% of the recovered capsules were intact anJ contained viable islets. Single transplants of 5 X i0 encapsulated Lewis rat islets into 12 BB rats restored normuglycemia for an average of 192+41 days, also without immunosuppression. These results demonstrate that properly constructed alginate-polylysine microeapsules can protect transplanted islets from immunorejection in spontaneously diabetic rodents.

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BLOOD GLUCOSE LEVELS IN DIABETIC RATS AFTER FREE AND I, iICROENCAPSULATED PANCREATIC ISLET TRANSPLANTATION WM Fritschy, GHJ Welters and R v Schilfgaarde. Dept. of Surgery, State University Groningen, The Netherlands.

MICROENCAPSULATION OF ISLETS IN BAg+-ALGINATE BEATS: IN VITRO AND IN VIVO EXPERIMENTS T.Zekorn, C.Klenke § U.Siebers, R.G.Bretzel, u. Z i m m e r m a n n § and K.Federlin, Medizinische K l i n i k III, Universitaet Giessen and+Lehrstuhl fuer Biotechnologie, Universitaet Wuerzburg, West-Germany Artificial membranes may prevent immune destruction of transplanted islets.---1. After encapsulation of 50 rat islets in Bariumalginate beats (diameter: 0.8 um) a glucose challenge w a s p e r f o r m e d (60 m i n s preincubation in 2.7 m m o l / l , 120 m i n s 1 6 . 7 m m o l / l , 60 m i n s 2.7 mmol/l, 1.5 ml Krebs-Ringer-buffer).--2. 50 encapsulated i s l e t s w e r e k e p t in c u l t u r e f o r 7 d a y s (RPMI 1 6 4 0 + I 0 % F C S , 37~ 5% C02) f o l l o w e d by a glucose challenge as described. 50 free floating islets served as controls in both experiments.-3. 600 e n c a p s u l a t e d r a t islets were transplanted intraperitoneally into Streptozotocin-diabetic CDl-mice. 600 free islets or empty capsules served as controls.--i. After 120 mins encapsulated islets (n=6) secreted 705.1 uU/ml insulin (controls: 1872.3 uU/ml, p<0.05) and after change to low glucose 176.6 vs 139.9 uU/ml (p=n.s.). 2. Precultured encapsulated i s l e t s (n=8) s e c r e t e d 3 6 . 4 % (120 mins, p~0.05) compared to controls and 158.2% (p~0.05) with low glucose. 3. At day 4 after transplantation 5/8 mice were postprandial normoglycemio (day i0: 6/7, d a y 21: 3/7). E m p t y capsules showed no and free islets a transient (day 2: 7/8, day 7: 0/8) normoglycemia.--Ba-alginate beats revealed a similar pattern of insulin secretion as free islets but on a lower level. This result was preserved in culture. Transplantation experiments demonstrated a marked prolongation of normoglycemia. Histol o g i c a l e x a m i n a t i o n s a r e in p r o g r e s s .

Microencapsulation of pancreatic islets has promise as a means of i~nunoisolation in allogenic transplantation. In this study we compared the effects of uneneapsulated isogenie islet transplantation (Albino-Oxford~AO) and alginate-polylysine microencapsulated isogenic and allogenic islet transplantation (Lewis~AO) on non-fasting blood glucose levels (BG) in streptozotoein diabetic rats (BG)20 mM). Each transplantation was performed with 8-12 ul islet tissue (2500-3000 islets), which equals the volume of the endocrine pancreas of a normal rat. All rats receiving free intraperitoneal isogenic islets (n=I0) became normoglyeemic for at least 6 months, with BG of 6.9 + 0.6 mM. Healthy normal control animals (NC) had BG of 7.2 + 0.6 mM. ~Tnen microencapsulated isogenic islets (n=5) were transplanted, also all rats became normoglycemic with BG of 8.4 + 0.6 mM for at least 3 months (p(0.005 compared to NC). Rats receiving allogenic microencapsulated islets (n=6) attained BG of 8.3 +_ 1.0 mM (p<0.02 compared to NC), for a maximum of 40 days. No relapse to hyperglycemy occurred. Microcapsules retrieved 3 weeks after isogenic transplantation contained viable islet tissue whereas no cellular overgrowth was seen. Conclusions: I. Intraperitoneal transplantation of one pancreas equivalent free islet tissue completely normalizes BG; 2. Transplantation of microencapsulated islets effeetivily reduces, but not completely normalizes BG; 3. Alginate-polylysine microencapsulation prevents allograft rejection for at least 40 days.

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125 AN IMPLANTABLE DIFFUSION CHAMBER FOR A BIOARTIFICIAL ENDOCRINE PANCREAS. H.Ohgawara, S.Karibe, N.Mochizuki, A.Mizuno and Y. Hi r at a Diabetes Center, Tokyo Women's Medical College, Tokyo, Japan. We p r e v i o u s l y described a method in which neonatal pig pancreatic i s l e t l i k e c e l l c l u s t e r s (ICCs) were embedded in an e x t r a c e l l u l a r matrix f o r long-term maintenance. When the IgCs were c u l t u r e d in the g e l - m a t r i x , they survived and functioned f o r a long-term c u l t u r e period in the presence of nicotinamide. An implantable d i f f u s i o n chamber f o r a b i o a r t i f i c i a l endocrine pancreas (Bio-AEP) was constructed by placing ICCn in an e x t r a c e l l u l a r matrix in the center of a r i n g holder sandwiched between two p o l y e s t e r membrane ( pore size : O.2um ), which were held in place by two other r i n g holders. Five NOD mice received Bio-AEPs containing appro xi mately 300-400 pig ICCs ( x e n o g r a f t i m p l a n t a t i o n ) . All d i a b e t i c NOD mice with Bio-AEPs survived without any immunosuppressant f o r over 7 weeks while the NOD mice which had received a chamber without ICCs ( c o n t r o l ) died w i t h i n 4 weeks. Our r e s u l t s i n d i c a t e t h a t the Bio-AEP shoul be useful f o r the implantation o f x e n o g r a f t i c i s l e t s in d i a b e t i c animals and may open a new f i e l d in the therapy of human d i a b e t i c s .

126 Reinnervation of transplanted islets in the mouse F. Sundler, O. Korsgren, L. Jansson, A. Andersson. Department of Medical Cell Research, University of Lund and Department of Medical Cell Biology, University of Uppsala, Sweden The pancreatic islets are richly innervated with both sympathetic, parasympathetic and sensory nerve fibers, and it is generally agreed that neuronal signals play important roles as modulators of islet hormone secretion. This study aimed to investigate the reinnervation of islet grafts by immunocytochemistry and acetylcholinesterase (ACHE) staining. For this purpose, syngeneic islets were grafted beneath the renal capsule of normoglycemic or alloxan-diabetic C57BL/6 mice. Also fetal porcine islets were implanted into alloxan-diabetic nude mice. In syngeneic grafts, neuropeptide Y (NPY) and tyrosin hydroxylase (TH) positive (presumably adrenergic) nerve fibers were seen already 6 or 8 weeks post-transplantation, but were more frequent after 12 and 20 weeks. VIP-fibers and fibers storing substance P and CGRP (probably sensory fibers) were few and scattered. AChE-positive fibers were regularly seen 12 and 20 weeks posttransplantation. In diabetic animals reinnervation was less pronounced up to 20 weeks post-transplantation. In porcine islet grafts VIP-containing fibers, originating from ganglia in the grafts, were numerous after 12 and 20 weeks. VIP-fibers originating from ganglia in the graft were often seen to invade the kidney cortex. Also NPY-, TH- and AChE-positive fibers were regularly seen, whereas CGRPand substance P-containing fibers were virtually absent. In conclusion, reinnervation of subcapsular syngeneic and xenogeneic islet grafts is established 12 weeks post-transplantation. The functional significance of this process remains to be defined.

OP 22 Hall A-15 HLA and Type I Diabetes 127

128

INCREASED FREQUENCY OF DELETED OR SHORT C4B GENES IN TYPE 1 DIABETIC PATIENTS. I. Deschamps, A. Marcelli-Barge, J.C. Pokier, R. Chantome and J.Hors. INSERM U30, H6pital Necker, and Laboratoire d'Histocompatibilit@ and INSERM U93, H6pital St. Louis, Paris, France. Type 1 diabetes is characterized by the high frequency of two HLA-DR3 haplotypes in linkage disequilibrium with C4 null alleles, C4AQ0 and C4BQ0, suggesting a possible relationship between 04 structural markers and susceptibility. Therefore, we have studied by Southern blot, using a 500 bp C4cDNA probe, the restriction fragment length polymorphism of the C4A, C4B genes in 3 5 unrelated caucasian diabetic and 40 control subjects genotyped for HLA-A,B,C,DR,Bf,C4A,C4B. No significant difference was observed at the C4A locus between patients and controls, the C4A deleted gene being mainly found with the extended haplotype A1BSDR3. At the 04B locus, the distribution of the two main structural markers, long and short, differed significantly. There was a decrease of C4B long in patients compared with controls (22% MS 49%, p<0.001) and a compensatory increase of both C4B short (52% vs 41%, respectively, n.s.) and C4B deleted (20% vs 8%, respectively, p<0.02). All genotypes containing C4B long (in the homozygous or heterozygous state) were markedly decreased in patients compared with controls (29% vs 77%, p<10"4). These findings suggest the specific involvement of a gene or gene product of the class III region, probably near the C4B locus, in susceptibility or resistance to Type .1 diabetes.

MAJOR SIZE DIFFERENCES DETECTED BY PULSED FIELD ELECTROPHORESIS MAPPING OF MHC HAPLOTYPES IN DIABETIC FAMILIES M.J.

Niven,

J.A. The

Sachs. London

G.A. Hitman, H. Pearce, B. Marshall and Medical Unit and Department off Immunology, Hospital

Medical

College,

London,

U.K.

Although the HLA-DQ genes appear to be of p a r t i c u l a r importance in determining susceptibility to type I (insulin-dependent) diabetes, there is mounting evidence that other MHC genes, such as HLA-DR, complement, TNF and class I , are i mpl i cat ed. The aim of t h i s study was to use pulsed f i e l d e l e c t r o p h o r e s i s mapping to i n v e s t i g a t e , in heterozygous f a m i l y members, the long range s t r u c t u r e of haplotypes defined by alleles of such genes; 21-hydroxylase, DRA, DQB, DOB and DPA probes were used. In one f a m i l y susceptibility to diabetes was l i n k e d to a DR3/DQw2 haplotype and resistance to DR2/DQwQ. In the other a DR4/DQw3 haplotype was associated with myxoedema and p e r n i c i o u s anaemia in two s i s t e r s , but only the one who a d d i t i o n a l l y possessed DR3/DQw2 also had diabetes. Major d i f f e r e n c e s between haplotypes were found in the size of the DQ/DR gene region: relative to the d i a b e t e s - p r o t e c t i v e DR2 haplotype, the DRw13 and two DR3 haplotypes studied are l a r g e r by 130 kb, and the two DR4 and two DR7 haplotypes are a f u r t h e r 140 - 150 kb l a r g e r . These v a r i a b l e lengths of DNA may car r y novel diabetogenic genes, may i n f l u e n c e expression o f nearby or d i s t a n t genes, and also suggest a mechanism f o r the maintenance of diabetogenic a l l e l i c combinations by recombination suppression.

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129 ESTIMATION OF HAPLOTYPE SPECIFIC IN T Y P E 1 D I A B E T E S I N F I N L A N D

130 ABSOLUTE

RISK

H L A - D Q A I and DQ B1 p o l y m o r p h i s m s and g e n e t i c s u s c e p t i b i l i t y to t y p e 1 d i a b e t e s m e l l i t u s .

E. Tuomilehto-Wolf, J. Tuomilehto, J. Lappivaara a n d A. C e p a i t i s and the DiMe Study Group. Department of Epidemiology, National Public Health Institute, Helsinki, Finland.

E.LARGERI, X.F.GUI, K.BESSAOUD2, R.KRISHNAMOORTHy3 J.ELION3, E.CLAUSER4, R.ASSANI. 1.Diabetes Dpt Hopital BICHAT, Paris, F r a n c e 2. Oran, A l g e r i a

In a n a t i o n w i d e study in Finland 751 families with a newly diagnosed Type 1 diabetic child aged 0-14 years were HLA genotyped. Whole HLA haplotypes (including HLA-A,C,B,DR and DQ loci) were used as markers for susceptibility to Type 1 diabetes. Haplotype frequencies (HPFR) and haplotype specific absolute risk per i00 000 per year (AR) were calculated for 1426 "diabetic" haplotypes found in the diabetic probands. Of the significantly increased "diabetic" haplotypes the recently described third most common haplotype in FinlandA2,CwI,Bw56,DR4,DQw8 - carried the highest AR (AR 210, HPFR 5.4%), followed by A28,Cw7,B8,DRwI7,DQw2 (AR 180, HPFR 2.1%), A3,Cw3,Bw62,DR4,DQw8 ( A R 151, HPFR 2.8%) and then the most frequently found "diabetic" haplotype A2,CwS,Bw62,DR4,DQw8 ( A R 141, HPFR 10.2%). The second most common "diabetic" haplotype - AI,Cw7,B8,DRwI7,DQw2 - (HPFR 9.3%) h a d a n A R o f 70. H a p l o t y p e specific absolute r i s k is a g o o d w a y o f a s s e s s i n g the importance and contribution of different "diabetic" haplotypes which are the most specific markers for susceptibility to Type 1 diabetes in Finland where the overall yearly incidence is t h e h i g h e s t i n t h e w o r l d (35 p e r i 0 0 0 0 0 ) .

Both t h e e [ a n d )B chains of t h e H L A - D Q a n t i g e n - p r e s e n t i n g m o l e c u l e a r e polymorphic and may c o n t r i b u t e to the g e n e t i c s u s c e p t i b i l i t y to t y p e 1 d i a b e t e s m e l l i t u s . Combined a n a l y s i s of a l l e l i c v a r i a t i o n s of the two coding g e n e s may help p r e d i c t i o n and u n d e r s t a n d i n g of the disease. We have studied 151 type 1 d i a b e t i c p a t i e n t s and 111 controls, using PCR a m p l i f i c a t i o n of g e n o m i c DNA, t y p i n g by ASO probes, RFLP a n a l y s i s of the a m p l i f i e d m a t e r i a l and d i r e c t n u c l e o t i d e sequencing. Among p a t i e n t s 9 1 % w e r e A s p a r t a t e n e g a t i v e a t codon 57 of the DQ )3 c h a i n (controls : 5 1 % ) , 84 % of t h e m being h o m o z y g o t e s ( c o n t r o l s : 28 %). The h i g h e s t risk was a s s o c i a t e d with h o m o z y g o c i t y for the two a l l e l e s with Alanine a t codon 57 : DQ B 2/3.2 (ie B 0201/0302) : RR = 21. H L A - D Q A polymorphism was c h a r a c t e r i z e d by p r e d o m i n a n c e , in p a t i e n t s , of DQ A 3/4.1 (ie : A 0301/0501) : 45 % (vs c o n t r o l s : 5 % ; p <.001 RR = 10). The c o m b i n a t i o n DQB 2/3.2 + DQA 3/4.1 was p r e s e n t in 25 % of p a t i e n t s , vs z e r o in c o n t r o l s (p < 0.001). No e s s e n t i a l d i f f e r e n c e a p p e a r e d e i t h e r b e t w e e n F r e n c h (n=I15) and Maghrebin p a t i e n t s , or b e t w e e n t y p e 1 a (n=110) and t y p e 1 b (polyendocrine) p a t i e n t s . Both the o[ and ]3 chains of DQ m o l e c u l e s a p p e a r involved in s u s c e p t i b i l i t y to type 1 d i a b e t e s , due p r e s u m a b l y to c o n t r i b u t i o n of both to the c o n f o r m a t i o n of the a n t i g e n - p r e s e n t i n g c l e f t .

131

132

THE DQA3 ALLELE IS ASSOCIATED WITH SUSCEPTIBILITY TO TYPE I (INSULIN-DEPENDENT) DIABETES. K.H. Jacobs, D. Jenkins, C. M i j o v i c , M. Penny, Y. Uchigata, T. Otani, Y. Hirata, J.A. Fletcher, A.R. 8radwell and A.H. Barnett. Dept. of Medicine and Immunology, U n i v e r s i t y of Birmingham, U.K. and Tokyo Women's Hospital, Japan.

HLA-DQBI GENE POLYMORPHISM CORRELATES WITH REMISSION IN RECENT ONSET TYPE I DIABETIC PATIENTS. O. BOUIX, C. PARER-RICHARD ~, J.F. ELIAOU ~e, A. ORSETTI, J. MIROUZE ~ and J. CLOT ~ , Service Exploration Physiologique des Hormones, e Service Maladies M@taboliques, e~Laboratoire d'Immunologie-Inserm U29, CHU MONTPELLIER, 34059 MONTPELLIER Cedex, France.

Inherited s u s c e p t i b i l i t y to Type I (insulin-dependent) diabetes may be determined by genes within the DQ subregion of the major h i s t o c o m p a t i b i l i t y complex. The DQA1 gene is such a candidate s u s c e p t i b i l i t y gene. A l l e l e - s p e c i f i c oligonucleotide gene probing was used to determine the frequencies of a l l e l e s of the DQA1 gene in d i a b e t i c and control subjects from three d i s t i n c t races. The DQA3 a l l e l e was s i g n i f i c a n t l y increased amongst Type I d i a b e t i c subjects in a l l investigated groups : Afro-Caribbeans - 30/38 diabetics v 12/93 controls, RR 23.4, pc<10-6; Asian Indians - 14/44 diabetics v 10/87 controls, RR 3.5, pc
The aim of this study was to look for an association between HLA-elass II alleles and the oecurenee of a remission of insulin-dependence in recent onset type I diabetics. We studied 22 unrelated caucasian type I diabetic patients ((i0 females and 12 males) aged 11-35 years at diagnosis (mean 20 years). Among them, two different groups could be defined after treatment by continuous intravenous insulin-infusion at the onset of the disease. One group of 12 patients underwent remission of insulin-dependence (R+) and the second group of i0 patients without remission (R-). No difference appeared in HLA serological typing between the two groups. We analysed the HLA-DQBI locus using polymerase chain reaction amplified genomie DNA hybridized with nod radioacZive labelled allele specific oligonucleotide. As expected, 18 of the 22 patients (82 %) were found to be homozygous for the absence of an aspartic acid residue at position 57 of the DQB chain. Moreover, DQBI~0201 all61e (DQw2) was more common in R+ than R- group (i0 of 12 (83 %) versus S of i0 (30 %) respectively, p = O.Oll), while an increase frequency of the DQBI~O302 allele (DQwS-2) was observed in the R- group (6 of 12 (50 %) versus 9 of lO (90 %), p = 0.045). These results demonstrate a genetic heterogeneity according to the oeeurenee of an insulin-induced remission in type I diabetic patients.

3. INSERM U 120 H o p i t a l R o b e r t DEBRE, Paris, F r a n c e 4. Coll~ge de F r a n c e .

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OP 23 Hall B-3 Fatty Acid and Glucose Metabolism 133

134

HYPOGLYCEMIC AND LIPOSTATIC EFFECTS OF CARNITINE-PALMITOYL-TRANSFERASE (CPT-I) INHIBITION IN TYPE-2-DIABETES. K.Ratheiser, B.Schneeweiss, P.Fasching, P.Nowotny, A.Korn, H.P.O.Wolf and W.Waldh~usl~ l.Med.Univ.['!inik, Lazarettg. 14, Vienna, Austria. To determine the therapeutic effect of the CPT-I-inhibitor etomoxir (ETO), 8 hospitalized obese NIDDM patients were studied (BMI: kg/m2=29.4+l.0; 48+5 yrs (means + SE)) at baseline (placebo = tl), and after oral ETO (50~g/day = t2, 100mg = t3, 1501~ = t4, 200rag = t5, placebo = t6). Fasting blood g]uc'ose (FBG, mmol/l), triglycerides (TGr rmaol/l), cholesterol (C, mmol/), FFA (umol/l), B-hydroxy-butyrate (B-<)HB, ~mol/l ) and GPT (U/l) were measured (tl-t6). Glucose utilization (M-value; indirect calorimetry) and hepatic glucose production (HGP) were examined during a 1OmU/kg.min euglycemic clamp (tl and t4). The dose-dependent fall in FBG (tlt6): 9.5+0.7, 8.7+1.0, 8.3+1.1", 7.8+0.9**, 7.9+1.1 was reversible in t6: 9.9+1.1. Plasma ii-pids were ~edueed: TG, 2.60+0.47 (tl), i.-21+0.19"* (t5); C, 6.19+0.31 (tl), 4.74+0.49-* (t5); B-OHB, ~20+33 (tl), 123+18"*--(t5); while F-FA increased: 485+42 ~tl), 737+I0~* (t5) as did GPT, 17+3 (tl)r 32+7- (t5)**. HGP-- (mg/min.m2) fell (tl/t4):--88+7/73+8*;-but M-values (mg/min.m2) remained unchanged: -263+2-7/ 268+29. Carbohydrate-oxidation (mg/min.m2) declined during the 10 mU/kg.min clamp: 90+8/74+9; while fat-oxidation (mg/min.m2) paradoxically tended to increase: 21+7/28+6. In conclusion, ETO (i) lowers HGP and fasting blood glucose, (2) reduces liDemia - TG, C and B-OHB included -, (3) does not affect glucose utilization (M-value), but (4) doubles plasma GPT. P vs tl: * <0.05; ** <0.01.

PLASMA FATTY ACID LEVELS INTERACT WITH NUTRITIONAL STATE IN PRODUCING INSULIN RESISTANCE YT Kruszynska', JG McCormack= and N Mclntyre'. 'Department of Medicine, Royal Free Hospital, London and ~Department of Biochemistry, Leeds University, UK. We examined the effects of increasing plasma NEFA with Intralipid on glucose metabolism during a 2h euglycaemic clamp (insulin 95+_.3mU/I) in conscious rats which were (a) fasted 24h, or (b) fed and infused with glucose for 4h, after food withdrawal, before the clamps. Clamp glucose requirement (GR) and 3-3H-glucose turnover were lower in fed than in fasted rats (turnover: 115+9 vs 139--+6 )Jmol/min/kg, p<0.05). Hepatic glucose output (HGO) was completely suppressed in both groups. Increased NEFA (,,,1.0 mmol/I) had no effect in fasted rats but further decreased GR in fed rats (67-+5 vs 107-+ 10~mol/min/kg, p<0.01). 3-3H-Glucose turnover (108-+4~umol/min/kg) did not decrease, indicating failure of suppression of HGO in fed rats with high NEFA levels. Without Intralipid, quadriceps muscle glycogen was increased by the clamp in fasted (28--+1 vs 19-+1 ~umol/g wet wt, p<0.001) but not in fed rats. Increased NEFA enhanced muscle glycogen deposition (fasted, 34-+2 vs 28-+1 ; fed 64-+6 vs 46-+2)Jmoi/g wet wt, both p<0.05) and inhibited muscle pyruvate dehydrogenase in both groups (fasted, i.8+0.4 vs 4.6+__0.7%active; fed 2.6-+0.9 vs 10.6-+3.4% active, both p<0.05) consistent with an inhibition of oxidative glucose metabolism. Conclusion: Although increased NEFA alters glucose metabolism in fasted rats, insulin resistance occurs only in fed rats, due to impaired suppression of hepatic glucose production.

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NON-ESTERIFIED FATFY ACIDS DO NOT IMPAIR GLUCOSE UPTAKE DURING HYPERGLYCAEMIA. M Walker, GR Fulcher and CF Sum. Department of Medicine, University of Newcastle upon Tyne, UK. Physiological plasma non-esterified fatty acid (NEFA) levels impair insulin stimulated glucose uptake (ISGU) during euglycaemia. By examining forearm and whole body ISGU and fuel oxidation we assessed whether this effect is reversed by hyperglycaemia. 7 normal males received 10% Intralipid (15ml/hr) and heparin for 210 minutes at euglycaemia and hyperglycaemia (EL and HL), with 0.154 mol/l NaCI as the control (EC and HC). Insulin (0.05 U/kg/hr), glucagon and somatostatin were given from 60 minutes, and blood glucose clamped at 4.1 _+0.1 (SEM)(EC and EL) and 7.0 -+ 0.2 (HC and HL) mmol/l. Plasma NEFA levels and forearm uptake were comparable with Intralipid (+61 -+ 10 and +52 _+8 nmol/lOOmlforearm[FA]/min, EL and HL), and were suppressed in the controls. Forearm ISGU decreased with Intralipid during euglycaemia but not hyperglycaemia (+3.34 + 0.25 v s +3.85 -+ 0.34 ~tmol/100mlFAJmin, EL v s EC; p<0.02), with comparable changes in whole body ISGU. Glucose oxidation decreased with Intralipid under both conditions (3.03 -+ 0.18 v s 3.35 = 0.14 [EL v s EC] and 3.33 -+ 0.3 v s 3.87 + 0.26 [HL v s HC] mg/kg/min; both p<0.05) as did forearm alanine release. Non-oxidative glucose uptake (NOGU) was enhanced during hyperglycaemia (7.84 -+ 0.74 v s 6.14 _+0.75, HL v s HC) with little change during euglycaemia. Physiological plasma NEFA levels decreased glucose oxidation and glycolytic flux under both conditions, but decreased ISGU only during euglycaemia. NOGU was enhanced during hyperglycaemia and appeared to reverse the inhibitory effect of NEFA on ISGU.

EFFECT OF INHIBITION OF UPOLYSIS ON GLUCOSE METABOLISM IN SKELETAL MUSCLES IN PATIENTS WITH TYPE 2 (NONINSULIN -DEPENDENT) DIABETES. H BECK-NIELSEN, A VAAG, P SKOTT, P DAMSBO AND MA GALL. HVID6RE HOSPITAL, KLAMPENBORG, DENMARK. Increased plasma non-esterified fatty acid (NEFA) levels may be important in causing insulin resistance in skeletal muscles in Type 2 (non-insulin-dependent) diabetes. The acute effect of the antilipolytic drug Acipimox on glucose metabolism was therefore studied in 12 patients with Type 2 (non-insulin-dependent) diabetes in a double blind cross-over study. Basal hyperglycaemia (PL vs AC, 10.2 _+ 1 vs 11.0 _+ 1 mM, NS) was maintained during insulin infusion (40mU/m2/min) and whole body glucose metabolism was assessed using 3-3H-glucose and indirect calorimetry. Biopsies were taken from the vastus lateralis muscle during basal and insulin stimulated steady state periods. Acioimox reduced NEFA in the basal state (0.12 _ 0.02 vs 0.70 -+ 0.05 mM, p< 0.01) and during insulin stimulation (0.05 -+ 0.01 vs 0.09 -+ 0.01 raM, p<0.05). Acipimox increased glucose oxidation in the basal state (76.2 _+ 7.3 vs 51.3 -+ 5.23 mg/m2/min, p<0.01). During insulin stimulation Acipimox increased both glucose oxidation (120.2 r 7.3 vs 96.2 -+ 5.2 mg/m~/mi n, p<0.01) and non-oxidative glucose disposal (245.8 _+ 47.3 vs 166.3 -+ 29.1 mg/m2/min, p<0.01). Acipimox enhanced basal and insulin stimulated activity of glycogen synthase (GS) in muscle biopsies (fractional velocity (0.1 and 10 mM G-6-P) = 0.31 _+ 0.03 vs 0.21 _+ 0.03 and 0.53 -+ 0.05 vs 0.42 + 0.04, p<0.05). In conclusion, inhibition of lipolysis acutely improves insulin action in patients with Type 2 (non-insulin-dependent) diabetes. This may be due 1o a stimulation of GS activity in skeletal muscles.

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THE ROLE OF F F A - G L U C O S E CYCLE ON G L U C O R E G U L A T I O N D U R I N G EXERCISE. Z. Shi, K. Yamatani, R. Gupta, A. Giacca, L. Lickley, and M. Vranic, Depts. of Physiology, M e d i c i n e and Surgery, Univ. of T o r o n t o We p r e v i o u s l y d e m o n s t r a t e d that in d e p a n c r e a t i s e d dogs, B-blockade, m a r g i n a l l y i n h i b i t i n g lipolysis, decreases g l u c o s e production, but fails to i n c r e a s e u t i l i z a t i o n during exercise. To d e t e r m i n e w h e t h e r greater s u p p r e s s i o n of F F A - g l u c o s e cycle can m i m i c insulin's effect also on g l u c o s e uptake, we treated depancreatised dogs with m e t h y l p a l m o x i r a t e (MP, inhibitor of FFA oxidation, 20 mg/kg/day p.o.) and MP+propranolol (5 ~ g / k g / m i n ) . MP effect on liver FFA o x i d a t i o n was c o n f i r m e d by m a r k e d l y d e c r e a s e d ketones. During exercise, glucose was constant in c o n t r o l s due to m a t c h e d increases in glucose u t i l i z a t i o n and production. W i t h MP, the rise in p r o d u c t i o n was b l u n t e d by 39J4% (p<0.01), and g l u c o s e d e c r e a s e d by 15+4%. U t i l i z a t i o n was unaffected. Infusion of o c t a n o a t e (oxidation u n a f f e c t e d b y M P ) reversed MP effects on glucose production. With M P + p r o p r a n o l o l , FFA, which i n c r e a s e d in all other groups, d e c r e a s e d by 38~3%(p<0.05). utilization was by 29!3% greater (p<0.05), and g l u c o s e fell by 29% more. Glucose p r o d u c t i o n was not further inhibited. Therefore, in insulin absence, d e c r e a s e d FFA a v a i l a b i l i t y or o x i d a t i o n inhibited g l u c o s e production, but both were required to increase utilization. Conclusion: Inhibition of F F A - g l u c o s e cycle can mimic insulin e f f e c t s during e x e r c i s e on liver and muscle. An important g l u c o r e g u l a t o r y role of insulin d u r i n g exercise may be indirect t h r o u g h the F F A - g l u c o s e cycle.

THE OPPOSITE EFFECT OF INSULIN AND FATTY ACIDS ON MUSCLE GLYCOGEN SYNTHASE AND GLUCOSE STORAGE IN MAN. R. Munger, J.P. Felber, H. Kleiber, D. Jallut, A. Golay, E. Temler, P. Frascarolo and E. J4quier. Division of Endocrinology, University Hospital, and Institute of Physiology, Lausanne, Switzerland. The purpose of the work was to study the regulatory mechanisms involved in glucose storage. 16 normal subjects participated in the study which was divided into 3 successive periods : A) basal; B) 120 rain preclamp with infusion of either saline, or Intralipid | to raise FFA; C) euglycemic hyperinsulinemic (89-94 ~U/ml) clamp. Continuous indirect calorimetry was used throughout. Muscle biopsies were obtained in 9 subjects in vastus lateralis at the end of each period. Two types of regulatory mechanisms were observed : 1) Stimulation of glycogen synthase (GS) activity (p<0.05 vs preclamp) and glucose storage following the glucose-insulin infusion (clamp), with a positive correlation between GS activity and muscle glycogen concentration (GLY) at the end of the clamp. 2) Inhibition of glucose storage after Intralipid (1.61+0.34 vs 2.99_+0.53 mg/kg.min after saline, p<0.05) with a negative correlation between GS activity at the end of the clamp (r=-0.89, p<0.001) and glucose storage (r=-0.79, p<0.01) vs GLY after Intralipid. The negative correlation of GS activity with GLY after Intralipid and the positive correlation of GS activity with GLY at the end of the glucose-insulin infusion show that this enzyme is stimulated by insulin and inhibited by an increase in plasma free fatty acids levels. This study confirms the important role of GS activity in the regulation of glucose storage in man.

OP 24 Hall A-18

Glycation 139

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FIBRINOGEN AND PLASMINOGEN ISOLATED FROM DIABETIC PATIENTS : CONSEQUENCES FOR FIBRINOLYSIS. A. Lutjensl~ H. Sanderson-Sellmeijer I, E.A.v.d. Veen 2 and J.v.d.'Meer 2.1Department of Clinical Chemistry, Andreas Hospital and 2Department of Internal Medicine, Free University Hospital, Amsterdam, The Netherlands. The influence of increased in vivo nonenzymatic glycosylation of fibrinogen and plasminogen on clot lysis was investigated. From blood from thirty healthy volunteers (aged 23 - 7 2 years, median 41 years; HbAlc 4.2 +_ 0.3 %, mean + SEM) and 37 type 1 (insulin dependent) diabetic patients (aged 21 - 76 years, median 48 years; HbAIc 9.5 +_ 0.5 %) plasminogen and fibrinogen were purified. Plasminogen activation was studied in a purified system, using plasminogen as substrate and streptokinase as plasminogen activator. Plasmin activity generated per m g plasminogen was 272 + 9 % of normal standard plasma in the diabetic patients (n = 6) and 117 +_ 16 % in the control subjects (n = 7, mean +_ SEM, p
CAPTOPRIL INHIBITS FLUORESCENCEDEVELOPMENT IN NONENZYMATICALLY GLYCOSYLATEDPROTEINS. C.A Le Guen, S. Bain, A.H. Barnett and J Lunec. Dept. of Clinical Chemistry and Medicine, University of Birmingham, U.K. Albumin and immunoglobulin G (IgG) show increased fluorescence in diabetic patients and fluorescent IgG correlates with retinopathy. We have previously demonstrated a role for oxygen radicals in fluorescence generation in non-enzymatically glycosylated albumin. Captopril, an angiotensin converting enzyme (ACE) i n h i b i t o r , has free radical scavenging a b i l i t y , a t t r i b u t a b l e to i t s thiol group. We investigated the effect of captopril, enalapril and perindoprilat (ACE inhibitors without scavenging a b i l i t y ) and two other thiol containing compounds, mercaptopropionyl-glycine (MPG) and N-acetyl-cysteine (NAC) (scavengers with no effect on ACE) on fluorescence generation by glycosylation in albumin and IgG. All three thiol containing compounds inhibited fluorescence development in IgG (by 73,54 and 54% at lOuM and by 74,64,64% at 100 uM, for captopril, MPG and NAC respectively,(p
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PLASMA PENTOSIDINE DETERMINATION REVEALS IMPAIRED PENTOSE METABOLISM IN DIABETES. P. Odetti, D.R. Sell, and V.M. Monnier. Institute of Pathology, Case Western Reserve University, Cleveland, OH, USA. Pentosidine is an advanced glycosylation endproduct and protein crosslink recently isolated from aging human extracellular matrix (J. Biol. Chem. 264:21597, 1989). Increased levels were found in skin from Type I and Type 1I diabetic subjects, particularly in presence of endstage renal disease (J. Clin. Inv. 85:380, 1990). To clarify the metabolic origin of the crosslink, pentosidine was assayed by a combination of C-18 reverse phase HPLC and ionexchange fluorescence chromatography in acid hydrolyzed hemolysate and plasma proteins from control (n= 16), diabetic (n= 16) and uremic nondiabetic subjects (n= 10). Mean -+ S.D. red blood cell levels were 0.15 + 0.03, 0.18 + 0.06 and 1.24 -+ 1.17 pmol/mg Hb respectively. Plasma pentosidine levels were 0.94 _+ 0.33; 2.91 + 1.63 and 18.22 _+ 4.29 pmol/mg respectively, i.e. 3 x (p<0.001) and 20 x (p<0.001) higher in diabetes and uremia than in controls. Plasma and RBC pentosidine did not correlate with glycohemoglobin level or age. These observations clearly demonstrate the metabolic origin of pentosidine and suggest that plasma pentosidine determination in diabetes might become a useful tool for assessment of impaired pentose metabolism. Although the therapeutic value of such measurements remains to be established, it is conceivable that they would help assessing overall cellular responses to interventions with aldose reductase inhibitors or aminoguanidine in face of poor metabolic control.

P R E V A L E N C E OF A U T O A N T I B O D I E S TO G L Y C A T E D LDL IN DIABETIC AND N O R M A L SUBJECTS T. Koschinsky, C.E. BUnting, U. Glahn, and J.L. Witztum, DUsseldorf, Germany, San Diego, CA, U.S.A. A u t o a n t i b o d i e s to n o n e n z y m a t i c a l l y g l y c a t e d LDL have been d e s c r i b e d in a few diabetic patients and normal subjects, but p r e v a l e n c e data are still missing. Therefore, sera from 281 d i a b e t i c patients (age range: 11-85 years, 142 Type I, 139 Type 2, 132 male, 149 female) and 169 normal subjects (10-80 years, 82 male, 87 female) were t e s t e d by d i l u t i o n and c o m p e t i t i o n RIA for binding to the f o l l o w i n g antigens: LDL from normal subjects incubated with g l u c o s e (80 mM) for 7 days (glc-LDL) and for 2-3 m o n t h s to generate advanced g l y c a t i o n endproducts (AGELDL). 51% of diabetic sere showed specific antibodies to glc-LDL (titer range 4-2048, m e d i a n 256) and in 29% antibodies to A G E - L D L (titer range 16-512, median 256). similar results were obtained with normal sera; 49% had specific antibodies to glc-LDL and 31% antibodies to A G E - L D L w i t h the same titer range and m e d i a n titer as for d i a b e t i c sera. The prev a l e n c e of positive titers increased w i t h age in diabetic (p<0.05) but not in normal sere. W i t h i n the diabetic sera, there was no r e l a t i o n to type of diabetes, diabetes duration or HbAlc. Thus, specific antibodies to glycated LDL are present in diabetics and normal subjects and could be involved in the pathogenesis of late complications, and the aging process.

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Csrboxymethylation of low density lipoprotein alters its catabolism vie the high affinity receptor K.E.Gempel, E.D.Schleicher, B.Olgem611er,K.D.Gerbitz, Munich, FRG Recently, it has been shown by Baynes et el. that oxidative cleavage of fructolysine residues generated by glycation leads to carboxymethylated proteins. Evidence has been provided that carboxymethylation occurs in vivo since carboxymethyllysine could be detecteo in urine and certain proteins and its elevation could be demonstrated in diabetes. To clarify whether carboxymethylation might alter the metabolism of low density lipoprotein, we have mimicked carboxymethylation by chemical modification of lysine residues with glyoxylic acid in the presence of Na(CN)BH 3. Carboxymethylation impaired the interaction of LDL with its high affinity receptor dependin 9 on the extent of modification. Carboxymethylation of approximately 50 % of lysine residues totally abolished high-affinity internalisation and degradation of LDL by cultured human fibroblasts. However, the scavenger pathway, as assessed by determination of cholesterol ester synthesis in mouse peritoneal macrophages, was not affected. These cells exhibited the well-known increase in --C-oleate incorporation into cholesterol esters upon incubation with acetylated LDL, but did not take up the carboxymethylated species faster when compared to unmodified LDL. Our results indicate that a non-brownin 9 modification of protein derived from glycation may alter its biochemical properties.

ADVANCED GLYCATION ENDPRODUCTS AND DEVELOPM E N T OF CATARACTS IN EXPERIMENTAL DIABETES.

M.Sensi, F.Pricci,G.Pugliese,D.Bellarosa, A.cristina a n d U. Di M a r i o . D e p a r t m e n t of Endocrinology, University of Rome,Italy. To assess the relation between AGE a c c u m u l a tion and cataract development in e x p e r i m e n tal d i a b e t e s , 56 S p r a g u e - D a w l e y rats were made diabetic by injection of S t r e p t o z o t o c i n (60mg/kg). Animals were followed either for 3 or 6 m o n t h s , monitoring changes in b o d y w e i g h t a n d i n i t i a l and final g l y c e m i a . At sacrifice, presence of cataract was o b s e r v e d in 5 o u t of 18 and 6 o u t of ii s u r v i v i n g animals respectively. No cataracts were o b s e r v e d in a c o n t r o l g r o u p of n o r m a l a n i mals. A G E relative levels were d e t e r m i n e d in water or urea-soluble lens proteins by m e a n s of spectrofluorimetry (excitation at 370 a n d emission at 430 nm). Diabetic rats (3 months) s h o w e d i n c r e a s e d A G E l e v e l s of b o t h w a t e r (p<0.005) a n d u r e a - s o l u b l e (p
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OP 25 Hall A-1 Nephropathy in Type 2 Diabetes 145 PREVALENCE AND CAUSES OF INSULIN-DEPENDENT DIABETIC

146 ALBUMINURIA IN N O N (NIDDM) P A T I E N T S .

H.-H. Parving, M.-A. Gall, P. Skett, H.E. J@rgensen, F. Jergensen, B. Nielsen a n d S. Larsen. Hvidere Hospital, Klampenborg, Denmark. The prevalence and causes of p e r s i s t e n t albuminuria (>300 m g / 2 4 h) w a s d e t e r m i n e d in NIDDM patients, age <66 years, attending a diabetic clinic during 1987. All eligible patients (n=370) were asked to collect at l e a s t one 2 4 - h u r i n e s a m p l e for a l b u m i n a n a l y sis. Urine collection was obtained in 224 males a n d 139 females (98%). F i f t y p a t i e n t s (seven women) suffered from persistent album i n u t i a (13.8%). The p r e v a l e n c e of a l b u m i n u r i a was significantly h i g h e r in m a l e s (19%) t h a n in f e m a l e s (5%), p < 0 . 0 0 5 . A k i d n e y b i o p s y w a s performed in 33 patients (66%). The k i d n e y biopsies revealed diffuse and/or nodular diabetic glomerulosclerosis in 24 p a t i e n t s (73%), while the r e m a i n i n g 9 patients (27%) had a v a r i e t y of n o n - d i a b e t i c g l o m e r u l o p a t h i e s , e.g. minimal lesion nephropathy and mesangioproliferative glomerulonephritis. Diabetic retinopathy was present in 15/24 p a t i e n t s (63%) with diabetic glomerulosclerosis, while none of the 9 p a t i e n t s w i t h a n o n - d i a b e t i c g l o m e r u lopathy had retinopathy. Our c r o s s - s e c t i o n a l study has revealed a high prevalence of albuminuria and of n o n - d i a b e t i c g l o m e r u l o p a t h y as a cause of this complication in N I D D M patients. Presence of diabetic retinopathy strongly suggests that a diabetic glomerulop a t h y is the c a u s e of a l b u m i n u r i a . A l b u m i n u r i c NIDDM patients without retinopathy require f u r t h e r e v a l u a t i o n , i.e. k i d n e y b i o p s y .

DIABETIC NEPHROPATHY IS RELATED TO DIFFERENT CARDIOVASCULAR RISK FACTORS IN TYPE 1 AND TYPE 2 DIABETES MELLITUS. P. N. KNOBL, C h . SCHNACK, P.PIETSCHMANN, R . P R A G E R , T h . V U K O V I C H , G. SCHERNTHANER. D e p t . M e d . II, U n i v . V i e n n a , A U S T R I A . P a t i e n t s with d i a b e t i c n e p h r o p a t h y (DN) a r e at a n i n creased risk for cardiovascular complications. In t h i s s t u d y we e v a l u a t e d c o n n e c t i o n s b e t w e e n a r t e r i a l blood p r e s s u r e , s e r u m lipids and coagulation factors a n d t h e d e g r e e of d i a b e t i c n e p h r o p a t h y in 54 t y p e 1 (DM 1) a n d 48 t y p e 2 (DM 2) diabetic p a t i e n t s . Patients were classified as follows: (a) normalbuminuria (<30 m g a l b u m i n / 2 4 h; DM 1: n=25; DM 2: n=17), ( b ) incipient nephropathy (30-300 m g / 2 4 h ; DM 1: n=lT; DM 2: n=16), (c) o v e r t n e p h r o p a t h y (>300 rag/24 h; DM 1: n = t 2 , DM 2: n;13). In p a t i e n t s with DM 1 we f o u n d s i g n i f i c a n t p o s i t i v e correlations between the stages of DN a n d t h e s e r u m levels of c h o l e s t e r o l (r=0.35, p < 0 . 0 2 ) , t r i glycerides ( r = 0 . 2 8 , p<0.05) ~ L D L - c h o l e s t e r o l (r=0.37, p < 0 . 0 1 ) , a n d t h e plasma levels of antithrombin III ( r = 0 . 3 7 , p<0.01) and fibrinogen ( r = 0 . 4 7 , p<0.001). In c o n t r a s t , in p a t i e n t s with DM 2 no significant c o n n e c tion b e t w e e n t h e s t a g e s of DN a n d t h e a b o v e m e n t i o n e d parameters was f o u n d . Systolic blood p r e s s u r e was c o r r e l a t e d to t h e d e g r e e of DN in b o t h DM 1 (r=0.36, p<0.02) a n d DM 2 ( r = 0 . 4 , p < 0 . 0 1 ) , whereas no c o r r e lation was o b s e r v e d for diastolic blood p r e s s u r e . O u r f i n d i n g s i n d i c a t e a close relationship between t h e d e g r e e of DN a n d a t h e r o g e n i c lipid fractions, f i b r i n o g e n levels a n d s y s t o l i c blood pressure in DM i , w h e r e a s in DM 2 o n l y systolic hypertension seems to be r e l a t e d to t h e d i f f e r e n t s t a g e s of DN.

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G L O M E R U L A R F U N C T I O N IN P I M A INDIANS W I T H T Y P E 2 ( N O N - I N S U L I N - D E P E N D E N T ) DIABETES. R.G. Nelson, P.H. Bennett, G.W. Williams, R.L. Berg, S.A. Hardy, W.C. Knowler, W.E. Mitch, and B.D. Myers for the Diabetic Renal Disease Study, Phoenix, AZ, Cleveland, OH, Atlanta, GA, and Palo Alto, CA, U.S.A.

CARDIOVASCULAR RISK FACTORS IN TYPE 2 (NON-INSULINDEPENDENT) DIABETIC PATIENTS WITH MICROALBUMINURIA. T. Kodama, T. Baba, T. Tomiyama and K. Takebe, Third Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki 036, Japan

Glomerular function was examined in 83 Pima Indians; 31 with normal glucose tolerance, 23 with diabetes <3 years duration, and 29 with Type 2 (non-insulin-dependent) diabetes of >_5 years duration and microalbuminuria (c. 30-300 mg/day). Glomerular filtration rate (GFR) (iothalamate clearance) averaged 122+4, 141+9, and 145+9 ml/min; renal plasma flow (PAH clearance) averaged 726+27, 826+62, and 800+39 ml/min; and filtration fraction averaged 17%, 17%, and 18%, respectively. GFR was significantly higher than in subjects with normal glucose tolerance only in those with diabetes of _>5 years duration and microalbuminuria (p=0.03). Neither renal plasma flow nor filtration fraction differed significantly among the groups. Similar results were obtained when clearance rates were corrected for body surface area. In contrast with Type 1 (insulin-dependent) diabetes, Pima Indians with Type 2 (noninsulin-dependent) diabetes of _>5 years duration and microalbuminuria have higher GFR than those with recent onset diabetes or normal glucose tolerance.

Relationships between blood pressure, serum lipids, fibrinogen levels and urinary albumin excretion rate were investigated in 25 Japanese Type 2 (non-insulindependent) diabetic patients with microalbuminuria (urinary albumin excretion rate [AER] 20-200 ug/min) and 25 individually matched Type 2 diabetic patients with normoalbuminuria (AER < 20 ug/min), matched for age, sex, body mass index, treatment and HbAlc level. Microalbuminuric diabetic patients had significantly higher systolic blood pressure (microalbaminuric group vs. normoalbuminuric group: 130 [4.0] vs. 116 [2.7] mmHg, mean and [SEM]) and serum fibrinogen levels (10.4 [0.3] vs. 8.8 [0.2] umol/l) and lower HDLcholesterol concentration (i.0 [0.i] vs. 1.2 [0.i] mmol/l) as compared with those in the normoalbuminuric patients. AER showed significant correlations with HDL-cholesterol concentration. No significant differences were found in diastolic blood pressure, total serum cholesterol, triglycerides, LDL-cholesterol, uric acid levels, glomerular filtration rate and urinary C-peptide excretion between the 2 groups. The results of this case-control study indicate that an elevation in systolic blood pressure and alterations in HDL-choleaterol and fibrinogen levels are present in Type 2 diabetic patients with microalbuminuria, which may contribute to an increased risk of cardiovascular disease. Nevertheless, it remains to be seen whether these alterations are the cause or consequence of diabetic incipient nephropathy in Type 2 (non-insulin-dependent) deiabetes.

A49

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MICROALBUMINURIA AS A PREDICTOR OF MORTALITY IN TYPE 2 (NON-INSULIN-DEPENDENT) DIABETIC PATIENTS: RESULTS FROM A 3-YEAR PROSPEC'FIVE STUDY.

24H AMBULATORY BLOOD PRESSURE AND URINARY ALBUMIN EXCRETION IN TYPE 2 DIABETES. A. Schmitz, M. Mau Pedersen and K.V~. Hansen, Medical Department M (Diabetes and Endocrinology), Aarbus Kommunehospital, DK-SO0O Aarhus C, Denmark. The relationship between blood pressure and microalbuminuria, b o t h a s s o c i a t e d with c a r d i o v a s c u l a r disease a n d d e a t h , is s p a r s e l y s t u d i e d in T y p e 2 ( n o n i n s u l i n - d e p e n d e n t ) d i a b e t e s , a n d r e s u l t s may be i n t e r f e r e d b y the p h e n o m e n o n of " w h i t e - c o a t - h y p e r t e n s i o n " . We t h e r e f o r e i n v e s t i g a t e d blood p r e s s u r e b y 24h a m b u l a t o r y r e c o r d i n g s (oseillometry) a n d examined w h e t h e r blood p r e s s u r e r e l a t e d to the level of u r i n a r y albumin e x c r e t i o n r a t e (UAER) b y s y n c h r o n o u s 24h collections. S e v e n t e e n diabetics (50-75 y e a r s of age) with mJeroalbuminuria (15
M. Mattock, N . M o r r i s h , R. G. Jackson*, G. C. V i b e r t i , M. EI-Gohari, A. Fitzgera Id, G. S. Scott, H. Keen. Unit f o r Metabolic M e d i c i n e , U M D S , G u y ' s Hospital,London and Lewisham* Hospital, L o n d o n , U K . T h r e e r e t r o s p e c t i v e studies have suggested that microalbuminuria p r e d i c t s all-cause (mainly c a r d i o v a s c u l a r ) mos in T y p e 2 ( n o n - i n s u l i n - d e p e n d e n t ) diabetic patients. Following a screening programme at Lewisham Hospital we d i v i d e d a g r o u p o f 142 Europid T y p e 2 patients (aged 35-64 y r s and without clinical p r o t e i n u r i a ) into a microalbuminuric cohort ( o v e r n i g h t u r i n a r y albumin e x c r e t i o n rate [AER] 20-200pg/min,n=36) and a cohort with AER<20pglmin (n=I06). Three years later 14 (10%) of these patients had died, 10 (27.7%) from the microalbuminuric cohort and 4 (3.8%) from the cohort with AER<2Opg/min (p<0.001). No patient with AER<20pglmin died o f c a r d i o vascular disease ( C V D ) . Seven o f 10 deaths in microalbuminuric patients were from CVD. No patient died of uraemia. Possible p r e d i c t o r s o f all-cause mortality were examined by a g e - a d j u s t e d p r o p o r t i o n a l hazards analysis. M i c r o a l b u m i n u r i a , c o r o n a r y heart disease,serum cholesterol and t r i g l y c e r i d e s were significant at the u n i v a r i a t e level (p<0~,05). In a multivariate stepwise model (p=0.10 f o r e n t r y ) only microalbuminuria (p=0.03} and serum cholesterol (p=0.001) e n t e r e d . For microalbuminuria the associated relative r i s k was 4.0 (95% CI 1 . 1 - 1 4 . 1 ) . This prospective s t u d y has shown microalbuminuria to be an independent r i s k - f a c t o r f o r all-cause mortality in T y p e 2 patients; CVD was the predominant cause o f death in the microalbuminuric cohort.

OP 26 Hall A-2 Immunology in Man II 151

152

HUMAN INSULIN AUTOIMMUNITY IS NOT ABNORMAL S.E. Fineberg, A.A. Biegel, K. Purr, N.S. Fineberg, and J.H. Anderson, Indiana University School of Medicine and Lilly Research Laboratories, Indianapolis, IN

Title: Insulinautoantibodies measured by Radioimmunoassay: H 1 g ~ p e c i f i c i t y for Type I Diabetes mellitus Authors: R. Kirkemm, F. Backer, H. Buschler*, H. Sauer*, R. G. Bretzel and K. Federlin, III. Medizinische Klinik und Poliklinik der Justus-Liebig-Universit6t Gie6en, Rodthohl 8, 6300 GieBen, FRG. *Diabetes-Klinik Bad Oeyn-

In nondiabetics, insulin autoimmunity may be an expression of "natural" immunity. Fifty healthy Caucasians (20 to 59 years, 27 males and 23 females) were studied for anti-insulin IgG (AI-IgG), islet cell antibodies (ICA), rheumatoid factor (RF), and HLA DR, DRW, and DQ. Nine had a parent and one a sibling with diabetes. AI-IgG were assessed by RIA and a quantitative ELISA using dilutions of 125I-IgG and unknowns with preincubation to determine specificity. Human, porcine and bovine insulin (HI, PI, BI) coated wells and loglogit plots are used to determine ng/ml (detectable + 3 SD=3.3 ng/ml). One individual had +I ICA and 2 had + ICA, 2 had +i RF and none had anti-insulin antibodies detectable by RIA. Binding to insulin was inhibited by preincubation with insulin but not with IgG, albumin, cpeptide, or glucagon. ELISA results are given as geometric means. AI-IgG binding to PI, BI, and HI were 3.0, 1.8, and 19.5 ng/ml; AI-IgG HI>PI>BI, all p<.02. Eighty-eight percent of individuals (44/50) had antibodies to HI. Haplotype DR4 DRW53 DQ3 correlated with increased AI-IgG binding to HI: haplotype present (n=20), 34.5 ng/ml, haplotype absent (n=30), 12.2 ng/ml. Healthy nond{abetic individuals usually express insulin autoimmunity, the level of which is correlated with HLA Class II haplotype.

hausen, 4970 Bad Oeynhausen, FRG I n s u l i n a u t o a n t i b o d i e s (IAA) as w e l l as i s l e t - c e l l a n t i bodies (ICA) have been described as markers o f the pred i a b e t i c period o f Type I Diabetes m e l l i t u s . The present study presents data from a prospective f a m i l y study i n cluding an o v e r a l l o f 879 subjects, 204 Type I d i a b e t i c probends, 378 o f t h e i r parents and 297 s i b l i n g s . The method applied was a hig~89 s e n s i t i v e competitive Radioimmuneessay (RIA) using i o d i n e - l a b e l l e d human i n s u l i n and excess cold i n s u l i n as competitor. 9 4 , i % o f a l l d i a b e t i c s had s i g n i f i c a n t amounts o f IAA, with 4,8 and 7,7 % o f t h e i r n o n - d i a b e t i c parents and s i b l i n g s r e s p e c t i v e l y p o s i t i v e . Thus, IAA, were found more of t en in the younger p o p u l a t i o n . I A A - p o s i t i v i t y in 218 normal c o n t r o l s was under 1%. When I A A - p o s i t i v i t y was compared t o the ICAstatus o f the n o n - d i a b e t i c popul at i on, a h i g h l y s i g n i f i cant c o r r e l a t i o n was found in the group o f the s i b l i n g s (P<.OOi). This held e s p+ e c. i a l l.y t .r u e . f o .r the male s i b l i n g s with almost 80 % o f ICA zndzvzduals also posl t zve f o r IAA, while only 40 % o f the female s i b l i n g s had both markers. In the group o f the parents, no c o r r e l a t i o n o f IAA and ICA could be demonstrated. In conclusion, IAA as measured by t h i s RIA appear t o be a good marker, h i g h l y s p e c i f i c f o r the p r e d i e b e t i c period o f Type I Diabetes m e l l i t u s i n the younger p o p u l a t i o n . The high percentage o f I A A - p o s i t i v e i n d i v i d u a l s in the ICA p o s i t i v e group and the low frequency i n the normal population make i t a u s e f u l l screening t e s t t o detect the e a r l y phase o f prec l i n i c a l diabetes in e young p o p u l a t i o n .

AS0

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ICA AND IAA IN RECENT ONSET TYPE 1 DIABETIC CHILDREN AND IN MATCHED CONTROL CHILDREN M Landin-Olsson, J Palmer, G Dahlqvist, L Blom, G Sundkvist, A Lernmark and The Swedish Childhood Diabetes Study Group Dept of Medicine, University of Lund, Malmo General Hospital, and Dept of Pediatrics, Sachs" Children's Hospital, Stockholm, Sweden and Veterans Hospital and Dept of Medicine, University of Washington, Seattle, USA Islet cell antibodies (ICA) and insulin autoantibodies (IAA) are frequent at diagnosis and may also preceed Type 1 diabetes. A population-based case-control study was carried out to define the sensitivity and specificity of ICA and/or IAA for Type 1 diabetes in childrerL During 16 months, 509 samples from consecutive new onset 0-14 year old Type 1 diabetic children in Sweden, and 420 age, sex, and geographically matched control children were collected. ICA were analysed in a two-colour immunofluorescence- assay and IAA in a radiobinding-assay. The upper limit of IAA (<2.30 %) was defined as the 97.5 percentile of the control population, and only patient samples taken within five days of insulin treatment (n=338) were included. ICA were present in 416/509 (82%) of the patients and in 17/420 (4%) of the control children, and IAA in 149/338 (44%) and 8/390 (2%), respectively. Among the patients, both ICA and IAA levels were negatively correlated with age (rs= -0.1; p=0.002 and rs= -0.4; p<0.001, respectively), and levels of ICA and IAA correlated (rs= 0.3; p<0.001). ICA and/or IAA were present in 309/338 (91%) of the patients and 22/390 (6%) of the controls, while ICA and IAA were present simultaneously in 139/338 (41%) and in 3/390 (0.8%), respectively. In conclusion, levels of ICA and IAA are correlated and age-dependent in new onset diabetic children. In control children the simultanous presence of ICA and IAA increases the specificity for Type 1 diabetes.

HETEROGENEITY OF IgG SUBCLASS ISLET CELL ANTIBODIES.

DISTRIBUTION

OF

N. Dozio, E. Bosi, S. Genovese, C. Belloni, A. M. Girardi, G. Pozza, G.F. B o t t a z z o and J.C. Sodoyez. San R a f f a e l e Institute, Milan, Italy Aim of the p r e s e n t study was to characterize the IgG subclass d i s t r i b u t i o n of islet cell antibodies (ICA) in sera of type-i diabetics with regard to the a s s o c i a t i o n or not with other autoimmunities. 31 sera w i t h ICA were studied: 15 sera had ICA o n l y (pure diabetics), 16 ICA and at least one other associated autoantibody (polyendocrines). The latter showed higher ICA titer (median 50 and 70.2 JDF-units, r e s p e c t i v e l y ) , but w i t h o u t r e a c h i n g statistical significance. All sera had ICAIgGl, 54.8% ICA-IgG2, 32.2% ICA-IgG3 and 51.6% ICA-IgG4. ICA-IgGI was the o n l y isotype in 32.3% patients; in 25.8% ICA w e r e of 2, in 12.9% of 3 and in 29.0% of 4 isotypes. The number ICA-IgG isotypes was significantly different b e t w e e n pure and p o l y e n d o c r i n e ICApositive sera: ICA-IgGI as single isotype was more frequent in p u r e d i a b e t i c s sera (60% vs 6.25%, p<0.005) w h i l e m u l t i p l e I C A - I g G isotypes (2 or more) w e r e more frequent in p o l y e n d o c r i n e sera (p<0.05). The f r e q u e n c i e s of each subclass were higher in the p o l y e n d o c r i n e p a t i e n t s as follows: ICA-IgG2 75.0 vs 33.3%, p<0.05), ICAIgG3 (50% vs 13.3%, p=0.07) and ICA-IgG4 (81.2% vs 20%, p<0.001). These findings suggest h e t e r o g e n e i t y of ICA, w i t h m o r e e x t e n d e d c l o n a l recruitment and trend to higher titers in p o l y e n d o c r i n e c o m p a r e d to d i a b e t i c sera.

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DETECTION ON THiN LAYER PLATES OF AUTOANTIBODIES TO HUMAN PANCREATIC FUCOGANGLIOSIDES IN THE SERA OF ICA POSITIVE TYPE I DIABETIC PATIENTS J. Portoukalian, A. Rebaa and C.H. Thivolet, INSERM U218 and INSERM U197, Lyon France Human pancreatic glycolipid extracts have been shown to block the

DISSOCIATION FUNCTIONS I N

binding of cytoplasmic islet cell autoantibodies (ICA) to islet cells

In order to study the cause of easy vulnerbility to infection of diabetic patients, peripheral neutrophil and monocyte functions were separately assessed in terms of oxygen radical formation and the e x p r e s s i o n of cell surface molecules including complement receptor (CR3) and lymphocyte function associated antigen-i (LFA-I). Neutrophils and monocytes were obtained from the blood of diabetic patients (n=57) and healthy controls (n:36) by centrifugation on Mono-Poly-Resolving-Medium (Flow). Oxygen radical formation in neutrophils and monocytes was d e t e r m i n e d with flow c y t o m e t r y after incubation of cells with 2 ' - 7 ' - d i c h l o r o fluorescin diacetate and subsequent stimulation with phorbol myristate acetate. Both CR3 and LFA-I antigens were d e t e r m i n e d with flow cytometry following incubation of cells with monoclonal antibodies. As c o m p a r e d with healthy controls, diabetic patients showed decreased oxygen radical generation in neutrophils (p<0.01) and in contrast its elevation in monocytes (p<0.05). CR3 e x p r e s s i o n was basically the same as the results for oxygen radical formation. However, monocytes from diabetic patients showed decreased e x p r e s s i o n (p<0.001) of LFA-I as compared with control monocytes. Thus, the activity of neutrophils and monocytes dissociated in diabetic patients as m a r k e d l y shown in oxygen radical formation. In addition, the e x p r e s s i o n of CR3 on monocytes from diabetic patients was increased while LFA-I on the cells was decreased.

(Colman et al, Diabetes I988). However, no direct binding to glycolipids was shown. We studied by immunostaining on thin-layer chromatographic plates the reactivity of sera from newly-diagnosed type I di~tbetic patients and first degree relatives to glycolipids recovered from several human pancreatic glands by chloroformmethanol extraction and anion-exchange chromatography. Thin-layer plates containing gangliosides and neutral glycolipid fractions were overlayed with patients' sera. Bound antibodies were visualized with a biotinylated second antibody and peroxydase-conjugated avidin. ICA content was determined by indirect immunofluorescence on human pancreas. Most ICA-positive sera reacted with several fucogangliosides which are minor components of human pancreas. Neuraminidase and/or fucosidase pretreatments abolished the reactivity of these autoantibodies. In conclusions, 1) in type I diabetes, the presence of ICA is correlated with the presence of autoantibodies to fucogangliosides, 2) the specific binding to gangliosides is often masked by the presence of natural antibodies to blood group antige,~s which implicate the separation of gangliosides from neutral glycolipids and 3) the limited distribution of fucogangliosides to pancreas and kidney reinforce the concept of organ specificity of this autoimmune disease.

OF NEUTROPHIL DIABETIC PATIENTS

AND

MONOCYTE

N. Aoki and M. Imamura, D e p a r t m e n t of Medicine, Kinki University School of Medicine, Ohsakasayama 589, Japan

A~

OP 27 Hall A-3 Endothelium 157

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SORBITOL ACCUMULATION IS NOT A GOOD INDEX OF POLYOL PATHWAY ACTIVITY IN THE ENDOTHELIUM CM Airey, JK Wales and PJ Grant. Department of Medicine, Leeds University, UK. Tissue sorbitol concentration has been used as an index of aldose reductase activity. However, sorbitol concentrations are alsodependent upon sorbitol dehydrogenase, the second component of the polyol pathway. We investigated the time course of sorbitol and galactitol (not a substrate for sorbitol dehydrogenase) depletion from human umbilical cord vein endothelial cells (HUVEC) in-vitro. Cells were preloaded with sorbitol or galactitol by incubation in 25 mmol/L glucose or 10 mmol/L galactose respectively. After 48 h this media was replaced by control media (5 mmol/L glucose, zero galactose). HUVEC polyols were assayed by GLC at intervals upto 24 h following media replacement. Results are expressed as mean(+_sd) and comparisons made using the Mann-Whitney U test. HUVEC sorbitol and galactitol concentrations following 48 pre-loading were 36.5 _+ 6.6 and 638.7 + 75.3 pmol/cm 2 respectively. 8orbitol levels fell significantly (22.8 +_ 4.2 pmol/cm 2 p<0.01) within 15 min of removal of external glucose load and after 2 h were not significantly greater than control values (11.3 _+ 2.8 v 6.8 + 2.3 pmol/cm2). HUVEC galactitol levels were not decreased until 8 h after media replacement (480.5 +_ 36.1 pmol/cm 2 p<0.01 ) and remained elevated at 24 h compared to control values (250.7 _+ 22.8 pmol/cm 2 v not-detectable). Thus the presence of high sorbitol dehydrogenase activity in HUVEC indicates a greater polyol pathway activity than may be concluded from sorbitol measurements alone.

L-TYROSINE CORRECTS DELAYS REPLICATION OF HUMAN ENDOTHELIAL CELLS. AS INDUCED BY HIGH GLUCOSE. La Selva M,Chiara P,Muccini E , M o l i n a t t i PA,Porta M and M o l i n a t t i GM. High glucose (HG) delays r e p l i c a t i o n of c u l t u r e d human umbilical vein e n d o t h e l i a l c e l l s (HUVEC) by prolonging the interval between DNA d u p l i c a t i o n and m i t o s i s . We h a v e p r e v i o u s l y reported t h a t microtubules of Huvec grown in HG develop c o l c h i c i n e r e s i s t a n c e (CR), suggesting a tubulin dysfunction i n t e r f e r i n g with mitosis. Maintenance of the microtubule c y t o s k e l e t o n and formation of the m i t o t i c spindle r e s u l t s from the balance between a c e t y l a t i o n of the l y s i n e aminogroups ( c o n f e r r i n g CR) and loss of the Cterminal t y r o s i n e from the t u b u l i n subunits. We i n v e s t i g a t e d i f t y r o s i n e c o r r e c t s HUVEC CR and r e p l i c a t i o n in HG. HUVEC were K e p t 18 days in media containing D-glucose 5.6 mmol/l, D-glucose 28 mmol/l or D-glucose 5.6 mmol/l and L-glucose 22.4 mmol/l, a non metabolized, g l y c o s y l a t i n g control agent . L - t y r o s i n e 14.0 mmol/l was employed. HUVEC grown in 28 mmol/l D-glucose and t y r o s i n e did not d i f f e r in number from those in 5.6 mmol/l (7232x102+/-650.13x10~ c e l l s culture well vs,8608x102+/-940.38xlO2, mean +/-SEM,n=5) whereas those in HG without tyrosine were reduced (4996. OxlO2+/-588.76x102 ,P=O. 01).HUVEC in L-glucose with and without t y r o s i n e did not d i f f e r s i g n i f i c a n t l y from those in 5.6 mmol/l Dglucose. L - t y r o s i n e abolished CR, as assessed e v a l u a t i n g c o l c h i c i n e - t r e a t e d c e l l s stained by immunofluorescence for tubulin. L-tyrosine counteracts the delayed HUVEC r e p l i c a t i o n caused by HG, which may r e s u l t from t u b u l i n a c e t y l a t i o n r a t h e r than g l y c o s y l a t i o n .

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ENDOTHELIN-I PRODUCTION IS REDUCED IN RETINAL CAPILLARY ENDOTHELIAL CELLS CULTURED IN HIGH GLUCOSE. P.A. Molinatti, M. Porta, K. Takahashi, S.M. Kanse, R.A. Brooks, S.R. Bloom and E.M. Kohner. Department of Medicine Royal Postgraduate Medical School London

DIABETIC PATIENTS WITH M I C R O A L B U M I N U R I A 1 1 2 2 A. Collier,1 J.P. Leach, A. McLellan, J.J. Morton and

Endothelin-1 (ET-I) is a potent vasoconstrictor synthesized by endothelial cells (EC). EC dysfunction in diabetic retinopathy may include reduced production of ET-I and, as an approach to verify this hypothesis, we measured it in the supernatants of bovine retinal EC cultured in the presence of glucose 5.6, 16.7 and 27.8 mmol/l. After 7 days in incubation, ET-I was 360 + 29.4 (mean + SEM of 8 duplicated experiments), 13278 + 16.5 (p<0.0~01) and 125.3 ~ 20.7 fmol/10 ~ cells (p< -0.0001), respectively. No significant differences were observed when mannitol was added to obtain final concentrations equismolar with those of glucose (391.4 61.4; 353.8 +45.7 and 357.3 ~ 39.3 fmoi/109 cells). Specific binding of ET-I to bovine retinal capillary" pericytes grown in the same concentrations of glucose did not show consistent variations. Impaired synthesis of ET-I from capillary EC may result from a direct toxic effect of glucose. Decreased ET-I tone on the capillary wall may account for increased retinal blood flow, as observed both in experimental hyperglycaemia and pre-clinical diabetic retinopathy.

INCREASED PLASMA E N D O T H E L I N LEVELS IN INSULIN-DEPENDENT

1M. Small.

~ i a b e t i c Department, Gartnavel General

Hospital and N C Glasgow

Blood Pressure Unit, Western Infirmary,

Endothelin is a peptide isolated from vascular endothelin. It is a potent vasoconstrictor and a growth factor for vascular smooth muscle. The aim of this study was to determine whether the concentration of plasma endothelin is abnormal in insulin-dependent diabetes, particularly in patients with microalbuminaria. Fifteen nor~oalbuminuric insulin-dependent diabetic patients (median (range); age 42(21-55)2rs ; duration of diabetes ll(8-20)yrs; plasma glucose 12.6(5.3-19.4)mmoi/1; HbA 1 9.1(7.8-13.6)%, 12 microalbuminuric insulin-dependent diabetic patients (age 44(20-54)yrs; duration of diabetes 13(9-22)yrs; plasma glucose 13.1(6.3-20.6)mmoi/i; HbA 1 9.6(8.0-14.7)%) and 12 comparably-aged (48(22-58)~rrs) controls were studied. Seven normoalbuminaric patients and ten microproteinuric patients had background retinopathy, while two microproteinuric diabetic patients had proliferative retinopathy. All patients had blood pressure <140/90 and easily palpable peripheral pulses. Plasma endothelin, measured by radio-immanoassay, was elevated in the normoalbuminuric diabetic patients (8.3(4.8-12.7)fmoi/i; p < 0.01) and the mieroalbuminuric patients (10.2(5.6-7.6) fmol/l; p < 0.OO1) compared with control subjects (6.1 (4.5-7.6)fmoi/I). Plasma endothelin was higher in the microalbuminuric patients compared with the normoalbuminuric group (p < 0.05). The increase in plasma endothelin confirms endothelial dysfunction in diabetes, and may contribute to the vascular disease and hypertension prevalent in diabetes.

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FAT DISTRIBUTION AND PLASNINOGEN ACTIVATOR ACTIVITY IN OBESE AND NON-OBESE TYPE 2 DIABETICS L. Van Gaal, G. Vansant, M. Van der Plancken, I. De Leeuw. Dept. of Endocrinology, Metabolism and Clinical N u t r i t i o n , U n i v e r s i t y of Antwerp (UIA), Antwerp, Belgium. Plasminogen activator i n h i b i t o r (PAl) is an important i n h i b i t o r of f i b r i n o l y s i s . I t has been shown that PAl levels are elevated in diabetics and obese subjects, in particular in patients with abdominal obesity. This paper reports on PAl a c t i v i t y levels in obese and nonobese type 2 diabetics, related to w a i s t / h i p - r a t i o (WHR) and insulin levels. PAl was measured in 86 male diabetics (mean age 59.7 • 8.7 y; BMI 26.6 • 2.1; WHR 0.98 • 0.06). PAl was measured in 4 age-matched subgroups according to BMI and WHR. Not only obese (OB) diabetics (p < 0.004) but also non-obese (NON-OB) d i a b e t i c s (p < 0.05) show increased PAl levels when compared to controls without abdominal adiposity. In patients with WHR < 1.0, PAl is s i g n i f i c a n t l y lower (OB: 15.8 • 4.7 AU/ml, NON-OB: 14.3 • 8.9 AU/ml) compared to diabetics with WHR > 1.0 (OB: 23.8 • 9.1AU/ml, NON-OB: 17.5 • 7.6 AU/ml). A close relationship exists between PAl and fasting insulin levels (r=0.48; p < 0.0001); also free testosterone (FT) is s i g n i f i c a n t l y correlated with PAl (r=0.21; p < 0.02). Multiple stepwise regression analysis indicate that PAl is in a f i r s t step dependent on insulin levels (p < 0.00001), further on WHR and on FT (up to 33%). This study indicates that PAl a c t i v i t y is related to fat topography with insulin and androgens as the most important key intermediates; this association not only occurs in obese but also in non-obese diabetic individuals and may contribute to a disturbed f i b r i n o lysis and an enhanced risk for athero(thrombo)sclerosis.

PLASMINOGEN ACTIVATOR INHIBITOR (PAl) AND TISSUE PLASMINOGEN ACTIVATOR (LPA) IN INCIPIENT DIABETIC NEPHROPATHY. D Carvalho-Brsgs, J Andrade, MC Nonteiro, JM Pina-Cabral, JL Medina and MP Hargreaves. Departments of Endocrinology and Physiology, Oporto Medical School, Oporto, Portugal. Several features of endothe]ial cell damage or disfunction have been described in incipient diabetic nephropathy. We investigated plasma tPA and PAI (systhetic chromogenie substract method, Diagnostics Stago Kits, France) in basal conditions and after fibrinolytie stimulation (venous occlusion) in 12 normoalbuminuric Type 1 (insulin-dependent) disbetics (IDDs) (Group 1), in lO IDDs with mieroslbuminurio (20-200 pg/min) (Group 2) and in 8 healthy controls. Basal tPA (u/ml) was slightly lower in controls (medion, 25-75th centiles; 0.27, 0.26-0.29) than in IDDs (Group l: 0.36, 0.29-0.47; Group 2: 0.44, 0.36-0.47); after 20 minutes of arm venous stssis, such differences were not seen, and the increase in LPA was significant (p
OP 28 Hall A-15 Oral Therapeutic Agents 163

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EFFECT OF METFORMIN ON LACTATE TURNOVER IN NON-INSULIN-DEPENDENT DIABETES R.A. Jackson, B.M. Sim, M.I. Hawa and L. DiSilvio The Middlesex Hospital, London WIN 8AA Metformin's effect on lactate turnover before and after glucose ingestion (75 g) and basal hepatic glucose output (HGO) were studied in I0 nonobese Type 2 (non-insulin-dependent) diabetics (age 54.3 -+ 1.8 yr) during randomised metformln (2 g daily) and placebo administration u~$ng simultaneous primed-constant infusions of i-[ C]lactate and 3-[3H]glucose. Basally metformin administration reduced plasma glucose levels from 9.4 -+ .9 to 6.4 -+ .7 rmaol/l (p <.001), while HGO (12.4 + 1.0 pmol/kg/min) and insulin levels (22 -+ 5 ~U/ml) were unchanged whereas lactate concentrations, lactate turnover and forearm lactate uptake (FLU) were increased, from 1.14 + .II to 1.75 -+ .21 ~ o I / i (p <.01), 11.7 + 1.2 to 15.6 -+ 1.4 pmol/kg/min (p <.05) and .08 + .07 to .33 • .ii pmol/100 ml forearm/rain (p <.05) respectively. After glucose ingestion metformin lowered the glucose tolerance curve but the incremental area under the curve and insulin levels were unchanged, while lactate levels and turnover increased. Metformin enhanced the incremental area under the lactate concentration curve (p <.05) and the increment in lactate appearance from 3.0 + 0.3 to 4.6 -+ 0.6 pmol/kg/min (p <.05). Increased FLU accompanied rising lactate concentrations after glucose loading. Conclusions: Metformin increases lactate turnover basally and after glucose loading by different mechanisms. Basally metformin augments lactate production by non-muscle tissues, probably the gut; after glucose ingestion probably hepatic lactate production is mainly increased. These data, with concurrent restraint of }{GO reflecting enhanced hepatic insulin sensitivity, suggest that metformin influences both non-insulin-mediated and insulin-mediated metabolism.

METFORMIN INCREASES INSULIN-STIMULATED GLUCOSE TRANSPORT IN INSULIN-RESISTANT HUMAN SKELETAL MUSCLE D Galuska, A Th6rne, and H Wallberg-Henriksson. Dept of Clin Physiol, Karolinska Hospital, Stockholm, Sweden. M e t f o r m i n is a b i g u a n i d e o r a l h y p o g l y c a e m i c a g e n t u s e d in t h e t r e a t m e n t o f n o n - i n s u l i n - d e p e n d e n t diabetes mellitus. The antihyperglycaemic effect o f m e t f o r m i n is n o t c o m p l e t e l y u n d e r s t o o d . T h e aim of the present study was to investigate the e f f e c t o f m e t f o r m i n (0.i mM) o n g l u c o s e t r a n s p o r t in n o r m a l a n d i n s u l i n - r e s i s t a n t human skeletal muscle. A newly developed human muscle strip p r e p a r a t i o n w a s u s e d . M u s c l e s a m p l e s ( 1 5 0 - 2 0 0 mg) w e r e o b t a i n e d f r o m m. r e c t u s a b d o m i n a l i s ( d u r i n g abdominal surgery) or from vastus lateralis of the quadriceps femoris muscle (by u s i n g o p e n biopsy technique) from 7 healthy controls (age 33• yrs, B M I 23• and from 5 insulin-resistant o b e s e s u b j e c t s (age 5 7 • yrs, B M I 3 3 • Metformin had no effect on basal or insulin-stimulated (i00 ~ U / m l ) 3 - 0 - m e t h y l g l u c o s e transport in incubated muscle strips from healthy subjects. Muscle tissue from the obese patients was markedly insulin-resistant (0.6930.19 for basal and 0.79• ~ m o l x m l "I x h "~ f o r i n s u l i n - s t i m u lation, N.S.). Metformin did not affect basal glucose transport in the insulin-resistant muscles, whereas insulin-stimulated (i00 ~ U / m l ) g l u c o s e t r a n s p o r t w a s i n c r e a s e d b y 56% ( 0 . 7 9 • vs 1.23• ~ m o l x m l I x h "I in t h e a b s e n c e o r presence of metformin, p<0.05). In conclusion, m e t f o r m i n c a n a b o l i s h i n s u l i n - r e s i s t a n c e in h u m a n skeletal muscle by increasing glucose transport across the muscle cell membrane.

A53

165

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INFLUENCE OF GLICLAZIDE ON GLUCOSE STIMULATED INSULIN SECRETION IN MAN

DIRECT C A R D I O V A S C U L A R EFFECT OF H Y P O G L Y C E M I C S U L P H O N Y L U R E A COMPOUNDS G. Ballagi-Pordany, M. Zs. Koltai, Z. Aranyi, and G. Pogatsa, National Institute of C a r d i o logy, Budapest, H u n g a r y

T.W. van Haeften, T.F. Veneman and E.A. van der Veen, Free University Hospital, Amsterdam. Sulfonylureas (SU) have been proposed to have their mechanism of action (closure of K+(ATP) channels) in common with glucose. This would imply that SU would augment insulin release at suhm~ximally stimulating blood glucose levels and not at maximally stimulating levels. We studied glucose-stimulated insulin release with hyperglycemic clamps (blood glucose levels: 8 and 32mMol/L; 60 minutes) after gliclazide (80mg, given 30 minutes before the clamp) or placebo, in 8 healthy volunteers; plasma C-peptide increments at 5 and at 60 minutes were used as assessment of first and second phase insulin release. Mean~S~ plasma C-peptide increments during clamps at 8 mMol/L were higher after gliclazide (1.07+0.10 vs 0.88+0.10 nMol/L and 1.36+0.13 vs 1.09+0.09 nMol/L, first and second phase, respectively, both p<0.05). During clamps at 32 mMol/L, no differences were observed between gliclazide and control days (1.23+0.13 vs 1.38+0.19 and 4.8+0.6 and 4.9+0.5 nMol/L, first and second phase respectively, both p>0.5). We conclude, that gliclazide enhances glucose stimulated insulin secretion at a suhmaximally stimulating blood glucose level, but not at a maximally stimulating glucose level; this is in accordance with the notion that SU have their mechanism of action in common with glucose.

The effects of first g e n e r a t i o n sulphonylurea compounds carbutamide, gliclazide and tolbutamide as well as second generation compounds g l i b e n c l a m i d e and g l i p i z i d e on the c a r d i o v a s c u l a r system were investigated in rabbits. Each compound was i n j e c t e d i n t r a v e n ously at cumulative doses of 74, 296, 592 ~ m o l / k g of c a r b u t a m i d e (n=6) and tolbutamide (n=6), 0.2, 1.0, 2.0 ~mol/kg of g l i b e n c l a m i d e (n=6) and glipizide (n=6), and 16, 32 and 48 ~mol/kg of galiclazide (n=6). M e a n arterial blood pressure, m y o c a r d i a l c o n t r a c t i l e force, cardiac output and heart rate were measured. The rate of change of m y o c a r d i a l c o n t r a c t i l e force development (positive dF/dt) and of myocardial r e l a x a t i o n (negative dF/dt) were also registered. The first g e n e r a t i o n sulphonylureas exerted a p o s i t i v e inotropic effect in c o n t r a s t to the second generation compounds. Thus the h y p e r t e n s i v e and inotropic effects of first generation sulphonylurea drugs m a y a g g r a v a t e the d y s f u n c t i o n of the cardiovascular system in type 2 diabetes secondary to diabetic cardiopathy, neuropathy, atherosclerosis, and obesity. This suggests, second generation sulphonylureas should be p r e f e r r e d in the t h e r a p y of type 2 diabetics, if s a t i s f a c t o r y m e t a b o l i c control cannot be achieved by d i e t a r y management alone.

167

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Effeet of tolbutamide on the myocardial tension in isolated heart from diabetic rat during ischemia and reperfusion.

SIMVASTATIN IMPROVES INSULIN ACTION IN HYPERCHOLESTEROLEMIC ,TYPE-2 (NON-INSULIN DEPENDENT) DIABETIC PATIENTS. G.Paolisso,G.Marrazzo,S.Sgambato,M.Verza,G.Gambardella , M.Varricchio and P.D'Onofrio. Institute of Geriatric Medecine and Institute of General Medecine .and Metabolic Diseases- University of Naples - Naples - Italy.

Koiehi Oshiro, Michio Shimabukuro, Yasuhiko Nakada, Fumio Nagamine, Ryuji Sunagawa, Keiji Murakami and Goro Mimura Second Department of Internal Medicine, School of Medicine, University of the Ryukyus, 0kinawa, Japan To examine the effect of tolbutamide on the myocardial developed tension (T) of the diabetic heart during isehemia and reperfusion, perfused isolated hearts were evaluated. The experimental rats were as follows : insulin dependent diabetic rats (IDDM; single intravenous injection of 60mg/kg streptozotocin (STZ) in male SpragueDawley rats), non insulin dependent diabetic rats (NIDDM; single subcutaneous injection of 90mg/kg STZ at 5 day neonates), tolbutamide-treated IDDM and NIDDM (T-IDDM, TNIDDM; giving tolbutamide 100mg/kg/day for 6 weeks via an orogastrie tube every day, respectively). At 14 weeks after birth, experiments were performed. After the equilibration, global ischemia was carried out followed by reperfusion. T was measured throughout isehemia and reperfusion. Basal values of T increased in both T-IDDM and T-NIDDM, compared with in IDDM and NIDDM, although chronic oral administration of tolbutamide did not ameliorate hyperglycemia in T-IDDM. Percent recovery of T in T-IDDM and T-NIDDM were significantly higher (P<0.01) than in IDDM and NIDDM throughout ischemia and reperfusion respectively. These results indicated that chronic oral administration of tolbutamide directly improved myocardial contractility throughout ischemia and reperfusion regardless of the improvement of glyeemia.

In order to study the possible effects of Simvastatin (S) on insulin action,12 type-2 (non-insulin dependent) diabetic (fasting blood glucose 7.4+0.4 mmol/l),hypercholesterolemic (fasting LDL-cholesterol 7.2+0.4 mmol/l) patients treated with weight-maintaining diet only were submitted to an euglycemic hyperinsulinemie (50 mU/kg x h ) glucose clamp in a double-blind ,cross-over procedure after 3 weeks treatment with either S (30 mg/day) or placebo (PBO) at 3 weeks intervals. Body mass index was similar after S and PBO (27.2+0.3 vs 27.2+0.6 p=NS).During glucose clamp, plasma g~ucose and insulin levels were similarly clamped at 6 mmol/l and 355 pmol/l in both occasions respectively. In the last 60 min of the experiment, S vs PBO strongly inhibited Hepatic Glucose Output (2.7+0.3 vs 5.2+0.4 umol/kg/min p<0.01) and increased both Glucose Disappearance rate (Hd) (26.3+0.6 vs 19.5+0.5 umol/kg/min p<0.05) and glucose Metabolic Clearance Rate (gMCR) (4.3+0.8 vs 3.6+0.7 ml/kg/min p<0.05).Plasma LDL-cholesterol (7.2+0.4 vs 4.3+0.3 mmol/l p<0.001) and triglycerides (2.9+0.4 vs 2.1+0.2 mmol/l p<0.01) levels were also lower after S than after PBO. In conclusion Simvastatin improves insulin action in type-2 (non-insulin dependent) diabetic,hypercholesterolemic patients.

A54

OP 29 Hall B-3 Metabolism in vivo 169

170

DECREASED HEPATIC GLUCAGON SENSITIVITY IN TYPE 1 (INSULINDEPENDENT) DIABETES: THE COUNTERREGULATORY ASPECT.

DOSE-RESPONSE RELATIONSHIP BETWEEN INSULIN AND COUNTERREGULATORY HORMONES DURING THE EUGLYCEMIC CLAMP IN RATS

C. ~ r s k o v ' , N. Mr l, O. R a s m u s s e n ~, O. P e d e r s e n 1, K . G . M . M . A l b e r t i ~ a n d O. SchmitzL D e p a r t m e n t of Medicine III, A a r h u s A m t s s y g e h u s , A a r h u s , D e n m a r k L Medical Department M (Diabetes & Endocrinology), Aarhus Kommunehospital, Aarhus, D e n m a r k ~. D e p a r t m e n t of Medicine, U n i v e r s i t y of N e w c a s t l e , Newcastle u p o n T y n e , UKL T h e e f f e c t of g l u c a g o n i n f u s i o n on h e p a t i c g l u c o s e p r o d u c t i o n (HGP) d u r i n g e u g i y c e m i a was e v a l u a t e d in 7 T y p e I diabetic p a t i e n t s a n d in 10 c o n t r o l s u b j e c t s . In d i a b e t i c s u b j e c t s n o r m o g l y c e m i a was m a i n t a i n e d d u r i n g t h e night preceding the study by a variable intravenous insulin and glucose infusion. During the study endogenous insulin s e c r e t i o n was s u p p r e s s e d b y s o m a t o s t a t i n (450 txg]h) a n d r e p l a c e d b y i n s u l i n i n f u s i o n (0.15 m U / k g / m i n ) . 3 H - g l u c o s e was i n f u s e d ( p r i m e d : 40 ~tCi, c o n t i n u o u s 0 . 4 txCi/min) f o r isotopic d e t e r m i n a t i o n of g l u c o s e t u r n o v e r . Plasma g l u c o s e was clamped at 5 mmol/1 f o r 2.5 h o u r s a n d g i u c a g o n (1.5 n g / k g ] m i n ) was t h e n i n f u s e d . B a s a l HGP was similar in d i a b e t i c s ( 1 . 4 + 0 . 4 m g / k g / m i n ) a n d c o n t r o l s (1.7 + 0.2 m g / k g / m i n ) . In diabetic s u b j e c t s HGP r o s e slowly to 2.2 + 0.5 m g ] k g / m i n d u r i n g t h e f i r s t h o u r s of g i u c a g o n i n f u s i o n a n d s t a b i l i z e d a t . t h i s level ( 2 . 4 + 0.5 m g ] k g / m i n in t h e t h i r d h o u r ) . In c o n t r o l s HGP i n c r e a s e d s h a r p l y to h i g h e r levels t h a n in t h e diabetic s u b j e c t s , 3.4 + 0.3 m g / k g / r o i n , d u r i n g t h e f i r s t a n d s e c o n d h o u r of g l u c a g o n i n f u s i o n ( p < 0 . 0 5 ) , a n d t h e n g r a d u a l l y fell (2.9 + 0 . 4 m g / k g / m i n : t h i r d h o u r ) . In c o n c l u s i o n : When s t i m u l a t e d with p h y s i o l o g i c c o n c e n t r a t i o n s of g h i c a g o n , T y p e 1 d i a b e t i c p a t i e n t s h a d a n overall r e d u c e d HGP r e s p o n s e a s well as a n a b n o r m a l response pattern. This may further attenuate an already compromised eounterregulatory response following a hypoglycemic episode.

S.J. Koopmans, S.F. de Boer, J.K. Radder, M. Fr~lich and H.M.J. Krans. Department E n d o c r i n o l o g y , University Hospital, Leiden, the Netherlands. It has been suggested that hyperinsulinemia per s~ might affect the levels of some counterregulatory hormones in the absence of hypoglycemia. We studied the effect of graded hyperinsulinemia and concomitant increased glucose metabolism on counterregulatory hormones by means of the 5-step sequential hyperinsulinemic euglyeemic clamp technique, combined with 3-3H-glucose infusion in 8 conscious rats. Results: (mean • sem). Insulin infusion rates (IIR) of O, 0.5, i, 3 and 16 mu.min-i resulted in plasma insulin levels of 23• 44• 85• 342• and 5535• gU/ml, tissue glucose uptake of 3• 3.7• 5• 9• and 12.3• mg.min-i and hepatic glucose production of 3• 2• 0.9• -0.2• and -0.3• mg.min-l, respectively. Plasma glucagon was half-maximally suppressed between IIR's of 0.5 and 1 mU.min-i and maximally suppressed at 3 mU.min-l. The suppression exactly paralleled the inhibition of hepatic glucose production. Plasma corticosterone and epinephrine were not influenced during the clamp. Moreover, the circadian rhythm of oorticosterone was unaffected. Plasma norepinephrine was increased (• p<0.05) at IIR's of i, 3, and 16 mU.min-l. The clamp-induced changes in counterregulatory hormones w e r e not observed during control-infusion experiments. Conclusions: The doseresponse clamp, i) decreases plasma glucagon in a dosedependent way, 2) has no effect on the measured adrenal hormones, 3) stimulates the sympathetic nervous system at IIR's exceeding 1 mU.min-l.

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DIFFERENTIAL EFFECTS OF A GRADED SELECTIVE SUPPRF~SSION OF INSULIN SECRETION ON GLUCOSE PRODUCTION AND REMOVAL IN DOG So J. Radziuk, S. Pye, J. Daviesl and T.J. McDonald, Ottawa aD~ London, Canada. The selective suppression of insulin decreases the metabolic clearance of glucose and increases its production. To determine the sensitivity of these processes to insulin, its secretion was inhibited to varying degrees using incrsasing doses of galanin. 24 normal 18hr-fasted dogs underwent an infusion of labelled glucose and a simultaneous one hour infusion of galanin. 5 galanin doses from 2 to 12ug/kg-hr were used. Portal blood was sampled using surgically preimplanted catheters. Glucose production (Ra) and its fractional disappearance rate were calculated. As the dose of galanin increased, the decrease in insulin area (peripheral) doubled. Neither peripheral nor portal glucagon changed significantly at any dose of galanin (from -3+2% to -6+3%)~ The maximal decrease in k ranged from 58~i0% (2ug7kg-hr galanin) to 52+7% (10ug/kg-hr) indicating that systemic glucose removal was maximally suppressed when peripheral insulin levels fell by only 50% (54_+5)~ In contrast, as insulin fell from 50% to 80% (80_+2) below basal, the integrated production of glucose rose from 0.69~0.12g above basal to 2.0+0.5g above basal - a threefold increase. This effect saturated at 7ug/kg-hr galanin.. These data indicate that systemic glucose utilization is very sensitive to small decrements in peripheral insulin whereas production appears to correlate with a wide range Of insulin: glucagon ratios as these fall below basal.

ADRENALINE'S EFFECT ON GLUCOSE- AND INSULINMEDIATED GLUCOSE DISPOSAL IN TYPE I DIABETES. F.P. Alford, G.M. Ward, J.M. Waiters, A. Kalfas, and J.D. Best Endocrinology Unit and University Department of Medicine, St. Vincent's Hospital, Fitzroy, 3065, Australia. The aim of this study was to determine the relative roles of changes in glucose-mediated glucose disposal (SG) and insulin sensitivity (SI), in the impairment of glucose disposal caused by adrenaline infusion in Type I (insulin dependent) diabetes. In 5 fasting young non obese Type I diabetic subjects with minimal endogenous insulin secretion, adrenaline (ADR) was infused at 25ng/kg/min for 5.5h, after a basal overnight insulin infusion (12mU/kg/hr). At 2.5h an intravenous glucose tolerance test was performed with supplemental exogenous insulin infusion to approximate the non-diabetic profde of endogenous insulin secretion, and analysed using a modification of the minimal model of Bergman et al. In random order each subject also had a control (CTR) infusion of basal insulin prior to the glucose tolerance test. Adrenaline infusion was associated with elevated basal plasma glucose (ADR vs CTR, 12.0 + 1.3 SE vs 8.1 + 0.9 mmolB) elevated plasma free fatty acids (0.9 + 0.I Vs 0.3 + 0.1 mmolfl), and profoundly reduced glucose disposal (Kg, 0.47 + 0.07 vs 1.93 + 0.33 min -1 X 102). The reduced glucose disposal was due to a large decrease in SI (0.9 + 0.5 vs 14.4 + 4.9 min -1. mU-1. L X 104, P<0.05), rather than the small decrease in SG (2.5 + 0.2 vs 3.1 + 0.5 rain -1 X 102, NS). In conclusion, adrenaline infusion in Type I diabetes impairs insulin- but not glucose-mediated glucose disposal.

A55

173 ~-FUJ~CE OF ~

174 ~

ON GUJCCSE CYCT~ AC~I~-~Z

AND INSULIN ACTION IN NORMAL MEN RDG Neely, DP Rooney, CN Ennis, B Sheridan, AB A t k i n s o n , P M B e l l a n d E R T r i m b l e , D e p a r t m e n t of Chemical Pathology, Queen's University of Belfast and Sir George E Clarke Metabolic Unit:, Royal Victoria Hospital, Belfast, United Kingdom. Increased activity of the hepatic glucose/gluoose-6phosphate cycle (GC) is associated with hepatic and p e r i p h e r a l i n s u l i n r e s i s t a n c e in a c r o m e g a l y . To determine whether a similar association occurs following short t e r m growth hormone (GH) elevation within the physiological range, two step hyperinsulinaemic glucose clamps were p e r f o r m e d in n o r m a l m a l e v o l u n t e e r s (n=7) a f t e r 1 2 h G H (4.4 mU.kg-l.h -I) and 12h saline infusions given in random order. GC activity was estimated by comparing hepatic total glucose output (h'l~) and hepatic glucose on (H~a) detertaJned s i m u l ~ l y by 2-JH - and 6- H glucose using labelled exogenous glucose infusions and selective enzymatic detritiation. GH infusion increased basal levels of ON (23.7 + 2.2 vs 3.0 + 0.5 mU/l; p<0.001) and insulin (11.6 _+ 1.3 vs 5.8 + 0.5 mU/l; p
~

IMPAIRMENT OF INSULIN ACTION ON GLUCOSE KINETICS BY MEDIUM AND LONG CHAIN FATTY ACIDS IN MAN I. Turkalj,D. eloesch,H. Bodmer, S. Vosmeer and U. Keller Department of Research, University of Basel, Switzerland Insulin sensitivity of glucose kinetics (6,6-2H-glucose infusion technique) was studied in normal subjects receiving either i) medium chain triglyceride (MCT) infusions 2 mg/kg/min (n:9), 2) long chain triglyceride (LCT) infusions 2 mg/kg/min (n=8), or 3) saline-glycerol (controls; n=6) during 6.5 hrs. During the last 2 hrs of lipid infusions an euglycaemie hyperinsulinaemio clamp (plasma insulin t%J 35~U/ml) was performed. During hyperinsulinaemia, suppression of hepatic glucose production was 71% diminished in the LCT group compared to controls (p<0.02) but unimpaired in the MCT group (LCT vs MCT: p<0.05). Metabolic clearance rate of glucose was 43% lower during MCT and 38% lower during LCT than in controls (MCT and LCT: p<0.01 vs controls; LCT vs MCT: n.s.). The amount of glucose infused during the clamp was 58% lower during LCT and 52% lower during MCT than in controls (both: p<0.01; LCT vs MCT: n.s.). Thus, insulin action on hepatic glucose production was diminished during elevated LCT but not during elevated MCT whereas insulin action on peripheral glucose clearance during LCT and MCT was similarly diminished. It is concluded that during increased supply of fatty acids, chain length influences their effect on insulin sensitivity of hepatic but not of peripheral glucose metabolism, presumably due to different intracellular handling of MC and LC fatty acids.

OP 30 Hall A-18 Pregnancy 175

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THE ECHOCARDIOGRAPHIC ASSESSMENT OF DIABETIC MOTHERS' FETUSES DURING INTRAUTERINE LIFE R.M. Botta, S. Pip#one* and M. Inguanta Clinica Medica B - University of Palermo * Department of Cardiology - ,~Casa del Sole,, Hospital - Palermo Aim of the present study was to evaluate during the intrauterine life echocardiographic diameters in fetuses of well-controned diabetic mothers. 20-insulintreated diabetic women (10 type 1 (insulin-dependent) and l0 type 2 (non insulin-dependent) diabetic pregnant women: mean age 28.88 -4- 8.12 years) were examined (first visit at 10.76 • 4.13 weeks). Their fetuses underwent an echocardiographic exam (Hewlett-Packard 77020AC) during intrauterine life at the 20 th, 28 t~ and 36th week of gestation: interventricular septum (IVS), ]eft ventricnlar wall (LVW), left ventricular diastolic dimension (LVDD), left ventricular systolic dimension (LVSD), right ventricular wall (RVW), right ventricular diastolic dimension (RVDD), right ventricular systolic dimension (RVSD) were determined. These parameters were compared to those of 183 normal pregnant women, divided for gestational age. Mean glycemic levels, glycosilated haemoglobin, triglycerid and cholesterol values, insulin-antibodies (ILIA) were evaluated. Moreover, fetal body weight index (FBWI) at birth was evaluated according to Babson's centiles. No fetus had congenital anomalies. We found inverse correlations between mean glycemic values and respectively IVS (r - - 0.534; p < 0.05), RVW (r - - 0.455; p < 0.05), RVDD (r - - 0.468; p < 0.05) and LVDD (r - - 0.578; p < 0.05) at 20 th week. A positive correlation exists between FBWI and IVS at 36 th week (r = 0.509; p < 0.05). Our results seem to indicate that in the first phase of pregnancy hyperglycemia may slower the growth of fetal heart.

TOKOLYSIS WITH NIFEDIPINE IN DIABETES COMPLICATED PREGNANCY. EFFICACY AND INFLUENCE ON FETAL WELLBEING. Gy.M.Csdkdny, M . I l l y d s , K.Hajdu, Zs. Tur i , A.Oobos, I . G d t i . Postgrad.Med.Schol, Budapest. Szabolos u 53. Hungary. 1035. Twenty seven normotensive d i a b e t i c pregnant p a t i e n t s (White-B:8, C:4, O:6, F:2, gestational diabetes:7) between 24-35 completed weeks of gestation were treated with nifedipine because of threatening preterm labor. The tokolysis was initiated with 2Q mg nifedipine given sublingually, and this dose was repeted in 6 hour intervals. Treatment was successful in 20 cases (74%). This success rate is identical with the earlier used terbutaIine. Carbohydrate metabolism was monitored by serum glucose profiles and fructosamine level (Baker's automated colorimetric method). Insulin was administered aiming normoglycaemia. During the nifedipine treatment a mild increase (5%) in insulin requirement was observed. Fetal biophysical profile and Doppler umbilical and aortic velocity waveform analyses were performed to assess fetal wellbeinB. Placental or fetal disturbance could not be found in normotensive patients only in those 2 oases who became hypertensive. We suggest that nifedipine is a safe tocolytic agent which does not alter the maternal normoglycaemia or fetal wellbeing and can be used routinely in diabetes complicated pregnancies.

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177 INSULIN RESISTANCE

IN THE OFFSPRING

OF DIABETIC

P I M A I N D I A N WOMEN. D.J. Pettitt, W.C.Knowler, P.H.Bennett, M.F. Saad, R.G. Nelson, M.A. Charles, and Q. Liu. N a t i o n a l I n s t i t u t e of D i a b e t e s and D i g e s t i v e and K i d n e y Diseases, and The C l e v e l a n d Clinic, Phoenix, AZ, U.S.A. Fasting serum insulin concentrations were c o m p a r e d in three g r o u p s of n o n d i a b e t i c Pima Indians aged 5 to 29 years - O f f s p r i n g of i) nondiabetic women, 2) p r e d i a b e t i c women (nondiabetic d u r i n g p r e g n a n c y but d e v e l o p e d d i a b e t e s later), and 3) w o m e n w i t h type 2 (noni n s u l i n - d e p e n d e n t ) d i a b e t e s d u r i n g pregnancy. Offspring of nondiabetic, prediabetic, and diabetic women had fasting insulin c o n c e n t r a t i o n s of 140 pmol/l, 162 pmol/l, and 195 pmol/l, respectively, a d j u s t e d for age and sex w i t h m u l t i p l e linear regression. Even a f t e r a d j u s t m e n t for f a s t i n g p l a s m a glucose, the o f f s p r i n g of p r e d i a b e t i c and d i a b e t i c w o m e n had s i g n i f i c a n t l y h i g h e r i n s u l i n c o n c e n t r a t i o n s than o f f s p r i n g of n o n d i a b e t i c w o m e n (p<0.02 for each). In spite of the p r e v i o u s l y d e s c r i b e d e f f e c t of the i n t r a u t e r i n e e n v i r o n m e n t on the risk for obesity and diabetes in this population, these data s u g g e s t t h a t i n s u l i n resistance, as e s t i m a t e d by the f a s t i n g i n s u l i n concentration, is an i n h e r i t e d trait and is not i n d u c e d simply by the e x p o s u r e to the d i a b e t i c i n t r a u t e r i n e environment.

A B N O R M A L G L U C O S E T O L E R A N C E IN P R E V I O U S G E S T A T I O N A L DIABETICS: A F O L L O W - U P STUDY. P. Damm, L. M ~ i s t e d - P e d e r s e n and C. K~hl. D i a b e t e s Center, Dept. of Obstet. & Gynecol., Rigshospitalet, and H v i d ~ r e Hospital, Copenhagen, Denmark. 318 w o m e n w i t h p r e v i o u s g e s t a t i o n a l d i a b e t e s mellitus (GDM) were offered a follow-up e x a m i n a t i o n 2-11 years after index p r e g n a n c y w i t h the aim of i n v e s t i g a t i n g the incidence of impaired g l u c o s e t o l e r a n c e (IGT) and diabetes m e l l i t u s and also to search for p o s s i b l e risk factors for the d e v e l o p m e n t of these disorders. All p a t i e n t s w e r e d i a g n o s e d at our Center and t r e a t e d only w i t h diet d u r i n g pregnancy. 256 (81%) a c c e p t e d to p a r t i c i p a t e in the study. 42 n o n - p o t e n t i a l diabetic women w i t h a n o r m a l OGTT d u r i n g p r e g n a n c y 2-11 years earlier served as controls. The m e a n age at f o l l o w - u p w e r e 35.8 and 33.6 years and the m e a n f o l l o w - u p time were 6.2 and 6.9 years for the p r e v i o u s GDM's and controls, respectively. A p p l y i n g the c r i t e r i a of WHO, 4% of the prior GDM's had Type 1 diabetes, 13% Type 2 diabetes, and 16% IGT w h e r e a s o n l y 7% of the controls had IGT. The area under the g l u c o s e curve (AUC) was s i g n i f i c a n t l y h i g h e r (P<0.Ol) for the normal weight previous GDM's with normal OGTT's c o m p a r e d w i t h the controls. Our results c o n f i r m that w o m e n w i t h p r e v i o u s GDM have an i n c r e a s e d i n c i d e n c e of m a n i f e s t diabetes and IGT few years after index pregnancy. N o r m a l w e i g h t p r e v i o u s GDM's w i t h a normal O G T T at f o l l o w - u p had h i g h e r AUC's than the controls.

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180

IMPAIRED G L U C O S E T O L E R A N C E IN THE THIRD T R I M E S T E R OF PREGNANCY: A S S C I A T E D F A C T O R S TO ITS P E R S I S T E N C E P O S T P A R T U M R.Lichiardopol, C.Dumitrescu, V.Nuteanu, M.Grigorescu, Z.Mirodon and I.Mincu. Clinic of Diabetes, Nutrition & Metabolic Diseases, Bucharest ROMANIA In a p r e v i o u s study, g l u c o s e t o l e r a n c e (75g. OGTT, WHO 1985) on 302 w o m e n during third trimester of pregnancy has been evaluated. Of these, 14 w i t h normal glucose tolerance (A group) and 23 with impaired glucose (IGT) tolerance (B group), two years after delivery, in a b s e n c e of p r e g n a n c y and lactation, have been r e i n v e s t i g a t e d . All group A females and 12 group B (BI group) have r e v e a l e d a normal g l u c o s e tolerance, r e m a i n i n g ii (B2 group) have presented IGT. B2 group has shown i n c r e a s e d values (x+SD) both concerning age (37.0~6.6 years as ~ o m p a r e d B1 group (30.2~5.5; p<0.02) and A group (29.9~5.9; p<0.02) and BMI (32.5~4.2 versus 26.4+5.2; p<0.01 and 23.3~4.4; p<0.001 respectively~. The ratio between basal insulinogenic index values (insulin (IRI) pM/Blood glucose (BG) mM) during pregnancy and two years p o s t p a r t u m has been 2.5~i.i A group, versus 1.4+0.8 El group and 1.5+0.6 B2 group (p<0.01)-- s u g g e s t i n g by comparison, persistence B1 and B2 group to an i n c r e a s e d level of insulin resistance. Ins~linogenic index (two years postpartum) after glucose (IRI l h + 2 h / B G lh+2h) has been lower in B2 group (44.1+10pM IRI/mMBG) as compared B1 group (68.6+26.8; p<0.01). Ours data suggest that, on increased insulin resistance background, reduction of glucose insulin response and i n c r e a s i n g age and BMI are a s s o c i a t e d to g l u c o s e tolerance worsening.

TITERS

OF ISLET-CELL ANTIBODIES DURING GESTATIONAL DIABETES ARE ASSOCIATED TO DETERIORATION OF POST-PARTUM GLUCOSE TOLERANCE. D. Mauricio, R. Corcoy, M. Codina, M. Puig-Domingo, J.M. Pou and A. de Leiva

Balsells,

M.

307 gestational diabetics (GD) investigated during pregnancy were screened f o r i s l e t - c e l l antibodies (ICA) t i t e r s ( I n d i r e c t immunofluorescence, human pancreas, prolonged incubation with a p r o t i n i n , JDF u n i t s ) . T i t e r s were compared t o those o f 22 newly-diagnosed type I d i a b e t i c s . Postpartum oral glucose tolerance (OGTT) was analyzed in 152 women (National Diabetes Data Group c r i t e r i a ) . Cases not c l a s s i f i e d as normal by s t r i c t criteria (diabetes, intolerant, borderline) were considered abnormal. Chi-square was used for comparison. 38 subjects were ICA p o s i t i v e (5 JDF:9; 10 JDF:16; 20 JDF:7; 40 JDF:4;>= 80 JDF:2). Titers d i f f e r e d from those o f newly-diagnosed d i a b e t i c s (5 JDF:I; 10 JDF:I; 20 JDF:6; 40 JDF:I; >=80 JDF:13) (p=20 JDF) GD p a t i e n t s (p
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OP 31 Hall A-1 Epidemiology II 181

182

I N C I D E N C E O F D I A B E T E S IN THE N A T I O N W I D E P A N E L O F 13 888 T W I N P A I R S IN F I N L A N D

IMPAIRED GLUCOSE TOLERANCE : RELATIONSHIP BETWEEN AGE OF DETECTION AND PROGRESSION TO DIABETES. L.Monnier, M. Zaki and C. C ol e t t e , D e pa rt me nt of Metabolic Diseases, Lapeyronie Hospital, 3#059 Montpellier Cedex France.

J.Kaprio, J,Tuomilehto, M.Koskenvuo, A.Reunanen, K.Romanov, J.Eriksson and J.Steng&rd. University of Helsinkl, Department of Public Health, Haartmanninkatu 3, 0 0 2 7 0 Helsinki, Finland.

The study included 13#2 subjects with impaired glucose tolerance. The metabolic abnormality was diagnosed between 1970 and 1979 by an OGTT (c ri t e ri a of the National Diabetes Data Group). The subjects aged 10-80 years were allocated to 7 groups defined by the decade of age at which the impaired glucose tolerance was de t e c t e d. The outcome was evaluated in 1985 : progression to either Type 1 or Type 2 diabetes. Among the #00 subjects aged i0-#0 years an overall figure of 6 % worsened to Type 1 diabetes : 15 % between 10 and 20 years, 8 % between 21 and 30 years and 1.5 % between 31 and #0 years. No subject older than #0 years progressed to Type 1 diabetes. Among the It #0 subjects aged 31-80 years 5 % worsened to Type 2 diabetes : 11 % between 31 to #0 years, 6.8 % between 41 and 50 years and less than 3 % for each decade after 51 years. No subject younger than 30 years progressed to Type 2 diabetes. These results show that the risk of progression from impaired glucose tolerance to overt diabetes is relatively small : 5 to 6 % a ft e r an average follow up of 10 years. Furthermore the type of progression depends on the age of de t e c t io n of the impaired glucose tolerance : Type 1 diabetes before 30 years, Type 2 between 30 and 50 years, and small risk of worsening to overt diabetes a ft e r 51 years.

The cumulative incidence of diabetes was assessed in the nationwide c o h o r t o f 13 8 8 8 Finnish same-sexed twin pairs born before 1958 and both co-twins alive in 1967. Data on diabetes were derived through computerized r~cord linkage from death certificates, the National Hospital Discharge Registry and National Drug Registry. Copies of original documents were collected for the suspect diabetic cases for the diagnostic classification in which the following number of cases were confirmed: 106 Type i diabetes, 499 Type 2 diabetes, 45 gestational diabetes and 24 secondary diabetes (mainly acute pancreatitis). Among monozygotic (MZ) twins 21 pairs with Type I diabetes were found of which 3(25%) concordant, and 140 pairs with Type 2 diabetes of which 29(34%) concordant. Among dizygotic (DZ) twins of 78 pairs with Type 1 diabetes 2(5%) and of 303 pairs with Type 2 diabetes 27(16%) were concordant. The proportion of twin pairs concordant for diabetes was clearly higher in MZ than DZ pairs, and it increased with age both in MZ and DZ pairs. These population based data are in agreement with the concordance estimates for Type i diabetes, but for Type 2 diabetes our concordance rates were lower than the previous clinic based estimates from other countries.

184

183 HYPERTRIGLYCERIDAEMIA MULTINATIONAL STUDY

AND MORTALITY IN DIABETIC SUBJECTS: THE

LK Stevens, JA Head and JH Fuller and the WHO Multinational Group. Dept of Community Medicine, University College London, London, U.K.

WHO Study

The relationship between serum triglyceride and subsequent mortality has been studied in 2660 middle-aged diabetic patients from 7 of the centres participating in the WHO Multinational Study of Vascular Disease in Diabetics. Subjects with non-insulin-dependent diabetes mellitus comprised 75% of the sample and 49% were males. Ageadjusted total and cause-specific death rates were calculated over a follow-up period of 6-7 years. Causes of death were assigned using standardised procedures based on information from death certificates, autopsy reports and clinical records. The distribution of serum triglyceride (TG) at base-line was divided into tertiles: (TGI < 1.58 rmmol/L ; TG2 1.59 to 2.56 rmmol/L ; TG3 > 2.57mmol/L). For subjects with non-insulin-dependent diabetes mellitus, total mortality rates were nearly 2.5 times higher for TG3 vs TGI for both males and females: Relative Risk (RR) was 2.4 for both sexes. For circulatory disease deaths (ICD 390-459), the mortality rates were doubled for TG3 vs TO1 : (RR males: 2.1 ; females: 2.0). These relative risks were similar when subjects with base-line coronary ischaemia were excluded from the analysis. There was no significant relationship between mortality and serum triglyceride in the smaller number of subjects with insulin-dependent diabetes mellitus. This prospective study demonstrates that serum triglyceride is an important predictor of mortality in patients with non-insulindependent diabetes mellitus.

,

HYPERINSULINAEMIA AS THE KEY FEATURE OF CARDIOVASCULAR AND METABOLIC SYNDROME. E. Ferrannini, S.M. Haffner, M.P. Stern, B.D. Mitchell, Natali, J.K. Patterson, and H. P. Hazuda, Department Clinical Epidemiology, San Antonio, Texas, USA and C N Institute of Clinical Physiology, Piss, Italy.

A A. of R

In a population-based survey of 2,930 subjects, prevalence rates of obesity (Ob), type 2 diabetes (D), IGT, high blood pressure (HBP), hypertriglyceridaemia (HTG = serum TG>2.9 mmol/1), and hypercholesterolaemia (HCh = serum Ch>6.5 retool/l) were 54.3, 9.3, 11.1, 9.8, 10.3, and 9.2%, respectively. The prevalence, however, of each of these disorders in its 'pure' form (=free of the other 5) was 29.0% (Ob), 1.3% (D), 1.8% (IGT), 1.5% (HBP), 1.0% (HTG), and 1.7% (HCh). Two-by-two associations were even rarer. Each 'pure' disease was characterized by byperinsulinaemia (either fasting or 2-br postglucose), suggesting insulin resistance. Furthermore, in any 'pure' disease several of the variables categorizing other members of the sextet ( e g blood pressure in 'pure' D, plasma glucose in 'pure' Ob or HTG, etc.) were significantly altered in comparison with 1,048 normal subjects. In the whole of the subjects who presented one or the other disorder (1,876 of 2,930 or 64%), marked fasting (128 v s 57 pmol/1, P<0.0001) and postglucose h y p e r i n s u l i n a e m i a (785 vs 364 pmol/l, P<0.0001) was associated with significantly higher body mass index (30.5 v s 22.8 kg/m2), waist:hip ratio (0.932 v s 0.848), fasting (5.5 v s 4.6 mmol/l) and postglucose (7.8 v s 5.0 mmol/l) glycaemia, systolic (123 v s 113 mmHg) and diastolic (74 v s 69 mm Hg) BP, serum TG (2.0 v s 1.1 retool/l) and Ch (5.3 v s 4.8 retool/l) concentrations. In conclusion, (1) insulin s e n s i t i v i t y , glucose tolerance, blood pressure, body adiposity, and serum lipids are a network of mutually interrelated functions; and (2) an insulin resistance syndrome underlies all 6 disorders carrying an increased risk of coronary artery disease, and accounts for their striking pattern of multiple overlap.

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186

IMPAIRED GLUCOSE TOLERANCE AND INCREASED RISK OF CARDIOVASCULAR DISEASE IN MAURITIANS

Genet~,c hemochromatosis and diabetes : a study in 474 hemocbxomatosis patients. J Yaouanq, J-Y Poitier, D Maugendre, P Btissot and H Allannic. Inserm U 49, Clim'que des Maladies du Foie et Clinique des Maladies Mdtaboliques. CHRU Rennes, France.

P.Z. Zimmet, G.K. Dowse, C.F. Finch, V.R. Collins, K.G.M.M. Alberti, J. Tuomilehto, D. Fareed, P. Chitson and H. Gareeboo for the Mauritius NCD Study Group, Lions-International Diabetes Institute, Melbourne, Australia. Risk factors for cardiovascular disease (CVD) were examined cross-sectionally in a random cluster sample of Indian (Hindu (n=2627) and Muslim (n=685)), Creole (n= 1351) and Chinese (n=415) subjects aged 25-74 years from the Indian Ocean nation of Mauritius. The national prevalence of both impaired glucose tolerance (IGT) and type 2 (non-insulin-dependent) diabetes is extremely high (16.6% and 11.9%, respectively) and varies little between ethnic groups. CVD risk factor levels were compared across categories of glucose tolerance within sex/ethnicity subgroups. Age-adjusted mean levels of body mass index; waist/hip ratio; fasting and 2-hour serum insulin; plasma triglycerides, total cholesterol and uric acid; and systolic and diastolic blood pressure were consistently lowest in subjects with normal glucose tolerance (NGT) while levels in subjects with IGT were intermediate to, or exceeded those associated with diabetes. Subjects with IGT were also less physically active, had lower levels of HDL-cholesterol, and had similar cigarette smoking habits to those with NGT. These results indicate that regardless of ethnic background, Mauritians with IGT have an adverse CVD risk factor profile, as reflected in an increased prevalence of electrocardiographic abnormalities. Longitudinal studies underway should confirm the relationship between IGT and CVD in Mauritians.

Diabetes, a frequent complication of genetic hemochromatosis ((3t/), reduces life expectancy ff present at diagnosis. To assess the underlying factors of diabetes in GH, we studied 474 unrelated GH patients (at least 5 g of iron removed by phlebotomy in males, 2 g in females) recruted between 1956 and 1990. At diagnosis we observed 191 diabetes (78 insulin-dependent), 63 glucose intolerance and 220 normal OGTT. Results: (1) No evidence for genetic factors was found: the HLA-A3 prevalence did not differ in all groups suggesting that all patients shared the same HLA-linked recessive gene; the DR3 or DR4 prevalence was not increased in insulin-dependent group (36.9% vs 43.7 in general population). (2) When compared with non-diabetic, diabetic patients were older (47.1+10.3 years vs 42.7+11.2 ; p<10-4), usually males (sex ratio M/H : 6.1 vs 2.5 ; p<10-3); iron overload was higher (23.4_+11.2 g vs 12.7+10 ; p<10-12), cirrhosis more frequent (59.4 % vs 12.4 ; p<10-12). In females diabetes was observed for minor degree of iron-loading than in males. (3) Since 1975 physicians awareness on GH frequency resulted in earlier detection of iron overload (25.6+11.5 vs 12.5_+9.1 g p<10 -7) and lower frequency of cirrhosis (47.7 % vs 29.6 ; p<10-3). Concurrently the frequency of diabetes decreased from 53.5% to 34.5 (p< 10-3). Conclusions: diabetes development is closely related to iron overload and should be efficiently prevented by GH large-scale screening and prophylactic phlebotomies in high prevalence areas of the hemochromatosis gene.

OP 32 Hall A-2 Metabolism in Type 2 Diabetes 187 I M P O R T A N T CONTRIBUTION OF G L U C O N E O G E N E S I S F O R INCREASED POSTPRANDIAL HEPATIC G L U C O S E O U T P U T IN TYPE 2 DIABETES. N. Nurjhan, A. Consoli, and J. Gerich. University of Pittsburgh, PA, U S A Impaired suppression of endogenous glucose output (EGO) is the major factor causing postprandial hyperglycemia in type 2 diabetes, Since increased gluconeogenesis is the process responsible for increased fasting E G O in type 2 diabetes, we undertook studies to determine whether gluconeogenesis was also responsible for the increased postprandial EGO. Eight normal volunteers (NV) and 8 matched type 2 diabetic subjects (DS) were infused with 2-3H glucose and UJ4C glycerol and were administered oral glucose enriched with 6-3H glucose, a4C glycerol incorporation into plasma glucose was used as an index of gluconeogenesis. Postabsorptive E G O and glycerol gluconeogenesis (GG) were increased 50% and 300% in DS (17.7__+0.6 and 1.13+0.16 vs 11.1+0.2 and 0.42+0.06/zmol/kg/min in NV, respectively, both p < 0.001). After glucose ingestion, E G O was twofold greater in DS (7.92+0.72 vs 3.98+0.5 umol/kg/min, p<0.002) and G G was six/old greater in DS (0.67+0.12 vs 0.13+0.02 umol/kg/min, p<0.004). Glycerol flux and its percent conversion into glucose were greater in 'DS (1.95+0.27 vs 0.98__+0.13 umol/kg/mirt, p<0.01 and 64__+11 vs 23 + 3%, p < 0.001) indicating involvement of both intrahepatie and extrahepatic mechanisms. Assuming that G G represented 15% of overall gluconeogenesis, the excess E G O in DS was completely accounted for by gluconeogenesis. CONCLUSIONS: Increased postprandial gluconeogenesis is largely responsible for the increased postprandial E G O in type 2 diabetes.

188 POSTPRANDIAL HEPATIC GLUCOSE CYCLING AND INITIAL SPLANCHNIC GLUCOSE UPTAKE IN DIABETES P.C. Butler and R.A. Rizza, Endocrine Research Unit, Mayo Clinic, Rochester, MN 55905, USA Excess glucose enters the circulation following glucose ingestion in patients with type 2 diabetes. This may be due to either increased hepatic glucose cycling (HGC) (of glucose to glucose-6-phosphate and/or glycogen), impaired initial splanchnic glucose uptake (ISGU) or excessive hepatic glucose release (HGR). We sought to determine the role of these processes by feeding a mixed meal containing both [2-3H]- and [6-~VI]glucose during a [6-14C]glucose infusion to 9 patients with type 2 diabetes, 6 with glucose intolerance and 12 controls. ISGU equals glucose ingested minus meal glucose appearance in circulation. Since [2-3H]- but not [63H]glucose is detritiated during HGC the difference in calculated HGR with these 2 meal tracers equals HGC. Postprandial HGR was increased with the severity of diabetes correlating with fasting glucose concentration (r=0.7, p < 0.01). In contrast, HGC was similar in subjects with diabetes, glucose intolerant and controls (361 + 67 vs 494 + 106 vs 322 -+ 44 ~,mol/kg/5hour). ISGU calculated with either [2-3H]glucose (17.8 + 1.4 vs 15.9 + 1.5 vs 10.7 + 1.2 g) or [6-3H]glucose (13.4 + 1.9 vs 7.9 + 2.4 vs 5.4 + 1.0 g) was increased (p < 0.01) in subjects with diabetes or glucose intolerance versus controls. In conclusion, excess postprandial glucose appearance in patients with type 2 diabetes and glucose intolerance is due to excess hepatic glucose release and not increased hepatic glucose cycling or decreased splanchnic glucose uptake.

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190

ENERGY EXPENDITURE IN NON INSULIN DEPENDENT DIABETES. A.M. Fontvieille, L. Schultz, R. Ferraro, R. Rising, and E. Ravussin. CDNS/NIDDK, Phoenix, Arizona, USA. To assess the impact of NIDDM on energy metabolism, 24hour energy expenditure (24EE), basal metabolic rate (BMR), and sleeping metabolic rate (SMR), were measured in 105 non-diabetic (70 M/35 F, 27_+7yr, 101_+25 kg, 32_+9% body fat) and in 37 diabetic Pima Indians (12 M/25 F, 33_+11 yr, 104_+32 kg, 39_+8% body fat) (mean _+SD) using a respiratory chamber, after at least 3 days on a weight maintenance diet. SMR was calculated as the mean of all 15-min periods from 11:30 p.m. to 5:00 a.m. during which spontaneous activity measured by a radar system was < 1.5%. The intra-subiect reproducibility of measurements was evaluated in 51 subjects measured twice: the intraclass correlation coefficients were 0.97, 0.89, and 0.95 for 24EE, BMR, and SMR respectively. When adjusted for differences in fat-free mass, fat mass, age, and sex, BMR and SMR were significantly higher in diabetics (80 kcal/day, p <0.01; 99 kcal/day, p <0.003 respectively) and 24EE presented a tendency to be higher in diabetics (63 kcal/day, p=0.08). Spontaneous physical activity was similar in both groups whereas the thermic effect of food, calculated as the mean of resting metabolic rate throughout the day above SMR and expressed as a percentage of energy intake, tended to be lower in NIDDM (17 vs 20%, p=0.08). We proposed that the higher BMR and SMR of Pima Indians with NIDDM may be due to the energy cost of gluconeogenesis. However, in response to food intake, NIDDM subjects seem to have a reduced thermogenesis.

CONTRIBUTION O F SKELETAL MUSCLE TO LACTATE AND ALANINE SYSTEMIC R E L E A S E IN NON DIABETIC AND TYPE II DIABETIC MEN. A. Consoli. N. Nurjhan, D. Bier and J. Gerich. Universities of Pittsburgh and St. Louis, U.S.A. Increased gluconeogenesis is the main cause of fasting hyperglycemia in type II diabetes. Although systemic appearance (SA) of lactate and alanine, key gluconeogenic precursors, is increased in type II diabetes, their sources are unknown. Previous net balance measurements suggest that muscle is a minor source; however, the net balance technique does not measure release of these precursors. Therefore, in the present study, we determined lactate and alanine SA and their forearm fractional extraction and release in 9 type II diabetic and in 9 nondiabetie subjects infused with [3-14C]Lactate and [3-13C]alanine to assess isotopically the contribution of muscle to the release of these precursors. Lactate and alanine SA were greater in diabetics (18.2 + 0.9 and 5.8 + 0.4 ~mol/Kg/min) than in nondiabetics (12.6 + 0.7 and 4.2 + 0.3, both p < 0.01). However, muscle lactate and alanine release in diabetics (6.9 + 0.7 and 3.4 + 0.3 ~mol/Kg/min) was not significantly different from that in nondiabetics (5.7 + 0.4 and 3.0 + 0.3 /~mol/Kg/min, both p=n.s.). Muscle release accounted for 40 + 4 and 45 + 3 percent of lactate SA in diabetic and nondiabetic patients, respectively (p = n.s.) and for 62 + 6 and 72 + 4 percent of alanine SA, respectively in diabetics and nondiabetics. CONCLUSIONS: i) In both diabetic and nondiabetic individuals, muscle is the major tissue supplying lactate and alanine to the systemic circulation; 2) However, tissues other than muscle are responsible for the increase SA of these substrates in type II diabetes.

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THE DIABETOGENIC EFFECTS OF GLUCOCORTICOIDSARE MORE PROtlOUNCED IN LOW THAN IN HIGH INSULIN RESPONDERS A. Wajngot, V. G r i l l , *M. Vrani~ and S. Efendie. Department of Endocrinology, Karolinska Hospital, Stockholm, Sweden and *Departments of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada. Insulin resistance may precipitate diabetes p r e f e r e n t i a l l y in low insulin responders (LIE). This hypothesis was tested by administration of dexamethasone (Dex) in six normal weight LIR and six matched subjects with high insul i n response (HIR). In a l l subjects oral glucose tolerance (OGTT), hyperalvcemic clamp (HC) and glucose turnover with 6-311-glucose was performed before and a f t e r Dex (15 mg o r a l l y during 48 hours). Dex evoked a more prominent increment in glucose area during OGTT ill LIR (control 21,5 3,2 and a f t e r Dex 39,7 ~ 3,9 mmol/I/2 hours, p
EFFICIENCY OF TRANSCAPILLARY INSULIN TRANSPORT IS A DETERMINANT OF INSULIN SENSITIVITY IN VIVO.

M. Adcr, Yd. Yang, and R.N. Bergman. University of Southern California, Department of Physiology,2025 Zonal Avenue, Los Angeles, CA, USA. We have reported a tight dynamic proportionalitybetween lymph insulin concentration (IL) (but not plasma insulin, Ip) and the rate of glucose utilization (Rd) during euglycemic clamps (JCI 84:1620, 1989). In the present study we asked whether "efficiency"of transcapillaryinsulin transport (IL/I P at steady-state) can account for variations in insulin sensitivity (SIP(clamp)). We did euglyccmieglucose clamps (n = 19) in normal dogs at 3 insulin infusion rates (3.6, 5.4, or 7.2 pmol/min/kg) and measured insulin in plasma and thoracic duct lymph. Ip rose from 108--+12to 294-+12, 468-+30, and 780+_._54pmol/L; IL was on average 29% lower at all doses (P<0.001). Rd increased with insulin dose (basal: 13.06+0.56; elevated: 32.50-+4.28, 54.06-+12.44, and 77.78__+10.39#mol/min/kg). Confirming previous results, during clamps Rd was more strongly correlated with lymph (r=0.96) than plasma insulin (r=0.78). SIP(clamp) varied from 0.90 to 3.45 x 102 ml/min/kg per pmol/L, and was correlated with efficiency of insulin transport (incremental ratio IL/Ip, r=0.52, P<0.03). CONCLUSIONS: Interstitial insulin determines glucose utilization in the physiologicrange of insulin, and efficiency of insulin transport from plasma to interstitium is partly responsible (25%) for the variance in insulin sensitivity in normal

animals. The possible role of transeaplnary insulin transport in pathological insulin resistance remains to be determined.

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ASSESSMENT OF THE METABOLIC ACTIONS OF BIOSYNTHETIC INSULIN ANALOGUES AND INSULIN ITSELF IN NORMAL MAN USING STABLE ISOT~PEC rTI~E~8.

DI-ARG INSULIN, AN INSULIN ANALOGUE: METABOLIC ACTION ON GLUCOSE AND LIPID METABOLISM IN DIABETIC PATIENTS. LD.Monti,A.Caumo,R.Poma,A.Picardi,M.Zoltobrocki P.Micossi and G.Pozza. Scientific Institute San R a f f a e l e , M i l a n o , Italy. Di-Arg(31-32) insulin (HOE 51 H) is an interesting insulin analogue since, when injected subcutaneously, it b e h a v e s k i n e t i c a l l y like a slow release insulin without the a d d i t i o n of e x t e r n a l d e p o t a g e n t s . H o w e v e r , its metabolic a c t i o n is s t i l l p o o r l y i n v e s t i g a t e d . A i m of t h e s t u d y w a s to e v a l u a t e t h e metabolic a c t i o n of d i - A r g i n s u l i n as c o m p a r e d to r e g u l a r insulin in 6 insulin treated diabetic patients. We studied i) t h e a b i l i t y of d i - A r g insulin to normalize early morning blood glucose after an o v e r n i g h t i n f u s i o n and the effects on hepatic glucose output (HGO) in postabsorptive s t a t e ; 2) t h e e f f e c t s o n HGO, peripheral glucose disposal, and lipid metabolism following an euglycaemic h!rperinsulinaemic (30 m U / k g / h ) c l a m p . Fasting blood glucose was equally normalized by comparable p l a s m a l e v e l s of d i - A r g a n d r e g u l a r insulin (15.7+5.9 vs 18.4+3.9 uU/ml, NS). Postabsorptive HGO w a s in the normal range (2.1+0.2 vs 2.1+0.1 mg/kg-min). Following clamp, HGO was equally suppressed, and M-value was not different (5.1+1.3 vs 3.5+0.7 mg/kg-min, NS). NEFA and triglycerides demonstrated s i m i l a r d e g r e e of s u p p r e s s i o n . In conclusion, as c o m p a r e d to r e g u l a r insulin, d i - A r g i n s u l i n i) is e q u a l l y a b l e to n o r m a l i z e both early morning blood glucose and postabsorptive HGO, and 2) has the same metabolic action on HGO, peripheral glucose disposal and lipid metabolism following euglycaemic clamp.

S.V.Gelding, K. Heslop, M.Rogers, V.Anyakou, D.Halliday* and D.G.Johnston. Department of Clinical Endocrinology, St. Mary's Hospital Medical School, Norfolk Place, London W2, England~*Clinical Research Centre, Harrow, Middlesex, England. The aim of the study was to compare the actions of two insulin analogues (Novo X2 and XI0) with insulin itself (Actrapid), using a stable isotope tracer technique. Three healthy non obese male volunteers, after an overnight fast, received primed dose constant intravenous infusions of the stable isotopes 6,6 2H glucose, 1,2,32H5 glycerol and I - ~ C leucine for 330 minutes, and a constant infusion of insulin or analogue (0.005units/kg/ hour) from 150 minutes. The tracers were quantified using gas chromatography - mass spectrometry. Each subject was studied with each insulin on three separate occasions. All flux results are expressed as mean (range) umol/kg/hour. After each insulin, glucose rate of appearance (Ra)fell (before Actrapid 745 (643-874), X2 841 (689-1098), XI0 669 (567-841): after Actrapid 692 (584-821), X2 785 (650-1032, XI0 617 (518-739)), but glycerol R fell more (before Actrapid 2.48 (2.09-2.99), X2 2.86a(2.07-3.81), Ki0 3.17 (2.77-3.56); after Actrapid 1.53 (1.10-2.00), X2 1.74 (1.33 -2.05), XI0 2.59 (2.03-3.14). Non-esterified fatty acids also decreased, particularly with X2.Prot~'in flux, measured as R a of alpha-ketoisocaproic acid, the deamination product of leucine, decreased after insulin, (before Actrapid 136 (131-144), X2 132 (123-143), XI0 142 (112-172); mean after Actrapid 125 (119-135), X2 109 (106-130), XI0 126 (99-154) and protein oxidation increased. Low dose infusionofinsulin and both analogues decreased glucose appearance, lipolysis and leucine flux, suggesting that modification of the insulin structure retained the major actions of insulin on carbohydrate, fat and protein metabolism.

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APPARENT INCREASE IN CORONARY HEART DISEASE WITH HUMAN PROINSULIN (HPI)--RANDOMIZATION FAILURE OR HPI EFFECT? C.T.Spradlin, J.A.Galloway and J.H.Anderson. Lilly Laboratories for Clinical Research, Indianapolis, IN USA. In February 1988 clinical trials with HPI were suspended because of an apparent increase in coronary heart disease (CHD) morbidity and mortality in one study. In this, 141 insulin-naive patients were randomized to treatment with NPH insulin or with HPI. Of 68 patients treated with HPI 2 (2.9%) had fatal and 4 (5.9%) had non-fatal myocardial infarctions (MI's) with none in the 73-patient control group (p = 0.018). All events occurred after one year or more (19.5 +/- 4.6 months) of HPI treatment. The mean pre-randomization percentile ranks of the six patients who had MI's among the other 135 persons in the study were--age: 76.7; diabetes duration: 80.3; body weight: 67.8; systolic BP: 80.3; diastolic BP: 67; total serum cholesterol: 74.7; LDL cholesterol: 75.5; triglyceride: 60.7 and HDL cholesterol: 51.0. (Other clinical details will be presented.) The overall frequency of CHD for all HPI studies combined was 2.2% of 224 patients on HPI and 1.3% of 231 patients on NPH (p = 0.449). Extrapolation of pharmacokinetic data generated in normal volunteers indicated that chronic treatment with HPI produces molar serum hormone concentrations which are 100-150 times normal. The experience with HPI raises important questions concerning the clinical evaluation of insulin analogues.

ABSORPTION OF S O L U B L E I N S U L I N A N D N P H - I N S U L I N F R O M D I F F E R E N T I N J E C T I O N SITES. J.E.Henriksen, A.Vaag, I.Ramsgaard Hansen, M.Lauritzen, H.Beck-Nielsen. Hvid~re Hospital, Klampenborg and Odense University Hospital, Denmark. T h e a i m w a s to i n v e s t i g a t e the absorption of s o l u b l e i n s u l i n d u r i n g o r d i n a r y d a i l y l i f e a n d of N P H - i n s u l i n d u r i n g r e s t ( m i m i c i n g t h e n i g h t ), w h e n i n j e c t i o n is p e r f o r m e d s u b c u t a n e o u s l y (SC) i n t o t h e t h i g h a n d into t h e a b d o m e n a n d i n t r a m u s c u l a r l y (IM) into t h e thigh. T h e t h i c k n e s s of t h e subcutaneous layer was measured using an ultrason a r s c a n n e r . A b s o r p t i o n w a s m e a s u r e d as d i s a p p e a r a n c e of 12SI-labeled i n s u l i n . S o l u b l e i n s u l i n injected SC into t h e a b d o m e n a n d I M i n t o t h e thigh was absorbed faster than soluble insulin i n j e c t e d SC into t h e thigh, T ~ ( time until 50 % of t h e i n j e c t e d i n s u l i n is a b s o r b e d ) b e i n g 156, 113 a n d 289 min. r e s p e c t i v e l y ( p<0.05, N = 6 ). N P H - i n s u l i n injected IM was absorbed faster than NPH-insulin injected SC i n t o t h e thigh , T~ b e i n g 8.0 a n d 10.3 h o u r s ( p < 0.05, N = ii ). T ~ for N P H - i n s u l i n i n j e c t e d i n t o t h e a b d o m e n w a s 9.6 h o u r s ( NS ). T h e i n t e r p a t i e n t coefficients of v a r i a t i o n was lower for NPHi n s u l i n i n j e c t e d SC into t h e t h i g h c o m p a r e d t o NPH-insulin i n j e c t e d SC i n t o t h e a b d o m e n , 20 % vs. 40 %, ( p < 0.05 ). T h e m o s t c o n s t a n t a b s o r p tionrate of N P H - i n s u l i n w a s o b t a i n e d a f t e r SC i n j e c t i o n into t h e thigh. W e c o n c l u d e , t h a t t h e i n j e c t i o n s i t e of c h o i c e for s o l u b l e i n s u l i n m u s t b e SC i n t o a b d o m e n or I M into t h e thigh, a n d f o r N P H - i n s u l i n SC i n j e c t i o n into t h e t h i g h .

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ODD PHENOMENA IN INSULIN ABSORPTION EXPLAINED BY INSULIN PHYSICO-CHEMICAL CHARACTERISTICS J.Brange, A.V~lund, S.Kang and D.R.Owens. Novo Research Institute~ DK-2880 Bagsv~rd, Denmark and Dept.of Med. U.W.C.M. University of Wales, UK. Monomeric and dimeric human insulin analogues were used to elucidate unexplained phenomena of subcutaneous absorption of soluble insulin, e.g. lag phase, effect of concentration, dose, etc. Seven normal subjects received, on separate days, s.c. injections (0.6 nmol/kg) of neutral soluble (0.6mM~UI00) , 125-I-labelled, human insulin, monomeric AspB9+GIuB27 and dimeric AspBl0 insulin analogues. Disappearance from the abdomen was followed for 8 hours by external gamma counting. The monomeric insulin had no lag phase and followed a monoexponential course throughout the absorption process. In contrast, two phases in rate of absorption were identified for the dimer and three phases for the normal hexameric human insulin. The mean disappearance rates (percent/h) calculated by log-linear regression analysis were: monomer 66, dimer 37 (0-2h) and 63 (>2h) and hexameric insulin 20 (0-2h), 36 (2-4h) and 61 (>4h) . The lag phase and the subsequent accelerated absorption of soluble insulin can now be explained by the associated state of native insulin in pharmaceutical for~lulation and the progressive dissociation into smaller units during the absorption process. The effects of insulin concentration, injected volume, temperature and massage on the absorption process are now also easily understood in the light of these results.

PHARMACOKINETICS OF INTRANASALLY A D M I N I S T E R E D INSULIN WITH PHOSPHOLIPID A S A B S O R P T I O N ENHANCER K Drejer*, A Vaag**, K Bech**, P E Hansen*, A R S~rensen* and N Mygind***. *Novo Research Institute, DK-2880 Bagsverd, **Hvid~re Hospital, DK-2930 Klampenborg, ***Rigshospitalet, DK-2100 Copenhagen. We assessed the pharmacokinetics of intranasal insulin with a medium chain phospolipid (Didecanoyl-L-alpha-phosphatidylcholine) as absorption enhancer by measuring plasma glucose, insulin, glucagon and C-peptide. Fasting normal volunteers (N=II) received 4U insulin intravenously, 6U insulin subcutaneously and 3 doses intranasally (0.3U/kg, 0.6U/kg, 0.8 U/kg). Intranasal insulin was absorbed in a dosedependent manner. Plasma insulin peaked 23• min after spraying and blood glucose reached a nadir after 46• min, which is a 20 min delay compared with intravenous injection. Compared with subcutaneous injection intranasal administration of insulin had the advantage of a quicker onset of action and, especially, a much more uniform time course of absorption. Bioavailability was 8.3% and 11.5% when compared to intravenously and subcutaneously administered insulin, respectively. When compared with subcutaneous insulin in the meal-relevant period (0-120 min) the bioavailability was 24.0%. Nasal irritation was slight and proportional to the insulin dosage. We conclude that intranasal insulin with phospholipid as enhancer has potential as a meal-related insulin adjunct both in Type I and in mild Type II diabetes. Further evaluation of reproducibility and long-term safety in diabetic patients need to be studied.

OP 34 Hall A-15 NOD Mice 199

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PREVENTION OF DIABETES IN NOD MICE BY NEONATAL GLUCOSE TREATMENT T. Bock, T.W. Kjaer, M. Jcrgensen, K.E. Josefsen and K. Buschard. Bartholin Institute, Kommunehospitalet, 1399 Copenhagen K, Denmark. The aim of this study was to investigate, whether neonatal glucose treatment influenced the incidence of diabetes in NOD mice. Neonatal NOD mice were injected subcutaneously with 50 ul 1.0 M glucose twice a day for 6 days. A group of untreated NOD mice served as control group. Animals were inspected daily and examined for glucosuria every 5 days. If + + glueosuria was detected, the animals were killed and blood glucose measured. The study was stopped at day 280. In the glucose treated group (n=39; 19 males, 20 females) 33% became diabetic compared with 58% in the untreated group (n=36; 20 males, 16 females). Kaplan-Meier estimation was used in calculation of cumulative diabetes incidence, the logrank test in significance evaluation. Mice receiving neonatal glucose treatment had a significantly lower diabetes incidence than the untreated group (x2=5.30, p<0.05). Among treated animals 50% of the females and 16% of the males became diabetic, compared with 69% and 50% in the control group. We conclude that neonatal glucose treatment prevents development of diabetes in NOD mice.

INSULIN TREATMENT PREVENTS T CELL TRANSFER IN THE AUTOIMMUNE NOD MOUSE C.H. Thivolet, E. Goillot, and P. Bedossa, INSERM U197 and INSERM US0 Lyon France, and Hospital A. B6clere, Paris France. Insulin has been shown to prevent diabetes onset in both BB rats and NOD mice, two experimental animal models where effector T cells cause beta ceil destruction. We studied the effects of insulin administration upon the ability of committed T ceils to transfer the disease in adult syngeneic recipients. Irradiated NOD males were i.v. injected with 107 spleen cells from diabetic females. A group of 20 male recipients were sub-cutaneously injected with 0.5 unit of rapid insulin twice daily during one month. Animals were monitered for glycosuria and compared with 18 male controls injected with the same amount of the insulin diluent. During the time of the study, none of the insulin treated animals developed diabetes whereas 15118 controls did. Histologic examination of pancreata from the insulin treated animals revealed 63 % normal islets, 22% with peri-insulitis, i5 % with insulitis and no atrophy, whereas in the control group only 13.6% of the islets were normal, 19.1% had perinsulitis, 29.7% had insulitis and 37.5 were atrophied. Mechanisms of this protection i.e. modulation of islet antigenic content or action on effector T cells are under investigation. These effects may have therapeutic implications in prediabetic patients.

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A NEW MARKER FOR THE DETECTION OF PANCREATIC INSULITIS IN THE NON-OBESE-DIABETIC MOUSE. A. Signore, M. Chianelli, E. Procaccini, G. Ronga, E.A.M. Gale, P. Pozzilli. Servizio Medicina Nucleare and Endocrinologia I, Clinica Medica II, Policlinico Umberto I, Rome, Italy; Dept of Diabetes and Metabolism, St. Bartholomew's Hospital, London, UK. We have previously shown in BB/W rats that the intravenous administration of 1231-labelled interleukin-2 allows the visualization of insulitis by gamma-camera imaging. We have now applied this technique to the NOD mouse. 1231-1L2 (100150 p.Ci) was injected intravenously in 65 NOD mice for in vivo gamma-camera imaging or for single organ counting at various times after injection (2, 5, 10, 20, 40, 60 and 90 minutes). Balb/c mice injected with 1231-1L2 and NOD mice with 1231-1actaibumin were used as controls. Results in NOD mice showed that IL2 binds to activated lymphocytes in vivo and rapidly accumulates in pancreas, liver and kidneys. After 15 minutes radioactivity declines in liver and kidneys but remains high in infiltrated panoreata. A positive correlation was found between counts in the pancreatic region and degree of infiltration calculated histologically (p<0.003). Pancreas radioactivity was also significantly higher in NOD injected with IL2 as compared to those injected with lactalbumin (3.87_+0.04 vs 0.75_+0.15, % of injected dose/gr of tissue, 5min after injection, p<0.001; 2.57_+0.15 vs 1.05_+0.22, 20min after injection, p<0.003). Autoradiography confirmed the binding of IL2 to infiltrating lymphocyles in pancreas. These results show that by injecting labelled cytokines, it is possible to image pancreatic insulitis in a prediabetic stage in the NOD mouse.

ROLE OF CD4 T CELLS B E A R I N G R E C E P T O R S ENCODED BY V~8 GENE SEGMENTS IN THE I M M U N O P A T H O G E N E S I S OF THE NOD MOUSE. B.F6rmby, N . M i l l e r and M . A . C h a r l e s * S a n s u m Medical R e s e a r c h Foundation, Santa B a r b a r a and *University of California, Irvine, USA. The NOD mouse develops type i diabetes secondary to ;~-cell d e s t r u c t i o n by i n f i l t r a t i n g lymphoid cells (autoimmune insulitis). We isolated the inf i l t r a t i n g lymphoid cells in the lesion of insulitis from 10-12 wk old female NOD mice and exam i n e d by c y t o f l u o r i m e t r i c analysis the T cell phenotypes. 76% were Thy 1.2+, 44% were CD4+, 5% were CD8+, 31% were Thy 1.2+CD4-CD8-. 34% of the CD4+ T cells were b e a r i n g V~8 gene segments. This p h e n o t y p e was o l i g o c l o n e d (>95%) from a T cell e n r i c h e d fraction isolated from diabetic NOD mouse spleens using i m m o b i l i z e d a n t i - F 2 3 . 1 mAb. The s t i m u l a t o r y indices of the p r o l i f e r a t i v e response of the o l i g o c l o n e d C D 4 + F 2 3 + T cells were: 24-+4 (n=3) to class II MHC e x p r e s s i n g s i n g l e - c e l l islet cells isolated from young male NOD mouse islets, 2.8+0.9 (n=6) to i r r a d i a t e d syngeneic control splenocytes, 1.3-+0.9 (n=4) to i r r a d i a t e d a l l o g e n e i c control splenocytes. C D 4 + F 2 3 + T cells were s t i m u l a t e d by p u r i f i e d p r o t e i n d e r i v a t i v e (PPD) of M y c o b a c t e r i u m t u b e r c u l o s i s (a major antigenic c o m p o n e n t is d e r i v e d from hsp65). A CD4+ F23+ T cell line was g e n e r a t e d by r e p e a t e d l y stim u l a t i n g w i t h PPD and clones were isolated by lim i t i n g d i l u t i o n of the line cells. W h e n a d m i n i s t rated i.p. into 4 wk old female NOD mice monthly, 0/i0 d e v e l o p e d diabetes at the age of 24 wk. 7/14 controls d e v e l o p e d diabetes. We conclude that aut o r e a c t i v e self MHC r e s t r i c t e d cloned CD4+F23+ T cells can m o b i l i z e r e g u l a t o r y m e c h a n i s m s e.g. an a n t i - i d i o t y p i c n e t w o r k capable of s u p p r e s s i n g s p o n t a n e o u s a u t o i m m u n e diabetes.

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MI{C CLASS I Kd-RESTEICTED CYTOTOXIC T-LYMPHOCYTES IN BETA CELL DESTRUCTION OF NON-OBESE DIABETIC ~IICE T.Taki, K.Yokono, Y.Hirao, A.Ando, N.Hatamori, M.Hayakawa, K. Suenaga and S.Baba. The 2nd Dep. of Internal Medicine, Kobe University School of Medicine, Kobe, Japan.

QUANTITATIVE AND FUNCTIONAL ANALYSES OF IN SITU ISLET I N F L A M M A T O R Y CELLS IN T H E N O D M O U S E A N D BB RAT. N. Hosszufalusi, B. Formby, H. Toyoda, E. Chan, N. Miller, M. Teruya, M.A. Charles, Irvine, Santa Barbara, a n d Los Angeles, California, U S A Inflammatory cells invading islets are thought to be m e d i a t o r s of islet destruction. H i s t o p a t h o l o g i c a l studies identify i n v a d i n g cell types, b u t are not q u a n t i t a t i v e or functional. Thus we examined prediabetic NOD and BB inflamed islets and diabetic N O D spleen cells using dispersed single cell preparations. C~11~ in NOD islets and spleen were phenotyped using Thy 1.2, CD4, CD8 and F23.1 antibodies and FACS. Thy 1.2+ CD4+ and C D 8 + cells were 63 and 25% of total in situ islet mononuclear cells and 25% of T cells were CD4CD8- (n=5). 16% of diabetic N O D spleen T cells were CD8- CD4- and 50% of C D 4 + T cells w e r e V ~ 8 + (n=5) compared to 8% V~8+ in C57BI controls (p<.01). Analysis of V@ gene expression (mRNA levels) in circulating leukocytes and in inflamed islets revealed transcripts for 6 of i0 V~ genes examined. Islet effector cells were purified using percoll and cytolytic assays used 51Cr labeled islet targets. N O D cell-mediated cytolysis (CMC) was 32--+3.7% (n=5) using a 20:1 Effector:Target (E:%] ratio; NOD spleen cells did not mediate cytolysis at 20:l(p<.001). At E:T ratios of i00:i, N O D (n=5) spleen cells had similar killing. In the BB rat, C M C was 62--+8.3% (n=5) at E:T i0:i. Spleen cells showed 10% killing at 20:l(p<.001). These data show i) in situ, spontaneously resident, unmodified islet a n d spleen cells have a significant presence of "double negative" T cells, 2) splenic and islet T cell s u b s e t s s h o w oligoclonality and differential expression of V~8 T cell receptors, and 3) islet killing by in situ islet immune cells in the NOD and BB models is very strong compared to s p l e n o c y t e killing.

We have previously reported that Lyt2 + cytotoxic Tlymphocytes (CTL) from non-obese diabetic (NOD) mice pancreas could destroy islet cells in NHC class I K drestricted manner in vitro. To clarify the role of this CTL in vivo, prevention of diabetes in NOD mice (H-2 K d, Db) was examined by administration of monoclonal antibodies (MoAb) to MHC class I molecules. Anti-Kd MoAb could attenuate the development of diabetes both in cyclophosphamide (CY)-treated male (3/38, 7.9%) and nontreated female NOD mice (0/9, 0%), whereas anti-D b MoAb (12/27, 44.4%) and anti-K b MoAb (13/22, 59.0%) could not. Immunohistochemical studies showed that islet-infiltrating cells in control mice were composed mainly of L3T4 + and Lyt2 + T-lymphocytes, while predominant L3T4 + and very few t2+ T-lymphocytes infiltrated within the islets in anti~ MoAb-treated mice. Furthermore, the administration of anti-Lyt2 MoAb could also diminish the development of diabetes in CY-treated NOD mice (2/13, 15.4%). These results suggest that MI{C class I Kd-restricted Lyt2 (CD8) positive CTL play an important role as direct effector cells in the destruction of beta cells in NOD mice.

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OP 35 Hall B-3 Cytosolic Calcium in B-cells 205

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P~OTEIN KINASE C AFFECTS BOTH GIiKX~E- AND ~ - M E DIATED ~ IN CYTOPLA._qvlICFREE CA 2+ IN MOUSE ~-~T.T~ P-O. Bemggren, L. Juntti-Berggren and A. SjOholm, Deparl]nents of Hbdocrinology, Karolinska Institute, Stockholm and Medical Cell Biology, i~miversity of Upp-

GLUCOSE INDUCES FAST OSCILLATIONS OF CYTOPLASMIC Ca 2+ IN SINGLE PANCREATIC B-CELLS

sala, Uppsala, Sweden

B. Hellman, E. Gylfe and E Grapengiesser. Department of Medical Cell Biology, University of Uppsala, Uppsala, Sweden

P r o t e i n kinase C (PKC) a c t i v a t i o n w i t h ~ phorbol ester TPA lowers the cytoplasmic free Ca2+ ccncentration ([Ca2+]i), by stimulating Ca 2+ efflux from the oell. Under similar ~ t i o n s thex~ is an ~ inward Ca 2+ current and a marked stimulation of insulin release. Glucose increases the activity of PKC but down-regulation of the enzyme^does not abolish gluoose-induoed either increase in [CaZ+]_, or insulin release. The aim of the present study was ~o further elucidate the role of pKC in the ~ u l i n secretory prooess. ~-cglls were obtained from obese hyperg!ycaemic mice. [Caz+] . was measured with fura-2 and inositol phosphates w ~ e separated by ion exchange ~ t o g r a ~ h y . Acute addition of i0 nmol/l TPA suppresuqed the carbamylcholine (Cch)-induoed (I00 ~nol/l) increase in [Caz+] : and inh/bited the production of inositol trispbosphat~ (Inspq). Subsequent to d o w n - ~ a t i o n of PKC, TPA was without-effect cn the Cch-induoed increase in [CaZ+]i. In P~C down-regulated cells, the Cch-induoed production of InsP~ and increase in [Ca2+]i were more acoentuated and the ~luoose-/_nduced increase in [Ca2+] . was slower and less prcr~mr~ed. The K+-induoed ~ in [CaZ+L was unaffected under t~_re conditions. It is ~ t e d that PKC indeed has a multifarious modulatory role in the insulin secretory process.

Glucose is known to induce slow rhy+thmic variations of the cytoplasmic Ca 2+ c o n c e n t r a t i o n (Ca 2 i) with a frequency of 0.2-0.5/rain in single pancreatic 13-cells. However, it has so far not been reported that such cells exhibit faster oscillations of Ca2+i corresponding to the electrical burst activity in intact islets. By monitoring Ca2+i continuously with dual wavelength fluorometry in fura-2 loaded mouse ~-cells we have now tested whether c~clic AIvIP may be a factor of importance for the type of CaL+i oscillations obtained in response to glucose. Characteristic slow oscillations with large amplitudes were observed at 8-20 mmol/1 glucose in single ~-cells cultured for 1-4 days. On addition of 1-100 nmo]/1 glucagon irregular transients and small amplitude oscillations with a frequency of 2-4/rain were superimposed on the slow Ca2+i waves. The effect of glucagon was mimicked by tile addition of 1 mmol/] of the dibutyryl or 8-bromo derivatives of cyclic AMP. When measuring Ca2+i only 2-4 hrs after [~-cell isolation, the fast oscillations were frequently observed also in the absence of agents increasing cyclic AMP. It is concluded that single ~-cells have the ability to respond to glucose both with slow and fast oscillations particularly when measures are taken to maintain their cyclic AMP content.

207

208

CALCIUM OSCILLATIONS IN ISOI2~TED RAT PANCREATIC ISLETS. B.E. Corkey, J.T. D e e n e y , Edk. Longo, Bdk. V a r n u m a n d K. T o r n h e i m , B o s t o n U n i v . S c h o o l of M e d i c i n e , B o s t o n , MA I n s u l i n s e c r e t i o n is p u l s a t i l e /n vivo, in t h e p e r f u s e d p a n c r e a s a n d in i s o l a t e d islets. Our p r e v i o u s findings h a v e led to a p r o p o s e d m e t a b o l i c m o d e l of g l u c o s e - i n d u c e d i n s u l i n s e c r e U o n involving A T P / A D P driven alt e r a t i o n s in electrical a n d m e t a b o l i c e v e n t s , r e s u l t i n g in Ca 2+ o s c i l l a t i o n s a n d i n s u l i n release. To t e s t t h e m o d e l w e u s e d fura-2 to investigate cytosolic free Ca 2+ r e s p o n s e s in single i s l e t s or g r o u p s of p e r i f u s e d islets. Ca 2+ o s c i l l a t i o n s w i t h a n average period of 5 m i n were s e e n at b o t h s t i m u l a t o r y ( 1 0 - 2 0 raM) a n d n o n - s t i m u l a tory (3 mM) g l u c o s e c o n c e n t r a t i o n s . Digital i m a g i n g of a large s e r i e s of i n d i v i d u a l i s l e t s i n d i c a t e d t h a t inc r e a s i n g g l u c o s e from 3 to 10 mM c a u s e d a n i n c r e a s e of a b o u t 2 0 0 n M in the a v e r a g e c y t o s o l i c free Ca 2+ v a l u e a n d a fivefold i n c r e a s e in t h e a m p l i t u d e of the o s c i U a t i o n s , w i t h n o c h a n g e in period. T h e i n c r e a s e in c y t o s o l i c free Ca 2+ a p p e a r e d to r e s u l t from recruitm e n t of ceils rather t h a n i n c r e m e n t a l Ca 2+ i n c r e a s e s in all ceils w i t h i n the islet, A g l u c o s e i n d u c e d rise in the average A T P / A D P ratio a n d oscillatory i n c r e a s e s in oxygen consumption and insulin secretion were also confirmed. T h e s e d a t a are c o n s i s t e n t w i t h a m e t a b o lic m o d e l of Ca 2+ oscillations.

SYNCHRONOUS INTRACELLULAR CALCIUM OSCILLATIONS D U E TO BURSTING ELECTRICAL ACTIVITY IN S I N G L E I S L E T S OF L A N G E R H A N S R.M.Santos, L.M.Rosario, D.Contreras, A.Nadal, J.Garcia-Sancho*, B.Soria and M. V a l d e o l m i l l o s . Department of Physiology, University of Alicante and *Department of Physiology and B i o c h e m i s t r y , U n i v e r s i t y of V a l l a d o l l i d , Spain. Glucose (llmM) induces an oscillatory electrical activity which is paralleled by oscillations in intracellular Ca 2§ concentration ( [ C a 2 * ] i ) z'2. T h e a i m s of this work were: (i) to establish the possible relationship between these oscillations (by s i m u l t a n e o u s l y r e c o r d i n g m e m b r a n e p o t e n t i a l and w h o l e islet Indo-i f l u o r e s c e n c e ) ; and (ii) to d e t e r m i n e t h e s p a t i a l o r i g i n of t h e Ca = § s i g n a l within the islet (by Fura-2 fluorescence imaging). We found that [Ca2§ oscillations occurred synchronously with the "bursts" of electrical activity. Raising glucose concentration from 11 t o 17mM i n c r e a s e d t h e d u r a t i o n o f t h e s e o s c i l l a t i o n s by a p p r o x i m a t e l y t h e same e x t e n t ( c . 60%). F u r t h e r m o r e , in t h e presence of the K+-channel blocker t e t r a e t h y l a m o n i u m (20mM), s h o r t - l i v e d [Ca2§ transients occurring simultaneously with large action potentials were r e c o r d e d . Thus, the [Ca2*]~ o s c i l l a t i o n s were l i k e l y to originate from p u l s a t i l e Ca2 " influx due to bursting electrical activity. Moreover, the calcium imaging experiments showed that [Ca=§ o s c i l l a t i o n s occurred synchronously throughout the i s l e t . This h i g h l y organized o s c i l l a t o r y calcium signalling system may subserve t h e islet b e h a v i o u r as a secretory "syncythium'. (1) V a l d e o l m i l l o s e t a l . (1989) FEBS L e t t . 259, 19; (2) R o s a r i o e t a l . (1990) Biophys. J. 57, 306a.

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L O C A L I Z A T I O N OF S E C R E T A G O G U E I N D U C E D C H A N G E S IN C'~TOSOLIC C A L C I U M IN S I N G L E RINmSF CELLS.

PROPAGATION OF Ca 2+ OSCILLATIONS IN CLUSTERS OF 8-CELLS STUDIED WITH A DIGITAL IMAGING TECHNIQUE

J.-M. Theler, P. Vacher*, C. Bartley, W. Schlegel* and C. B. Wollheim. Div. de Biochimie Clinique and Fondation pour Recherches M6dicales*, Dpt. of Medicine, University of Geneva, Switzerland

E. Gylfe, E. Grapengiesser and B. Hellman. Department of Medical Cell Biology, Uppsala University, Sweden

To define the role of calcium in insulin secretion, spatial and temporal changes in cytosolic calcium ([Ca2 + ]i) were monitored in single RINm5F cells. [Ca2 + ]i was measured in fura-2 loaded cells by real-time image processing (25 frames/sec). Nuclei in uustimulated ceils displayed lower [Ca2 + ]i than the eytosol, in the cytoplasm, areas of apparently elevated [Ca2 + ]i (hot-spots) were observed. Spontaneous oscillations occured occasionally. The initial rise in [Ca2 + ]i in response to carbachol was fast (0.5 to I sec) and localized in well defined areas close to the plasma membrane and subsequently spread throughout the cell. The rise due to depolarization with high K + (0.5 to 1 sec) was distributed along the entire surface of the cell. In response to vasopressin and ATP the rise in [Ca2+]iwas slower (1 to 10 secs). The fuel secretagogue D-glyceraldehyde caused a slower onset (0.5 to 2 min) with an initial disseminated phase of [Ca2 + ]i rising to about 250 nM followed by a generalized increase reaching mieromolar levels. Oscillations were encountered after all types of stimuli. Each cell exhibited a characteristic latency and each stimulus elicited given response patterns. This approach allows to delineate the role of localized [Ca2 + ]i changes in secretagogue induced exocytosis.

Based on electrophysiological data it has been proposed that the pancreatic islets are equipped with particular pacemaker cells. Digital image analysis combined with sensitive dual-wavelength microfluorometry has now been used to study whether cytoplasmic Ca 2+ ([Ca2+]i) oscillations originate from such B-cells in clusters loaded with the Ca 2+ indicator fura-2. Single mouse B-cells exposed to 8-20 mmol/l glucose exhibited large amplitude oscillations of [Ca2+]i. In addition to the previously reported frequency range (0.2-0.5/rain), even slower responders with one [Ca2+]i peak per 10-15 rain were identified. In small clusters of 6-]4 cells the average frequency was 0.6/min. The clusters were found to contain micro-domains of electrically coupled cells with synchronized oscillations. During glucose stimulation adjacent domains became functionally coupled. The oscillations originated from different cells in the cluster. The results indicate that coupling of B-cells tends to increase the frequency of the glucose-induced oscillations, and that the oscillatory characteristics are determined collectively rather than by particular pacemaker cells.

OP 36 Hall A-18 Experimental Nephropathy 212

211 ABNORMAL P O L Y ~ 4 I N E M E T A B O L I S ~

IN

THE GLOMERULUS

OF THE D I A B E T I C RATS K . U t s u n o m i y a , N . Y o s h i z a w a , H. Kurata, K . N o m u r a and Y.Ikeda, Tokyo, Japan G l o m e r u l a r h y p e r t r o p h y occurs as the first s t r u c t u r a l change in d e v e l o p m e n t of d i a b e t i c nephropathy. It is r e p o r t e d that e x o g e n o u s a d m i n i s t r a t i o n of polyamine, the e s s e n t i a l substance for cell p r o l i f e r a t i o n , causes the similar g l o m a r u l a r h y p e r t r o p h y to the d i a b e t i c k i d n e y in normal rats. In order to assess the p o s s i b i l i t y that a b n o r m a l p o l y a m i n e m e t a b o l i s m m a y p a r t i c i p a t e in d e v e l o p m e n t of d i a b e t i c nephripathy, we e x a m i n e d the r e l a t i o n s h i p b e t w e e n g l o m e r u l a r p o l y a m i n e synthesis and g l o m e r u l a r h y p e r t r o p h y in s t r e p t o z o t o c i n ( S T Z ) d i a b e t i c rats. Male W i s t a r rats, w e i g h i n g 200g, were m a d e d i a b e t i c w i t h STZ 40mg/kg, After 3 months, the g l o m e r u l i w e r e isolated f r o m normal (n=5) and diabetic(n=5) rats by the stepwise s i e v i n g method. G l o m e r u l a r p o l y a m i n e content was m e a s u r e d by HPLC. The r e m n a n t k i d n e y was fixed for PAM staining. The m e a n g l o m e r u l a r volume' was c a l c u l a t e d in the light m i c r o s c o p i c p i c t u r e s u s i n g a c o m p u t e r i z e d planimeter. All polyamine, e s p e c i a l l y p u t r e s c i n e ( 1 . 2 3 + 0 . 7 2 vs 0.26• pmol/mg, p<0.01) and spermine(5.50+1.15 vs 3,16+0.14 pmol/mg, p<0.01), was s i g n i f i c a n t l y i n c r e a s e d in d i a b e t i c rats c o m p a r e d w i t h normal rats. The m e a n g l o m e r u l a r v o l u m e was also s i g n i f i c a n t l y larger in d i a b e t i c rats than normal rats(1.270• vs 0 . 9 5 + 0 . 0 7 u m 3, p<0.01). These data s u g g e s t the p o s s i b l i t y that there is the profound relationship between accelerated g l o m e r u l a r p o l y a m i n e synthesis and g l o m e r u l a r h y p e r t r o p h y in d i a b e t i c rats.

SODIUM RESTRICTION PREVENTS HYPERFILTRATION AND RENAL HYPERTROPHY IN STREPTOZOTOCIN DIABETES. T.L Alien, M.E. Cooper and G. Jemms. Endocrine Unit, Department of Medicine, Austin Hospital, Heidelberg, Auslralia. This study has evaluated the effects of dietary sodium restriction on the renin-angiotensin system and glomerular filtration rate (GFR) in streptozotocin diabetes. Diabetic and control male Sprague-Dawley rats weighing 225-275 g received either a low sodium (LS) [0.005%] or normal sodium (NS) [0.4%] diet.

Four weeks later, GFR, right

kidney weight (KW) and plasma angiotensin II (AII) were determined. Diabetes was associated with a 48% increase in GFR, a 45% increase in KW and 76% reduction in AII when compared with controls (p<0.01). Sodium restriction in diabetic rats reduced GFR (LS, n=10, 3.5 + 0.2 [mean + SEM] vs NS, n = l l , 4.6 + 0.3 ml/min, p<0.01), restored plasma A n to conlrol values (LS, 153 [geometric mean] vs NS 37.3 pg/ml, p<0.01) and retarded kidney growth (LS, 1.20 + 0.02 vs NS, 1.59 + 0.08 g, p<0.01). GFR correlated negatively with plasma AII (r=-0.52, p--0.0009) and positively with KW (r=-0.66, p=0.0001). The reduction in GFR associated with sodium restriction may be secondary to altered intraglomerular haemodynamics mediated by increased AII or due to a decreased renotropic effect.

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214

SIX MONTHS SANDOSTATIN~ TREATMENT IN DIABETIC RATS: EFFECTS ON KIDNEY GRO~I~ AND URINARY ALBUMIN EXCRETION.

Renal Hyperperfusion in 30% Galactose-Fed Rats Is Prevented by Two Structurally Distinct Aldose Reductase Inhibitors

A . Flyvbjerg, S.M. Marshall, J. F r y s t y k , K.W. Hansen, R.

~ s t e r b y , A.G. Harris and H. ~ r s k o v . Medical Department M (Diabetes & Endocrinology), Aarhus Kommunehospital and Institute of Experimental Clinical Research, University of Aarhus, DK-8000 Aarhus C, Denmark; Department of Neuroendocrinology, Clinical Research, Sandoz, Basle, Switzerland. The initial renal h y p e r t r o p h y in experimental diabetes is p r e v e n t e d b y administration of a somatostatin analogue (Sandostatin| To evaluate the long-term effects of Sandostatin| streptozotocin-diabetic rats and non-diabetic controls were treated with two daily subcutaneous injections for 6 months. Untreated diabetic and non-diabetic animals were used as reference groups. Body weight, blood glucose output, u r i n a r y glucose, u r i n a r y albumin excretion (UAE) and fodder intake were measured monthly. At the termination of the s t u d y (after 6 months) occurrence of cataracts was r e g i s t e r e d and the kidneys were weighed. There were no differences in body weight, fodder intake, blood or urine glucose levels between the two diabetic g r o u p s , but cataracts score (55 vs. 86%, p<0.05), kidney size (896 vs. 1000 mg, p<0.025) and UAE (417 vs. 1098 ~tg/24h, p<0.02) were all reduced in the diabetic group receiving Sandestatin| when compared to u n t r e a t e d diabetic group. In non-diabetic animals Sandostatin| reduced body weight (274 vs. 293 g, p<0.01), kidney g'2owth (695 vs. 764 rag, p<0.01) and UAE (83 vs. 364 ~g/24h, p<0.02) compared to u n t r e a t e d controls. Thus, UAE in Sandostatin-treated diabetic animals was identical to that of untreated nondiabetic controls. These results show that long-term Sandostatin| treatment in experimental diabetes reduces certain functional abnormalities r e g a r d e d to be prognostic of late diabetic complications, without interference with metabolic control.

215 REDUCED PROGRESSIONOF ALBUMINURIAIN DIABETIC RATSBYTHEALDOSEREDUCTASEINH~ITOR,TOLRESTAT. M.L. MCCALEBand M.L. MCKEAN,Princeton,NJ, USA. The progressive increase in urinary albumin excretion (UAE), which precedes the development of diabetic nephropathy, can be prevented in diabetic (DM) rats if the aldose reductase inhibitor (ARI), tolrestat, is administered at the initiation and throughout the duration o f hyperglycemia. The efficacy of an ARI in the clinic, however, will be assessed after patients exhibit a symptom of DM nephropathy, such as albuminuria. We therefore determined the ability of tolrestat to intervene in the further progression of already established UAE of streptozocin (STZ)-diabetic female Wistar rats. Two months after STZ injection, DM rats were ~ o u p e d as Low-UAE (0.2-1.0 m g a l b u m i n / d a y ) or H i g h - U A E ( 1 . 9 - 5.9 mg albumin/day), then intervention with tolrestat (25 mg/kg per day) was begun for half DM rats in each UAE greup. After six months of treatment, reductions of UAE by tolrestat were observed in both groups [Mean + SEM of mg/day: non-DM, 14 + 7; DM Low-UAE, 32 _+ 8 vs 15 + 4* ( - 5 3 % ) with tolrestat; D M High-UAE, 103 _+ 19 vs 66 _+ 11 (-36%) with tolrestat], which when adjusted for baseline levels, was significant (p<0.05) in the L o w - U A E group only. DM-indtmed rise of urinary protein of both groups was also significantly (p<0.05) reduced by tolrestat. In c o n c l u s i o n , interventional therapy with the ARI, tolrestat, can reduce the progression of established UAE in long-term diabetic rats.

P.J. dates, C.A. EIlery, and L.R. Pustilnik. Dept. Metabolic Diseases, Pfizer Central Research, Groton, Connecticut, USA Renal hyperperfusion may be an important pathogenic factor in diabetic nephropathy. To clarify the role of polyol pathway activity in hyperglycemia-induced renal hyperperfusion, we studied the effects of two structurally distinct aldose reductase inhibitors (ARI's), sorbinil and CP-73,850, on renal blood flow in galactosemic rats. ARI-sensitive renal hyperperfusion in rats fed 30%-D-galactose was demonstrated by intracardiac infusion of 15 gm 57Co-microspheres in Inactin-anesthetized rats. Compared with normal-chow-fed rats of similar weight (~300 g) and cardiac output (~95 ml/min), rats fed galactose for 21+2 days had elevated renal perfusion: 5.3+0.8 (10) vs. 4.6_+0.8 (26) ml/min/g-kidney (+15%, ~+_SD(n), p<0.0S). However, rats fed galactose and sorbinil (~50 mg/kg body weight/day) had normal perfusion: 4.5_+0.6 (7) ml/min/g-kidney, (p<0.05 vs. galactose-fed, NS vs. normal). ARI-sensitive renal hyperperfusion in 30%-galactose-fed rats was confirmed by laser Doppler flowmetry (LDF). In Inactin-anesthetized rats previously fed 30% galactose for 10-16 days, LDF-measured renal cortical blood flow was elevated 11%: 307+22 (10) vs. 277_+27 (10) perfusion units (p<0.05). However, cortical flow in rats fed galactese and CP-73,850 (~25 mg/kg body weight/day) was normal: 283_+15 (11) perfusion units (p<0.05 vs. galactose-fed, NS vs. normal). Similar data were obtained with sorbinil (~50 mg/kg body weight/day). Neither drug effected galactose-induced renal hypertrophy or body weight. We conclude that renal hyperperfusion in galactosemic rats can be completely prevented by structurally distinct inhibitors of the

poJyol pathway.

216 THE SPECIFIC ~'~'ECT OF ~g{G-CoA ~crf~qTf INHIBITOR ON DIABETIC NEPHROPAT~/ H. Kurata, K. T~tsunQmiya~ X. Nc~nura, N. Yoshizawa, T. Sasaki, J. Yok~a~s: yo Kkeda, Tokyo, Japan On 25th FASD, we re~rted that treatment with ~iG-CoA reductase inhibitor Q0ravastatin) has the preventive effect on the nephro~et!lv cf diabetic rats without changes of serl~ lipi~~ levels 9~d sorbitol content of kidney. In this stud_v: ~,e cfm~are~5 the effect of pravastatin with that of probuco I on diabetic nephropathy in streptozotocin-diabetic rats. Male Wistar rats (250g) were made diabetic with STZ 40mg/kg. These animals were divided into 4 groups. Group i: normal controls (n=8). Group 2: diabetic rats without treain~nt (n=6). Group 3: diabetic rats treated with pravastatin 2 5 0 m ~ in water (n=6) . Group 4: dJ.abetic rats treated with 1% probucol eh(~ (n=10). Seru~ lipid, blood glucose levels and urinary alb~mlin excretion were measured until 3 months. Cholesterol and triglyceride were significantly higher in Group 2 and 3 than in Group i. However, group 4 showed normalization of ser~n lipids. Urinary albtmJ_n excretion was r~arkedlv reduced by pravastatin (group 3: 2.4-+1.3 vs group 2:5.i+l.3mg/day, p<0.01), but no change by probucol (group 4: 5.5+_3.6mg/day vs group 2, NS). The activity of k'MG-CQg~reductase of kidney was significantly higher in diabetic rats, but probucol didn't affect it. Our data suggest that pravastatin has the specific effect on the progession of diabetic nephropathy, through the improvement of increased ~ G - C o A reductase activity in kidney.

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OP 37 Hall A-1

Nephropathy 217

218

GLYCOSAMINOGLYCAN SYNTHESIS IN FIBROBLASTS OF PATIENTS WITH DIABETIC NEPHROPATHY.

THERE IS A G E N E T I C S U S C E P T I B I L I T Y TO THE MICROVASCULAR C O M P L I C A T I O N S OF DIABETES. BA Millward, DEH Llewelyn, NJ Caplen, S Wong, A Patel, PJ Watkins and AG Demaine. Departments of Diabetes and Medicine, King's College Hospital, London SE5 8RX, ENGLAND. Only a p r o p o r t i o n of type 1 diabetics develop m i c r o v a s c u l a r complications; 89 get nephropathy and 89 retinopathy. Whilst h y p e r g l y c a e m i a and h y p e r t e n s i o n are partly responsible for their development, genetic factors are also important in p r o v i d i n g differences in b a c k g r o u n d susceptibility. We have studied three groups of type 1 diabetics, i. DIABETICS WITH N E P H R O P A T H Y - 50 diabetics with p e r s i s t e n t protelnurla, retinopathy + neuropathy; 2. DIABETICS WITH R E T I N O P A T H Y 25 patients with r e t i n o p a t h y and no microa l b u m i n u r i a or neuropathy. These two groups were c o m p a r e d with 3. DIABETIC CONTROLS - 50 diabetics with no microalbuminuria, r e t i n o p a t h y or neuropathy after at least 20 years of diabetes. We have used RFLP analysis to look at several genetic loci on chromosomes 6 (MHC and related loci) and 14 (Immunoglobulin and r e l a t e d loci). Using a new m e t h o d of analysis which employs logic, we have shown genetic differences between these three groups of patients, which allows d i s c r i m i n a t i o n between types of complications. For example, RFLPs af c o m p l e m e n t C4 and immunoglobulin Sal are a s s o c i a t e d with p r o Y i f e r a t i v e retinopathy. Genetic factors are, therefore, important in the development of m i c r o v a s c u l a r c o m p l i c a t i o n s of type 1 diabetes, and RFLPs can be used in c o m b i n a t i o n to predict those p a r t i c u l a r l y at risk of developing them.

A. Kofoed-Enevoldsen ~, T. Deckert 1, I.M. Horowitz ~, L. Kjelldn 2, M. Deckert ~ and C. Lykkelund 1. ~Steno Memorial Hospital DK-2820 Gentofte, 2Biomedical Center, Uppsala. Fibroblast cultures from type 1 (insulin dependent) diabetic patients with and without diabetic nephropathy were studied in order to identify possible differences in the synthesis of heparan sulfate and other proteoglycans. Seven patients with more than 26 years diabetes duration and normal urinary albumin excretion, 11 with clinical diabetic nephropathy, and 6 healthy controls were included. Confluent cultures (3rd-6th subculture) were incubated for 72 h with 35S-sulfate and 3H-glucosamine in the presence of high glucose concentration (16 mM). In both diabetic groups the incorporation of 3H-glucosamine into hyaluronic acid was significantly increased compared to controls (p<0.02), but related to total glycosaminoglycan (GAG) synthesis the increase was not significant. The ratio of heparan sulfate to GAG synthesis was significantly lower in patients with nephropathy compared to controls (median(range): 0.15(0.11-0.25) vs. 0.20(0.17-0.21), p=0.031), whereas normoalbuminuric patients, 0.18(0.12-0.23), did not differ significantly from controls (p=0.33). No significant differences were found in the incorporation of 35S-sulfate in heparan sulfate, or degree of N-sulfataUon. The biosynthesis of glycosaminoglycans seems to be altered in type 1 diabetic patients, most so in patients with nephropathy.

219

220

C A R D I O V A S C U L A R D I S E A S E IN THE P A R E N T S OF T Y P E 1 (INSULIN-DEPENDENT) D I A B E T I C S W I T H N E P H H R O P A T H Y .

DIET AND GLYCAEMIA AS MODULATORS OF RENAL RESPONSES TO PRO TEIN LOAD IN HYPERFILTERING DIABETIC PATIENTS. S.L.Jones,P~S.Kontessis,J.Pinto,R.Dodds,E.Bognetti,M.Wiseman,R.Nosadini and G.C Viberti.Unit for Metabolic Medicine, UMDS,Guy's Hospital,London,UK and Univers,of Padova,Italy. We have studied the effects of recent protein intake and prevailing glyeaemia on renal haemodynamic responses to a protein challenge.Eight type l(insuluin-dependent)diabetics with glomeruiar hyperfiltration (HF; GFR 135ml/min/l.73m2), mean (range)age (21-36)yrs,duration of diabetes 14 (10-26) yrs were compared with 8 patients with normal glomerular fi Itration rate (NF),matched for age and duration of diabetes during euglycaemic (5mmol/l) and hyperglycaemic(12mmol/l) clamps after a 3-week period of low protein diet (LPD,0.5g/ kg/day) or of normal protein diet (NPD,ig/kg/day).In HF on NPD, GPR and RPF (inulin and PAH clearances) failed to increase significantly after meat ingestion during either hype rglycaemia GFR (144~7 to 149~8 ml/min/l.73m2),RPF (716~40 to 760~65 ml/min/l.73m 2) or euglycaemia (140~6 to 150~9;676 +_.46 to 737• lowered basal GFR and restored the respo nse to protein challenge during both hyperglycaemia (126~5 to 147~i0,p<0.05;647~56 to724~60,p<0.05) and euglycaemia (122~9 to 137~9,p<0.001;674!48 to 713~45,p
K.A. Earle, J.D. Walker, S.L. Jones, C. H i l l and G.C. Viberti. U n i t for M e t a b o l i c Medicine, U M D S G u y ' s Hospital, L o n d o n SEI 9RT. Type 1 (insulin-dependent) diabetics with p e r s i s t e n t p r o t e i n u r i a have a h i g h f r e q u e n c y of hypertension and early mortality from cardiovascular disease (CVD) . This study examined the frequency of positive family history for CVD in a group of 122 type 1 (insulin-dependent) diabetics: 61 n o r m o a l b u m i n u r i c (NA) and 61 p r o t e i n u r i c (P) a l b u m i n e x c r e t i o n rate (AER) <20 u g / m i n and > 3 0 u g / m i n r e s p e c t i v e l y . B o t h groups w e r e m a t c h e d for a g e , s e x and d u r a t i o n of diabetes. The P group consisted of 30% microalbuminuric (AER<200ug/min) , and 70% persistently p r o t e i n u r i c - (AER>200ug/min). 116 p a r e n t s from the NA group ( 5 7 M , 5 9 F ) , m e a n age 65 years (range 28 - 87 years) w e r e c o m p a r e d w i t h 117 p a r e n t s from the P g r o u p (59M,58F) m e a n age 66 years (range 37 - 84 y e a r s ) . T h e f r e q u e n c y of d i a b e t e s in the p a r e n t s of the NA and P g r o u p s w a s 5% and 10% r e s p e c t i v e l y (X z [with Yates correction] = 0.88,p<0.5). The f r e q u e n c y of p o s i t i v e f a m i l y h i s t o r y of CVD in the NA group was 23% compared w i t h 46% in the P group, (X2=6.33,p<0.01). There was an equal contribution of p o s i t i v e CVD histories from the parents of the microalbuminuric and p e r s i s t e n t l y p r o t e i n u r i c patients. Thus familial, possibly genetic, factors m a y c o n t r i b u t e to the excess of CVD in type 1 (insulin-dependent) diabetics with nephropathy.

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A resistance to the hormonal effects of atrial ~triuretic peptide (ANP) characterizes insulin~Jependent diabetic patients. R. Trevisen, C. Giorato, Fioretto P.,A. Morocutti,DeDon~ C., G. Opocher, F. Mantero, Abbruzzese E., A. Tiengo end R. Nosadini. Padua, Italy. We have shown that euglycemicdiabetic.~have high ANP levels end normal natriuresis suggesting a resistance to Ak~ action. We infused ANP in 8 diabetics at euglycaemic levels during continuous subcutaneous insulin infusion. 6 normal subjects served as control. Pla~ra free insulin levels were constant throughout the study (diabetics: 17_+2 mU/l) controls: @~_2;p<0.01). Baseline ANP concentrations were higher (35~_8 pg/ml) in diabetic than control subjects (18~3; p 0.01); ANP infusion (0.06/~/Kg/min) raised ANP plasma concentrations to 300 pg/ml in subjects. Natriuresis was greater in controls (from Z14+_82 to 4~161 7mD1/min) than in diabetics (frcm 190~_20to 250~20, p<0.Ol). GFR rose from 106/45to 120/6 ml/min/l.T8m2 (p
I M P A I R E D G L O M E R U L A R CHARGE S E L E C T I V I T Y IN MICROALBUMINURIA AND DIABETIC NEPHROPATHY J G Fox, J D Quin, K R Paterson, D StJ O ' R e i l l y and J M Boulton-Jones. Royal Infirmary, Glasgow, UK. A b n o r m a l i t i e s of g l o m e r u l a r c a p i l l a r y wall charge may be i m p o r t a n t in the p a t h o g e n e s i s of d i a b e t i c nephropathy. U s i n g the ratio of the c l e a r a n c e of p a n c r e a t i c i s o a m y l a s e to that of the s i m i l a r - s i z e d (approx. 54 kd) but m o r e a n i o n i c s a l i v a r y i s o a m y l a s e (CPAm/CSAm) as an index of g l o m e r u l a r charge selectivity, we s t u d i e d g r o u p s of Type 1 (insulin-dependent) d i a b e t i c s w i t h a l b u m i n e x c r e t i o n rate (AER) <30 m g / 2 4 h r and d i a b e t e s of <5 years d u r a t i o n (n=13), A E R <30 m g / 2 4 h r and d i a b e t e s of >15 years d u r a t i o n (n=15), m i c r o a l b u m i n u r i a (AER 30 -300 mg/24hr, n=13) or e s t a b l i s h e d n e p h r o p a t h y (AER >300 mg/24hr, n=9). I s o a m y l a s e s in serum and u r i n e w e r e m e a s u r e d by an i m m u n o i n h i b i t i o n m e t h o d w h i c h e m p l o y s two m o n o c l o n a l a n t i b o d i e s a g a i n s t s a l i v a r y isoamylase. All p a t i e n t s had serum c r e a t i n i n e <160 )/mol/l and BP <160/95. HbA 1 was not s i g n i f i c a n t l y d i f f e r e n t b e t w e e n the groups. C P A m / C S A m was s i m i l a r in the two groups w i t h A E R <30 m g / 2 4 h r (medians 2.8 and 2.5) i n d i c a t i n g that it is not r e l a t e d to d u r a t i o n of d i a b e t e s if A E R remains normal. CPAm/CSAm was s i g n i f i c a n t l y lower in the m i c r o a l b u m i n u r i a (median 2.0, p<0.01, M a n n Whitney) and e s t a b l i s h e d n e p h r o p a t h y g r o u p s (median 1.9, p<0.001) c o m p a r e d w i t h e i t h e r of the groups w i t h normal A E R s u g g e s t i n g that g l o m e r u l a r c a p i l l a r y wall a n i o n i c c h a r g e is r e d u c e d in m i c r o a l b u m i n u r i a and n e p h r o p a t h y due to Type 1 diabetes.

OP 38 Hall A-2 Immunology in Man Ill 223

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VIRUS A N T I B O D I E S IN TYPE 1 D I A B E T E S IN F I N L A N D H. Hy6ty, P. Leinikki, M. Hiltunen, T. H u u p p o nen and the Study Group of C h i l d h o o d D i a b e t e s in Finland. U n i v e r s i t y of T a m p e r e and U n i v e r sity Central H o s p i t a l of Tampere, Tampere, and National Public Healths Institute, Helsinki, Finland.

C O X S A C K I E V I R U S A N T I B O D I E S A N D S T U D Y OF ISLET C E L L A N T I B O D I E S IN T Y P ~ I D I A B E T I C C H I L D R E N

In the p r e s e n t investigation serum IgGand I g A - c l a s s a n t i b o d y levels to mumps, C o x s a c k i e B4, rubella, measles, respiratory syncytial, E p s t e i n - B a r r and h e r p e s s i m p l e x type i v i r u s e s w e r e a n a l y s e d by enzyme i m m u n o a s s a y in F i n n i s h type 1 d i a b e t i c children. Serum samples were o b t a i n e d from 471 p a t i e n t s and m a t c h e d c o n t r o l s d u r i n g the years 1981-89. I g G - c l a s s CB4 antib o d y levels did not d i f f e r b e t w e e n p a t i e n t s and controls. P a t i e n t s had also normal level s of IgG-class mumps antibodies in 1981-83 but d e c r e a s e d levels in 1985-89. IgG levels to all o t h e r v i r u s e s w e r e d e c r e a s e d in patients. IgAclass CB4 a n t i b o d i e s w e r e e l e v a t e d in patients. IgA-class m u m p s and rubella antibodies were e l e v a t e d in 1981-83 but w e r e at normal levels afterwards. IgA-class antibodies to other viruses did not s i g n i f i c a n t l y differ between p a t i e n t s and controls. The r e s u l t s s u g g e s t that the o v e r a l l a n t i b o d y r e s p o n s e to viral antigens is d e f i c i e n t in type 1 diabetes. However, antibody levels to CB4, mumps and rubella v i r u s e s w e r e e l e v a t e d (IgA-class) or at normal levels (IgG) p o s s i b l y r e f l e c t i n g t h e i r role as e t i o l o g i c a l agents.

~ichalkov~ A.Petrovi~ovd, M.Ko~{~l,J.Raj~dn~ ~ .9Batik. M . N I k ~ and V . D r o b n g . l s t P e d i a t r i c C l i nie,Com@nius

University,Bratislava,Czechoslovakia

The a i m of this s t u d y was to f o l l o w up P r o s p e c tive!y the p o s s i b l e role of c o x s a c k i e v i r u s inf e c t i o n in the e t i o l o g y and h e t e r o g e n e i t y of typ e 1 d i a b e t e s m e l ! i t u s . In a l o n g i t u d i n a l f o u r y e a r study w e f o l l o w e d 162 n e w l y d i a g n o s e d d i a betics. I n p a i r e d s e r u m s a m p l e s the n e u t r a l i z i n g a n t i b o d i e s and s p e c i f i c I g M c l a s s a n t i b o d i e ~ a g a i n s t e i g h t types of c o x s a c k i e v i r u s e s w e r e e s t i m a t e d and c o m p a r e d w i t h a c o n t r o l g r o u p of 460 h e a l t h y children. R e c e n t i n f e c t i o n w i t h c o ~ s a c k i e v i r u s e s , in p a r t i c u l a r w i t h types B 3 , B 4 , BS~ w e r e f o u n d in 56,2%/n=91/ ~s c o m p a r e d w i t h 18%/n=84/in healthy children/~=O,1483,p<0,O01~ In a g r o u p of 92 j u v e n i l e d i a b e t i c s - H L A t y p e d the p r e v a l e n c e and p e r s i s t e n c e of c y t o p l a s m i c a n t i b o d i e s to islet c e l l s / I C A and C F - I C A / w e r e d e m o n s t r a t e d . F o u r - y e a r p r o s p e c t i v e study show e d s i g n i f i c a n t l y l o n g e r p e r s i s t e n c e of ICA in d i a b e t i c s w h o s u f f e r e d f r o m c o x s a c k i e v i r u s inf e c t i o n p r i o r to d i a b e t e s m a n i f e s t a t i o n / p < O , 0 2 5 ~ T h e c o n c u r r e n t a c t i o n of c o x s a c k i e v i r u s i n f e c t i o n b e f o r e the m a n i f e s t a t i o n of d i a b e t e s cont r i b u t e s to the h e t e r o g e n e i t y of 8 u t o i m m u n e p r @ cess in our ~ u v e n i l e d i a b e t i c s .

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I:'ROI~IN C ~ T I O N S IN C ~ I L D ~ W I T H 5~TRLY DIAGNOSED ~ 1 (INE#J~IN-I~K~K~f) DIAm~7=.~ A~K) IN ' I I ~ I R SIBLINGS M.Knip, S.G. Hartling, J. Karjalainen, P. Penttil&, C. Binder, H.K. Akerblc~ and the Study Group an Childhood Diabetes in Finland. Department of Pediatrics, University of Oulu, Children's Hospital, University of Helsinki, National Public Health Institute, Helsinki, Finland and StenoMemorial Hospital, Gentofte, Deramark

LACK OF STANDARDIZATION OF THE INTRAVENOUS GLUCOSE TOLERANCE TEST IN PREDICTION OF TYPE 1 (INSULIN-DEPENDENT) DIABETES.

To examine the pcssible asscciatic=% between circulating proinsulin (PI) ccclcentraticrls in diabetic probands and their siblings and t o e valuate the relaticcl between PI levels, metabolic decccapensaticcl and autoimmune markers at the diagnc~isof ID[lMwemeasured the PI ~ t r a tions with an enzyme-linked i ~ b e n t assay in the peripheral circulation of 65 newly diagnc~ed diabetic childre=% (mean age 9.0 years) and 95 siblings (mean age 10.6 y ~ r s ) . The d i a b e t i c c h i l d r e n h a d h i g h e r P I levels (median 11.0 pmol/l, range <1.8-184 pmol/l) than their siblings (median 3.8 pmol/l, range<1.8-310 Imttol/l; p<0.001). The PI ~ t r a t i ( i n s in the probands were not related to those in the siblings (rs~-0.07; NS). In the diabetic children there was an inverse correlation between the PI levels and blood gluccse ccrce~traticm~s (r~=-0.38; p<0.01) and degree of ketoacidosis (rs=-0.25; p
The intravenous glucose tolerance test (1VGTF) is increasingly used as an adjunct to immunological markers in prediction of Type 1 (insulin-dependent) diabetes and may soon be used to monitor intervention therapy. The test has developed independently in many centres and never been standardized. Questionnaire on IVGTI" procedure in 9 centres screening pre-diabefics revealed major differences. The glucose bolus was 0.3g or 0.5g/Kg up to 25, 35 or 50g or no maximum, given as 20-66% solution, with accepted infusion time 20 seconds-6 minutes. Time-zero was variously defined as the beginning, mid-point and end of infusion giving a potential range from start of infusion to "+ 1 minute" of 1-7 minutes. Not all centres participated in external insulin assay quality control (QC). Two used no QC material at physiological concentrations. Mean inter-assay coefficient of variation < 15mU/L: 15% (range 6.3-31); 15-60mU/L: 7% (3.8-14); >60mU/L: 7% (4.1-12.8). This lack of consensus in performance of the IVGTT severely limits its usefulness. High-risk, islet-cell-antibody positive subjects are rare and widely scatttered. The numbers necessary to improve prediction and evaluate intervention therapies will demand pooling of data. Standardization of measurement of immune markers is well advanced; agreement on a common protocol for additional tests used in pre-diabetes must now follow.

P.J. Bingley and E.A.M. Gale, Department of Diabetes and Metabolism, St. Bartholomew's Hospital, London EC1, U.K.

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ACUTE INSULIN RESPONSE (AIR) TO IV GLUCAGON (AIRGin) AND ARGININE (AIR-Arg) IN SUBJECTS AT RISK FOR TYPE I DIABETES "LOW" RESPONDERS TO IV GLUCOSE AND IN HEALTHY CONTROLS. S. Bardet, V. Rohmer, H. Stetieh, M. Matte, H. Allannic, B. Charbonnel and P. Sa~'. Nantes, A n g e r s , R e n n e s - F R A N C E . The time-relationship of the loss of AIR to glucose (AIRG) with alterations of AIR to other stimuli is not well documented. AIR-Gln and AIR-Arg were compared in 11 first degree relatives ( I C A + / I A A +) of type I diabetes with a "low" AIR-G (IRI 1+3 rain<3 rd percentile of 68 controls) (Group I) and in 20 healthy volunteers. These 2 groups were comparable : age (23_+7 vs 25+4 yrs), sex (M/F : 4/7 vs 9/11), B.M.I. (101.5+3.5 vs 102.3+40%), fasting glycaemia (5.6+0.3 vs 5 . 4 + 0 . 2 mmol/1). Tolerance tests to IV G (0.5 g/kg ; 2 min), Gin (2 m g ; 2 min) and Arg (5 g ; 1 min) were performed after overnight fasting. AIR-G, AIR-GIn and AIR-Arg (104+7, 105+13, 96+11 I.tU/ml) were similar in controls. In group I, AIR-GIn and AIR-Arg (42+12 ; 60+10 gU/ml) were lower than in controls (p<0.01 ; p<0.04). When the ratios of AIR to the 3 stimuli (Gln/G ; Arg/G ; Arg/Gln) were c o m p a r e d , A I R - A r g / G and A I R - A r g / G l n were higher in subjects at risk than in controls (p<0.0001 ; p<0.01), while AIR-Gln/G were not statistically different. Four subjects only displayed a loss of AIR-G. A m o n g seven subjects with decreased AIR-GIn and AIR-Arg, one (22 yrs, DR 3/4, ICA +, IAA-) developed type I diabetes after 9 months. A) Losses of AIR-G and -Gln seem rather c o n c o m i t a n t while a loss of A I R - A r g may be more dissociated : a functional defect of B-cells may exist at early stages in some risk subjects B) When drawing near diabetes, a loss of AIR to all stimuli may be detectable.

COMPARISON OF ACUTE INSULIN RESPONSES TO GLUCOSE AND GLUCAGON IN A POPULATION AT RISK OF TYPE I (INSULIN DEPENDENT)DIABETES.B.VIALETTES,C.MATTEI-ZEVACO,X.THIRIONH.PIERON,V.LASSMANN-VAGUE,Ph.VAGUE.Department of diabetology. University of Marseille. France. Low acute insulin response to glucose (AIR glucose) is characteristic of preclinical type I (insulin-dependent) diabetes.To compare this marker to response to glueagon (AIR glucagon) we designed a test sequentially associating glucose (O.3g/Kg at to),glucagon (img at tlO) IV loads, applied to 141 negative,18 positive ICA relatives of type I (insulin-dependent) diabetics,5 prediabetics selected by transient hyperglycaemia. AIR glucose was:sum of plasma insulin at 2 and 5mn,AIR glucagon:subsequent plasma insulin increment after i0 th mn. Low responses were defined by value under 5th percentile of ICA-population (respective]y 40,4SuU/ml).Both parameters were correlated (r=O.S21).Significant (p < 0.003) increase of both parameters occured during puberty (< i0 yr : 79.3 and 86.7,10-15 yr : 105.1 and IS5.9 ~ iS yr : 77.8 and lO0.S uU/ml).Eoexisting abnormal responses occured in only 3 I C A - ( 2 . 1 % vs 5 % for one test alone).In ICA+ abnormal responses were associated in 2 (ii %),isolated low AIR glucose in l.ln 6 ICA+ and i0 ICA-,test was repeated 2-7 times (24-78 months).Both responses remained normal in I0 ICA-,4 ICA+.In i still non diabetic (low titer ICA+),transient low AIR glucose was not associated with abnormal AIR glucagon.ln i (high titer ICA+) low AIR glucose and glueagon appeared respectively 15 and i0 months before diabetes. In 5 "hyperglyeaemic prediabebics",both responses were permanently abnormal.Conclusion : AIR glucose and glucagon are related and probably reflect functionnal beta ce]] mess.Addition of glucagon improves specificity of IVGTT as marker of pretype I diabetes.

A69

OP 39 Hall A-3 Eye Disease I 229

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BLOOD GLUCOSE CONTROL AND PROGRESSION OF DIABETIC RETINOPATHY: SEVEN YEARS RESULTS FROM THE OSLO STUDY. K.Dahl-J~rgensen, O.Brinchmann-Hansen, LoSandvik and K.F.Hanssen. Aker Diabetes Research Center, Dept. of Ophthalmology, UllevAl University Hospital and MedstatCenter for Medical Statistics, Oslo, Norway. Forty-five type 1 (insulin-dependent) diabetic patients (age 18-42 years, diabetes duration 7-23 years) were nreated either with CSII, multiple insulin injections (x4-6 daily) or conventional (x2 daily) treatment and followed prospectively for seven years. Mean HbA1 during seven years 9.5+I.5(SD)%, range 7.3-13.2% (normal<7.6%). Hetinopathy was assessed by counting the number of microaneurysms and haemorrhages (Ma/H) from colour photos from both eyes. Retinopathy worsened from start to seven years (p<0.01). Mean HbAI>I0% through seven years indicated a critical level for developing severe background retinopathy (HbAI>I0% vs <10%: p<0.01). Multivariate linear regression analysis identified the following independent determinants for retinopathy (Ma/H) seven years later: Number of Ma/H at the time of inclusion into the study (p=0.046), HbAI at the time of inclusion (p=0.027), reduction of HbAI during seven years (p=0.041) and duration of diabetes (p=0.044). On the basis of these four variables we have developed a predictive model for the development of diabetic retinopathy (number of Ma/H). In conclusion: Less retinopathy was demonstrated in the group with improved blood glucose control after seven years. Blood glucose levels as measured by glycosylated hemoglobin is a main determinant of progression of diabetic retinopathy. We have developed a new predictive model for the progression of diabetic retinopathy.

HBAIC, D U R A T I O N OF DIABETES, AGE, A N D C - P E P T I D E A R E R E L A T E D T O THE D E V E L O P M E N T OF C O M P L I C A T I O N S IN T Y P E 1 DIABETES. L.H. Lassen, O. S n o r g a a r d and C. Binder. Steno Memorial Hospital, DK-2820 Gentofte, Denmark. Metabolic control and the prevalence of diabetic retinopathy and microalbuminuria was studied prospectively f o r 5 - 1 0 y e a r s in a c o h o r t o f 192 newly diagnosed Type 1 diabetic patients. Mean age at onset was 29.9• years (Mean• S u b j e c t s a t t e n d e d t h e c l i n i c a t e n t r y , i, 3, 6, 9, 12, a n d t h e n e v e r y 6 m o n t h s . 13% h a d d e v e l o p e d retinopathy after 7.5• years. Analysis of var i a n c e (ANOVA) s h o w e d s i g n i f i c a n t l y h i g h e r H b A l c v a l u e s a f t e r t h e f i r s t y e a r in t h i s g r o u p c o m pared to subjects without retinopathy, p<0.0001. I n c i p i e n t o r o v e r t n e p h r o p a t h y w a s o b s e r v e d in 12 subjects. Compared to the normoalbuminuric patients, they had been through a period of poorer m e t a b o l i c c o n t r o l , p < O . 0 1 (ANOVA). S u b j e c t s w i t h either retinopathy or nephropathy showed higher H b A l c levels a f t e r t h e f i r s t y e a r , b o t h c o n s i d e r i n g t h e i n d i v i d u a l m e a n s (p<0.05) a n d i f A N O V A is a p p l i e d (p<0.0001). However, in a s t e p w i s e multiple regression analysis for the outcome of the total complication score, long diabetes duration (p<0~ old age (p<0.0001), high Cpeptide at onset (p<0.Ol), but only to a minor d e g r e e h i g h m e a n H b A l c (p=0.056) w a s r e l a t e d t o the appearance of complications. We conclude, t h a t m e t a b o l i c c o n t r o l is only one o f s e v e r a l p a r a m e t e r s o f s i g n i f i c a n c e in t h e d e v e l o p m e n t o f c o m p l i c a t i o n s in T y p e 1 d i a b e t e s .

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H Y P E R T E N S I O N IS NOT THE M A J O R RISK F A C T O R FOR DEVELOPING DIABETIC RETINOPATHY B. F e l d t - R a s m u s s e n , K. N ~ r g a a r d a n d T. D e c k e r t . Steno M e m o r i a l H o s p i t a l , D K 2 8 2 0 G e n t o f t e , Denmark.

LENS AUTOFLUORESCENCE AND TRANSMITTANCE IN DIABETIC PATIENTS. EFFECTS OF AN ALDOSE REDUCTASE INHIBITOR. J.Tamsma, J.M.A. van Gerven, L. Vrij, J.P. Boot, J.A. van Best, H.H.P.J. Lemkes and J.A. Oosterhuis, Departments of Endocrinology and Metabolic Diseases and Ophthalmology, University Hospital, Leiden, The Netherlands

W e t e s t e d w h e t h e r h y p e r t e n s i o n is a n independent r i s k f a c t o r of r e t i n o p a t h y o r r a t h e r a c t s s e c o n d a r y to a l t e r a t i o n s in t h e f i l t e r i n g p r o p e r t i e s o f r e n a l a n d e x t r a r e n a l c a p i l l a r i e s . To d o t h i s w e s e p a r a t e d t h e i m p a c t of h y p e r t e n s i o n (AH) a n d o f p r e s e n c e o f a n i n c r e a s e d u r i n a r y a l b u m i n e x c r e t i o n r a t e (UAE) o n t h e p r e v a l e n c e of r e t i n o p a t h y in 273 t y p e 1 d i a b e t i c p a t i e n t s . D e f i n i t i o n s u s e d : (AH): s u p i n e b l o o d p r e s s u r e > 1 6 0 / 9 5 m m Hg. N o r m a l UAE: <30 m g / 2 4 h. C l i n i c a l n e p h r o p a t h y : U A E > 3 0 0 m g / 2 4 h. F o u r g r o u p s w e r e e s t a b l i s h e d . G r o u p 1 (n=55): N o r motensive and normoalbuminuric. G r o u p 2 (n=51): Hypertensive, normoalbuminuric. G r o u p 3 (n=33): Normotensiv with nephropathy. Group 4 (n=134): H y p e r t e n s i v e w i t h n e p h r o p a t h y . Age: 41 y e a r s (i0) ( m e a n (SD)) a n d d i a b e t e s d u r a t i o n : 2 3 ( 1 0 ) w a s s i m i l a r in t h e g r o u p s . B l o o d p r e s s u r e w e r e ; G r o u p I: 1 2 3 ( 1 2 ) / 7 5 ( 5 ) m m Hg. 2: 1 4 6 ( 1 9 ) / 8 7 ( 1 2 ) . 3: 1 3 3 ( 1 4 ) / 8 1 ( 6 ) . 4: 1 4 7 ( 1 8 ) / 8 7 ( 8 ) . T h e p r e v a l e n c e o f r e t i n o p a t h y was: ( n i l / b a c k g r o u n d / a d v a n c e d / b l i n d ) ; G r o u p 1 (%): 5 4 / 3 7 / 9 / 0 . 2: 46/ 3 7 / 1 7 / 0 . 3: 1 9 / 3 2 / 4 2 / 7 . 4 : 3 / 2 4 / 4 5 / 2 8 (ANOVA, p < 0 . 0 1 , G r o u p 1 v s 2 (NS)). R e t i n o p a t h y w a s w o r s e in G r o u p 3 ( n e p h r o p a t h y , n o r m o t e n s i v e ) v s Group 2 (normoalbuminuric, hypertensive) ( p < 0 . 0 0 1 ) , a n d f u r t h e r a g g r a v a t e d in g r o u p 4 ( G r o u p 3 v s 4, p < 0 . 0 0 1 ) . C o n c l u s i o n : H y p e r t e n s i o n p e r se is n o t a s s o c i a t e d w i t h i n c r e a s e d r e t i n a l c h a n g e s b u t a g g r a v a t e s t h e s e w h e n sup e r i m p o s e d to t h e i n c r e a s e d a l b u m i n s i e v i n g o f patients with clinical nephropathy.

The development of cataract is enhanced in diabetes mellitus. Lens opacification and autofluorescence can be monitored in vivo, by the Fluorotron Master. Thus, preclinical cataract was evaluated in 33 diabetic patients. The relationships of these parameters to age, diabetes duration and HbA 1 were assessed by multiple regression analysis. Age was significantly associated with both lens autofluorescence and transmittance. Diabetes duration was independently related to anterior lens autofluoresce (partial correlation coefficient R: 0.47, p = 0.023) and to lens transmittance (R: -0.53, p < 0.002). Lens autofluorescence in diabetes mellitus increases twice as fast as normal. Lens transmittance diminishes 0.5% per year of diabetes mellitus, above the normal age-related opacification. No significant correlations were found with HbA I. In animal models, cataract develops soon after induction of diabetes mellitus. This is reversible by aldose reductase inhibitors, within weeks. Therefore, the effects were studied of six months treatment with placebo or tolrestat, an aldose reductase inhibitor, on lens autofluorescence and transmittance. No significant changes occurred during this half year, in either group. These results do not support the relevance of experimental diabetic cataract to the accelerated development of senile cataract in diabetic patients. They suggest that a slow pathogenic mechanism is responsible for the enhanced cataractogenesis in diabetes mellitus.

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GROWTH OF RETINAL CAPILLARY PERICYTES, THOUGH NOT RETINAL ENDOTHELIAL CELLS, IS IMPAIRED BY HIGH GLUCOSE. M. Porta, P.A. Molinatti, R.A. Brooks and E.M. Kohner. Department of Medicine Royal Postgraduate Medical School

A MOLECULAR TITRATION ASSAY TO MEASURE mRNA LEVELS IN HUMAN DIABETIC RETINAL MICROVE S SELS . E. Cagliero, M.B. Grant and M. Lorenzi. Eye Research Institute and Departments of Ophthalmology and Medicine, Harvard Medical School, Boston, and Department of Medicine, University of Florida, Gainesville, USA. Since our previous in vitro studies have shown that high glucose induces a program of altered gene expression in human umbilical vein endothelial cells (HUVEC), we have begun to investigate if similar changes occur in human diabetic retinal microvessels. Similar amounts of RNA were recovered from diabetic (69+32 pg, mean~SD) and control (62+32 pg) microvessels ~solated from each pair of eyes. Northern blot analysis of total RNA failed to show transcripts other than ~-actin. Thus, the more sensitive molecular titration assay was used to study the expression of plasminogen activator inhibitor-i (PAI-I) in a pool of 5 controls (mean age 64+5 years) and 5 type 2 diabetics (mean age 67+3 years). The assay is based on liquid hybridization of ~ncreasing amounts of RNA to excess labeled anti-sense RNA probe. We observed that Human retinal microvessels express PAI-I, that the levels are 10-fold lower than in HUVEC (311+159 copies/cell versus 3590+2245 in 3 experiments) and that the transcript appears more abundant in diabetic microvessels (601 copies/ cell versus 424 in controls), mimicking the findings obtained in endothelial cells exposed to high glucose in vitro. This technique should provide a sensitive mean of quantitating changes induced by diabetes even in low-abun dance transcripts.

Capillary pericytes are selectively lost in early diabetic and galactosaemic retinopathies, while endothelial cells (EC) are still spared. The effects of hexoses on bovine retinal pericytes (BRP) and EC (BREC) replication were studied by growing them in media with 5.6 mmol/l glucose alone and enriched with extra glucose, marmitol or galactose to obtain final concentrations of 16.7, 27.8 and 50.0 ~mol/l. After 7 days in culture, there were signficantly less BRP/culture well in 16.7 mmo!/l glucose (341 + 78 cells x 103 , mean + SEM, p = 0.0028) and galactos~ (304 ~ 55, p = 0.0032~, not mannitol (344 ~ 52), than in the 5.6 mmol/l controls (417 h 98, 355 + 75 and 337 + 52, respectively). Higher glucose and galactose further depressed BRP counts. BREC did not change in ~umber, except at 50.0 mmol/l glucose (533 + 66 vs 629 + 67 at 5.6 mmol/l~ p = 0.032). Confluent [RP were the~ treated with mitomycin-C, a non lethal inhibitor of replication, and left 7 days in 16.7 mmol/l glucose and galactose but did not decrease in number when compared with the 5.6 mmol/l controls, suggesting that both sugars impair replication without causing cell death. Retinal pericytes appear more vulnerable than retinal EC to hexoses metabolised through the polyol pathway and metabolic damage may mediate their early dropout in diabetic retinopathy.

OP 40 Hall A-15 Insulin-substitutes? 235

236

PEROXOVANADATES STIMULATE MUSCLE GLYCOGENESIS AND REDUCE ISOPRENALINE BUT NOT AMYLIN EFFECTS

VANADATE INCREASES INSULIN SENSITIVITY AND KEVERSES INSULIN RESISTANCE IN R A T A D I P O C Y T E S J. E r i k s s o n , P. L 6 n n r o t h , U. S m i t h . D e p t . of M e d i c i n e II, U n i v e r s i t y of G o t h e n b u r g , Sweden. Vanadate (Va) e x e r t s s e v e r a l i n s u l i n - l i k e effects in r a t a d i p o c y t e s . In t h e p r e s e n t s t u d y w e e v a l u a t e d t h e e f f e c t of V a o n i n s u l i n s e n s i t i v i t y in isolated rat adipocytes.1~he cells were incubated at 370C for 20~n when C-3-0-methyl-glucose transport and ~zbI-insulin binding were determined. Receptor recycling was prevented by treating the cells with KCN. I Va and insulin increased 25I-insulin binding ~5f o l d (p<0.01) w i t h o u t c h a n g i n g r e c e p t o r a f f i n i t y . This enhancement was dose-dependent with a maxim a l e f f e c t s e e n at 4 m M V a or I 000 u U / m l i n s u lin. T h e e f f e c t of V a w a s s t i l l d e m o n s t r a b l e in insulin-resistant cells (amiloride- or cAMP-treat e d c e l l s ; c e l l s f r o m o b e s e rats) (p<0.05) w h e r e n o i n s u l i n e f f e c t w a s seen. 5 m M V a i n c r e a s e d g l u c o s e t r a n s p o r t to t h e s a m e e x t e n t as a m a x i m a l c o n c e n t r a t i o n of i n s u l i n (p<0.001). Furthermore, submaximal concentrations of V a (0.5-2 m_M) i n c r e a s e d t h e c e l l u l a r s e n s i t i vity to insulin stimulation of g l u c o s e t r a n s p o r t ( p < 0 . 0 1 ) . T h i s w a s a l s o s e e n in c e l l s f r o m o b e s e r a t s a n d in a m i l o r i d e - t r e a t e d cells (p<0.05), where the impaired insulin sensitivity was essentially normalized b y Va. In c o n c l u s i o n : Va enhances insulin sensitivity in rat adipocytes partly through an effect on the insulin-receptor interaction. Va can reverse ins u l i n r e s i s t a n c e w h i c h m a k e s it p o t e n t i a l l y usef u l in t r e a t i n g i n s u l i n r e s i s t a n t s t a t e s .

B. L e i g h t o n , G.J.S. Cooper*, C. D a C o s t a and E.A. F o o t . D e p a r t m e n t of B i o c h e m i s t r y , University of O x f o r d , U K a n d * A m y l i n C o r p . , San Diego, USA Sodium orthovanadate, at potentially toxic l e v e l s , g i v e n to s t r e p t o z o t o c i n diabetic rats normalises glucose homeostasis. Other less toxic vanadate compounds with greater potency m a y be m o r e u s e f u l . We compared the insulinl i k e e f f e c t s of o r t h o v a n a d a t e a n d p e r o x i d e s of vanadate (p e r o x o v a n a d a t es, P V s ) on glucose metabolism in i s o l a t e d incubated rat soleus muscles. Orthovanadate (i0 mM) s e l e c t i v e l y and maximally stimulated glycolysis (182 + 19%) a n d g l u c o s e oxidation (127 + 33%) b u t only partially increased glycogen synthesis (32 -+ 18~. T h e r a t e s of g l y c o l y s i s , glucose oxidation and glycogen synthesis were stimulated maximally b y e i t h e r P V s (imM) o r insulin (1000 ~U/ml); e.g.', PVs (imM) a n d insulin (1000 m U / m l ) increased t h e r a t e s of glycogen synthesis f r o m 1 . 7 3 -+ 0 . 7 2 (basal]; n = 4 ) to 6 . 5 1 _+ 0 . 9 a n d 7.4 + 0 . 5 9 ' ~ m o l / h / g , respectively. Glycogen synthesis was i n h i b i t e d b y r a t a m y l i n (i00 nM; 45 + 59) a n d isop~enaline (I00 nM; 62 -+ 5%). However, only amylin, but not isoprenaline, inhibited (27 -+ 6%) t h e r a t e of g l y c o g e n e s i s stimulated by PVs. These results demonstrate that peroxovanadates: I) f u l l y m i m i c t h e e f f e c t s of insulin on glucose metabolism in skeletal muscle in vitro; 2) a r e m o r e p o t e n t than orthovanadate.

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VANADIUM : A KEY TO THE PROBLEM OF INSULIN RESISTANCE ? S.M. Brichard, L.N. Ongemba, A.M. Pottier, M. Grrard and J.C. Henquin. Unit6 de Diabetologie et Nutrition, University of Louvain, Brussels, Belgium.

Effects of recombinant human i n s u l i n - l i k e growth factor I infusions on glucose tolerance and i n s u l i n secretion

The trace element Vanadium improves glucose homeostasis in animal models of type 1 and type 2 diabetes. Oral administration of Vanadate (V) slightly lowered plasma glucose and markedly decreased plasma insulin levels in genetically obese and insulinresistant f_.a/~ rats. We have studied the mechanisms of these effects of V. The glucagon responses to insulin-induced hypoglycaemia or to iv arginine were not attenuated in V-treated rats and the daily urinary excretion of catecholamines was not decreased. During euglycaemic-hyperinsulinaemic clamps with [6-3H]-glucose, the glucose infusion rate required to maintain glycaemia at basal levels was 45 % higher in V-treated than in control rats. This did not reflect a greater inhibition of hepatic glucose production by insulin, but corresponded to a larger increment in peripheral glucose disposal. Similar experiments with [2-3H]-deoxyglncose showed that the increase in glucose uptake occurred in white and brown adipose tissue and in various types of skeletal muscles. The action of insulin was particularly impressive on the heart of V-treated rats. In conclusion, the beneficial effects of V on glucose homeostasis in insulin-resistant rats are not due to a decrease in counterregulatory hormones but involve correction of the impaired sensitivity to insulin in peripheral tissues.

P.D. Zenobi, S. Graf, H. Ursprung and E.R. Froesch Metabolic Unit, Department of Internal Medicine, U n i v e r s i t y Hospital, 8091 Zurich, Switzerland Glucose tolerance during an oral glucose tolerance t e s t and a mixed meal (3980 kJ, 80% carbohydrates) was determined in 6 healthy volunteers 24 and 29 hours, respect i v e l y , a f t e r s t a r t i n g an infusion with saline, 7 and 14 ~g of recombinant human i n s u l i n - l i k e growth factor I (rhIGF I ) / k g . h , r e s p e c t i v e l y . There was no major change of the glucose levels during rhIGF I infusions although the glucose peak was s l i g h t l y retarded and the curves shifted to the r i g h t (not s i g n i f i c a n t ) . However, both i n s u l i n and C-peptide levels were lower ( i n s u l i n : 64• [mean• during 7 pg rhlGF I/kg.h [not s i g n i f i c a n t ] and 38• during 14 pg rhlGF I/kg.h [p < 0.005]; C-peptide: 64• [p < 0.02] and 46• [p < 0.005]). I t can be concluded that glucose tolerance is e s s e n t i a l l y unchanged during the infusions of rhlGF I but is achieved at much lower i n s u l i n secretion and i n s u l i n l e v e l s . I t appears, therefore, that rhlGF I increases glucose disposal per se and/or p a r t i a l l y suppresses i n s u l i n secretion. These offects of rhlGF I were more marked in the subjects with high fasting and high stimulated i n s u l i n l e v e l s . Thus, rhlGF I appears to lead to a state where normal glucose metabolism, both fasting and a f t e r glucose loading, occurs at s i g n i f i c a n t l y decreased i n s u l i n levels. Therefore, rhIGF I becomes an i n t e r e s t i n g i n v e s t i g a t i v e and possibly therapeutic tool in states characterized by i n s u l i n resistance such as obesity and type 2 diabetes.

239

240

IGF-I NORMALIZES GLUCOSE METABOLISM IN INSULIN-RESISTANT DIABETIC RATS L. Rossetti, A. Giaccari and S. Frontoni. University of Texas Health Science Center at San Antonio, U.S.A. We compared the effects of IGFol and insulin on glucose metabolism in control and diabetic (90% pancreatectomized) awake, unstressed rats. Low/high IGF-I (0.65/1.96 nmol/kg-min; plasma concentration ~ 100/160 nmol/L) or low/high insulin (21.6/28.7 pmol/kg.min; plasma concentration ~ 540/720 pmol/L) were infused with [3-3H]-glucose, while maintaining euglycemia. In controls the total glucose uptake, glycolysis, and muscle glycogen synthesis were similar during low/high IGF-I compared with low/high insulin, respectively. In diabetic rats the glucose uptake was reduced by 30% vs control during the insulin infusions (88.3+6.7 vs 121.1+1.7 and t08.9+3.9 vs 156.1+5.0 I~mol/kg.min; p
EFFECTS OF I G F - I ON THE RATES OF GLUCOSE TRANSPORT AND UTILIZATION IN RAT SKELETAL MUSCLE IN-VITRO G. D i m i t r i a d i s , M. P a r r y - B i l l i n g s , T. Piva, D. Dunger, G. Wegener, U. Krause and E. Newsholme. Departments of Biochemistry and Zoology, U n i v e r s i t i e s of Oxford, U.K. and Mainz, W.Germany. IGF-I has a s t r u c t u r a l homology with i n s u l i n but i t s hypoglycemic e f f e c t i n - v i v o is due s o l e l y to an increase in glucose u t i l i z a t i o n (GU). Since skeletal muscle is of primary importance in GU, the e f f e c t s of IGF-I (20ng/ml) and i t s i n t e r a c t i o n with i n s u l i n were investigated on t h e r a t e s o f 3 - O - m e t h y l g l u c o s e transport (GT), l a c t a t e formation ( g l y c o l y s i s , GL) and glycogen synthesis (GS), in r a t soleus muscle i n - v i t r o .

IGF-I vs controls increased GT (558• 461• 569• 18, 634• vs 395540, 344• 489532, 631• dpm/min/g), GL (13.3• 12.0• 13.5f0.65, 13.455 0.58 vs 9.0250.44, 9.14• 11.42• 13.7950.47 umol/h/g) and GS (4.55• [email protected], 5.08• [email protected] vs 1.92• 2.1750.19, 3.55• 5.9350.59 pmol/h/g) at 1, 10, 100, 1000 mU/l insulin, respectively (p
A72

OP 41 Hall B-3 Insulin Secretion in vivo 241

242

E f f e c t of g a s t r i n - r e l e a s i n g peptide (GRP) on the insulin r e s p o n s e to i n t r a v e n o u s and oral g l u c o s e in man. R. Ebert, J. A s c h e n b e c k and W. Creutzfeldt. Department of Medicine, University of G6ttingen, G6ttingen, FRG

GALANIN P O T E N T I A T E S GROWTH HORMONE AND BLUNTS INSULIN R E S P O N S E TO A R G I N I N E IN MAN M.Maccario, E.Ghigo, V.Martina, E.Arvat, M.R. Valetto, F.Valente, M.Nicolosi, S.Bertaina, M.C. Ghigo and F.Camanni Div. of Endocrinology, Dept. of Clinical Physiopathology. Univ. of Turin, Italy.

It was e x a m i n e d w h e t h e r GRP a f f e c t s insulin s e c r e t i o n after an oral or i n t r a v e n o u s g l u c o s e load. In 9 subjects the g a s t r i c e m p t y i n g rate of 60 g g l u c o s e was determined. GRP was i n f u s e d i.v. in two doses (1.5 and 0.75 p m o l / k g / m i n ) . P l a s m a glucose, insulin, C-peptide, gastrin, GIP and CCK w e r e m e a s u r e d over 180 min. The initial d e l i v e r y of g l u c o s e from the s t o m a c h was i n h i b i t e d by GRP (1.5 pmol), w h e r e a s GRP (0.75 pmol) had no effect. The i n s u l i n r e s p o n s e was a t t e n u a t e d only by the high dose of GRP (integr. IRI: 17.3• control vs. 10.1• nmol/l x min GRP, p
Galanin (GAL), a 29 a m i n o a c i d neuropeptide, is present w i t h i n p a n c r e a t i c islets and inhibits glucose-stimulated insulin release in animals but not in man. In the latter GAL increases both basal and G H R H - i n d u c e d GH secretion. We studied the effect of GAL (80 p m o l / k g / m i n infused iv over 90 min) on the arginine (ARG, 30g infused iv over 30 min)- s t i m u l a t e d GH and insulin s e c r e t i o n in 8 h e a l t h y v o l u n t e e r s (aged 20-30 yrs). Plasma glucose levels were also studied. When given alone, GAL induced an increase of GH s e c r e t i o n (GAL vs placebo, AUC, mean • gEM: 316.5 • 73.9 vs 76.1 • 18.3 ~g/L/h; p<0.02) but failed to m o d i f y insulin secretion (AUC: 476.6 • 85.9 vs 544.0 • i08.8 mU/L/h). GAL p o t e n t i a t e d the A R G - i n d u c e d GH s e c r e t i o n (AUC: 1635.0 • 294.2 vs 618.4 • 161.2 ~g/L/h; p<0.01) and blunted the insulin response to ARG (AUC: 838.7 • 85.6 vs 1300.0 248.1 mU/L/h; p<0.05). Glucose levels were not modified by GAL and ARG both alone or in combination. In conclusion, these results show the i n t e r a c t i o n of GAL w i t h ARG to potentiate GH and to blunt insulin s e c r e t i o n in humans. The additive effect of GAL and ARG on GH secretion suggests that these drugs, in spite of other data f a v o u r i n g their somatostatins u p p r e s s i n g effect, may influence somatotrophs also through different mechanisms. T h e b l u n t i n g effect of GAL on the A R G - i n d u c e d insulin response suggests that this neuropeptide may m o d u l a t e beta cell s e c r e t i o n also in man.

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ADENOSINE-5 '-O- (2-THIODIPHOSPHATE ) STIMULATES INSULIN SECRETION AND REDUCES GLYCAEMIA IN THE RAT. G. Bertrand, J. Chapal, G. Ribes and M.M. Loubati~resMarian• Faculty of Medicine, Laboratory of Pharmacology, CNRS URA 599, Montpellier, France. ATP and ADP stimulate insulin secretion by activating P2y purinoceptors on pancreatic B cells. 2-methylthio ATP, a more specific P2y agonist, infused directly into the pancreatiooduodenal artery because of its rapid metabolization, induced an insulin response and a slight reduction of glycaemia in dogs. In this study we have investigated the insulin secretory effect of adenosine-5 '-O- (2-thiodiphosphate ) (ADP-B-S), a more stable P2 agonist. Experiments were performed in vitro and in vivo in fed anesthetized rats. In vitro, in isolated pancreas perfused with 8.8 mmol/l glucose, ADP-8-8 induced a biphasic insulin response in the range 5.10 -8 and 5.10 -7 mol/l and was about iO0 times more potent than ATP or ADP. In vivo, ADP-S-8 was injected into a jugular vein ; at 0.I mg/kg ADP-B-S induced an immediate but transient increase of plasma insulin levels (p < 0.O1) whereas at 0.2 mg/kg, this P2 agonist evoked an immediate biphasie increase in plasma insulin (p < 0,O1) and a delayed decrease in plasma glucose concentrations, significant from min 30 (p < O.01). In conclusion, ADP-B-S is a potent insulin stimul atory P2 agonist which is also able to reduce glycaemia in the rat.

EFFECT OF PROLONGED IN V r V O G L U C O S E I N F U S I O N O N I N S U L I N S E C R E T I O N IN R A T S W I T H P R E V I O U S G L U C O S E INTOLERANCE. C. THIBAULT, A. KTORZA, B. PORTHA. Labomloirede Physiolog~edu D~veloppement, C N R S U R A 30?, Universit~ Paris 7, F-75251 Paris,

France.

We have inve~a~ed the effect of prolonged hype~lyc~mia on the subsequent insulinsecre~n m vivo in ratswith various degrees of glucose imokrance. 4 groups of m~s receiveda ~mJque h~ectionof a low concentra~on of s~ep~ozolocin(ST'Z):20, 27, 30 or 35 mg/Kg m s ~ y

8"I'Z 20, S'IZ

27, STZ 30 or gTl 35 mrs. Controlmrs (C) were mjectsd with cleatsbuffer. In all STZ groups, nonual basel~lyc~mm but impeimd glucose toleranceand insulinsecretionwere observed. These defects ~ m coucentralion.3 ~eks e_earSTZ i n c h ,

pmponionsl to STZ

baselhype~lycemia (17 mmo~l)

was produced by a ~ h - glucose infusion.Glucose fnduced secretionw ~ im,es~atsd 3h aftsrs~oppir~ the infueion,by intrevenoueglucose ~okmr~e tests.Glucose - infusionimproved ~

secretionin (C) rats (AI~AG w~a

two - foldh~her than beforeinfusion)end stillmore in ratswith mild glucose imole~oce (AI/~ were 3 and 4 - fold Nwber in STZ 20 aud SZ 217 rats, ~l~C~vely). But, itiucree.~l~

secm~on only slightlyin STZ 30 ra~s

and not atallm STZ 35 rats. These data show that pmloi'%,edhype~lycaemia he~ a beneficialeffect on JDsulinsecretion in ratswith mild glucosein~okran~ whereas ithas no effectin ratswith more severeglucoseintolerance.

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DISTRIBUTION OF (GLP)-I AND INFLUENCES OF GLP-I AND GLPi<~-se) ON INSULIN AND GLUCAGON SECRETION IN THE MOUSE

GLUCAGON-LIKE INSULINOTROPIC PEPTIDE APPROACH TO TREATING DIABETES?

T. Fridolf. G. BOttcher, F. Sundler, B. Ahr@n, Depts. of Pharmacology, Medical Cell Research and Surgery, Lund Univ. , Sweden

li. Gutniak, B. *B. Ahr'~n and S. EfendiC. Department of Endocrinology, Karolinska Hospital, Stockholm, Sweden and *Department of Pharmacology, Lund U n i v e r s i t y , Lurid, Sweden. The amino acid sequence 7-36 of glucagon-like i n s u l i n o t r o p i c peptide I (CLIP) stimulates i n s u l i n release. Therefore we investigated the e f f e c t of this peptide on isoglycemic meal related i n s u l i n requirement (IMIR) in 6 Type 2 (non-insulin-dependent) and 4 Type I ( i n s u l i n dependent) d i a b e t i c s . Insulin requirement was determined during 180 min a f t e r the meal using Biostator(R) at simulated i d e n t i c a l glycemic responses. CLIP was given as an infusion (0.75 pmol/kg/min) 3C min p r i o r to and 18C min a f t e r a meal. In Type 2 patients IMIR was decreased by as much as 89% (from ZC.5 t 3.3 to 2.2 • 0.5 U,p
Proglucagon, occuring in islet A cells and gut L cells, is processed to several peptides. One of these is GLPl<7-se~, being released after a meal and stimulating insulin secretion. Its interaction with basal and stimulated islet hormone secretion is, however, not established. We studied the cellular distribution of GLP-I and the effects of GLP-I and GLP-icT_s6)amide on islet hormone secretion in the mouse. Immunofluorescence studies revealed that GLP-l-immunoreactivity occurred within peripheral islet cells and in cells located mainly distally in the small intestine and in the entire large intestine. These cells were identical with glucagonfglicentin cells. In in vivo studies, basal insulin secretion was stimulated by GLP-1cT-s6)amide when injected i.v. at 8 and 32 nmol/kg, and by GLP-I at 3Z nmol/kg. GLP-IcTse~amide also showed additive stimulatory influence on insulin secretion with glucose, carbachol, and CCK-8, at 8 or 52 nmol/kg. In contrast, stimulated insulin secretion was unaffected by GLP-1. Basal and carbachol- and CCK-8-stimulated glucagon secretion were inhibited by GLP-icT_~amide but unaffected by GLP-I. Thus, GLP-Iinm%unereactive cells are identical with glucagon/glicentin cells, and GLP-lc~_s,~amide stimulates insulin secretion and inhibits zlucagon secretion. Therefore, GLP-Ic~_ s~ might be both an incretin and a paracrine modulator of islet function.

I (7-36) - NEW

OP 42 Hall A-18 Endothelium & Rheology 247

248

DIABETES MELLITUS AFFECTS RED BLOOD CELL PLASMA MEMBRANE PROPERTIES EITHER DIRECTLY OR BY DECREASING LIFE-SPAN

INCREASED

E. Faloia, Staffolani, *Istituto UniveI~itA UniversitA

PE Jennings, M Mclaren, J Brown, N Scott and JJF Belch. Department of Medicine, Ninewells Hospital and Medical School, Dundee, Scotland.

*L. Mazzanti, 0p. Borboni, ~R.A. Rabini, *R. ~ L~uro, R. De Pirro di Biochimica e Cattech~a di ~docrinologia, di Ancona; o Cattedra di I~Idoerinologia, II di Ronm - Italy

In order to clarify if diabetes mellitus induces alterations per se or by decreasing the red blood cell (RBC) life-span, RBCs from 23 in good control insulin-dependent diabetic patients (DM) (24-47 years) and 26 healthy subjects (N) (26-47 years) were separated in five age-dependent fractions (I: early ir~tur~ PdBCs; Voldest RBCs). I, 6 diphenyl-l,8, 5 hexatriene mobility, inversely related to membrane fluidity (P), lipid peroxidation (LP), Na K -ATPase (NA) mad Aeetyleholinesterase (AchE) were studied on plasma membranes. During RBC aging both N and DM showed deeressed NA (p 0.001, fraction I vs fraction V), increased P (p 0.OO1) and increased LP (p O.OO1). In comparison to N, DM showed higher P and LP in all the fractions (p 0.001) except in fraction I, thus suggesting that diabetes alters these parsmeters by decreasing life-span; conversely, N and DM snowed different NA at all RBC ages (p 0.001) thus suggesting a direct effect of the disease state. During RBC aging, AchE decreased in N but it increased in DM; moreover, N and DM showed similar values in fractions I, but not in the other four fractions (p O. OO1 ), thus suggesting an effect appearing only during aging. In conclusion diabetes mellitus alters RBC plasma membrane properties through different mechanisms.

WHITE CELL AGGREGATION

IN TYPE i

DIABETES:

A POSSIBLE FACTOR IN MICHOANGIOPATHY.

The white cell is fundamental in inflammatory vascu]itis therefore we examined whether it may contribute to the abnormalities in free radical activity and endothelial cell function in diabetic microangiopathy. We studied 19 type 1 diabetic patients (mean age 40 yr and duration of diabetes 18 yr; i0 patients with retinopathy) and 12 age-matched non-diabetic controls. We used stsndardised techniques of whole blood white cell aggregation (WCA) and measured the oxidative effects of free radicals as plasma lipid peroxides (MDA-LM) and plasma thiols (PSH). Endothelial cell function was assessed as Factor VIII vW:Ag (vWF). The diabetics had increased MDA-LM: median 9.3 umol/l (range; 8-11.1) compared to controls 6.5 umol/l (5.5-8.2, p<0.001) and increased vWF 116% (35-219) cf 85% (60-144, p<0.05); together with reduced PSH 428 umol/l (260-559) cf 481 umol/l (441-535, p
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249 EVIDENCE FOR A KEY ROLE OF HYPERGLYCAEMIA-DEPE~DENT~ITHPg~BIN Iii ALTERATIONS IN THE COAGULATION ABNOR~Z/ETTESOF DIABETES MELLITUS g. Ceriello,D. Giugliano,A. Quatraro,E. Marchi,M. Barbanti,P. Lefebvre Cart. Diabetologia,IFae. Med. ,Napoli-CentroDiabetol. Casa di Cura S.Rita Tarsnto,Alfa Ric.-Bologrm,Italy-Div.ofDiabetes,Univ. Li@ge,Li6ge,Belgium The existence of a direct link between abnormal glucose level and increased aotivity of cce~ulation system in diabetes r~nains tmclear.In T.his study we measured ~bin-antithranbin complex (TAT),fibrinopep%ideA (FPA),factor X concentration (FXo) and activatsble factor X (FXa),anti~in III concentration (ATIIIc) and activity (ATIIIa) in SO type I diabetic subjects and in SO matched healthy cantrols.In the presence of knereased levels of FPA (3._0~O.43 vs 1.7+O.21 nE/ml,p {0.01;M+SE),decrea6ed ATIIIa (89._6~4.12vs lOS.9_+2.22~o,p~O.Ol) ,FXa (81.8+2.2 vs 97.3~2.1%, p
in diabetic and normal subjects decreased ATIIIa,FXa;TAT and increasedFPA and FXa/ATIIIa ratio,while ATIII c and FXc did not change.Hepanin mud low molecular weight hepanin administrationwas shown to reduce,these phenomena.The FPA increase revealed the presence of hyperglycaemiaLdependeat ~fmombin hyperaetivation.Moreover,theexistence of decreased TAT and ATIIIa in pr~ence of increased FXa/ATIIIa ratio and FPA,stcesses the central role of hyparglycaemia-depandantATIII alteratieas in precipitating three,in hyperactivity in diabetes.

250 EPIDEP~4ALGRO~TfHFACTORANDPLATELET-DERI-VI3D GROgTfH EACTOR]I~ DLOOD OF ADULT DIAE.L~IC FATIEIqTS. A. LEV-RAN, and D.L. HWhNG. City of Hope National i~dical Center, Duarte, California, USA. Vascular pathology, especially prominent in diabetes, is accoF~oaniedby abnom~alities of blood platelets. Alphagr~lules of platelets contain and release smooth muscie cell mitogens epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). We aimed at assaying them by specific radioir~nunoassays in b l o o d o f 82 diabetic patients over age 30 and in 53 age- and sex-matched controls. In diabetes there was increase in the serr~ levels of EGF (191+43 vs 155+64 pM, p.O002) and PDGF (222+47 vs 160+26 pM, p . 0 0 0 2 T a n d in plamma level of EGF ( 5 3 + ~ v s 38~14--~, p.O001). %~lese levels did not correlate with age, sex, body build, type of diabetes, its duration, control(plasmla glucose, glycohemoglobin), albt~ainuria, hypertension, retinopathy or severe coronary disease. Negative (!) correlation with serum creatinine was found for sert~ EGF (r-.373. p.0008) and PDGF (r-.564, p.0285) but not for plasma EGF. Increase of s ~ levels of EGF and PDGF independent of diabetes control or cc~plications corresponds to reported increase of other alpha-granule factors (beta-fihrcmboglobulin, platelet factor 4). ~]]us, diabetes itself and not its ccmplications is associated with increased releasability of platelet growth factors, and this release is related to excretory kidney function; increase in plascaa EGF points to the effect of diabetes on other organ(s) of EGF production and/or release. All these changes may play a role in the increased vulnerability of diabetic vessels.

251

252

INCREASED PLATELET Na+/H + EXCHANGE RATES IN DIABETIC MICRO-ANGIOPATHY. P. BARBE, J.P. SALLES*, J,P. LOUVET, H. CHAP*. Department of Endocrinology and INSERM U326", CHU PURPAN, 31059 TOULOUSECedex,FRANCE.

THE FIBRINOLYTIC ACTIVITY IN TYPE l (INSULIN-DEPENDENT) DIA BETIC PATIENTS WITH NO EVIDENCE OF VASCULAR DISEASE. F.Cristiano,D.Cucinotta,M.A.Pizzoleo,M.Quartarone,O.Mangana ro and G . S q u a d r i t o . l n s t i t u t e of Internal Medicine,University of Messjna:Italy. In d i a b e t i c subjects a depression of plasma f i b r i n o l y t i c a~ t i v i t y may contribute to the development of vascular damage. However,previous i n v e s t i g a t i o n s of f i b r i n o l y s i s in diabetes have yelded c o n f l i c t i n g results.We studied 26 type 1 (insulin-dependent)diabetic patients (mean age 24.3~6.4 years, mean diabetes duration 4.9+2.4 years,HDAlc 7.8+2.1%),accur a t e l y selected in order to exclude vascular complications or other concomitant diseases.ln each of them we assessed plasma levels of:D-dimer of f i b r i n (ELISA method);tissue plasminogen a c t i v a t o r , t - P A (ELISA method),both at rest and a f t e r venous occlusion;plasminogen a c t i v a t o r i n h i b i t o r , P A l (spectrophotometry).The same parameters were also eva]uated in a control group (20 normal subjects aged 27.4~3.2 years). In d i a b e t i c patients plasma levels (m~S.E.) of D-dimer were 221+30 ng/ml,those of t-PA 4.95+ 0.4 ng/ml and those of PAl 3.13+0.36 IU/ml and did not s i g n T f i c a n t l y d i f f e r from what observed in the control group (173+__22, 5.86+_0.38 and 3.76~ O.25,respectively);moreover no c o r r e l a t i o n was found between these parameters and HbAlc levels or diabetes duration. After venous occlusion t-PA increase was s i m i l a r in both groups.These results suggest that in type 1 (insulin-dependent) d i a b e t i c patients with no evidence of vascular disease e x t r i n s i c f i b r i n o l y t i c a c t i v i t y is within normal l i m i t s , both at precocious steps (plasma levels of t~PA and PAl) and at the l a t e ones (D-dimer release from f i b r i n ) .

The plasma membrane Na+/H+ exchanger is a ubiquitous system which plays a role in the regulation of intracellular pH and the control of cell growth. In order to assess the potential role of this system in the pathogenesis of diabetic microangiopathy, we investigates 33 insulindependent diabetic patients (age: 38.4+12 years (m_+sd), diabetes duration: 16.5+5.4 years). We tested the platelet Na+/H+ exchange as the rate of amiloride sensitive and sodium dependent volume gain of cells suspended in sodium propionate; the volume changes coupled to Na uptake were measured by cell sizing with a Coulter counter and Channelyser. There was no significant correlation between the Na+/H+ exchange rate constant and age, diabetes duration, blood pressure, glycated hemoglobin and fructosamin levels on lhe day of the test. The exchange rates were clearly enhanced in presence (n=14) vs absence (n=19) o1 diabetic nephropathy (permanent microalbuminuria or macroalbuminuria) (0.30+0.6 vs 0.24• p<0.02), in presence (n=18) vs absence (n=15) of proliferative diabetic retinopathy (0.29• vs 0.23• p
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OP 43 Hall A-1 Therapy of Hypertension and Nephropathy 253

254

PERINDOPRIL VERSUS NIFEDIPINE: EFFECTS ON MICROALBUMINURIA IN HYPERTENSIVE AND NORMOTENSIVE DIABETIC SUBJECTS. M.E. Cooper, A.E. Doyle, T.J. Allen, G. Jerums, F. Alford, M.L. Mashford, J. Hammond, M. de Luise. The Melbourne Diabetic Nephropathy Study Group, Departments of Medicine, Diabetes and Clinical Pharmacology, St. Vincent's, Austin and Repatriation General Hospitals, Melbourne, Australia.

ANGIOTENSIN CONVERTING ENZYME INHIBITION POSTPONES THE DEVELOPMENT OF DIABETIC NEPHROPATHY.

A comparison between the ACE inhibitor perindopril and the calcium

The effectiveness of angiotensin converting enzyme inhibition in preventing diabetic nephropathy (albuminuria >300 mg/24h), was evaluated in a randomized long-term follow-up study of 44 normotensive (127/78 (12/10) mmHg) insulin-dependent diabetic patients with persistent microalbuminuria (30-300 mg/24h). Twenty-one patients received captopril (25-100 mg/day) throughout the whole study period (36 months) with thiazide added during the final 6 months. The remaining 23 were left untreated. The urinary excretion of albumin was gradually reduced from 82 (33-204) to 47 (8-134) mg/24h (geometric mean (range), p<0.05) in the captopril treated group, while an increase from 105 (34-282) to 139 (9-1949) mg/24h (p<0.05) occurred in the control group. Seven of the untreated patients, while none of the captopril treated patients had progressed to diabetic nephropathy (p<0.02). Systemic blood pressure, glomerular filtration rate, haemoglobin A,o, and urinary excretion of sodium and urea remained practically unchanged in both groups during the 3 year trial. Captoprii postpones development of diabetic nephropathy in normotensive insulin-dependent diabetic patients with persistent microalbuminuria. The effect is independent of changes in systemic arterial blood pressure.

antagonist nifedipine was perfomaed over 12 months in diabetic

subjects with microalbuminufia (urinary albumin excretion rate [UAE] 20-200 ~g/min).

Hypertensive (BP>160/95 mmHg, n = l l ) and

normotensive subjects (n=36) were stratified separately and randomized to receive either perindopfil (n=23) or nifedipine (n=24). There were no baseline differences between the two groups in mean blood pressure

(perindopril 102+2, nifedipine 109+4 mmHg) or UAE (perindopril 35x/+1.1, nifedipine 36x/+1.2 ktg/min, geometric mean x/+ tolerance factor). There was a reduction in albuminuria during therapy in hypertensive (-23+7%) but not in normotensive subjects (0+3%) [hypertensive vs normotensive, p=0.003]. Over the 12 month period, there was no difference in mean blood pressure (perindopril, 98+_+_2vs nifedipine, 99+_2 mmHg) or UAE between perindopril (31x/+1.2) and nifedipine (29x/+1.3 pg/min) treated groups. After 1 month cessation of treatment there was a marked increase in UAE in perindopril (53x/+1.3) and nifedipine (55x/+1.3 ~g/min) groups. Anfihypertensive

ER Mathiesen, E Hommel, J Giese, and H-H Parving. HvidQre Hospital, Emiliekildevej 1, Copenhagen, Denmark.

therapy may prevent the rise in albuminuria in normotensive microalburninuric subjects but ACE inhibition does not confer a specific advantage.

255

256

COMPARISON OF CAPTOPRIL, METOPROLOL AND HYDROCHLOROTHIAZIDE IN HYPERTENSIVE TYPE 2 (NONqNSUMN-DEPENDENT) DIABETIC PATIENTS. M-A Gall, P Rossing, E Hommel, ER Mathiesen, P Skirt, U Gerdes, H Beck-Nielsen and H-H Parving. Hvidbre Hospital,Denmark.

RENAL EFFECTS OF ACE-INHIBITION IN TYPE i (INSULINDEPENDENT) PATIENTS TREATED WITH BETABLOCKER AND THIAZ!DE.

The aim was to compare antihypertensive efficacy, safety, and tolerance of captopril (C), metoprolol (M) and hydrochlorothiazide (H) in Type 2 diabetics with essential hypertension. In a double blind, placebo-controlled crossover trial 23 patients were treated for four 2 months periods with C 25 mg twice daily, M 50 mg twice dally, H 12.5 mg twice daily and placebo (P). Dose was doubled at 4 weeks if supine diastolic pressure >90 mmHg. After an initial 6 weeks wash-out period arterial blood pressure was 188_+14/101_+7 mmHg. Mean arterial blood pressure was lowered during all treatments compared to the placebo-period (C: 114_+2mmHg (p<0.05), M: 112_+1 mmHg (p<0.001), H: 111_+1 mmHg (p<0.001)). Haemoglobin A,o increased from 7.5% to 8.2% by H (p<0.001), while no significant changes were observed with C (7.8%) or M (7.8%). HDL-cholesterol was lowered from 1.19 to 1.10 mmol/I by H (p<0.05) while C and M had no adverse effects on lipid profile. Glomerular filtration rate and fractional clearance of albumin were unchanged. Orthostatic blood pressure response and digital systolic blood pressure in the lower limb remained unchanged. Subjective side-effects was rare during all treatments and not significantly different compared to placebo. We conclude that antihypertensive treatment with captopril or metoprolol in Type 2 diabetic patients is well tolerated and causes no deteriorationin metabolic control and kidney function, while hydrochlorothiazide has adverse effects on glycaemic control and lipid profile.

C.K. Christensen, M.Mau. Pedersen, K.W. Hansen, A. SchmJtz and C.E. Mogensen, Medical Department M (Diabetes and Endocrinology), Aarhus Kommunehospital, DK-8000 Aarhus C, Denmark. The aim of the study was to investigate the effect of supplementary ACE-inhibition to conventional antihypertensire treatment (AHT) in diabetics with early nephropathy. Inclusion criteria were i) ongoing treatment with metoprolol and bendroflumethiazide. 2) Urinary albumin excretion (UAE) below i000 ~g]min and 3) age below 50 years. Study design: double blind (Ramipril 5 m g or plaeebof24h) crossover (four months in each period). T e n Type 1 patients were studied (mean age 38 yrs, diabetes duration 25 yes). Test parameters at the end of each study period were: BP, GFR, R P F (clearance of ]1~-iotha]amate} I~'-hippuran) and U A E measured from clearance samples. A slight but significant fall was seen in B P (placebo versus RamJpri]), mean B P I01 versus 95 m m H g , 2p<5.0%). No significant changes were seen in G F R (120 versus 121 ml/mJn) or R P F (484 versus 497 mlfmJn). A sig~nificant fall in renal resistance was seen (0.397 versus 0.333, 2p
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257

258

SANDOSTATIN~ REDUCES INCREASED KIDNEY FUNCTION A N D SIZE IN TYPE I DIABETES. O. Serri, H. Beauregard, P. Brazeau, A. Harris, and K. Gallant. Centre de Recherche, H6pital Notre-Dame, U n i v e r s i t ~ de Montr4al and Sandoz.

THROMBOXANE SYNTHASE INHIBITION AND RENAL F U N C T I O N IN DIABETIC NEPHROPATHY. P.S. Kontessis, A.E. Hayward, A.C.G. Collins, S. De Cosmo and G.C. Viberti. U n i t for M e t a b o l i c Medicine, UMDS, Guy's Hospital, London, UK. S t u d i e s in n o n - d i a b e t i c renal d i s e a s e h a v e suggested that thromboxane (Tx) may be an important m e d i a t o r of abnormal renal function. The role of Tx in d i a b e t i c n e p h r o p a t h y is p o o r l y known. In a one week, double-blind, randomized p l a c e b o control study of Tx synthase i n h i b i t o r (TxSI) (FCE-22178, 400 mg b.i.d, or t.i.d.) we m e a s u r e d g l o m e r u l a r filtration rate (GFR, inulin clearance), renal plasma flow (RPF, PAH clearance), fractional clearance of albumin (SAlb) and r e a b s o r p t i o n rate of @z-microglobulin (~2-m) in 12 type 1 (insulin-dependent) patients with diabetic nephropathy. In 5 additional patients, the effect of TxSI 400 m g b.i.d, was c o m p a r e d w i t h that of TxSI 400 mg t.i.d. G F R (52 • 2.8 vs 52.6 • 3.5) m l / m i n / l . 7 3 m 2, RPF (306.6 • 28 vs 293.2 • 25) m l / m i n / l . 7 3 m 2 and renal v a s c u l a r resistance (RVR) (224 • 38 vs 222 • 26 mmHg/min/l) were similar after p l a c e b o or TxSI. eAlb was s i g n i f i c a n t l y lower after TxSI (5.66 • 1.37 vs 4.03 • 1.15 x i0 "4, p=0.028) but the reabsorption rate of ~2-m was u n c h a n g e d (97 • 11.8 vs 104 • 24.7 ~g/min). S e r u m f r u c t o s a m i n e and mean blood pressure were similar after p l a c e b o or TxSI. TxSI t.i.d, r e s u l t e d in a lower eAlb than TxSI b.i.d. (3.98 • 1.14 vs 2.89-+ • 1.15 x 10 .4, p=0.041) while GFR, RPF and R q R r e m a i n e d unchanged. These data indicate that TxSI reduces a l b u m i n u r i a but has no e f f e c t on renal haemodynamics. The a n t i - p r o t e i n u r i c effect of TxSI m a y be dose dependent.

G l o m e r u l a r h y p e r f i l t r a t i o n can c o n t r i b u t e to the i n i t i a t i o n and p r o g r e s s i o n of d i a b e t i c n e p h r o p a pathy. To evaluate w h e t h e r c h r o n i c t h e r a p y w i t h Sandostatin, a s o m a t o s t a t i n analogue, can reduce i n c r e a s e d g l o m e r u l a r f i l t r a t i o n rate (GFR) and k i d n e y size in i n s u l i n - d e p e n d e n t diabetes, we studied for a p e r i o d of 3 months, ii p a t i e n t s w i t h type I diabetes and GFR >~ 135 m l / m i n / l . 7 3 m 2. The p a t i e n t s w e r e r a n d o m l y assigned in a b l i n d d e s i g n e i t h e r to c o n t i n u o u s sc S a n d o s t a t i n infusion (5 patients) or to p l a c e b o (6 patients). The p a t i e n t s w e r e aged 23-53 yr, normotensive, h a d no proteinuria, and h a v e had diabetes for 120 yr. G F R and k i d n e y v o l u m e w e r e m e a s u r e d by T c - 9 9 m DTPA and CT scan, respectively, b e f o r e and 3 m o n t h s after the start of therapy. V a l u e s w e r e c o r r e c t e d to 1.73 m 2 body surface. R e s u l t s are expressed as m e a n +_ SE. In the S a n d o s t a t i n group, the G F R fell from 173 +_ 15 to 136 + 12 ml/min, k i d n e y v o l u m e (right + left) d e c r e a s e d from 426 +_ 42 to 379 + 30 ml, I n s u l i n - l i k e g r o w t h factor I (IGF-I) d r o p p e d from 0.96 +_ 0.18 to 0.57 +_ 0.i U/ml (p < 0.05). In the p l a c e b o group, GFR, kidney volume, and IGF-I were not s i g n i f i c a n t l y m o d i f i e d d u r i n g the study period. Glycosylated hemoglobin did not change significantly in both groups. The results show that l o n g - t e r m t r e a t m e n t w i t h S a n d o s t a t i n reduces i n c r e a s e d G F R and kidney size in type I d i a b e t i c p a t i e n t s i n d e p e n d e n t l y of the glycemic control.

OP 44 Hall A-2 Glucose Metabolism in Man 259

260

HYPERGLYCEMIA DECREASES GLUCOSE STORAGE BY >50% IN TYPE 1 DIABETES H. Vuorinen-Markkola, V.A. Koivisto and H. Yki-J~=rvinen, Second Department of Medicine. Helsinld University, Holsinld, Rnland We evaluated the mechanisms underlying hyperglycemia induced insulin resistance ("glucose toxicity"). Eight type 1 diabetic patients (age 36+2 yrs, BMI 24-+1 kg/m 2) were studied twice, after 24 hours of hyper- (20+1 mM, i.v. glucose) and normoglycemia (7-+1 mM, saline) while receiving identical diets and insulin doses. In the morning after hyper- and normoglycemic days, serum insulin was raised to 50 mU/I (0300 min). Plasma glucoses were maintained similar during the 300 min by increasing, or maintaining it at 17-+1 mM (30-150 min) after 24 hrs of normo- and hyperglycemia, respectively, and by allowing it thereafter to fall to normoglycemia in both studies (180-300 min). During normoglycemia, total (4.5-+0.5 vs 6.1 -+0.7, p<0.05) and nonoxidative (1.6-+0.4 vs 3.5-+0.6 mg/kg.min, p<0.01) glucose disposal were lower after 24 hrs of hyper- than normoglycemia whereas glucose oxidation rates were similar. In response to transition from hyper- to normoglycemia, serum growth hormone and glucagon concentrations increased significantty, but similarly after 24 hrs of hyper- and normoglycemia. Serum cortisol concentrations remained unchanged. In conclusion: 1) Hyperglycemia can induce a marked defect in nonoxidative glucose disposal. 2) Thus, part of the decrease in insulin-stimulated glucose storage in type 1 diabetic patients can be secondary to hyperglycemia per se.

INSULIN RESISTANCE, BUT NORMAL BASAL GLUCOSE PRODUCTION RATES IN PATIENTS WITH MILD DIABETES. O. Hother-Nielsen and H. Beck-Nielsen. Medical department 3, Aarhus amtssygehus, Aarhus, Denmark. Fasting hyperglycaemia in Type 2 (non-insulin-dependent) diabetes has been suggested to be due to hepatic overproduction of glucose and reduced glucose clearance. We studied 22 patients (10 lean and 12 obese) with newly diagnosed mild diabetes mellitus (fasting plasma glucose <15 mmol/I, urine ketone bodies <1 mmol/I) and two age and weight matched groups of non-diabetic control subjects. Glucose turnover rates and sensitivity to insulin were determined using adjusted primed-continuous 3JH-glucose infusion and hyperinsulinaemic euglycaemic clamp technique. Insulin stimulated glucose utilization was reduced in both diabetic groups (lean patients: 281-+33 vs 444-+21 mg/m 2, rain, p<0.01, obese patients 292-+27vs 408-+21 mg/m 2, min, p<0.01 ). Basal glucose concentrations in diabetic patients decreased 0.43-+0.05 mmol/I per min (p<0.01). Glucose production rates were smaller than glucose utilization rates (lean patients: 87-+3 vs 94-+3 mg/m%min, p<0.01, obese patients: 79-+5 vs 88-+5 mg/m 2. min, p<0.01), were not correlated to basal glucose or insulin concentrations, and were not different from normal (lean controls: 87-+4 mg/m2.min, obese controls: 80-+5 mg/m2.min). These results suggests that the basal state in diabetic patients was a compensated condition where glucose turnover rates were maintained near normal despite defects in insulin action.

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EFFECT OF HYPERGLYCEMIA ON THE PATHWAYS OF LIVER GLYCOGENEStS IN DIABETIC RATS A. Giaccari and L. Rossetti. University of Texas Health Science Center at San Antonio, U.S.A. We examined the effect of hyperglycemia on hepatic glycogenesis by the direct vs indirect pathway in awake, unstressed, control and diabetic (90% pancreatectomy) rats, Euglycemic (6.0 mM) and hyperglycemic (11.7 and 17.2 mM) clamp studies were performed with [3-3H] - and [U-14C]-glucose under postabsorptive conditions and identical hyperinsulinemia (~3.3 nM). Total glucose uptake and muscle glycogen synthesis were decreased in diabetic vs control during all studies, while glycolytic rates were similar. Hepatic UDPGlucose (measured by HPLC) was increased in diabetic vs control and unmodified by hyperglycemia. The glycogen synthesized by the direct pathway (hepatic 3H-UDPG/portal vein 3H-glucose specific activities ratio) was similar in diabetic (7+2%) and control (8+2%) under euglycemia, and increased in both groups during hyperglycemia. However, the direct pathway was decreased in diabetic vs control during hyperglycemia (11.7 raM: 27_+3% vs 45_+2%; 17.2 raM: 39_+3% vs 65___4%; p<0.01). During the hyperglycemic (11.7 mM) clamp, the minimal contribution of the indirect pathway (14C/3H ratios in UDPG and infusate) was 59_+3% in diabetic and 38_+4% in control. These results suggest that: hyperglycemia is a potent regulator of the pathways of liver glycogen formation in both control and diabetic rats; the gluconeogenic pathway of liver glycogen synthesis in diabetic rats is increased.

SYNTHESIS OF HEPATIC GLYCOGEN BY THE INDIRECT PATHWAY DETERMINES THE AMOUNT FORMED DIRECTLY FROM GLUCOSE Z. Zhang and J. Radziuk, Ottawa, Canada The formation of glycogen by the indirect or gluconeogentic pathway is necessary for normal hepatic glycogen repletion. To determine whether this pathway also influences the direct formation of glycogen from glucose, 8 rat livers were perfusedwith oxygenated blood for two hours. The initial perfusate glucose and lactate concentrations were 257_+Smg/dl and 50+2mg/dl respectively, cl4-l-glucose was added to the perf~sate to assess direct glycogen formation. Either glucose (group I, n=4) or lactate (group II, n=4) was infused into the perfusate at img/min. Initial lactate uptake and glucose formation were identical: 2.1+0.2 and 1.5+0.3mg/min respectively. Both lactate uptake and glucose production decreased to zero (-0.3+0.3 and -1+0.2 mg/min) within 15 minutes with glucose (I)--but not wi~h lactate (II): 1.2+0.1 and 0.4+0.1mg/min after 120 minutes. The final plasma gTucose and l~ctate levels were 445+16 vs 338+33mg/dl and 8+1 vs 65_+15mg/dl in groups I and II respectively. At th~ end of the study total, directly and gluconeogenetically formed glycogen were 0.84+0.11, 0.45+0.09 and 0.39+0.08mg/g liver vs 2.44+0.47 (~<0.01), 1.03T0.17 (p<0.02)--and 1.41+0.33 (p<0.02)--mg/g liver for glucose (I) vs (II) lactate--infusion. These data indicate that continued lactate uptake by the liver with gluconeogenetic glycogen formation determine not only the amount of glycogen formed by this route, but also by direct synthesis from glucose.

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QE~Vr.L'.tm.TIVE CC~/PARISCN OF ~HE PA~}Z~m~S OF HEPATIC GLYfDGEN REPLETION IN FED AND FAb'rmu MAN G.I. Shulman, G. Cline, W.C. Schumann, V. (~landramouli, K. Kumaran, B.R. Iandau. Division of Endocrinology, Yale University School of Medicine, USA. The effect of fasting versus refeeding on hepatic glycogen repletion by the direct pathway, i.e. glucose -> glucose-6-P -> glycogen, was determined. Acetaminophen was administered during an infusion of glucose labeled with [I-13C]- and [6-14C]glucose into four healthy volunteel~ follmwing an c~ernight fast and in the same subjects four hours after breakfast. 13C enrichments in carbons 1 and 6 of the glucose formed frcm urinary acetaminophen glucuronide cc~pared to enrichments in carbons 1 and 6 of plasma glucose provided an estimate of glycogen foliation by the direct pathway. ~he specific activity of glucose f-rcm the glucuronide c c ~ to the specific activity.~f the plasma glucose, along with the percentages of " C in carbons 1 and 6 of the glucose frc~ the glucuronide, also provided an estimate of the amount of glycogen formed by the direct =Pa_14t~y. The estimates were similar. Those f r c ~ [6-• C]glucose would have been higher than frcm [l-~C]glucose, if the pentose cycle contribution to overall glucose utilization had been significant. After an overnight fast, during the last hour of infusion, 49+3% of the glycogen formed, was formed via the direct pathway. Following breakfast, at similar plasma glucose and insulin concentrations, the percentage increased to 69+7% (p <0.02). Thus, the contributions of the pathways to hepatic glycogen formation depend on the dietary state of the individual. Under normal conditions, where individuals consume multiple meals per day, most hepatic glycogen repletion prebably occurs by the direct pathway.

INSULIN INHIBITS GLUCOSE PRODUCTION M A I N L Y AS A RESULT OF ITS PERIPHERAL ACTION IN DIABETES A. Giacca, S. Fishe{, R. Gupta, Z. Shi, L. Lickley, and M. Vrani~. Depts. of Physiology, Medicine and Surgery, Univ. of Toronto. It is believed that peripheral h y p e r i n s u l i n a e m i a is required in treating Type 1 diabetes to suppress glucose production (Ra), because of absent p o r t o - p e r i p h e r a l insulin gradient. To determine whether peripheral infusion is less efficient than portal in suppressing Ra, we infused equimolar insulin (5.4 pmol/kg/min) p e r i p h e r a l l y (n=8, i n s u l i n = 380 pM) or p o r t a l l y (n=6, 280 pM) in depancreatised dogs, m a i n t a i n i n g a 3-hour hyperglycaemic ( - 1 0 m M ) glucose and specific activity clamp (matched stepped tracer infusion = MSTI). Peripheral infusion was more effective than portal on glucose uptake (Rd, two-fold higher increase) and, surprisingly, also on Ra (82+9% suppression vs 62~6%, p<0.01). This could Feflect larger suppression of gluconeogenic precursors (alanine, 45~4 vs 30~4%, p<0.05; glycerol, 57+3 vs 44~7%, p<0.01) and FFA (78~3 vs 6 6 + 6 % ~ p<0.01). In diabetes, glucose cycling (difference between HPLC-purified T2 & T6glucose) was increased three-fold; paradoxically, peripheral hyperinsulinaemia augmented it further. Portal (5.4 pmol/kg/min) and p e r i p h e r a l (2.7 pmol/kg/min, n=5) infusions, resulting in comparable insulin and Rd, suppressed Ra equally (62!6 vs 70!4%) without affecting cycling. Therefore, peripheral h y p e r i n s u l i n a e m i a is not required to adequately suppress Ra with peripheral infusion. Conclusions: using MSTI, in diabetes, i) Insulin inhibits Ra m a i n l y by peripheral mechanisms (suppressed gluconeogenic precursors and FFA), 2) Hepatic cycling was increased three-fold and, acutely, did not decrease with insulin.

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OP 45 Hall A-3 Sulphonylurea and K-channels in B-cells 265

266

MGZ+ATP CONTROLS GLIBENCLAMIDE- AND DIAZOXIDEBINDING TO THEIR RECEPTOR IN PANCREATIC B-CELLS

ADP STIMULATES

M.Schwanstecher, S.Ltser, I.Rietze and U.Panten. Institute of Pharmacology and Toxicology, University of Gtttingen, FRG

K. Bokvist, F.M. Ashcroft , P.-O. Berggren and P. Rorsman. Department of Medio~l Physics, Gothenburg University, Gothenburg, ,Sweden, University Laboratory of Physiology, Oxford, UK and Department of Endocrinology, Karolinska Institute, Stockholm, Sweden

Evidence was presented recently that sulfonylurea-induced closing and diazoxide-induced opening of the ATP-sensitive K+-channnel require phosphorylation of the drug receptor and/or channel. We investigated in microsomes obtained from mouse pancreatic islets whether binding of glibenclamide to its receptor and displacement of glibenclamide-blnding by diazoxide are altered by the presence of both Mg2+ and ATP or related nucleotides. Equilibrium binding revealed that MgATP [0.1 mmol/l) increased the dissociation constant (Kr~) for binding of ['H]-g!ibenclamide fourfold (p<0.01). This effect on [3H]-glibenclamide-binding was not observed in the presence of the nonhydrolysable ATP-analogue 5'-adenylylimidodiphosphate (AMP-PNP) or in the absence of Mg2+. When added in the presence of glucose and hexokinase to prevent formation of ATP, MgADP, MgGTP or MgGDP did not inhibit binding of [3H]-glibenclamide (0.2 nmoi/1). In the absence of MgATP dlazoxide concentrations up to 200 gmol/l did net displace specifically bound [3H]-glibenclamide. However, when the affinity of [3H]-glibenclamide for its binding site was decreased by adding MgATP, the half maximally displacing concentration of diazoxide was 150-+8 gmol/l. The data suggest that the affinity of the sulfonylurea receptor is controlled by protein phosphorylation and diazoxide exerts its effect by interaction with the phosphorylated state of the receptor.

AND INHIBITS THE ATP-REGULATED POTASSIUM CHANNEL IN MOUSE f~-CELLS BY BINDING TO SEPARATE SITES

ATP-regulated K+-channels (K-ATP) determine the membrane potential of pancreatic B-cells. Glucose metabolism increases cytoselic ATP/ADP resulting in channel closure, membrane depolarization, initiation of electrical activity and insulin secretion. We have investigated the interactions of ATP and ADP in controlling channel activity by recording single K-ATP channel currents from inside-out patches of mouse ~-cells. All experiments were performed in the presence of 1 mM intracellular Mg 2+. i00 gM ATP blocked K-ATP by 9SZ. Immediately after patch isolation, ADP (I00 gM) stimulated K-ATP activity in the absence of ATP and activated channels inhibited by ATP. At higher concentrations, ADP inhibited channel activity (Ki ~ S mM). Whereas the ability of ADP to increase channel activity, both in the absence or presence of ATP, declined with time after patch excision, the blocking effect of ADP became more pronounced (Ki ~ 0.2 mM after 40 minutes). These results suggest the activating and inhibitory actions of ADP are mediated at separate sites. They further indicate that ADP does not relieve ATP-induced inhibition by competing with ATP for a common inhibitory site because: i) activating cohcentrations of ADP have no inhibitory effect; 2) the stimulatory effect of ADP is lost with time whereas its inhibitory action persists.

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PERFORATED PATCH RECORDINGS OF ATP-REGULATEDK-CURRENTS AND ELECTRICALACTIVITYIN MURINEPANCREATICB-CELLS FM Ashcroft, M Faehling, CMS Fewtrell, P Rorsman and PA Smith. University Laboratory .of Physiology, Parks Rd., Oxford, OX1 3PT, UK. The effects of sulphonylureas and diazoxide on B-cell membrane potential suggest that glucose (0-20raM) controls B-cell electrical activity, and insulin secretion, by graded inhibition of ATP-regulated K-channels (K-ATP channels). Single channel recordings, however, suggest that most K-ATP channels are closed above 7raM glucose. We have used the perforated patch configuration, which retains cellular metabolism intact, to investigate the effects of glucose on membrane potential and whole-cell K-ATP currents in mouse B-cells at 30-32 ~ Glucose (8raM) decreased K-ATP currents and stimulated bursts of electrical activity resembling those recorded from intact islets. Subsequent addition of tolbutamide (25gM) or glucose (20raM) further decreased K-ATP currents and induced continuous electrical activity. The intensity of electrical activity was closely correlated with K-ATP current amplitude suggesting that regulation of K-ATP channels controls glucose-induced electrical activity in B-cells. The relationship between glucose concentration and both K-ATP current and electrical activity showed a marked heterogeneity between cells (Ki for channel inhibition; 3 to 10raM glucose). This may account for the heterogeneity of insulin secretion found in single B-cells using the reverse haemolytic plaque assay.

TOLBUTAMIDE STIMULATES Na + E N T R Y INTO P A N C R E A T I C

~-CELLS L. Ali, E. Grapengiesser, E. Gylfe, B. H e l l m a n and P.E. Lund. Department of Medical Cell Biology, University of Uppsala, Sweden Hypoglycemic sulfonylureas differ from glucose and other secretagogues, which inhibit the K+ permeability, in increasing rather than decreasing the sodium content in pancreatic islets. Dual wavelength fluorometry and the indicator SBF1 have now been employed for measuring the concentration of free Na+ in single B-cells. Also with this approach tolbutamide (100 ]Jmol/1) acted oppositely to glucose and raised intracellular Na+ (60%). To evaluate whether this effect was due to increased influx and/or inhibited efflux, integrating flame photometry was used for analyzing how tolbutamide affected the sodium content after removal of, and subsequent re-introduction of, extracellular Na+. In the Na+-deficient medium glucose accelerated the loss of sodium both in the presence and absence of ouabain~ whereas tolbutamide lacked such an effect. However, tolbutamide differed from glucose in effectively promoting the accumulation of Na+ when the ion was re-introduced into a previously depleted medium. The results provide additional arguments for a sulfonylurea-induced increase of islet sodium and indicate that this effect is mediated by stimulated entry into the B-cells. The increased influx of Na+ may contribute to the insulin secretory response by augmenting the rise of cytoplasmic Ca 2+ induced by the depolarization of the ~-cells.

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SOLUBILIZATION AND CHARACTERIZATION OF THE g-CELL SULFONYLUREA RECEPTOR. S.J.H.Ashcroft and I. Niki. Nuffield Department of Clinical Biochemistry,John RadcliffeHospital, Headington, Oxford, U.K. There is evidence that the high affinity sulfonylurea binding sites in HIT T15 t~-cell membranes can be equated with the ATP-sensitive K-channel (or closely associated protein). The aim of this study was to solubilize the f~-cell sulfonylurea receptor in an active form and compare the properties of the soluble receptor with those previously determined in m e m b r a n e preparations and intact cells. HIT cell membranes were solubilized in a Tris-buffer c o n t a i n i n g 2% CHAPS a n d t h e n i n c u b a t e d w i t h

TISSUE DISTRIBUTION OF THE ENDOGENOUS LIGAND FOR THE CENTRAL SULFONYLUREA RECEPTOR IN RAT A. Virsolvy-Vergine & D. Bataille, Centre CNRS-INSERM de Pharmacologie-Endocrinologie, 34094 Montpellier, France

[3H]glibenclamide (Hoechst). Bound [3H]glibenclamide was separated from free by rapid gel filtration. The solubilized extract showed time- and protein concentration-dependent [3H]glibenclamide binding activity. More than 85% of the binding was displaced by unlabeled glibenclamide or tolbutamide (half maximal displacement with 14nM and 125~tM, respectively). The curve relating specific [3H]glibenclamide binding by the solubilized receptor to glibenclamide concentration was hyperbolic; Scatchard analysis indicated a K d of 7nM and t~max of 90 f m o l / m g protein. These to those seen with microsomes or p r e s e n t findings s h o w that the receptor can be solubilized with sulfonylurea binding activity.

values are comparable whole HIT cells. The t~-cell glibenclamide retention of specific

We have p r e v i o u s l y s h o w n (F.E.B.S. Lett. 242: 65-69, 1988) that the rat central n e r v o u s system (CNS) contains an endogenous ligand, of pepfidic nature, for the hypoglycemic sulfonylurea receptor present in the CNS. We analyzed the tissue distribution and molecular forms of this ligand in the f o l l o w i n g rat tissues: p a n c r e a s , s t o m a c h , liver, small intestine, k i d n e y and in different areas of the CNS. 3 H G l i b e n c l a m i d e b i n d i n g on m e m b r a n e s p r e p a r e d from the cortex and HPLC of acetic acid extracts from boiled tissues w e r e the analytical tools. The ligand was undetectable in s t o m a c h , liver, intestine and kidney. In h y p o t h a l a m u s , striatum, h i p p o c a m p u s , cerebeUum, medulla oblongata and cortex, the respective contents (pmol Glibenclamide e q u i v . / g w e t w e i g h t + s.e.m., n=4) were : 0.02+0.01, 0.028+0.001, 0.06+0.01, 0.02~0.01, 0.05+0.005, 0.11_+0.03. A d e s c e n d i n g a n t e r o - p o s t e r i o r g r a d i e n t was n o t e d in the cortex. In pancreas, the content was =3-fold lower than in the cortex. Two sharp peaks of activity, displaying different isoelectrie points, were o b s e r v e d in the CNS, whereas the activity in pancreas was more heterogeneous. We conclude that, in rat, the e n d o g e n o u s ligand is present, u n d e r several molecular forms, essentially in the CNS and the pancreas.

OP 46 Hall A-15 Epidemiology III 271

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DNA POLYMORPHISMSOF THE INSULIN-RECEPTORGENE IN TYPE 2 DIABETES:ASSOCIATIONWITHFASTINGBLOODGLUCOSE

GLUCOSE TOLERANCE IN A TANZANIAN COMMUNITY

M. Sten-Linder, S. Vilhetmsdotter, A. Wedell, I. Stern, T. Pollare, P. Arner, S. Efendic, R. Luft and H. Luthman. Dept. of Clinical Genetics and Endocrinology, Karolinska hospital, Stockholm, Dept. of Geriatrics, Uppsala University, Uppsala, and Dept. of Medicine, Huddinge hospital, Stockholm, Sweden.

DG McLarty, ABM Swai, N Mlingi, H Kitange, H Rosling and KGMM Alberti.

CHRONICALLY EXPOSED TO CASSAVA TOXICITY

Muhimbili Medical Centre, Dares Salaam, Tanzania. To determine glucose tolerance in a rural community with a limited protein intake and long exposure to cassava toxicity, fasting and 2-

The significance of insulin receptor gene variants in the etiology of non-insulin-dependent diabetes has been investigated by an analysis of restriction fragment length polymorphisms in a genetically homogeneous Swedish population. Seven polymorphisms at the insulin receptor locus were analyzed, and the genotype distribution was compared in 76 diabetic patients and 84 healthy controls. The control subjects were selected for fasting blood glucose below 5.2 mM and age above 44 years. The distribution of genotypes defined by an RsaI insertion polymorphism showed some difference between controls and diabetic patients (Z2=5.22, p=0.07). Homozygotes for the RsaI 6.2 kb 2-allele were overrepresented among the controls. In addition, 2/2 Rsal homozygotes among the diabetic patients treated with diet showed lower fasting blood glucose levels than 1/1 homozygotes defined by this marker (p=0.009). The remaining six DNA markers failed to reveal any association with non-insulin-dependent diabetes. These results support the hypothesis that the 6.2 kb RsaI 2allele is in linkage disequilibrium with a functionally important variant at the insulin receptor locus. The Rsal 2-allele might constitute a .predictc,r for fasting glucose levels in patients with non-insulin-dependent diabetes mellitus.

hour blood glucose levels (following administration of 75 g glucose) were measured in 1062 subjects 15 years and above in a village in North-west Tanzania (74% response rate). Diabetes and impaired glucose tolerance rates were 0.6% and 7.1% respectively, and were not significantly different from those found in 6 other villages whose major carbohydrate source was not cassava. In a random sample of subjects, most blood cyanide levels (n=102, mean=l.18 umol/1, sd=l.41), urinary thiocyanate levels (n=192, mean=517 umol/l, sd=458.4) and serum thiocyanate levels (n=198, mean=295 umol/1, sd=185.3) were elevated. Significant correlations were found between serum thiocyanate and fasting blood glucose (Kendall's tauc = 0.14, p=0.003) and urinary thiocyanate and fasting insulin levels (Kendall's tauc = -0.16, p=0.044).The significance of these correlations requires further study, but chronic cyanide exposure appears as unlikely cause of islet-cell damage in this community.

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PREVALENCE OF DIABETES AND IMPAIRED GLUCOSE TOLERANCE I N F I N N I S H M E N A G E D 70 T O 89 Y E A R S .

DIABETES

J . H . S t e n g ~ r d I, J . P e k k a n e n I, J . T u o m i l e h t o I, P . K i v i n e n 2, E. K a a r s a l o 3, A . N i s s i n e n 2 a n d M. J . K a r v o n e n 4. i. ) N a t i o n a l P u b l i c H e a l t h I n s t i t u t e , H e l s i n k i , 2. ) U n i v e r s i t y of K u o p i o , 3. ) L o i m a a D i s t r i c t H o s p i t a l , F i n l a n d , 4. ) P i o p p i , I t a l y . At the 30-year follow-up of the Finnish cohort of Seven Countries Study a standardized two-hour glucose tolerance test was performed to all the responders without antidiabetic medication. The study population consisted of 175 men aged 70-89 years in East and 228 in West Finland. The overall age and body mass index (BMI) adjusted mean fasting blood g l u c o s e (BG) l e v e l w a s 6 . 0 m m o l / l i n t h e E a s t and 6.3 mmol/l in West. The mean value of two-hour BG was significantly higher in the West (8.3 mmol/l) than in the East (7.4 mmol/l) (p<0.01). The age standardized prevalence of diabetes (DM, W H O c r i t e r i a ) w a s 20% in the West and 14% in the East. The prevalence increased with age in both areas a n d i n m e n a g e d 80 y e a r s o r m o r e i t w a s 2 7 % i n the East and 26% in the West. The age standardized prevalence of impaired glucose tolerance (IGT) w a s h i g h e r (32%) i n t h e W e s t t h a n i n t h e E a s t (19%). T h i s p r e v a l e n c e did n o t v a r y c l e a r l y w i t h age. B M I r o s e w i t h a g e from 25.4 to 27.6 in the East and decreased from 27.1 to 25.7 kg/m2 in the West. In diabetics a g e a d j u s t e d m e a n B M I w a s 27.1, i n IGT 25.8 and in normals 26.1 kg/m2 (p=0.15 between groups).

ISLET

CELL ANTIBODY NEGATIVE, INSULIN-DEPENDENT MELLITUS: TYPE I OR E A P ~ Y - O N S E T T Y P E II?

J.K. Radder, G.E. Ching-Yong, J. Wind, M. Fr~lich, *H.G.M. Geertzen and R.R.P. de Vries. University Hospital, Leiden and *Laboratory of Blood Transfusion Service, Amsterdam, The Netherlands. For investigational and immunotherapeutic purposes a clear classification of type I diabetes is necessary. In islet cell antibody negative (ICA-), insulindependent diabetics (IDD) we found a higher body mass index (BMI) and endogenous insulin reserve than in ICA positive (ICA+) IDD. Therefore, we questioned, whether ICA- IDD had type I or early-onset type II diabetes. On the basis of the prevalence of type II diabetes in the families the following division was made: group i: without or with type II diabetes in second and/or third degree relatives (n=20: Ii ICA+, 9 ICA-; type II diabetes in the families did not influence ICA, BMI, waist hip ratio (WHR), fasting C-peptide or HLA-DR typing in the patients); group 2: with type II diabetes in first degree relatives, including one or both parents (n=4; 4 ICA-). Compared to group I, group 2 had higher BMI, WHR and fasting C-peptide. In group 2 Cpeptide increased, while it decreased in group 1 during follow-up. Compared to controls, HLA-DR 3 and/or 4 was increased and HLA-DR 2 decreased in group i; in group 2 HLA-DR did not differ from controls, so group 1 has all the characteristics of type I diabetes and group 2 of early-onset type II diabetes. Conclusion: In ICA- IDD the prevalence of first degree relatives with type II diabetes, especially in one or both parents, point at early-onset type II diabetes.

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SLOW GLUCOSE REMOVAL RATE AND HYPERINSULINEMIA ARE PREDICTORS OF THE RISK OF TYPE II DIABETES. B.C. Martin, J.H. Warram, A.S. Krolewski, J.S. Soeldner and C.R. Kahn. Research division, Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, U.S.A. Although both insulin resistance and insulin defiency contribute to the pathogenesis of Type II diabetes, there is considerable debate as to which defect is primary. In order to gain insight into the temporal order of these two defects, we evaluated 155 offspring of two parents with Type II diabetes with respect to glucose clearance and insulin secretion and then followed these individuals for an average of 13 years to observe the development of Type II diabetes. For comparison, we evaluated in a similar manner 186 controls with no family history of diabetes. The groups had similar age range (16-60) and percent males. Two phenotypic characteristics distinguished offspring of diabetic parents from controls. The offspring had slower glucose removal rates (Kg) (2.1 vs 2.3 %/min, p<0.01) and higher insulin levels, both fasting (19 vs 14 gU/1, p<0.0001) and after intravenous glucose. Significance persisted after adjustment for obesity. During 13 years of follow-up 16% (25) of the offspring developed Type II diabetes. This is eight times the general population risk. The risk of diabetes increased 2 fold with decreasing Kg and 4 fold with increasing fasting insulin when the groups were divided at the medians of these variables. If both slow Kg and high fasting insulin were present, the risk was multiplied eight fold, so the two factors acted independently. Thus, one to two decades before diagnosis of diabetes, reduced glucose clearance is already present. This is a c c o m p a n i e d by c o m p e n s a t o r y h y p e r i n s u l i n e m i a , n o t hypoinsulinemia, suggesting that the primary defect is in peripheral tissue response to insulin and glucose rather than in the pancreatic beta-cell.

VERY LOW INCIDENCE OF TYPE I (INSULIN-DEPENDENT) DIABETES IN PERUVIAN POPULATION. S. S e c l ~ n , M . l . Rojas~ B.Millones, H.Valdivia~ O.NuSez~ H.Medina, W.Venturo~ F . B u s t a m a n t e and N. S~nchez. Diabetes Epidemiology Research Peruvian Group(Contributor-DERI Group), Diabetes C l i n i c , U n i v . Peruana Cayet. H e r e d i a . L i m a - P e r d . The enormus g e o g r a p h i c a l v a r i a b i l i t y o f Type I (insulin-dependent) diabetes i n c i d e n c e has been r e c o g n i z e d . Comparison of registries have shown that this type o f diabetes is predominantly a chronic autoimmune d i s e a s e that affects the w h i t e race or p o p u l a t i o n s w i t h a substantial white genetic admixture. This report describes t h e e p i d e m i o l o g y o f Type i (Insulin-dependent) diabetes in peruvian population as a representative pattern of an mixed race. In Lima~ PerO with 5"659,200 hab. from which 1"944~057 hab. are less t h a n 15 y e a r s old~ a Registry of Type I ( i n s u l i n - d e p e n d e n t ) d i a b e t e s was m a d e based i n primary data s o u r c e f r o m 20 hospitals, of a l l cases o c c u r r e d between 19801988. The c r i t e r i a f o r s e l e c t i o n were= d i a g n o s e d as d i a b e t i c (WHO), be l e s s t h a n 15 y e a r s o l d a t the time o f onset of disease, be on insulin t h e r a p y and reside in Lima during 1980-1988. The cases" number were 54 f r o m w h i c h 25 were l e s s t h a n 15 y e a r s o l d i n 1988 w i t h a prevalence 1.2/i00~000 hab. The annual age-adjusted i n c i d e n c e was 0.7/100,000 hab. The s e c o n d a r y data source was the Diabetes Peruvian Association with SO% v a l i d a t i o n . The v e r y low p r e v a l e n c e and i n c i d e n c e of Type I (insulindependent) diabetes in peruvian population comparated w i t h c a u c a s i c p o p u l a t i o n f r o m E u r o p e , USA, and S p a n i s h - h e r i t a g e p o p u l a t i o n s (Cuba, San Diego~ Colorado)~ indicates an mixed race probably without a substantial white genetic admixture.

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OP 47 Hall B-3 Lipids and Lipoproteins 277

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LACK OF LIPID A B N O R M A L I T I E S IN TYPE I D I A B E T I C S WITH M I C R O A L B U M I N U R I A IN GOOD M E T A B O L I C CONTROL A. Lala, A. Scoppola, M.G. Felici, E. Civetta, R. G a t t a , S. F r e n t o n i , S. G a m b a r d e l l a a n d G. Menzinger - Malattie del Ricambio - II U n i v e r s i t & di Roma - Italy. The relationships between serum lipid, apolipoprotein levels and urinary albumin excretion were investigated in 25 t y p e I diabetic patients, 18 n o r m o a l b u m i n u r i c s (N; AER= 2.7 +1.9 ug/min) and 7 m i c r o a l b u m i n u r i c s (M; AER =56+59 ug/min) m a t c h e d for age, BMI, duration of disease and HbA 1 levels (N=8.4%; M=8.5%). The patients were admitted to the m e t a b o l i c ward and studied after several days of optimized metabolic control (mean of three daily plasma glucose determinations: N=7.6+1.5 mM; M = 7 . 6 + 1 . 8 mM) . Serum cholesterol and t riglycer-ide concentrations were assayed enzimatically. High density lipoprotein was isolated after orecipitation of V L D L a n d L D L by M n C I 2 a n d heparin. LDL cholesterol was calculated by Friedewald formula. Serum apolipoprotein concentrations were d e t e r m i n e d by immunoturbidimetry. Results were as follows: Total cholesterol (mM) 4.4+0.8 4.4+0.3 LDL cholesterol (mM) 2.9~0.8 2.6T0.7 HDL cholesterol (mM) 1.3T0.3 1.2~0.3 Chol HDL : Chol LDL 0.46~0.2 0.52T0.5 T r i g l y c e r i d e (mM) 1.03T0.2 1.2T0.3 Apo B (mg/dl) 69T9 74TI0 Apo A-I (mg/dl) 98~26 i01~15 Apo A-I : Apo B 1.38T0.4 1.37~0.2 Our study provides evidence that type 1 diabetic patients w i t h m i c r o a l b u m i n u r i a m a i n t a i n e d under optimized metabolic control do n o t s h o w a n y differences in lipid and lipoprotein levels when compared to n o r m o a l b u m i n u r i c ones.

DOES OVERNIGHT SUPPRESSION OF PLASMA NONESTERIFIED FATTY ACIDS (NEFA) REDUCE H E P A T I C GLUCOSE P R O D U C T I O N IN TYPE 2 DIABETES C.Saloranta,M-R.Taskinen,E.Widen,K.Laakkonen and L.Groop, Helsinki University Hospital, Helsinki, F i n l a n d To test this hypothesis, 20 Type 2 diabetic patients (age 60 • 3 yrs, BMI 28.3 • 0.8 kg/m 2) participated in a double-blind, placebo controlled c r o s s - o v e r study w i t h a d m i n i s t r a t i o n of a n i c o t i n i c acid derivative, A c i p i m o x (500 mg sustained release, A), or p l a c e b o (P) at 9 p.m. for 4 weeks, w i t h an intervening 2 w e e k w a s h - o u t period. At the end of each t r e a t m e n t p e r i o d hepatic g l u c o s e p r o d u c t i o n (HGP) was m e a s u r e d by 3-JH-glucose and s u b s t r a t e o x i d a t i o n by indirect calorimetry. From m i d n i g h t to 7 a.m. NEFA conc. were lower d u r i n g A vs P (195 • 14 vs 472 • 74 #mol/L, p< 0.001). However, at 8 a.m. N E F A conc. were similar (A:520 • 115, P:514 • 59 #mol/L, NS), and after Ii a.m. N E F A conc. were continuously h i g h e r d u r i n g A than P (p<0.05), reaching m a x i m u m v a l u e s at 8 p.m. (A:572 • 97, P:278 • 25 #mol/L, p<0.001). The rebound rise of N E F A was a s s o c i a t e d with a rise in lipid oxidation rate (A: 3.72 • 0.31 vs P: 2.98 • 0.41 #mol/kg.min, p<0.05) in the evening. There was no d i f f e r e n c e b e t w e e n A and P in HGP d u r i n g night (1.90 • 0.20 vs 2.02 • 0.23 mg/kg.min) or early m o r n i n g (2.03 • 0.20 vs 2.13 • 0.19 mg/kg.min). Thus, A c i p i m o x did not result in lower fasting B-glucose (A: 9.0• vs P: 8.7• mmol/L) or HbAlc (A: 8.2• vs P: 8.1fi.9 %). CONCLUSIONS: O v e r n i g h t lowering of NEFA by A c i p i m o x is a s s o c i a t e d with a s u s t a i n e d rebound rise in N E F A during the day. The question rises w h e t h e r it is p h y s i o l o g i c a l l y possible to c h r o n i c a l l y suppress lipolysis in diabetic individuals.

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U P T A K E O F FREE F A T T Y A C I D S IN P E R F U S E D S K E L E T A L M U S C L E IS A S A T U R A B L E F U N C T I O N O F U N B O U N D F A T T Y ACID CONCENTRATION.

VLDL SUBFRACTIONS IN TYPE I D I A B E T I C P A T I E N T S L.Patti, G.Romano, A . R i v e l l e s e and G.Riccardi. Institute of Internal M e d i c i n e and M e t a b o l i c Diseases, 2nd M e d i c a l School, University of Naples, Italy. VLDL lipoprotein is e n r i c h e d in e s t e r i f i e d cholesterol in type I d i a b e t i c patients irrespectively of their blood glucose and lipid values. Furthermore VLDL is h e t e r o g e n e o u s in terms of p a r t i c l e size and composition. T h e r e f o r e we e v a l u a t e d the fasting lipid composition of VLDL subfractions (A,B,C,D), isolated by d i s c o n t i nuous density gradient ultracentrifugation, in 9 male n o r m o l i p i d e m i c (cholesterol 4.43• mmol/l; t r i g l y c e r i d e s 0.88• mmol/l) (M• type I diabetic patients (age 32• yrs; BMI 25• kg/m 2) in m o d e r a t e glycemic control (HbAIc 7.6• and in 8 m a l e n o r m o g l y c e m i c controls m a t c h e d for age (31• yrs), BMI (25• kg/m2), cholesterol (4.48• mmol/l), t r i g l y c e r i d e s (0.84• mmol/l). Free and e s t e r i f i e d cholesterol (FC, EC), t r i g l y c e r i d e s (TG) and phospholipids (PL) were e v a l u a t e d in total V L D L and in each V L D L subfraction. Each c o n s t i t u e n t was e x p r e s s e d as % of the total VLDL lipid mass. As expected VLDL was e n r i c h e d in TC(17• vs15• p<0.025) and EC (9• vs 7• p<0.025) and dep l e t e d in TG (60• vs 64• p<0.05) in type 1 diabetic patients. This composition a b n o r m a l i t y was confined only to the smallest p a r t i c l e s (VLDL D) enriched in TC(23• vs 20• EC (13• vs I0• p<0.05) and PL (29• vs 25• p < 0 . 0 0 1 ) . I n conclusion, V L D L composition abnormalities in type i diabetic patients are present only in the subfraction D c o n t a i n i n g the smallest and most a t h e r o g e n i c particles. Cholesterol enrichment of V L D L D might contribute to the increased c a r d i o v a s c u l a r risk of type i diabetic patients.

N

M

L.P.Turcotte, B.Kiens and E.A.Richter. A u g u s t Krogh Institute, U n i v e r s i t y of C o p e n h a g e n , Copenhagen, Denmark. Recent evidence has shown that cellular uptake of free fatty acids by isolated hepatocytes, a d i p o c y t e s and c a r d i a c m y o c y t e s i s a s a t u r a b l e f u n c t i o n o f the unbound plasma f r e e f a t t y acid c o n c e n t r a t i o n . We i n v e s t i g a t e d the e x i s t e n c e o f s i m i l a r uptake k i n e t i c s in a p h y s i o l o g i c a l s k e l e t a l muscle p r e p a r a t i o n by using the i s o l a ted perfused r a t h i n d q u a r t e r . Gluco~9 (6 mM), t r a c e r amounts o f albumin-bound (U-'~C)palmi r a t e and v a r y i n g amounts o f albumin-bound p a l m i t a t e (30 t o 2200 uM) were added t o the p e r f u s i o n medium. F r a c t i o n a l uptake o f palmitate across the hindquarter d e c r e a s e d f r o m 0.11 t o .024 w i t h i n c r e @ s i n g p a l m i t a t e d e l i v e r y (6 t o 60 nmol/min-gm- ~). Palmitate uptake was linearly related to both total plasma palmitate c o n c e n t r a t i o n (r=0.97) and palmitate delivery (r=0.95). However, when plotted against the calculated unbound palmitate concentration, palmitate uptake fitted a simple MichaelieMenten relation. The calculated Vmax (19.1 + 0.97 nmol/min.sm-') and Km (0.08 + 0.02 umol/l) did not vary over 40 minutes of perfusion at rest. Over the range of palmitate concentrations s t u d i e d , p e r c e n t palmitate oxidation remained unchanged. Increasing plasma palmitate c o n c e n t r a t i o n s did not a~fect glucose uptake (59.6 + 3.1 nmol/min.gm-'). These results show that, as in isolated cell systems, uptake of free fatty acids in perfused skeletal muscle f o l l o w s saturation k i n e t i c s c o n s i s t e n t with c a r r i e r - m e d i a t e d membrane t r a n s p o r t o f f r e e f a t t y acids.

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D N A R E S T R I C T I O N P O L V M O R P f 4 I S M IN A P O L I P O P R O T N I N A I - C I l I - A I V G E N E C L U S T E R A S S O C I A T E D WITH M A C R O ANGIOPATHY IN T Y P E 2 (NON-INSULIN-DEPeNDENT) OIABETES E. Renard, A . M . Ounuy, L. Monnier and A. C r a s t e s de Paulet Laneyronie Hosoital - ~" 34059 Montoellier Cedex - France.

SIMVASTATIN INCREASES ALPHA- AND GAMMA-TOCOPHEROL IN RED BLOOD CELLS OF PATIENTS WITH NON-INSULIN-DEPENDENT DIABETES MELLITUS. A. Diemers, A. Alberda, R. Zwertbroek, H.J.R. Velvis and J.J. van Doormaal. Dept of Endocrinology and Central Lab for Clinical Chemistry, University Hospital, Groningen, The Netherlands.

The disparity of cardiovascular complications in tyoe 2 (non-insulin-dependent) diabetes and their frequent association with low HDL and high triglycerides levels led us to look for a genetic determinism exores~ed by ONA restriction polymornhism in apoliDonrotein AI-CIII-AIV 9ene cluster. Genomic DNA from 27 individuals without personnal or familial metabolic or cardiovascular diseases and from 45 type 2 (non-insulin-deoendent) diabetic patients, all Caucasians, was restricted by Sstl, Msol and Pstl enzymes and hybridized with a radiolabelled aoolipoprotein A1 gene probe. 30 diabetic oatients showed no cardiovascular complication and 15 were affected by coronary heart disease or arteriopathy. The frequency of polymorphic sites (expressed by ,92 allele for Sstl, M2 for Mspl and P2 for Pstl) was consistent with previous studies in the healthy group : 5 % $2, 4 % M2, 9 % P2, and not different for the whole diabetic @rouo : 3 % $2, 6 % M2, 5 % P2. However $2 allele and S2M2PI haplotype were respectively 4.9 and 5.1 times more frequent in the macroanqiopathic subgroup (Woolf ratio) ; the relative incidence of M2 and P2 alleles was respectively 1.5 and 1.3. No plasmatic lipid abnormalities could be related to the presence of an heterozygous qenotype. A genetic predisposition to macroangiopathv in type 2 (non-insulin-dependent) diabetes seems likely even if its mechanisms remain unprecise.

In diabetes mellitus, increased lipid peroxidation and lowered cellular contents of the antioxidant vitamin E have been found. Vitamin E is delivered to cells by plasma lipoproteins. To evaluate the effect of the lipid-lowering drug simvastatin (S) on alpha (~)- and gamma (y)-tocopherol (T) in red blood cells (RBC), 9 patients (ages 40-67 years) with Type 2 (non-insulindependent) diabetes mellitus and hypercholesterolaemia (cholesterol at baseline after >4 weeks on diet: median 7.31, range 6.69-8.65 mmol/l) were subsequently treated with placebo (P)(4 weeks) and S in a daily dose of 10 mg (6 weeks), 20 mg (6 weeks) and 40 mg (6 weeks). Diabetes was treated by diet and sulphonylureas alone. RBC aT and yT increased after use of S during 18 weeks in all (medians: at baseline 5.34 and 0.40 nmol/1010 cells, change after P: 0.0 and +19.5% (NS), change after S: +35.5 and +55.6% (p<0.01)). Plasma ~T and 7T, expressed as ratios to the sum of cholesterol and triglycerides did not change. Serum cholesterol decreased in all (change after P : +1.2% (NS), change after S: -30.9% (p<0.01)). Serum triglycerides, glycosylated haemoglobin and fasting blood glucose did not change. Conclusion: S increases aT and 7T in RBC of Type 2 diabetic patients with hypercholesterolaemia.

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OP 48 Hall A-18 Novel G e n e s and Diabetes 283

284

REGULATION OF ISLET GROWTH IN THE PREDIABETIC BB/S RAT: (II) THE ROLE OF NOVEL ISLET REGENERATING GENES STUDIED BY NORTHERN/DOT BLOT ANALYSIS A.J. Bone, K.A. Webster and S.H. Banister, Endocrine Section, Medicine II, Southampton General Hospital, Southampton, UK The presence of a novel regenerating gene ( 'Reg' ) has been reported in regenerating islets in an animal model of surgical diabetes. We have used serial pancreatic biopsies to investigate the presence/expression of preproinsulin and 'Reg' genes. Polymerase chain reaction (PCR) amplified DNA from prediabetic rat pancreatic sections with increased islet cell proliferation confirmed the presence of the 'Reg' gene. RNA was prepared from snap-frozen pancreatic biopsies of diabetes prone (DP) and diabetes resistant (DR) BB/S rats by homogenisation in guanidinium thiocyanate followed by ultracentrifugation in caesium chloride. Northern blot analysis showed expression of 'Reg' mRNA. Dot blot analysis was used to semi-quantify amounts of mRNA for 'Reg' and preproinsulin. Increased amounts of 'Reg' mRNA (compared to normal Wistars) were detected in serial biopsies from a DP prediabetic rat 90 and 65 days prior to hyper glycaemia, levels subsequently ~ec lined in third and fourth biopsies taken 45 days before and seven days after onset of diabetes. Levels of preproinsu!in mRNA showed a similar pattern with peak amounts in the' second biopsy. 'Reg' mRNA levels in age-matched biopsies from a DP animal that remained normoglycaemic appeared higher than those observed in the prediabetic rat. We suggest that a relationship may exist between 'Reg' gene expression and development of diabetes in the BB/S rat.

E X P R E S S I O N O F H U M A N C O M P L E M E N T R E C E P T O R 2 (CR2) ON RINm5f CELLS INDUCES REJECTION BY SYNGENEIC RATS, J . - C . C a r e l , V . M . H o l e r s , L. F a l q u i a n d P . E . L a c y . W a s h i n g t o n U n i v . Sch. of M e d . , s t L o u i s , MO 63110 and INSERM U188, Paris. To study the consequences of neoantigen expression on an insulinoma line, we used retroviral constructs to transduce a full length CR2 (C3d/Epstein-Barr virus receptor) c D N A in s e n s e (~) o r a n t i s e n s e (B) o r i e n t a t i o n s to the rat insulinoma cell line RINm5F. CR2 was detected by FACS analysis on 98% of RINm5FCR2~ cells but not on parental or RINm5F-CR2B cells. CR2 allowed EBV infection of ~0.5% of RINm5F-CR2~ cells. Insulin secretion was decreased in R I N m 5 F - C R 2 ~ c e l l s as c o m p a r e d t o parental c e l l s (-60% a t b a s e l i n e , -78% after stimulation w i t h K+) a n d w a s n o t a f f e c t e d in RINm5F-CR2B cells. RINmSF-CR2B cells transplanted to syngeneic NEDH rats produced tumors, hypoglycemia and hyperinsulinemia before d a y (d.) 14 p o s t t r a n s p l a n t a t i o n and these tumors were devoid of inflammatory infiltrate. In c o n t r a s t , RINm5F-CR2~ cells were heavily infiltrated with mononuclear cells at d.8 and d.ll and produced tumors and h y p o g l y c e m i a a r o u n d d.28. These tumors did not express CR2 and arise f r o m a CR2 negative population p r e s e n t in R I N m 5 F ~ C R 2 ~ cells. We c o n c l u d e t h a t e x p r e s s i o n of a h u m a n x e n o a n t i g e n on an insulinoma cell line induces rejection by syngeneic rats but does not break tolerance to revertant cells which do not express this xenoantigen or to pancreatic islets.

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C O M P L E M E N T C4 AND HEAT SHOCK PROTEIN 70 G E N O T Y P E S AND TYPE I DIABETES A.G. Demaine, N.J. Caplen, R.D. Campbell and B.~. Millward. Depts. of Medicine and DiAbetes, Kings College School of Medicine, London SE5 8RX and MRC I ~ n u n o c h e m i s t r y Unit, Oxford. Type I diabetes is strongly a s s o c i a t e d with genes of the Major H i s t o c o m p a t i b i l i t y Complex (MHC) class II (DR and DQ) and to a lessor extent class III (complement C4). Recently, a number of new genes have been localised between the C4 and TNF loci of the class III region. We have used r e s t r i c t i o n fragment length polymorphism (RFLP) analysis to investigate the C4 and heat shock p r o t e i n (HSP)-70 loci of 167 patients with type I diabetes and 92 normal controls. In the patient p o p u l a t i o n there was a large excess of deletions of the C4A locus (48.5% vs. 22.1% p=0.0005). The HSP-70 probe i n c o n j u n c t i o n with Pst-I detects two alleles of 9 or 8.5 kilobase (kb). There was a significant increase of the 8.5 kb allele in the patient group compared to controls (0.551 vs. 0.353 p=0.005). Further, there was only one case in either the patient or control group of the C4A deletion occurring without the 8.5 kb HSP-70 allele. Finally, the 3 2 ; 1 5 ; 8 . 5 / 9 ; 8 . 5 C4/HSP-70 genotype occurred in 55% of those patients with r e t i n o p a t h y without n e p h r o p a t h y c o m p a r e d to 17.0% of patients with no m i c r o v a s o u l a r c o m p l i c a t i o n s after 20 years of diabetes (p=0.005), suggesting that this region may contain gene(s) which p r e d i s p o s e to retinopathy.

H E A T S H O C K P R O T E I N I N D U C T I O N BY I N T E R L E U K I N - I B IN RAT P A N C R E A T I C ISLETS. S.Helqvist, B.Polla, J . J o h a n n e s e n and J.Nerup. Steno M e m o r i a l Hospital, D e n m a r k and H o p i t a l Cantonal, Geneva, Switzerland. The m e c h a n i s m of a c t i o n b e h i n d the c y t o t o x i c effects of the b e t a - c e l l toxin i n t e r l e u k i n - I B (ILl) is not known. We p r e v i o u s l y h y p o t h e s i z e d that IL-I e x e r t s b e t a - c y t o t o x i c i t y t h r o u g h t o x i c free o x y g e n radical (FOR) formation. Since FOR act i v a t e s h e a t shock p r o t e i n (HSP) s y n t h e s i s d u r i n g n o n - l e t h a l c y t o t o x i c i t y , we s t u d i e d the p r o t e i n s y n t h e s i s p a t t e r n in i s o l a t e d rat p a n c r e a t i c islets e x p o s e d to h e a t (43 ~ , 30 min) or r h I L - ! B (150 pg/ml, 24 h) by use of S D S - P A G E and a u t o r a diography. R h I L - I B i n d u c e d the s y n t h e s i s of t h r e e p r o t e i n s of 32, 73 and 90 kDa. By W e s t e r n - b l o t t ing and p a r t i a l p e p t i d e mapping, the 73 and 32 kDa p r o t e i n s w e r e d e m o n s t r a t e d to be an H S P and h e m e oxygenase, respectively. H e a t shock i n d u c e d the s y n t h e s i s of 65, 72, 73, 90, ii0 kDa HSPs. The IL-I specific, islet-specific p a t t e r n of p r o t e i n s y n t h e s i s indicates that r h I L - I B e x e r t s FOR f o r m a t i o n in islet cells. The r h I L - I B i n d u c e d 73 kDa p r o t e i n is e s p e c i a l l y interesting, since the gene of HSP 73 was r e c e n t l y d e m o n s t r a t e d to be l o c a t e d in the MHC class III g e n e region. Since HSPs r e p r e s e n t c e l l u l a r d e f e n s e and a HSP 73 gene p o l y m o r p h i s m is r e l a t e d to IDDM s u s c e p tibility, these o b s e r v a t i o n s m a y be p a t h o g e n e t i c a l l y important.

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PRELIMINARY IDENTIFICATION OF THE PRIMARY BETA CELL ANTIGEN AS BEAT SHOCK PROTEIN 65 D.B.Jones and N.R. Hunter, Department of Medicine, Western General Hospital and Scottish National Blood Transfusion Service, Edinburgh, Scotland, U.K. The p r i m a r y beta cell antigen of insulindependent diabetes is considered to be a 64kD protein identified by Baekkeskov I. We have demonstrated that the 64kD protein may be fnduced in rat i n s u l i n o m a (RIN) cells by incubation with the cytokines interleukin I (10 u/ml), y interferon (]0 u/ml) and tumour necrosis factor (2.5x10-*mol) but also by c u l t u r i n g the cells at 42~ for I hour. Furthermore the RIN cell 64kD protein showed cross-reactivity on Western blots with monoclonal antibody directed against Mycobacterium Tuberculosis heat shock protein 65 but not with antibodies directed against a similar epitope of Mycobacterium Leprae heat shock protein 65. Indirect immunofluerescence studies demonstrated increased binding of monoclonal antibodies directed against Mycobacterium Tuberculosis heat shock protein 65 to RIN cells cultured with interleukin IB (10 u/ml). The 64kD protein appears to be a heat shock protein. The epitope specificity of the monoclonal antibodies used suggests that the amino acid region 200-230 confers autoreactivity of the heat shock p r o t e i n 65 in insulindependent diabetes. I. Baekkeskov, Nature 1982.

INSULIN-DEPENDENT DIABETES - A POLYGENIC DISEASE? IMPLICATIONS OF TUMOR NECROSIS FACTOR 8 AND HEAT SHOCK PROTEIN 73 POLYMORPHISMS. F.Pociot, J.Mglvig, LWogensen, C.Milner, R.D.Campbeil and J.Nerup. Steno Memorial Hospital, Gentofte, Denmark, University of Oxford, UK. We suggest that IDDM is a polygenic disease, where susceptibility may be conferred by normal alleles in rare, unfavourable combinations. This hypothesis is substantiated by the examples of the TNFB-gene and the heat shock protein 73 (HSP73), both located in the MHC class III region. We show that a diallelic Ncol polymorphism of the TNFBgene exists (5.5 and 10.5 kb), and demonstrate that the TNF8 10.5 allele associates with IDDM on DR4-haplotypes. TNFB 10.5 homozygosity is 3 times more frequent in patients than in DR-, DQ-matched controls. Furthermore, IDDMpatients have a significantly higer in vitro monokine response than controls, when matched for TNFB-genotype. In addition, a diallelic PsU polymorphism of the HSP73-gene consisting of fragments of 8.5 and 9.0 kb is identified. A significant difference was observed between diabetics and controls (p<0.03). This is clue to a strong association of the 8.5 allele to IDDM on DR3-haplotypes. Fifty of 54 IDDMpatients had this allele versus 14 of 24 random controls (p = 0.0013). In conclusion, TNFB-genotypes reflect secretory capacity of monokines involved in the pathogenetic process. HSP's are important scavengers of free oxygen radicals, which may act as secondary mediators in B-cell destruction. Thus, through quantitative control of elements of IDDM pathogenesis, polymorphisms of TNFB and HSP73 genes may contribute to IDDM susceptibility.

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OP 49 Hall A-1

Hypoglycaemia 289 COGNITIVE

290 FUNCTIONS

AND

CEREBRAL

BLOOD

FLOW

IN TYPE

I

Psychological

causes for

severe

hypoglycemias

DIABETIC PATIENTS DURING HYPOGLYCAEMIA

L. Heinemann, W. Rautenberg*, A.A.R. Starke, I. M~hlhauser, M. Hennerici , and M. Berger. Dept. of Nutrition, University of D~sseldorf and Neurological Depts., Universities of Mannheim and D~sseldorf (FRG)

A. H i r s c h and U. B s k k e r - G r e v e , Krankenhaus Bethanien, Martinistr. 4 4 - 4 6 , 2000 Hamburg 20, FRG

Efficiency of brain performance depends critically upon glucose uptake; therefore cognitive functions may become deteriorated during hypoglycaemia. In 8 type-I (insulindependent) diabetic patients in good metabolic control (GDiab) (mean HbAlc (SD) 6.3 (0.4) %), 8 patients in moderate metabolic control (MDiab) (8.8 (0.7) %) and 8 healthy normals (NOR) blood glucose concentration (BG) was lowered stepwise and clamped for 45 min each at 5.0, 3.6, and 2.5 mmol/l. Blood flow through the arteria cerebri media was measured by a transcranial doppler and latency (P300 response) of acustically evoked potentials as a parameter of cognitive function was registered. At 3.6 mmol/l mean latency of the P300 response increased by 27.3 ms in NOR (p<0.04), by 1.8 ms in GDiab, and by 10.5 ms in MDiab. Lowering to 2.5 mmol/l resulted in an additional increase of the latencies to a total of 42.8 (p<0.01), 9.0 (NS), and 21.2 ms (p<0.02)o Peak velocity in arteria cerebri media rose by 4.6, 5.2 and 4.2 cm/s at 3.6 mmol/1, and by 12.0 (p<0.004), 22.8 (p<0.004) and 25.3 cm/s (p<0.003) at 2.5 mmol/1 BG vs basal levels. NOR showed the lowest increase of cerebral blood flow while the latency of the P300 response rose by 15 %, as compared to 8 % in MDiab, and 3 % in GDiab. Conclusions: effective glucose transport due to increased cerebral blood flow may have caused a significantly lower increase in the latency of the P300 response in diabetic patients. Significant differences of the P300 latency between GDiab and MDiab were not found.

Severe hypoglycemias are a serious problem, even for diabetes-educated patients. I n an a t tempt to evaluate the reasons for these difficulties we s t u d i e d 15 p t s who had a t l e a s t o n e severe hypoglycemia in the year before their two-week inpatient diabetes education. Six months after education pts were interviewed for 3 - 5 hs t o f i n d o u t i f t h e e d u c a t i o n was s u c cessful in reducing hypoglycemias, A short questionnaire was g i v e n c o n c e r n i n g t h e i r perception, evaluation, and b e h a v i o r w i t h r e s p e c t t o severe hypoglycemias, Results: The c o n t e x t o f severe hypoglycemias was a s s e s s e d i n d i v i d u a l l y . S i x p t s who c o n t i n u e d t o h a v e s e v e r e h y p o glycemias after education (non responding group) were compared to the other 9 pts without further hypoglycemias (responding group). There w e r e no d i f f e r e n c e s in self-attributed awareness to hypoglycemia. Significant differences in questionnare s c o r e s f o r t h e non r e s p o n d i g g r o u p w e r e : s e e i n g h y p o g l y c e m i a s as a c h a n c e f o r a v o i d i n g demands ( m o r e ) ; a v o i d i n g t o t a k e g l u c o s e when h y p o g l y c e m i c ( m o r e ) ; d e p r e s s i o n ( m o r e ) . Two p t s r e p o r t e d p l e a s a n t f e e l i n g s before, while or after b e i n g h y p o g y c e m i c , and they tolerated or even actively caused hypoglycemias. Severe hypoglycemias in educated patients were m a i n l y a c c o u n t e d f o r by p s y c h o l o g i c a l causes.

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EFFECT OF INTERMITTENT HYPOGLYCAEMIA ON COUNTERREGULATOR~ HORMONE SECRETION. B. Widom and D.C. Simonson, Joslin Diabetes Center, Boston, USA. To determine whether intermittent hypoglyeaemia contributes to the impaired counterregulatory hormone secretion and diminished autonomic symptoms seen in intensively treated diabetes, we performed hypoglycaemic insulin clamps (2 mU/kg.min) on 4 consecutive days in 7 healthy, non-diabetic subjects. A stepwise glucose decline of 0.56 mmol/L each 30 minutes was produced on days 1 (pre) and 4 (post), and a sustained 1 hour hypoglycaemic stimulus was produced on days 2 and 3 (mean glucose nadirs each day = 2.3-2.6 mmol/L). Basal hormone levels were similar in pre and post studies (epinephrine--295• vs 175• pmol/L; cortisol--417iSlvs 319• glucagon--49• vs 41+13 ng/L; growth hormone--2.8• vs 3.4• #g/L). However, glucose levels at which sustained increases in epinephrine (p=0.06), eortisol (p=0.01), glueagon (p-0.02), and growth hormone (p=0.06) occurred were all decreased on day 4 following episodic hypoglycemia. Glucose thresholds for autonomic symptoms (2.9 vs 2.4 mmol/L; p-O.04), but not neuroglycopenic symptoms (2.4 vs 2.2 mmol/L; p=NS), were also reduced. Conclusions: l) Glycaemic thresholds for counterregulatory hormone secretion are decreased following 3 episodes of hypoglycaemia in healthy volunteers. 2) Autonomic but not neuroglycopenic symptoms are similarly reduced. 3) Intermittent hypoglyeaemia may be an etiologic factor in the attenuated counterregulatory and symptomatic responses seen in intensively treated diabetes.

DOES SLEEP INTERFERE WITH HYPOGLYCEMIA-INDUCED GROWTH HORMONE SECRETION IN TYPE I DIABETIC PATIENTS? H. Walter, I. Faerber, I. Karg, and H. Mehnert, II1. Med. Dept. City Hospital Munich-Schwabing, Munich, F.R.G. Peaks of human growth hormone (HGH) are found in the first third of the night, timely related to slow wave sleep. Hypoglycemia also is a powerful stimulus for HGH secretion, and the role of HGH in hormonal counterregulation is well established. We studied the pattern of HGH secretion in 8 type I diabetic patients (age 24.3 + 5.4 y, 6.7 + 4.1 y of diabetes, Rb A1 10.3_+ 1.7 %) in a sleep laboratory (polygraphic sleep recording) during 2 nights (10 p.m.- 8 a.m.) of hyperglycemia (hyper) and insulin induced hypoglycemia (hypo) in random order. Blood was drawn from an indwelling i.v. catheter every 10 minutes. 8 healthy, age matched males served as controls. In contrast to the elevated baseline HGH levels (2.3 + 1.1 ng/ml) sleep related (sr) HGH secretion was not increased in D and could not be detected in 2 patients with a hypoglycemic episode before the start of the investigation. No HGH peak could be observed after insulin-induced hypoglycemia during sleep, when hypo occured within 2 hours after sr HGH secretion (4 patients). There was an exaggerated response of HGH, when hypo coincides with the sr HGH burst. Only two patients in sleep stage 1 and 2 perceived the hypoglycemia, while those in stage 3 and 4 (deep sleep) and REM did not. Our data indicate, that perception of nocturnal hypo depends on the present sleep stage , that HGH response to hypo is altered by the preceeding sr HGH secretion, and that there is a refractory period after hypo induced and sr HGH secretion.

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I N C R E A S E D GLUCOSE T R A N S P O R T z CAUSE OF RECURRENT HYPOGLYCEMIA I N I N S U L I N - D E P E N D E N T DIABETICS.

METABOLIC RESPONSE TO INSULIN INDUCED HYPOGLYCAEMIA IN PATIENTS AFTER PANCREAS AND KIDNEY TRANSPLANTATION. L Luzi,A Battezzati,A Secchi,E Larocca,C Staudaeher,V Di Carlo, G. Pozza. DeparSments of Medicine and Surgery,H San Raffaele, University of Milan, Italy. To assess the metabolic response to insulin induced hypo_ glycaemia(HVPO) , 7 Type ] (insu] Jn dependent)d~abetic !Jrae mic patients after kidney pancreas transplant(KPTx:age=S6+ Sy;IBW=SS+2%;transplant age=lO+_2mo. ;HbAlc=5.6+_O.2%;prednis_o ne:10mg/day ;oyclosporin:5mg/kg, day ;azathioprine=img/kg, day) and 6 controls(CON)were studied by means of 3--3H glucose in fusion combined with 0.SmU/kg.min insulin(I) clamp for 2h (plasma G=S.2mmol/l).Basal plasma G(4.8+O.2vs4.6+O.l) NEFA (42+2vs4823 ymol/dl ),glycerol (63+_18 }]mol/l )lactate (458+54 pmol/l), ALA(225+It)LEU(128+_9) ILE(65+4),VAL(256235),PHE(45 +6}_~mol/l)were comparable in KPTx and CON.Basal freelRl (15 vsSpU/ml)was higher in KPTx.Hepatic glucose production was similar in the basal(2.1+O.2vs2.0+O.2mg/kg.min)but more sup_ pressed in KPTx during l(60min=O.7+O.ivsl.2+O.l;p
F.J. A r r i e t a , A. C a s l a , F. R a m o s , C. G r a n t , B. Casanova, J.L. Herrera-Pombo and A. Rovira. FundaciSn Jim@nez Diaz, Universidad AutSnoma de Madrid, Spain. The mechanisms by which severe and recurrent hypoglycemia may occur in insulin-dependent diabetic patients requiring low doses of insulin, without secondary diabetic complications or failure of other endocrine glands, are of unknown origin. In six insulin-dependent diabetic patients, insulin binding and glucose transport w e r e s t u d i e d in i s o l a t e d a d i p o c y t e s o b t a i n e d f r o m subcutaneous fat tissue of gluteal region. All patients had basal plasma C-peptide <300 pmol/l, non-stimulated by i.v. g l u c a g o n (i mg). Two patients presented recurrent hypoglycemic e p i s o d e s a n d w e r e t r e a t e d w i t h 0.,3 a n d 0 . 5 U o f i n s u l i n / k g BW/d, t h e r e m a i n i n g p a t i e n t s f o l l o w e d t r e a t m e n t w i t h 0 . 5 t o 1.0 U o f i n s u l i n / k g B W / d , atd they did not present hypoglycemia. Insulin binding to adipocytes in patients having recurrent hypoglycemia was within the range o b s e r v e d in t h e r e s t o f t h e p a t i e n t s . B a s a l a n d insulin-stimulated glucose transport (U[14C]D-glucose) were higher in d i a b e t i c s with hypoglycemia (Basal: 183 and 62 vs 33• fl/cell/s, mean• 1 0 ~ m o l / l i n s u l i n : 342 a n d 127 vs 73• fl/cell/s). Conclusion: In some insulindependent diabetic patients, an increased glucose transport may be the cause of recurrent hypoglycemia.

OP 50 Hall A-2 Insulin Secretion in Man 295

296

Obesity plays a critical role in the modulation of pulsatile insulin secretion. H Flax, M F r a n k a n d D R M a t t h e w s , D i a b e t e s Research Laboratories, Rdcaliffe Infirmary, W o o d s t a c k R o a d , O x f o r d 0 X 2 6HE. The pulsatile secretion of insulin has not been well characterised in obesity. Both a longitudinal and a cross-sectional study were undertaken to assess the reponse of insulin oscillations 9o weight reduction and to cheracterise the pulsatile insulin secretion w i t h d i f f e r e n t l e v e l s o f o b e s i t y , i) ii normal glucose-tolerant subjects with median BMI 30.4 were followed up for 6 months during which a median weight loss of 9.3kg was obtained decreasing their BMI to 25.7. F o u r i e r T r a n s f o r m (FT) a n a l y s i s s h o w e d initial 30min long term-trends which normalised to 15min (p=.02) by 6 months and were of less amplitude. Autocorrelation (AC) showed initial irregular oscillations which r e g u l a r i s e d w i t h w e i g h t loss. 2) 62 s u b j e c t s were categorised in 4 groups according to B M I : < 2 2 (GI), 2 2 - 2 6 . 9 (G2), 2 7 - 2 9 . 9 ( G S ) , > 3 0 (G4). A d o m i n a n t 1 5 m i n s p e c t r a l p e r i o d ( p = . 0 2 ) w a s s e e n in G1 a n d G 2. A s e c o n d s p e c t r a l p e a k o f 3 0 m i n w a s s e e n i n G2, m o r e i n G S a n d d o m i n a n t l y i n G4. Significant (p=<0.001) regular oscillations were only s e e n i n GI. C o n c l u s i o n s . l ) T h e l e v e l o f obesity critically influences the pattern of i n s u l i n o s c i l l a t i o n s a n d is r e v e r s i b l e w i t h w e i g h t loss. S) Obese subjects (particularly BMI>30) have longer term trends in insulin oscillation which may underlie the glucose i n t o l e r a n c e o f o b e s i t y . 3) L o w e r i n s u l i n oscillations were associated with pustaile insulin secretion, suggesting greater efficacy.

PULSATILITY OF BASAL PLASMA INSULIN CONCENTRATION CAN BE EXPLAINED BY RANDOM SECRETORY PROCESSES D.Overkamp, W.Renn, B.Graf, M.Beck and M.Eggstein Med. U n i v . - K l i n i k , Abt. IV, D - 7 4 0 0 T u b i n g e n It is a m p l y d o c u m e n t e d t h a t b a s a l p l a s m a i n s u l i n c o n c e n t r a t i o n v a r i e s n o n r a n d o m l y in n o r m a l s u b jects. Results from time series analysis of such d a t a w e r e i n t e r p r e t e d as b e i n g i n d i c a t i v e o f a n underlying periodic process, causing speculations about the nature of a synchronizing pacemaker. The present study aimed at further characterizing the pattern of basal insulin concentration in normal subjects, and to provide an explanation f o r its g e n e r a t i o n . 7 n o r m a l v o l u n t e e r s (6 m a l e , 2 f e m a l e ) w e r e s t u d i e d in t h e b a s a l p o s t a b s o r p tive state. Heparinized blood was withdrawn cont i n u o u s l y f r o m a h e a t e d b a c k h a n d v e i n f o r 80 t o 160 m i n u t e s , a n d a l i q u o t e d i n t o o n e m i n u t e f r a c tions. Plasma insulin concentration was determ i n e d u s i n g a d o u b l e a n t i b o d y E L I S A (VK 2 - 10%). All subjects exhibited nonramdom fluctuations of insulin concentration (runs test, p<0,05). Regression analysis of power spectra amplitudes derived from the timeseries versus frequency s h o w e d , t h a t t h e s e m e a s u r e s a r e r e l a t e d b y i/f s, w i t h 0,5 < B < 1,5. D a t a e x h i b i t i n g the same characteristic could be generated by using gaussian variables as i n p u t into a mathematical compartmental model representing insulin distribution and degradation. We conclude, that the fluctuations of basal plasma insulin concentrat i o n c o r r e s p o n d t o a i/f n o i s e , a n d t h a t r a n d o m secretory processes within the pancreas are s u f f i c i e n t t o e x p l a i n its o c c u r r e n c e .

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P R O I N S U L I N R E S P O N S E TO A H Y P E R G L Y C E M I C CLAMP IN TYPE 2 DIABETES MELLITUS AND IN T H E I R RELATIVES M . R ~ d e r , J . E r l k s s o n , S . H a r t l l n g , L.Groop and C . B l n d e r . Steno Memorlal Hospltal , Gentofte, Denmark and Fourth D e p a r t m e n t of M e d l c l n e , Helsinki U n i v e r s i t y Hospital, Helsinki, Finland. Proinsulin(PI) and C-peptide(CP) levels w e r e followed in 9 patients with type 2 diabetes, 17 f i r s t - d e g r e e relatives (7 with impaired(IGT) and i0 w i t h normal glucose tolerance(NGT)), and 8 h e a l t h y c o n t r o l s d u r i n g a 120-150 minutes h y p e r g l y c e m i c g l u c o s e clamp ( BG=7 mmol/l) a n d a e u g l y c e m i c insulin clamp (BG=5.5 mmol/l). At the end of the h y p e r g l y c e m i c clamp 1 m g of g l u c a g o n was g i v e n i.v. M e a n fasting PI v a l u e s were 17.0, 8.6 and 11.5 pmol/l in diabetics, IGT and NGT relatives respectively, compared to a sign i f i c a n t l y lower mean value: 6.1 pmol/l in the controls. M e a n fasting CP was not s i g n i f i c a n t l y different among groups. During h y p e r g l y c e m i c clamp PI and CP response from b a s e l i n e v a l u e s were s i g n i f i c a n t l y impaired in the diabetics. D u r i n g e u g l y c e m i c clamp the PI level d e c r e a s e d c o m p a r a b l y in all groups. A f t e r g l u c a g o n stimulation PI levels were 13.9, 3.9 and 8.4 pmol/l in diabetics, relatives and controls r e s p e c t i v e ly, not s t a t i s t i c a l l y different. CP response was s i g n i f i c a n t l y lower in diabetics compared with controls: 0.90 vs. 2.57 nmol/l, relatives h a v i n g i n t e r m e d i a t e response: 1.80 nmol/l. The results further s u b s t a n t i a t e s a b n o r m a l i t i e s in B cell function in type 2 diabetes. The a t t e n u a t e d r e s p o n s e to g l u c o s e but p r e s e r v e d response to g l u c a g o n suggests a defect of the glucose signal in the B cells of type 2 diabetics. *

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P. M. Clark, M. Burnett, L. Cox, J.C. Levy, R.C. Turner and C.N. Hales. Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge and Diabetes Research Laboratories, Radcliffe Infimary, Oxford, UK. Two-site immunoradiometric assays (IRMA) for plasma insulin and precursors may allow improved detection of impaired beta-cell function. Plasma insulin-like peptides were measured in 6 diet-treated, weightmaintaining diabetic subjects (fasting plasma glucose 6.0_+SD0.7mmol/l) and in an age/weight matched group of 9 non-diabetic subjects during 60minute 5mg-kg IBW-tmin -'r glucose infusion. Insulin was also assayed by less specific radioimmunoassay (RIA). Biological variability of insulin between consecutive 5 minute blood samples measured by IRMA and RIA was similar fasting (coefficients of variation 15.8% and 16.6%, respectively) and after glucose infusion (CVs 10.8% and 9.3%). Fasting IRMA insulin, 32-33 split-proinsulin, intact proinsulin and RIA insulin did not differ between diabetic and normal subjects. Sixty-minute insulin response to glucose was impaired in diabetic compared with normal subjects when measured by IRMA (geometric mean: 53 (SD range: 27-104)pmol/I and 126(76-211)pmol/I, respectively, p<0.05) and RIA (72(32-160) and 179(100-324)pmol/I, p<0.05). Though stimulated proinsulin and 32-33 split-proinsulin did not differ between the two groups, the proportion of proinsulin to total insulins was higher in diabetic than normal subjects (12_+SD5% and 6_+3% respectively, p<0.05). Conclusion: Mild type II diabetic subjects have impaired insulin response to glucose measured by either IRMA or RIA. 32-33 splitproinsulin is normal but the proportion of proinsulin is raised.

300

299 CcmNmarison of 2 insulin assays in homeostasis model assessment of insulin resistance and insulin deficiency in type 2 (non-insulin-dependent) diabetes DK Nagi, TH Hendra, *RC Temple, *P Clark, *A Schneider, *CN Hales, JS Yudkin, Academic Unit of Diabetes, Whittington [~spital, Archway Road, London NI9 5NF, UK. *Departlent of Clinical Biochemistry, Addenbrooke's Hospital, Hills Road, Cambridge CB2 Homeostasis model assessment (HOMA) is a computer solved model to calculate insulin resistance (IR) and beta cell function (BCF) from fasting plasma concentrations of insulin and glucose. The observation that some insulin assays may cross-react with proinsulin-like molecules, which are present in high concentrations in type 2 diabetes, means that the assay employed may have a profound effect on these calculations. The aims of this study were to compare estimates of IR (normal=l) and BCF (normal=100%) in type 2 diabetic subjects, using concentrations of insulin measured by specific monoclonal two-site irxmnoradiometric assay (IRMA insulin) with those measured by standard radioir.nunoassay ( IRI ). The HOMAequatlon was modxfled ~aploylng the observation that IRMA insulin comprises 76 (SD 3)% of all insulin-like molecules in non-obese non-diabetic subjezts. 50 type 2 diabetic subjects, mean duration of diabetes 3 (0.5-16) years, fasting glucose 9.4 (SD 3.2)~m~ol/l, glycated haemoglobin 9.1 (SD 1.9)% showed fasting IRHA insulin concentrations of 38 (SD 15)% of "IRI" concentrations. IR calculated using I ~ insulin in the [:!Odelwas 3.8 (SD 2.6) compared to 8.3 (SD 6.5) using "IRI" (P<0.OO01). BCF uring IRMA insulin was 41.4 (SD 35.2)% compared to 86.1 (SD 74.2)% using "IRI" (P<0.0001). Specific assays for insulin employed in the HO~iAmodel suggest that insulin deficiency may play a more important role than insulin resistance in type 2 die~etes. H

Immunoradiometric assay of insulin and proinsulins and radioimmunoassay of insulin compared in type II diabetic and normal subjects

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FEEDBACK INHIBITION OF INSULIN SECRETION IN TYPE ll(NON-INSULIN-DEPENDENT) DIABETES. C.Baynes, V. Anyaoku, D.G. Johnston and R.S.Elkeles, D e p a r t m e n t of C l i n i c a l E n d o c r i n o l o g y , St M a r y ' s H o s p i t a l Medical School, N o r f o l k P l a c e , London, W2, England9 I n s u l i n is k n o w n to i n h i b i t its own s e c r e t i o n in n o r m a l s u b j e c t s b u t w h e t h e r it does so to t h e s a m e d e g r e e in t y p e II d i a b e t e s is u n c l e a r . The a i m of this study was to assess w h e t h e r exogenous insulin is a b l e to s u p p r e s s i n s u l i n s e c r e t i o n n o r m a l l y in t y p e II d i a b e t e s , i n d e p e n d e n t of p r e v a i l i n g p l a s m a g l u c o s e c o n c e n t r a t i o n . P l a s m a C - P e p t i d e (CP) r e s p o n s e s to s u s t a i n e d h y p e r i n s u l i n a e m i a ( ~ 600 p m o l \ l ) w e r e a s s e s s e d d u r i n g a 4 h o u r i s o g l y c a e m i c c l a m p in 9 p a t i e n t s w i t h t y p e II d i a b e t e s (D) and 5 a g e and w e i g h t - m a t c h e d c o n t r o l s (C). A r t e r i a l i s e d v e n o u s blood was clamped at ~0.3mmol\l below fasting in each s u b j e c t and w a s n o t p e r m i t t e d to r i s e a b o v e t h e f a s t i n g level. F a s t i n g CP was 906 +_ 133 in D vs. 553 + 200 p m o l \ l in C, p =NS. CP d e c l i n e d to 560 +_ 90 p m o l \ l in D vs. 226 +_ 76 p m o l \ l in C, p = .015 (a c h a n g e of 65 +_ 6.4% vs. 37 +_ 4.3%, p = .01). T h e g l u c o s e c o n c e n t r a t i o n s during t h e s t u d i e s , h o w e v e r , were 9.8 +_ 0.9 mmol\l in D vs. 4.6 _+ 1.4 mmol\l in C. T h e r e f o r e , 4 of t h e d i a b e t i c s u n d e r w e n t a s e c o n d clamp study at e u g l y c a e m i a (5.7 +_ 0.5 m m o l \ l ) . Under t h e s e conditions, CP s u p p r e s s e d to t h e s a m e e x t e n t as t h e c o n t r o l s ( f r o m 866 + 133 to 366 + 90 p m o l \ l , a c h a n g e of 58 + 7.3 %, n o t s i g n i f i c a n t l y d i f f e r e n t f r o m C ). We c o n c l u d e t h a t in t y p e I1 ( n o n - i n s u l i n dependent) diabetes : i) E l e v a t e d p l a s m a g l u c o s e c o n c e n t r a t i o n s m a i n t a i n insulin secretion despite physiological hyperinsulinaemia but ii) E n d o g e n o u s i n s u l i n s e c r e t i o n is s u p p r e s s e d n o r m a l l y by insulin f e e d b a c k under euglycaemic c o n d i tions.

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OP 51 Hall A-3 Signal Transduction in B-cells 301

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SUBCELLULAR DISTRIBUTION OF SMALL GTP-BINDING PROTEINS DURING STIMULATION OF INSULIN SECRETION R. Regazzi, S. Ullrich and C.B. Wollheim Division de Biochimie Clinique, University of Geneva, Switzerland

BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERISATION OF GTP-BINDING PROTEINS IN ISOLATED RAT ISLETS OF LANGERHANS

The ras-related small molecular mass GTP-binding proteins (SMGs) have been implicated in the regulation of secretion. The role of these proteins in insulin secretion was analysed by examining their subcellular distribq~ion in RINm5F and HIT-T15 cells. SMGs can be detected by ["~P]GTP binding after gel electrophoresis and transfer to nitrocellulose membranes. Using this technique, several such proteins were found in cytosol, plasma membrane and granule-enriched fractions. Except for a 27 kDa protein that was found exclusively in RINm5F cells, the SMGs in the two cell lines were practically identical. One of these proteins was identified as smg25A/rab3A using a specific antibody, while a second one, rho, could be detected in insulin secreting cell fractions by ADPribosylation with the Clostridium betulinurn C3-exoenzyme. The subcellular localization of SMGs was not altered when intact cells were incubated with different secretagogues. However, stimulation of insulin secretion from permeabilized cells with non-hydrolysable GTP analogues, that maintain GTP-binding proteins in their activated state, increased the labelling of a 20 kDa SMG in the plasma membrane-enriched fraction. Thus, the simultaneous measurements of insulin secretion and distribution of SMGs have revealed one potential candidate, whose involvement in the regulation of exocytosis merits further investigation.

303 MODULATION

N.S. Berrow and N.G. Morgan, Cellular Pharmacology Group, University of Keele, Keele, Staffs., U.K. The presence of a number of distinct GTP-binding proteins has been demonstrated in secretory cells but these have not been well characterised in the endocrine pancreas. In this study we have charaeterised rat islet GTP-binding proteins using biochemical and immunological methods. Isolated islets were disrupted with ultrasound, the proteins separated by SDS-PAGE and transferred to nitrocellulose for analysis. Islet proteins were screened for the presence of the hetero-trimeric proteins G i and G o by the use of antisera specific for their sub-units. Immunoreactive bands corresponding to proteins of approximately 40kDa were observed with each antiserum, consistent with the presence of both G i and G o in islet cells. In addition several novel GTP-binding proteins were identified in islet cells by incubation of the blots with [~-32P]GTP and autoradiography. Six proteins with molecular weights in the range 14-28kDa were evident together with a protein of 49kDa. Unlabelled GTP analogues (I-10~M) displaced the bound [~-32p]GTP but other nucleotide triphosphates were ineffective at similar concentrations, suggesting that these proteins have a higher affinity for guanine nucleotides. These results demonstrate that rat islets contain a number of GTP-binding proteins that may have roles in intracellular signalling events associated with the control of insulin secretion.

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L-TYPE CALCIUM CURRENTS

IN MOUSE B-CELLS

BY GTP-BINDING PROTEINS

CA 2+ MODULATION OF REDOX STATE IN SINGLE B-CELLS MONITORED BY MICROFLUORIMETRY

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P. Rorsman, K. Bokvist and P-O Berggren . Department of Medical Physics, GSteborgs Universitet, GSteborg and Department of Endocrinology, Karolinska Institute, Stockholm, Sweden. L-type Ca-channels participate in the depolarizing phase of the H-cell action potential and mediate the Ca-influx required for insulin secretion. We have investigated whether these channels might represent a target for regulation by GTP-binding proteins. Ca-currents were recorded using the whole-cell configuration of the patch-clamp technique. Whereas addition of GTP (I mM) to the cytoplasmic solution did not affect the Ca-current, inclusion of the non-hydrolyzable analogue GTP~S (0.5-I mM) reduced the maximum peak Ca-current density from 21 pA/pF to I0 pA/pF (P4 hours) and the current density rose from ii pA/pF to lS pA/pF (P
W.-F. Pralong & C. B. Wollheim Division de Biochimie Clinique, University of Geneva, Switzerland

An increase in pyridine nucleotide fluorescence has been shown to precede Ca 2+ influx in nutrient stimulated single B-cells loaded with fura 2. We examined whether intracellular ionized calcium ([Ca2+]i) elevation, which is a prerequisite for insulin secretion, also participates in the stimulation of the metabolic flux in B-cells. Rat islet monolayer cultures were studied by fluorescence microscopy at the optimal wavelengths (excitation 360 nm; emission 460 rim) for reduced pyridine nucleotides (NAD(P)H). In order to raise [Ca2+]i, the muscarinic agonist carbachol was used, which remains efficient in Ca 2+ depleted medium. Carbachol (100 uM) was tested at different glucose concentrations, with other nutrients (pyruvate, 2ketoisocaproate), and with or without external Ca 2+. The latter condition blocks nutrient-induced [Ca2+]i rises and insulin secretion. At 4 mM glucose, carbachol did not increase NAD(P)H fluorescence but did so at stimulatory glucose concentrations or in presence of pyruvate and 2-ketoisocaproate, 10 mM each. In Ca2+-depleted medium, carbachol was still capable of enhancing NAD(P)H formation in presence of the nutrients. Thus, the nutrient dependent alteration in redox state by Ca 2+, reflects one of the metabolic adjustments of the B-cell allowing the generation of coupling factors, including ATP, sustaining and potentiating insulin secretion.

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CALCIUM-DEPENDENT ACTIVATION OF THE KREBS CYCLE IN ISLETS STIMULATED BY NON-GLUCIDIC NUTRIENT SECRETAGOGUES W.J. Malaisse and A. Sener. Laboratory of Experimental Medicine, Brussels Free U n i v e r s i t y , Brussels, Belgium.

ISLET Ca2+/CALMODULIN-DEPENDENT PROTEIN KINASE IS NOT IDENTICALTO Ca2+/CALMODULIN-DEPENDENTPROTEINKINASEII Hughes, S.J. & Ashcroft,S.J.H. Nuffield Departmentof Clinical Biechemislry, John RadcliffeHospital,Headington,Oxford,U.K. We attempted to identify islet Ca2+/calmodulin-dependent protein kinase (CaM ldnase) by comparing its activity with purified brain CaM kinase II. We followed [7-32p]ATP incorporation into islet homogenate proteins and exogenous glycogen synthase (GS) by SDS-PAGE and autoracliography. Islet CaM kinase in the presence of calmodulin and Ca 2+ phosphorylated major endogenous substrates of 104, 57 and 53kDal and GS. The inhibition by lmMalloxan of phosphorylation of the 104kDal substrate (95+5%) was significantly (P<0.05) greater than that of GS (45+4%) or of the 57kDal (51+10%) or 53kDal (63+7%) substrates. The phosphorylation of GS by brain CaM kinase II was not inhibited by alloxan. The Ca2+ dependencies of phosphorylation of the endogenous islet substrates differed. Half maximal phosphorylation was attained at Ca2+ concentrations of 0.13+0.01, 0.33_+0.06 and 0.45+0.04gM for the 104, 57 and 53kDal substrates respectively. Differential centrifugation located the 104kDal substrate in the post100,000g supernatant and the 57 and 53kDal substrates in the particulate fraction. These data suggest that i) islet CaM kinase is not identical to brain CaM kinase II; ii) the phosphorylation of endogenous substrates by islet CaM kinase shows differing sensitivities to calcium.

An e s s e n t i a l f e a t u r e o f the metabolic response of i s l e t s to D-glucose consists in the p r e f e r e n t i a l s t i m u l a t i o n of mitochondrial o x i d a t i v e events. To explore whether t h i s phenomenon is a t t r i b u t a b l e to a c t i v a t i o n of mitochondrial dehydrogenases, we examined the e f f e c t of n o n - g l u c i d i c n u t r i e n t secretagogues upon both D - [ 6 - z 4 C ] g l u c o s e o x i d a t i o n and D-[5-3H]gluccse u t i l i z a t i o n in i s l e t s exposed to a low concentration (6 mmol/l) of the hexcse. L - l e u c i n e , 2-ketoisocaproate, 2-aminobicyclo[2,2,1]heptane-2-carbox y l i c acid and 3-phenylpyruvate f a i l e d to a f f e c t D-[5-3H] glucose u t i l i z a t i o n but augmented the z"CO2/aH20 production ratio. N o n - n u t r i e n t secretagogues, such as theophyll i n e , carbamylcholine, g l i c l a z i d e and glibenclamide did not a l t e r the ~"C02/aHzO r a t i o . The a c t i v a t i o n of the Krebs cycle b y . n u t r i e n t secretagogues was impaired in the 2 t . . . absence o f Ca . Likewise, CdCI2,. n ] f e.d l p l n.e , verapamll and the absence of Caz decreased the ~4C%/3H20 r a t i o in i s l e t s exposed to 16.7 mmol/l D-glucose. Aminooxyacetate, however, f u r t h e r increased such a r a t i o . Non-glucidic n u t r i e n t s stimulated the o x i d a t i o n of D - [ 6 - z 4 C ] g l u c o s e 14 despite unaffected D - [ 3 , 4 C ] g l u c % e o x i d a t i o n . These f i n d i n g s support the view t h a t a Ca2 -dependent a c t i v a t i o n o f 2 - k e t c g l u t a r a t e dehydrogenase p a r t i c i p a t e s in the o x i d a t i v e response of i s l e t s to n u t r i e n t secretagogues. The~ also suggest t h a t the mitochondrial accumulation o f Ca2may be caused by changes in ATP a v a i l a b i l i t y and mitochond r i ~ l redox s t a t e , and not s o l e l y by a r i s e in c y t o s o l i c Caz- a c t i v i t y .

OP 52 Hall A-15 Eye Disease II 307

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ELECTROPHYSIOLOGICAL AND PSYCHOPHYSiCAL TESTING OF VISION IN EARLY TYPE 1 (INSULIN-DEPENDENT) DIABETES. M.A.S. Di Leo I, S. Caputo I, G. Ghirlanda I, B. Falsini 2, M. Caesar 2, V. Porciatti 3 and A.V. Greeo I. Institute of Internal Medicine ! and Ophthalmology 2, Rome; Institute of Neurophysiolog~, CNR, Pica, I t a l y To reveal early macular dysfunctions in 14 Type 1 (insulin-dependent) diabetic patients without retinopathy and duration of disease <5 years, we used the pattern electroretinogram (PERG) and dynamic contrast sensitivity (CS), in response to sinusoidal gratings of 5 spatial frequencies, between 0.6-4.8 o/deg. Stimuli had identical spatio-temporal (8 Hz) characteristics and field size (24" X 14"). No patient had neither macular edema nor concomitant diseases. PERGs were found to be abnormal below 2 SD of control means in i0 patients (71.4%) on at least one spatial frequency in one or both eyes, CS was abnormal in 5 patients (35.7%). Compared with control subjects, PERG amplitudes were significantly reduced in patients at 0.6 c/deg (ANOVA, meanISD: 0.61 i 0.25 vs 0.76 9 0.22 ~V; p=0.01), 1.0 c/deg (0.57 * 0.24 vs 0.84 9 0.18 ~V; p=0.0001), 1.4 c/deg (0.60 • 0.23 vs 0.90i0.23 ~V; p=0. 0001) spatial frequencies and CS values were significantly reduced at all spatial frequencies (0.6 c/deg: 48.6 9 2.1 vs 51.1 i 2 dB, p=0.0001; 1.0 cldeg: 48.7 ~ 1.9 vs 50.9 • 2.1 dB, p=0.0003; 1.4 c/deg: 47.5 9 1.7 vs 49.7 9 2.4 dB, p=0.0006; 2.2 c/deg: 45.4 i 1.9 vs 48.2 • 2.7 dB, p=0.0001; 4.8 c/dog: 36.2 • 2.0 vs 38.6 i 3.4 dB, p=0.003). Our results indicate that a combined electrophysiological and psychophysical study may help to detect in early Type 1 diabetes macular abnormalities before the onset of retinopathy, although the PERG is found to be a more sensitive test.

COLOUR VISION ABNOPd~ALITIES IN INSULIN-DEPENDENT DIABETIC PATIENTS WITH LITTLE OR NO RETINOPATHY K.J.Hardy J.Lipton M.O.Scase D.H.Foster and

J.H.B.Scarpello North Staffordshire Royal Infirmary, Stoke-on-Trent, Staffordshire, England. Colour vision abnormalities are common in diabetics with retinopathy. The aim of the present study was to determine whether such abnormalities also occur in non-complicated diabetics without clinical evidence of retinopathy. Fifty-one insulin-dependent diabetics (33 males): age 26.8• y (mean • S.D.), diabetes duration 7.5• y, were compared with 23 non-diabetic controls (12 males), age 25.5• y. Snellen-acuity was better than 6/9 in all subjects, Ophthalmological examination of controls was normal. Diabetics were judged free from retinopathy if fundoscopy and retinal photographs were normal. Thirty patients had fluorescein angiography: 24 were normal, 5 had < I0 microaneurysms, and only I had> i0 microaneurysms. Ishihara 38-Plate Test and the City University Test were normal in all subjects. The diabetics' mean Farnsworth-Munsell lO0-Hue Test score was 91.9• compared with 27.3• for controls (p <<0.001); age-corrected scores were also highly significantly different, 43.6• for diabetics and -11.3• for controls (p << 0.001). No relationship was found between colour vision changes and duration of diabetes, blood glucose at the time of colour testing, or glycosylated haemoglobin. We conclude that colour vision is often abnormal in diabetics without overt retinopathy and may provide a marker for assessment of early intervention therapy in diabetic eye disease.

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EFFECTS OF TOLRESTAT, AN ALDOSE REDUCTASE INHIBITOR, ON DIABETIC RETINOPATHY. A SIX MONTH DOUBLE BLIND TRIAL. E. van Oosterhout, J.M.A. van Gerven, H.H.P.J. Lemkes, J.P. Boot and J.A. van Best, Departments of Endocrinology and Metabolic Diseases and Ophthalmology, University Hospital, Leiden, The Netherlands

Influence of IGF I in the progression of diabetic retinopathy. J. Larra~aga~ F. Cordido~ B. Romero, J. Fernandez-Vigo~ J. Castro and J.L. Herrera Pombo. Servicio de Endocrinologia~Fundaei6n Jim@nez Diam. Univ. Aut6noma. Madrid; Unidad de Diabetes Ocular.Departamento de Oftalmologia. Univ. de Santiago. SPAIN.

A six months placebo controlled trial of the aldose reductase inhibitor tolrestat was performed in 31 diabetic patients with various degrees of retinopathy. Diabetic retinopathy was investigated by fundus photography, fluorescein angiography and vitreous fluorophotometry (Fluorotron Master). Separate morphological features of diabetic retinopathy were scored on a decimal scale. No changes occurred in microaneurysms and in lesions that reflect vaso-obliteration (soft exsudates, capillary closure, intraretinal microvascular abnormalities (IRMA's) and new vessels). Signs of increased vascular permeability (hemorrhages, hard exsudates and focal fluoreseein leakage) increased during placebo and decreased during tolrestat. The difference was significant for focal fluorescein leakage (0.5 + 0.8 units vs -0.3 + 0.5 units (M + SD), p = 0.02). Thus? some improvement of enhanced vascular permeability in diabetic retinopathy occurred during tolrestat treatment. In apparent contradistinction, fluorescein permeability into the vitreous, measured by fluorophotometry, did not change. Further analysis revealed that fluorescein accumulation into the vitreous was only increased in (pre)proliferative diabetic retinopathy. The permeability of fluorescein into the vitreous was closely related to the extent of both IRMAs (r = 0.66, p < 0.001) and new vessels (r = 0.77, p < 0.001), by multiple regression analysis. Thus, vitreous fluorophotometry reflected advanced diabetic retinopathy, whereas the limited improvement of diabetic retinopathy during tolrestat became apparent in earlier signs of enhanced vascular permeability.

The pathogenesis of diabetic retinopathy is not clear.The aim of this study sam to evaluate the presence of IGF I in vitreous and aequeous humor from diabetic patients with proliferative retinopsthy. The patients (n = 57) were grouped according to the activity of angioproliferation evaluated by f u n d u s e o p y a n d ret~nography in two groups: low activity (LA) n=25 and high activity (HA) n=32. Vitreous from such patients (n=36)~ were obtained by vitrectomy. Samples sere lyophilized and before assayed~desslted on Sephadex G 25 disposable columns in assay buffer. IGF I was assayed by RIA using non-equilibrium technique from Furlanetto. IGF I was 21• mU/ml in LA vs 30• mU/ml in HA, p<0.05.Acqueous humor was obtained from patients who underwent ocular surgery due to glaucoma or cataracts. IGF I levels were 24• mU/ml in LA, similsr values were obtained in HA=27• mU/ml. Compaired with controls ( 12 sex-age matched non-diabetie patients) IGF I=24• mU/ml~ no differences were found Pith each acs stage nor all diabetics. Conclusion: Our results show the presence of small quantities of IGF I in vitreous and aequeous humor. In spite of this small quantities, IGF I could be one of the factors involved in the progression of retinal neovascularization in diabetes. At the moment,the levels of IGF I found in scqueous humor could net be related with angioproliferative activity in these patients.

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LONG-TERM EVIDENCE FOR IMPAIRED RETINAL FUNCTION IN TYPE 1 (INSULIN-DEPENDENT) DIABETIC PATIENTS S. Caputo I, M.A.S. Di Leo I, G. Ghirlanda I, B. Falsini 2, V. Porciatti 3 and A.V. Greco I. Institute of Internal Medicine t and Ophthalmology 2, Rome; Institute of Neurophysiology 3, CNR, Piss, Italy. The retinal function, was prospectively investigated

NORMOGLYCEMIC RETINOPATHY

u s i n g the s t e a d y - s t a t e focal e l e c t r o r e t i n o g r a m (ERG) of the macula in 25 Type 1 (insulin-dependent) d i a b e t i c p a t i e n t s w i t h no or early r e t i n o p a t h y on entry into the study and a g a i n after 3 y e a r s Focal ERG was e v o k e d by u n i f o r m field (flicker ERG, F-ERG) and s i n u s o i d a l g r a t i n g stimuli (pattern ERG, P - E R G ) The F-ERG a n a l y s i s y i e l d s first (IF) and second (2F) components; the P-ERG is d o m i n a t e d by the second component ( 2 P ) IF has a p h o t o r e c e p t o r o r i g i n 2F and 2P represent subsets of generators in the inner retina 2P, but not 2F, is c o r r e l a t e d w i t h g a n g l i o n cell activity. M e a n 2F a m p l i t u d e (paired t test=6 8; mean~SD: 0.42 i 0.14 vs 0 28 i 0 12; p=0 0001), and m e a n 2P a m p l i t u d e were s i g n i f i c a n t l y r e d u c e d (t=2 2; 0 41 ~ 0 Ii vs 0 37 , 0 13; p=0 03) No m o d i f i c a t i o n of IF r e s p o n s e s was found after 3 y e a r s V a r i a t i o n s of 2F r e s p o n s e s were s i g n i f i c a n t l y c o r r e l a t e d w i t h those of 2P r e c o r d i n g s (r=0 47, p=0 0006) No c o r r e l a t i o n was found w i t h H b A l c values and r e s p o n s e s of the P - E R G and F-ERG Our results show a progression of postreceptoral dysfunctions, but not in the p h o t o r e c e p t o r activity, d u r i n g the course of Type 1 diabetes. Changes in the n e u r o s e n s o r y retina may play a key role in the d e v e l o p m e n t of d i a b e t i c r e t i n o p a t h y

INTERVALS

DELAY

THE

ONSET OF

DIABETIC

H. van Houten, H.H.P.J. Lemkes, H. Arps. University Hospital Leiden, Department of Endocrinology and Metabolism, P.O. Box 9600, 2300 RC, Leiden, The Netherlands. 25 Type 1 (insulin-dependent) diabetic patients were studied from diagnosis by annual fluoresceinangiography (FA) and threemonthly HbAlc (N<6.7%) to analyse the characteristics of cumulative hyperglycemia in relation to the appearance of the first definite microaneurysm (MA). Follow-up was closed in i0 patients at the appearance of first MA (MA+group), mean duration 2.8 years, and in 15 patients at the last MA free FA (MA-group), mean duration 5.7 years. Groups were not different with regard to age, sex, insulin dose, blood pressure, smoking, cholesterol or triglycerides. Mean HbAlc differed significantly between MA~groups and MA+group: 7.8% vs 9.1% p<0.02. However, cumulative hyperglycemia (area under curve of excess HbAlc in each patient for individual follow-up) was identical in both groups. There was a significant difference between both groups in both frequency and duration of normoglycemic intervals (HbAlc<6.7), e.g. more than 20% of followup: MA+ O/lO, MA- 7/15. In conclusion: in our study group, it is not cummulative exposure to hyperglycemia per s~, which predicts the onset of retinopathy. Combined results of cumulative hyperglycemia and normoglycemic intervals suggest compensation or reversibility for subclinical previous hyperglycemic damage by normoglycemic intervals.

Ago

OP 53 Hall B-3 Cardiovascular Disease 313

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AN EXPLANATION FOR THE INCREASED RISK OF ATHEROSCLEROSIS IN THE HYPERCHOLESTEROLAEMIC DIABETIC PATIENT?

ASYMPTOMATIC ATHERDSCLEROSIS AND INSULIN RESISTANCE

Tomkin GH,Owens D, Johnson A, Collins P. Dept. of Metabolic Medicine, Adelaide Hospital and The Royal College of Surgeons in Ireland, Dublin 2. Low density iipoprotein (LDL) control of intraeellular cholesterol synthesis in mononuclear cells is disturbed in diabetic patients. This study describes the normalization of this defect when serum cholesterol levels are reduced. Hypercholesterolaemic diabetic patients (group i, n=ll) were compared with hypercholesterolaemic non-diabetic (group 2, n=ll) and normocholesterolaemic non-diabetic subjects (group 3, n=ll). Serum cholesterol values were 7.8~.26, 7.70+0.28 and 5.52• mmol/l respectively. [~C]-acetate incorporation into mononuclear cells, a test of cholesterol synthesis~ was 226• 149• and 122• nmol/mg cell protein for groups i, 2, and 3 respectively. LDL from group i patients inhibited acetate incorporation into mononuclear cells from normal volunteers significantly (25%) less than LDL from group 2 (p<0.02) and (46%) from group 3 (p<0.002). Reduction to normal of serum cholesterol using simvastatin, a HMGCoA reductase inhibitor, was accompanied by a (39%) decrease in cholesterol synthesis in mononuclear cells in group i (p<0.0O5) and 29% in group 2 (p>0.05). Inhibition of cholesterol synthesis by LDL increased to 48.8• (p
M. Laakso, H. Sarlund, R. Salonen, M. Suhonen, K. Py~r~l~. J.T. Salonen, and P. Karhap~. Department of Medicine, Kuopio, Finland High insulin levels h a v e been shown to be risk indicators for coronary heart disease in nondiabetic subjects in prospective population studies. Furthermore, insulin resistance as measured by the glucose clamp technique has been shown to be associated with l i p i d and lipoprotein changes favouring atherosclerosis and with high blood pressure. No study has, however, demonstrated that insulin resistance per se is d i r e c t l y associated with atherosclerosis. To this aim we studied 22 middle-aged lean subjects with asymptomatic a t h e r o s c l e r o s i s in the femoral or c a r o t i d a r t e r i e s , and 13 corresponding c o n t r o l subjects. In the 2-hour oral glucose t o l e r a n c e t e s t , glucose and i n s u l i n l e v e l s were similar in both groups. During the euglycaemic hyperinsulinaemic (1200 p m o l / l i t e r ) clamp st u d i e s , subjects with a t h e r o s c l e r o s i s had s i g n i f i c a n t l y reduced whole body glucose uptake (60+3 vs. 71+4 pmol/kg/min, p=O.O19). Glucose o x i d a t i o n , I Tpid o x i d a t i o n , i n h i b i t i o n of l i p o l y s i s , and potassium disposal were s i m i l a r in both groups. In c o n t r a s t , n o n o x i d a t i v e glucose disposal was s i g n i f i c a n t l y reduced in the p a t i e n t s compared to t h a t in the c o n t r o l s (39+2 vs. 50+4 pmol/kg/min, p=O.Ol4). When glucose uptakes were mashed during the hyperglycaemic clamp s t u d i e s , the r a t e of n o n o x i d a t i v e glucose uptake normalized in the p a t i e n t s . These r e s u l t s provide the first evidence that asymptomatic a t h e r o s c l e r o s i s i s an i n s u l i n r e s i s t a n t s t a t e even without the presence of hyperinsulinaemia. This i n s u l i n r e s i s t a n c e is c h a r a c t e r i z e d by reduced whole body and n o n o x i d a t i v e glucose uptake, normal glucose and l i p i d o x i d a t i o n and potassium d i s p o s a l , and normal suppression of l i p o l y s i s during hyperinsulinaemia.

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LONG-TERM PROGNOSIS OF PATIENTS WITH NON-INSULINDEPENDENT (Type 2) DIABETES MELLITUS AFTER ACUTE MYOCARDIAL INFARCTION. A FIVE YEAR FOLLOW UP STUDY.

A SIX MONTHS' FOLLOW-UP OF DIABETIC MIOCARDIAL INFARCTION: INCREASED RISK OF CONGESTIVE HEART FAILURE V. Manicardi, V. Malavasi, Lo~neri, G. Bellodi and~C. Cosce~i 2nd Medical Division, Hospital of Guastalla, Reggio Emilia 1st Medical Division, Hospital of Parma, Italy

T. Melchior, P. Hildebrandt, N. Gadsb~ll, C. TorpPedersen, and L. K~ber. Depts. o f Cardiology and Physiology, Glostrup H o s p i t a l , DK-2600 Glostrup, Denmark. L e f t and r i g h t v e n t r i c u l a r e j e c t i o n f r a c t i o n (LVEF, RVEF) was measured by r a d i o n u c l i d e cardiography in 575 consecut i v e p a t i e n t s with acute myocardial i n f a r c t i o n (MI) before discharge from the coronary care u n i t . 45 were noni n s u l i n - d e p e n d e n t (Type 2) d i a b e t i c s t r e a t e d with o r a l a n t i - d i a b e t i c therapy or on d i e t alone. Residual v e n t r i c u l a r f u n c t i o n estimated by mean LVEF (45%) and RVEF (61%) was s i m i l a r in Type 2 d i a b e t i c s and in n o n - d i a b e t i c p a t i e n t s . During 5 years f o l l o w up m o r t a l i t y r a t e i n Type 2 d i a b e t i c p a t i e n t s was 52% as compared with 39% among p a t i e n t s with no diabetes m e l l i t u s . This increased m o r t a l i t y r a t e was h i g h l y s i g n i f i c a n t . M u l t i v a r i a t e anal y s i s showed t h a t Type 2 diabetes m e l l i t u s was an i ndependent p r e d i c t o r o f m o r t a l i t y (p=O.O071). Divided i n subgroups according t o e j e c t i o n f r a c t i o n (~< 45%) the f i v e year m o r t a l i t y was: +Type 2, LVEF > 45% : 37% : +Type~LVEF < 45% : 64% : -Type 2, LVEF > 45% : 26% : -Type~LVEF < 45% : 52%. I t i s concluded t h a t p a t i e n t s with Type 2 diabetes m e l l i tus have s i m i l a r r e s i d u a l l e f t v e n t r i c u l a r f u n c t i o n as n o n - d i a b e t i c p a t i e n t s when discharged from the coronary care u n i t a f t e r MI. The f i v e year m o r t a l i t y r a t e was s i g n i f i c a n t l y increased in d i a b e t i c s , net only among p a t i e n t s with more severe but also among p a t i e n t s with only a s l i g h t r e d u c t i o n o f l e f t v e n t r i c u l a r performance.

The mortality rate at follow-up (6 mo-3yr) as clinical fe~ tures of the acute phase of Myocardial Infarction (AMI)was examined in 364 survivors (1985-87). In 67 Diabetic patients (D) (age 71• 37M/30F) the mortality rate was significantly (29.9 vs 19.8% p < O . 05) higher than in 296 Non-diab~ tics (ND) (age 64• 226M/70F) and the difference was m~ re evident in younger (<65y: 16 vs 8.2%), uncomplicated p~ tients (26 vs 11.8%, p ~ O . 02). In ND, age (p~O. O05), infarct site (anterior) (p~O. 05) and congestive heart fail~ re (CHF) (p~O. O001) were predictive of poor prognosis in the follow-up, while in D none of the above variables was significantly correlated with mortality. In a subgroup of 193 subjects (44 D and 149 ND) a routine lab-analysis and a complete recording of clinical events were performed at I and 6 mo. after AMI. 24% of ND showed CHF (significantly correlated with age and anterior site) vs 48% of D (without relationship with age and infarct site); 33% of D vs 18% of ND developped CHF ex-novo: The incidence of post-AMI angina, arrhythmias, reinfarction was similar in the two groups. No correlation of metabolic parameters with the mortality rate and CHF was observed in the two groups: In the follow-up of AMI, Diabetic patients have a poorer prognosis than Non-diabetics, due to the development of CHF, independently of age, infarct site, and metabolic control.

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ACE-INHIBITION IMPROVES ON A LONG-TERM BASIS THE INSULIN RESISTANCE OF ESSENTIAL HYPERTENSION. E.Torlone,A.-M.Rambotti, M.Britta, G.Perriello, F.Santeusanio, P. Brunetti, and G.B.Bolli. University of Perugia, Italy. In order to assess the long-term metabolic effects of ACEinhibition in essential hypertension, 8 subjects (6M, 2F) with mild hypertension but normal glucose tolerance and body weight, were studied after a run-in (1 month), and at the end of 3 month treatment with captopril (25 mg x 2 daily, p.o.) and placebo in a single blind, cross-over fashion, with a euglycemichyperinsulinemic clamp (step 1, plasma insulin - 1 7 0 pmol/1 for 2h, followed by step 2, plasma insulin -600 pmol/1 for additional 2h) combined with tracer infusion (3-3H-glucose) and indirect calorimetry. Blood pressure decreased more after captopril than after placebo (142 -!-- 83 vs 160 _ 98 mmHg, p<0.001). Insulin sensitivity improved after captopril, but not after placebo, both at liver level (step 1, hepatic glucose production 0.10 -+ 0.09 vs 0.29 _+ 0.11 mg/kg/min), mad at muscle level( step 2, glucose utilization 6.41 --- 1.23 vs 5.34 -+ 1.06 mglkg/min -a result primarily due to a 60% increase in non-oxidative glucose disposal, i.e. glucose storage), captopril vs. placebo, respectively, both p<0.001. Basal and insulin-suppressed free fatty acid concentration was no different after captopril and placebo. Conclusions. ACE-inhibition improves the insulin resistance of essential hypertension on long-term basis, both at liver and muscle level, the latter primarily by partially reversing the specific abnormality of intracellular fate of insulin-stimulated glucose disposal in essential hypertension, i.e. storage.

I N S U L I N RESISTANCE: R I S K F A C T O R FOR C A R D I O V A S C U L A R D I S E A S E IN R H E U M A T O I D A R T H R I T I S . A. R o v i r a , C. G r a n t , A. C a s l a , F.J. A r r i e t a , G. Herrero-Beamount and J.L. Herrera-Pombo. Fundacion J i m e n e z Diaz, U n i v . A u t o n o m a d e M a d r i d , s p a i n . Hyperinsulinemia is c o n s i d e r e d a possible common factor of d i s o r d e r s in c a r b o h y d r a t e and lipid metabolism, as w e l l as o f h y p e r t e n s i o n . In this work we studied the presence of the abovementioned alterations in r h e u m a t o i d arthritic patients with and without inflammatory activity. P a t i e n t s w i t h i n f l a m m a t o r y a c t i v i t y (erytrocyte sedimentation r a t e > 30 m m H g , n = 1 7 ) , c o m p a r e d to p a t i e n t s o f s i m i l a r age, s e x a n d b o d y m a s s i n d e x , w i t h o u t a c t i v i t y (n=14) had: i) H i g h e r p r e v a l e n c e of abnormal glucose tolerance (41% v s 7%) a n d h i g h e r i n c r e m e n t a l a r e a s of g l u c o s e ( 8 9 6 9 • vs 5304• p<0.05, mean• after an oral glucose l o a d (OGTT). 2) H i g h e r p r e v a l e n c e o f h y p e r i n s u l i n e m i a ( m o r e t h a n o n e v a l u e along the O G T T a b o v e t h e m e a n + 2 S D f o r a control group, 6 1 . 1 % v s 3 8 . 9 % . 3) L o w e r i n s u l i n s e n s i t i v i t y i n d e x o b t a i n e d b y the modified minimal model (1.40• vs 2.96• x l 0 "4. m i n 1 . ~ U 1 . m l "I, p < 0 . 0 1 ) . 4) H i g h e r serum levels of t r i g l y c e r i d e s (107• vs 79• mg/dl, p<0.05) and lower serum levels of total cholesterol ( 1 8 2 • vs 202• mg/dl, p<0.05), LDL-C (118• vs 135• mg/dl, p<0.05) and HDL-C (42• vs 52• mg/dl, p<0.001). 5) Higher atherogenic index Chol/HDL-C (4.4• vs 3.9• p < 0 . 0 5 ) . 6) H i g h e r systolic (133• vs 124• mmHg, p<0.05) and diastolic (85• vs 76• mmHg, p<0.001) blood pressure. The high incidence of c a r d i o v a s c u l a r disease among patients with rheumatoid arthritis can b e e x p l a i n e d b y t h e c o - e x i s t e n c e of m u l t i p l e r i s k factors s e c o n d a r y to h y p e r i n s u l i n e m i a and insulin resistance.

OP 54 Hall A-18

Neuropathy (Intervention) 319

320

THE EFFECT OF MEXILETINE HYDROCHLORIDE ON DIABETIC PAINFUL

LONG TERM EFFECTS OF TOLRESTAT, AN ALDOSE HEDUCTASE INHIBITOR, ON SYMPTOMATIC DIABETIC SENSORY POLYNEUROPATHY. J.M.A. van Gerven, H.H.P.J. Lemkes, J.G. van Dijk, G.W.A.M. Padberg and H.M.J. Krans, Departments of Endocrinology and Metabolic Diseases, Clinical Neurophysiology and Neurology, University Hospital, Leiden, The Netherlands

NEUROPATHY Y.Suzuki and K.Matsuoka,

1-4-17, Mita, Minato-ku, Tokyo

A two-week, double-blind study of mexiletine hydrochloride and a placebo was conducted using 21 diabetic patients with Dainful neuropathy( 18 males and 3 females, mean age 54.3+ i0.0 ).

Mexiletine hydrochloride( 300mg

daily) was given orally in three divided doses. evaluate pain, we used original auestionnaire. results showed that

To The

1 ) the overall pain' s improvement

rate was significantly higher(p<0.01) during trea~nent with Mexitil (Phase M) than during trea~nent with the placebo (Phase P);

efficacy rate was 76.2% for Phase M.

2) Changes in symptoms were tested for significant differences.

In Phase M, a smarting pain in the calf signifi-

cantly improved from the pre-trea~nent state.

Calf smart-

ing and pin-prick-like pain and cramps relatively improved.

In Phase

calf cramping.

P, improvement was observed only for

3) In terms of nocturnal acceleration of

pain and insomnia, there was no difference between the two phases.

We concluded that mexiletine hydrochloride,

developed as an antiarrythnJc drug, was effective for treating Dainful neuropathy in diabetes melitus, with inmediate action in short-term treatment.

As this drug

produces no serious side effects, it will likely be widely used.

The effects of the aldose reductase inhibitor to!restat on symptomatic diabetic polyneuropathy were examined in a six months double blind investigation of 37 patients followed by an extension phase of six months tolrestat treatment, without breaking the double blind code. Masked tolrestat treatment, relative to placebo, improved the median motor nerve conduction velocity of four nerves (0.6 (-0.7/3.2) m/sec vs -1.4 (-3.0/0.9) m/sec (median (50%CI)), p = 0.009), the median sensory nerve conduction velocity of two nerves (0.0 (-12.4/2.9) m/sec vs -3.6 (-20.4/0.7) m/sec (median (50%CI)), p = 0.081) and the metatarsal vibration perception (-0.01 + 0.i0 logVolts vs 0.06 + 0.07 logVolts (mean +-SD), p = 0.035). The same effects were observed during tolrestat treatment of the control group, in the extention phase. During this period, prolonged tolrestat treatment further improved the average conduction velocity of four motor nerves ((2.7 (0.3/5.1) m/sec (median (95%CI)), p = 0.045) and of the ulnar F-response (3.8 (1.1/6.4) m/sec (median (95%CI)), p = 0.025). Neuropathic symptoms did not improve during the study. Dermal sensitivity increased in the second half year of tolrestat treatment, evidenced by amelioration of the carpal vibration perception (-0.07 (-0.14/-0.05) logVolts (median (95%CI)), p = 0.05) and by an increase of hypersensitivity to touch and pain (p < 0.05). This study has shown gradual improvement of nerve conduction velocity during one year of tolrestat treatment in symptomatic diabetic polyneuropathy, with indications of sensory recuperation during the second half year.

A92

321

322

BIOCHEMICAL AND MORPHOMETRIC RESPONSE TO TOLRESTAT IN HUMAN DIABETIC NERVE D. A. Greene, W. Bochenek, Y. Harati, A.A.F. Sima, T. Hohman, D. Hicks, M. Beg, B. Gonen and the Tolrestat Study Group. Ann Arbor MI USA, Radnor PA USA and Winnipeg MN Canada. Hydantoin-derived aldose reductase inhibitors (ARI's) increase nerve conduction, lower nerve sorbitol, promote nerve fiber regeneration and repair, and improve symptoms in selected diabetic neuropathic patients; this response pattern has not been shown for tolrestat, a less lipophilic carboxylic-acid-based ARI with a proven safety record. Eighteen neuropathic diabetic subjects treated open-label with tolrestat 200-400mg/day for 3-5yrs were randomized to 12mos of additional double-blind therapy with tolrestat (n=9) or placebo (n=9) after which sural nerve biopsies were performed. Tolrestat was undetectable in placebo nerves, but attained therapeutic nerve levels of 0.82+0.28[SD](n=2) and 1.27+0.35(n=7)ng/mg protein at doses of 200-400mg/day respectively. Sorbitol and fructose levels.were decreased by >50% in tolrestat vs. placebo nerves (0.84+0.22 vs. 1.52_+0.41nmol/mg protein, and 1.73+0.96 vs 4.07+l.98nmol/mg protein, p<0.01 and 0.02 respectively) while nerve glucose and myoinositol were unchanged. Regenerating fibers were 2-fold higher with tolrestat (42+_15%[n=4] vs 22+_10%[n=5]). Tolrestat traverses the blood-nerve barrier and improves nerve biochemistry and morphometry in human diabetic neuropathy without inducing hydantoin-like side effects.

EFFECTS OF myo-INOSITOL AND ITS 1,2,6-TRISPHOSPHATE ON NERVE CONDUCTION IN DIABETIC RATS A.L. Carrington, C.B. Ettlinger, N.A. Calcutt and D.R. Tomlinson, Department of Pharmacology, Medical College of St. Bartholomew's Hospital, London, U.K. We studied the effects of PP56 (D-myo-inositol 1,2,6trisphosphate; M.J.Siren, 1984, U.S. Patent No. 4735936; Perstorp Pharma, Sweden) or myo-inositol on resistance to hypoxic conduction block in diabetic rat sciatic nerves. 30 male Wistar diabetic (streptozotocin 50 mg/kg i.p.) rats formed 3 groups ufitreated, PP56-treated (1 mg/h s.c.; Alzet osmotic pump 2ML4) and myo-inositol-treated (500 mg/rat/day in drinking water); 10 non-diabetic rats formed a control group. Treatments were maintained for 3 weeks. Motor nerve conduction velocity (MNCV) was then measured under halothane anaesthesia in the sciatic/tibialis system; 48-72 h later rats were killed by a blow to the head and left sciatic nerves were desheathed and compound action potentials recorded in vitro (in Krebs-Henseleit bicarbonate buffered saline containing 0.5 mM rnyo-inositol). Preparations were gassed initially with 95% 02/5% COz, followed by a 40 min hypoxic period during which the 02 content was reduced to 8% (balance N2). MNCV (m/sec _+ SEM) in diabetic rats (45.3 +-1.2) was slowed compared to controls (57.7+-1.6; p<0.05). This slowing was attenuated by PP56 (53.4_+2.0) and by myo-inositol (50.0+-1.5; both p<0.05 untreated diabetics). The decline in action potential amplitude (% of initial value +- SEM) during the 40 rain period of hypoxia was greater in controls (54.8+-4.9) than in diabetics (76.1+-3.0; p<0.01) or in treated groups (PP56 69.4+-5.1, p<0.05; myo-inositol - 79.9+-5.2, p<0.01). This implicates different mechanisms in the development of altered MNCV and resistance to hypoxia in diabetic nerve.

323

324

A DOUBLE-BLIND CROSSOVER STUDY OF XAMOTEROL IN POSTURAL HYPOTENSION DUE TO DIABETIC AUTONOMIC NEUROPATHY

EFFECTS OF CHRONIC ALPHA-ADRENORECEPTOR BLOCKADE ON NERVE FUNCTION AND VASCULAR SUPPLY IN DIABETIC RATS. M.A. Cotter, N.E. Cameron, K. Ferguson and S. Robertson, School of Biomedical Sciences, University of Aberdeen, Scotland, U.K. The aim was to examine effects of chronic vasodilator treatment on nerve function and vessel growth in experimental diabetes to help elucidate the contribution of vascular factors to early nerve dysfunction. Two groups of streptozotocin-diabetic (45mg/kg) and one group of non-diabetic male Sprague-Dawley rats were used. One diabetic group was untreated for 8 weeks, the other was given prazosin (5mg/kg/day). In final experiments, sciatic motor and sensory nerve conduction velocity were measured in vivo. Hypoxic resistance was measured on sciatic trunks in vitro, monitoring compound action potential amplitude while the preparation was gassed with nitrogen. Sciatic trunk vessel density was measured on frozen sections stained for alkaline phosphatase. Motor and sensory conduction velocity deficits (15-30%) seen with diabetes were largely prevented by treatment (p
P J Leslie, C J Thon~oson, B F Clarke and D J Ewing Diabetic Department and University Department Medicine, Royal Infirmary, Edinburgh

of

PrelimJ_nary reports have suggested that xamoterol, a beta I partial agonist improves symptomatic postural h y p o t e n s i o n in a u t o n o m i c failure. We undertook a double-blind randomised crossover trial of one month's treatment with xamoterol (200 mg bd) in eleven diabetic subjects with established autonomic neuropathy and a postural systolic blood pressure (SBP) drop of more than 20 n~nHg. Blood pressure was measured for i0 minutes supJ]le and i0 minutes after standing at 0900, 1130, 1330 and 1530 using a random zero sphygmomanometer. Heart rate was measured by 24 hour ambulatory EC~. During the placebo run-in period the difference between maximum and minimum recorded SBP falls ranged from 22-70 nmHg. There were no differences in blood glucose, HbAI, waking heart rate or diastolic blood pressure. Sleeplng heart rate was increased (xamoterol 86 (8 SD), placebo 77 (9) bpm, p = 0.003). Although there was a significant increase in supine SBP (156 (12) vs 149 (17) nmHg, p = 0.036), there was no corresponding rise in standing SBP (113 (19) vs 120 (14) nmHg, NS) and consequently SBP fall was greater on treatment (42 (16) vs 29 (13) nm~Hg, p = 0.003). These results indicate that xamoterol fails to improve postural hypotension secondary to diabetic autonomic neuropathy and demonstrate the n e e d for m u l t i p l e BP m e a s u r e m e n t s in r a n d o m i s e d controlled trials.

A93

Poster Presentation PS 1 Islets: Biosynthesis and Desensitisation P325

P326

Glucocorticoid regulation of proinsulin production by transfected FAO (hepatoma) cells

LIPOSOME-DELIVERED GLUCOSE 6-PHOSPHATE STIMULATES IIgSULIN BIOSYNTHESIS IN ISOLATED RAT ISLETS

F. Vollenweider, D. Gross, J. Philippe, L. Villa-Komaroff and P. Halban Labs. Jeantet and Dpt. microbiology, Geneva School of Medicine, Switzerland; Hadassah University Hospital, Jerusalem, Israel; Children's Hospital, Boston, USA Expressing the insulin gene in cells devoid of the regulated (granular) secretory pathway typically results in low levels of proinsulin production without means of modulation. We have transfected FAO (hepatoma) cells with the rat insulin II gene driven by muMLV-LTR (DOL vector) and selected stable (G418-resistant) clones. Of 4 clones studied, the best only produced approx, lng/106 cells/24h of insulin immunoreactive material (IRI), with low cellular IRI. 24h exposure to dexamethasone resulted in a striking, dose-related, increase in IRI released to medium :- control : 31.9; 10-8 tool/l: 756; 10-7 mol/I : 959 ng/dish/24h. Higher doses were less effective. The time-course of stimulation was (ng IRI/dish released over given time; control vs dexamethasone):- lh : 5.8 vs 6.7; 3h: 12.0 vs 32.9; 6h: 17.4 vs 139; 12h: 21.0 vs 377; 24h: 29.5 vs 966. In keeping with cells secreting via the constitutive pathway, HPLC analysis of released IRI (basal and stimulated) showed predominantly proinsulin-like material rather than native insulin. Northern blot analysis of mRNA levels showed a dramatic increase in preproinsulin mRNA after dexamethasone treatment. The data show a) dexamethasone can stimulate proinsulin production > 30-fold over 24h, b) stimulation is slow in onset and accompanied by increased preproinsulin mRNA, implicating augmented proinsulin synthesis and subsequent constitutive release rather than stimulated release per se. Transfected FAO cells are thus a versatile model for studying ectopic proinsulin production.

C. /Jberg and N. Welsh. Department of Medical Cell Biology, Uppsala University, Sweden. It is well established that glucose 6-phosphate is required for active protein synthesis in reticulocyte lysates. We therefore decided to investigate the putative role of glucose 6-phosphate in insulin biosynthesis. Glucose 6-phosphate was introduced into the cells of freshly isolated rat islets by means of a liposome technique (pH-sensitive liposomes), and the biosynthesis of insulin and total protein and the release of insulin were determined. It was found that liposomes containing 0.2 mol/l glucose 6-phosphate stimulated insulin biosynthesis during an 1 h incubation period at 1.67 mmol/l glucose (580+140 vs 220-+50 dpm/islet x h, P<0.05), at 5.6 mmol/l glucose (1460+!90 vs 860-+160, P<0.O5) and at 16.7 mmol/l glucose (1840-+290 vs 970-+210, P<0.05) when compared to control liposomes. The glucose 6-phosphate containing liposomes stimulated also total protein biosynthesis at 1.67 mmol/l glucose (4910-+970 vs 3330-+720 dpm/islet x h, P<0.05) and at 5.6 mmol/1 glucose (7130-+1670 vs 5640-+1260, P<0.05), but not at 16.7 mmol/l glucose (7630-+1240 vs 7060_+2000, P>0.05). Islet insulin release was significantly stimulated by the glucose 6-phosphate containing liposomes at 1.67 mmol/l (6.4-+0.9 vs 5.0• ng insulin/10 islets x h, P<0.05) and 5.6 mmol/l glucose (24.2-+4.8 vs 18.3+-3.5, P<0.05) but not at 16.7 mmol/l glucose (39.1-+8.0 vs 47.4-+9.2, P>0.65). These results suggest that glucose 6-phsophate may play a significant role, direct and/or indirect, in the regulation of insulin biosynthesis.

P327

P328

REGULATION OF INSULIN GENE EXPRESSION BY ENKEPHALINS IN ISOLATED RAT ISLETS OF LANGERHANS

SODIUM BUTYRATE INCREASES INSULIN GENE EXPRESSION, INHIBITS GLUCOSE-STIMULATED INSULIN RELEASE IN RIN CELLS. A. E. Karlsen, W. Y Fujimoto, S. Dube and /~. Lernmark University of Washington, Dept. of Medicine, Seattle, WA,,USA. Effects of sodium butyrate (NAB) on insulin expression and glucose-stimulated insulin release were determined in RIN5AH-T2-B cells. Cultures in log phase were grown until confluency or arrested by 2mmol/1 NaB. Insulin mRNA and secretion of insulin to the culture medium in cells approaching confluency were markedly decreased. In contrast, NaB arrested cells showed insulin mRNA and secretion levels indistinguishable from log phase RIN cells. RIN cells in log phase showed dose-dependent glucosestimulated insulin release in Krebs-Ringer medium (KRM) both without and potentiated by amino acids (AA)(-AA: 5.2 vs 9.6 p_U x m1-1 x ~tg DNA'I; p<0.05; +AA: 9.9 vs 52.7; p<0.0025). RIN cells approaching confluency did not release insulin in response to glucose in KRM without AA but showed a 3.6-fold increase with AA ( - A A : 4.4 vs 4.8; n.s.; + A A : 7.6 vs 27.5; p<0.001). In contrast, cells arrested by NaB did not respond to glucose irrespective of AA (-AA: 12.3 vs 13.0; n.s.; + A A : 15.1 vs 17.1; n.s.). It is concluded that: l) insulin expression in RIN cells is closely associated with growth rate; 2) although cells growth-arrested by NaB maintain insulin gene expression and secretion, their ability to release insulin in response to glucose is inhibited, hence growth arrest induced by confluency or NaB has different effects on insulin release and gene expression.

R. Salazar ~ad H. Z~hlke, Institute of Biochemistry, University of Greifswald, GDR 0pioid peptides (~-endorphin, enkephalins) are both present and synthesized in islets. They may play a role in the formation of insulin. The aim of this study was to determine the effects of enkephalins on the transcriptional and trar.slational level of the insulin gene expression. Isolated rat islets of Langerh~us were maintained under various culture conditions for 26 hr. The content of islet preproinsulin mRNA was assayed by dot-blot hybridization using a cloned rat proinsulin cDNA as probe. The amount of (pro)insulin biosynthesis was determined by the incorporation of (JH)leucin into (pro)insulin. In the presence of 5 mmol/l glucose~the preproinsulin mRNA content was i n creased by 10-~mol/l Met-ankephalin to 198 % (p(0.001) and by Leuenkephalin to 145 % (p <0.001) of the control without enkephalins. Preproinsulin mRNA content was raised in the presence of 5 m~ol/l glucose by 10-10mol/1 Met-enkephalin to 146 % (p = 0.004) whereas the effect of Leu-enkephalin at this concentration was not significant. These effects corresponded with ~ increase in the (pro)insulin biosynthesis under the same conditions. The biosynthesis of (pro)insulin was strongly decreased by actinomycin D at the glucose and enkephalin concentrations tested. The results suggest that there is a dose-dependent increase of ~nsulin gene transcription by enkephalins.

A94 P329

P330

The levels of m o u s e i n s u l i n p e p t i d e s I and II do not r e f l e c t those of their r e s p e c t i v e m R N A s D. Gross, B. Wentworth, C. Rhodes, B. Schnetzler, P. H a l b a n and L. V i l l a - K o m a r o f f Hadassah University Hospital, Jerusalem, Israel; Labs. Louis Jeantet, G e n e v a School of Medecine, Switzerland; Joslin Diabetes Center and C h i l d r e n ' s Hospital, Boston, U S A

INSULIN GENE EXPRESSION IN RATS: EFFECT OF PROLONGED HYPERGLYCEM I A, D, GAUGUIER, S, DAROUYL P, FROGUEL#, J, CAPEAU#, G, REACH', J PICARD#, L, PICON and A. KTORZA,Phys, Dev, Lab,. University Paris 7, CNRS URA 307; *Service de Diabetologie Hotel Dieu Hospital: #Fac. Med, St, Antoine, INSERM U 181, Paris, France. The effect of prolonged hyperglyc~emia on preproinsulin (PPl) gene transcription remains unclear. We investigated the effect of long-term hyperglyc~emia induced by continuous glucose infusion on B-cell PPI mRNA content in catheterized unrestrained rats. Rats were starved 2 days and received a glucose infusion during I, 2, 6, 20 or 48 hours, Starved rats, catheterized but not infused, ~'ere used as controls, PPI mRNA levels were determined by Northern blot analysis followed by hybridization with a 32p-labelled cDNA insulin probe. During infusion, glycmmia increased to be maximal at 30 minutes and remained stable afterward (19,3._0,7 retool/t), Insulinemia increased continuously during glucose infusion ( 1 hour: 3403_+277 pmol/l; Z hours: 3800+_457 pmol/l; 20 hours: 7049+_386 pmol/l; 48 hours: 8817~517 pmol/l ). PPI mRNA levels were not modified by 1 hour of glucose infusion but were increased 1 S7-fold after 2 hours and 2,35-fold after 6 hours compared to controls and remained stable until 48 hours, These results indicate a rapid effect of hyperglycemia on B-cell PPl mRNA contentwhich was however delayted compared to its effect on insulin secretion. The absence of alteration of PPl mRNA concentration with the prolongation of glucose infusion suggests a persistent effect of glucose in rive on PPI gene transcription.

Pancreatic preproinsulin mRNA levels are p r e s u m e d to r e f l e c t insulin i m m u n o r e a c t i v i t y . We h a v e t e s t e d this for the two n o n - a l l e l i c m o u s e i n s u l i n genes. P r e p r o i n s u l i n I and II m R N A was q u a n t i f i e d by S1 n u c l e a s e p r o t e c t i o n a s s a y u s i n g s p e c i f i c 3'-end probes. In m o u s e p a n c r e a s p r e p r o i n s u l i n m R N A I was 73.4 ~ 4.8 % of total (I + II). HPLC separation of p a n c r e a t i c insulin I and II s h o w e d i n s u l i n I as only 27.2 • 0.5 % of total (I + II). M o u s e islet m R N A and p e p t i d e levels s i m i l a r l y s h o w e d I:II ratios of 84:26 for m R N A and 24:76 for peptides. The latter was not explained by selective loss of insulin I during extraction/HPLC nor by e x c e s s i v e a m o u n t s of n o n - c o n v e r t e d mouse p r o i n s u l i n I. T r a n s f o r m e d (~-TC) m o u s e B - c e l l s w e r e s i m i l a r to n a t i v e m o u s e B - c e l l s w i t h excess p r e p r o i n s u l i n I m R N A and i n s u l i n II peptide. Thus, in the mouse, p r e p r o i n s u l i n m R N A levels do not a c c u r a t e l y reflect i n s u l i n p e p t i d e levels. The u n u s u a l amino acid s e q u e n c e of m o u s e p r e p r o i n s u l i n I signal p e p t i d e c o m p a r e d w i t h p r e p r o i n s u l i n II and most other species, could lead to inefficient transfer into the RER thereby accounting (at least in part) for the relatively i n e f f i c i e n t p r o d u c t i o n of i n s u l i n II in m o u s e cells.

P331

P332

MEASURING THE BALANCE BETWEEN INSULIN BIOSYNTHESIS AND RELEASE

IMPAIRED MITOCHONDRIAL OXIDATIVE RESPONSE TO D-GLUCOSE IN ISLETS FROM 5TREPTOZOTOCIN-INJECTEDRAT&

F Schuit and D Pipeleers* Dept of Biochemistry and Dept of Metabolism and Endocrinology* Vrije Universiteit Brussel, Brussels, Belgium

M.-H. GIROIX1, A. SENEP.2, D. BAILBEI , B. PORTHA1 and W.d. MALAISSE2. 1Laboratory of Developmental Physiolo~, University of Paris 7, Paris, France, and 2Laboratory of Experimental Medicine, Brussels Free University, Brussels, Belgium.

A reduction in panGreatic insulin content can result from an imbalance between the rate of insulin production and that of insulin release. Since glucose stimulates both processes, it is unclear whether the sugar alone can induce such imbalance. In the present study we have measured net insulin biosynthesis and release in purified B ceils that were incubated for 120 min at varying glucose concentrations. At 2.5 mmol/l glucose, no difference was noticed between the rates of insulin production (4.4_+0.8 pmol/106 ceils) and release (9.7+5.3 pmol/106 cells - P>0.3). At 7.5 mmol/1 glucose, 2.5-fold more hormone was produced (56 + 9 pmol/106 cells) than released (21_+3 pmol/106 ceils - P<0.01), whereas the reverse occurred at 20 mmol/1 glucose (67+10 vs 181_+36 pmol/106 cells - P<0.02). At glucose concentrations where the rates equilibrated (10 mmol/1) addition of glucagon (10-8 mol/1) caused an imbalance in favor of release (P<0.02), whereas adrenaline (10-7 mol/1) induced the reverse (P<0.01). It is concluded that the glucose regulation of the rates of insulin biosynthesis and release permits buildup of the hormonal stores at physiological glucose concentrations. This process can be disturbed by excessive glucose levels and/or other secretagogues which lack a stimulatory effect on hormone synthesis.

Islets from adult rats injected with streptozotocin during the neonatal period display an impaired secretory response to D-glucose and, to a lesser extent, to L-leucine. Despite normal hexokinase and glucokinase activity and normat binding of these isoenzymes to mitocnondria, the metabolic response to a high concentration of D-glucose is severely affected, especially in terms of oxidation in the Krebs cycle. Thus, the ratio of O-[6-14C]glucose oxidation/D-[5-3H]glucose utilization is less markedly increased in response to a rise in hexose concentration and, at high glucose concentration, less markedly decreased by the absence of Ca2+ and presence of cycIoheximide in islets from diabetic than control rats. This metabolic defect contrasts with (i) an unaltered capacity of the islets to oxidize D-[6-14C]glucose, [2-14C]pyruvate, L-[U~4C]glutamine and L-[U-14C]leucine at non-insuIinotropic concentrations of these nutrients, (tI) a lesser impairment of the oxidation of L-[U-14C]leucine tested in high concentration (20 mmol/1), (iii) an unaltered transamination of [214C]pyruvate and L-[U-14C] leucine, and (iv) a close-to-normal glycolytic flux and fractional contribution of anaerobic and aerobic glycolytic modalities. It is speculated that the preferential impairment of the metabolic and secretory response to D-glucose is mainly attributable to an altered coupling b e t w e e n Ca 2+ a c c u m u l a t i o n and the s t i m u l a t i o n o x i d a t i v e events in B - c e l l m i t o c h o n d r i a of d i a b e t i c rats.

of

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CHRONIC HYPfi~3LYCfiFffADESENSITIZES B-CELLS THROKK~ EXCESSIVE ST/2g31ATION V. Grill and Y. Sako, Department of Endocrinology, Karolinska Hospital, S-I04 01 Stockholm, Sweden. Chronic hyperglycemia is associated with insensitivity of ~ t i c B-cells to glucose. Insensitivity could be linked to "glucotoxicity", i.e. hyperglycemia per se (prevailing view) or 15~ excessive stimulation of insulin release. These possibilities were irfce~tigated in nondiabetic rats which were infused for 48 h with enough gluoose to produoe moderate hyperglycemia (~4G) or high hyperglycemia (HHG). Mean blood glucose during MHG was 13.0+0.9 and during HHG 22.7+2.4 mM. Insulin respcnses to 30 min of 27 ram glucose were subsequently measured in per• pancreas. Glu~ose-stimulated release was similar after ~Drmoglyoemia and MHG but reduced by 41% after HHG. Infusion with tolbutamide (8.3 mg/kg/h) during ~qG (to further stimulate B-cell secretion in vivo) inhibi~ ted gluoose-induced release from per• pancreas by 71%. Co-lnfusion of diazoxide (5 mg/kg/h) completely inhibited the several (mean 6.5)-fold rise in insulin levels during FHG and HHG alone. Infusion of diazoxide during MHG potentiated 3.6-fold subsequent insulin response to 27 ~4 glucose (35.0+5.7 mU/30 rain after F~G, 126+18.6 mU/30 min after F~G + diazoxide). Diazoxide with HHG further increased (8-fold) the glucose response in per• ~ to 280.5+4.9 mU/30 min. -Inhibition of insulin secretion demasks a positive effect of increasir~ degrees of hyperglyoemia on glucose-induoed insulin secretion. Insensitivity is linked, at least in part, to exoessive stL~ulation of the B-cell.

A R G I N I N E - I N D U C E D D E S E N S I T I Z A T I O N OF INSULIN RELEASE: INTERACTION BETWEEN SUBSTRATES AND SIGNAL AMPLIFYING PATHWAYS R. Nesher a n d E. Cerasi. Department of Endocrinology

and Metabolism, Hebrew University Hadassah Medical Center, Jerusalem, Israel. Biphasic insulin release (IR) results from interaction between amplifying and time-dependent inhibitory (TDI) signals. Repeated 10-rain 5 mmol/l Arginine (Arg) stimuli were utilized in the isolated rat pancreas to study metabolic events involved in TDI. Arg failed to induce IR in the absence of glucose or with 10 mmol/1 lactate + 3 mmolA pyruvate as fuel. KIC (5 or 10 mmolB) induced brisk IR, but amplification of Arg-induced IR was minimal. TPA (100 nmol/1) or carbachol (10 gmol/l) + monooleolyl-glycerol (t00 gmol/l) did not support Arg-induced IR at 0 glucose, whereas 50 gmolB IBMX or 10 gmolB forskolin could replace glucose. TDI was abolished by glucose (8.3 but not 3.3 retool/l) or glyceraldehyde (5 retool/l) and reduced by KIC (5 retool/l). IBMX, forskolin, TPA and carbaehol abolished TDI at 3.3 retool/1 glucose. At 0 glucose, IBMX and forskolin failed to block TDI. Thus Arg-induced IR requires minimal energy-generating metabolic flux while stronger flux is required for the abolishment of TDI. Elevation of cAMP alone, activation of phosphoinositide hydrolysis and activation of PKC are not sufficient to abolish TDI, but may amplify subthreshold glucose effects on TDI.

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HUMAN ISLETS C H R O N I C A L L Y E X P O S E D IN V I T R O TO DIFFERENT STIMULI BECOME DESENSITIZED TO THE SAME STIMULI G I V E N ACUTELY. A.M. Davalli, A.E. Pont• C. Socci, S. Braghi, F. Bertuzzi, B. Fattor, V. Di Carlo and G.Pozza. I s t i t u t o S c i e n t i f i c o San R a f f a e l e , M i l a n o , I t a l y .

DIFFERENT SENSITIVITY TO GLUCOSE COMPARED TO OTHER B-CELL SECRETAGOGUES IN FEMALE NOD MOUSE ISLETS. E. Strandell. D e pa rt me nt of Medical Cell Biology, Uppsala University, Uppsala, Sweden. The aim of the present study was to i nve s t i ga t e the B-cell response in islets of prediabetie NOD mice. Insulin release to glucose and other seeretagogues was examined in islets i m m e d i a t e l y a f t e r isolation from female NOD mice, aged 5and 13-week. Islets isolated from 13-week-old mice had a significantly lower insulin response to 16.7 mmol/L g h e o s e as compared with islets obtained from 5-w e e k-ol d mice: 43.0 • 7.2 vs 62.8 • 7.7 ng insulin/10 islets x 60 rain (p<0.01). The insulin response to all the other seeretagogues was similar in the two age groups: 5.6 mmol/L glucose + 10 mmol/L arginine: 22.9 • 4.5 vs 18.6 • 3.6, 2 mmol/L glutamine + 10 mmol/L leucine: 40.9 • 7.6 vs 36.8 • 7.8, 5.6 mmol/L glucose + 1 lamol/L glipizide: 16.8 f 2.2 vs 23.1 • 3.3 ng insulin/10 isIets x 60 rain. The islet DNA content was higher in the 13-week-old mice, 639 • 122 vs 319 • 42 (5-week) ng insulin/10 islets (p<0.05) probably due to i nfi l t ra t i ng lymphoeytes. The insulin content was not affected, 1220 • 128 (13-week) vs 1067 + 144 (5-week) ng insulin/10 islets. Thus, the present results show a resemblance to the early phases of insulin-dependent diabetes mellitus in which a p r e f e r e n t i a l loss of the g l u c o s e - s t i m u l a t e d insulin secretion has been reported. It could be t ha t the i m m u n e - m e d i a t e d suppression is specifically harmful to the mechanism causing insulin release induced by glucose.

Chronic exposure to h i g h glucose media desensitizes islets to an acute glucose stimulus. In this s t u d y we e v a l u a t e d the e f f e c t of chronic exposure of human islets to d i f f e r e n t s e c r e t a g o g u e s . A l i q u o t s of 100 islets were c u l t u r e d for 48 hours in standard culture m e d i a s u p p l e m e n t e d w i t h 5.5mM glucose; 16.7mM glucose; 10mM L - a r g (+5.5mM glucose); 100~M tolbutamide(+5.SmM glucose). Insulin release declined with time in all c u l t u r e media: from 0.24• ~ U / i s l e t / m i n (at 6h) to 0 . 0 9 • (at 24h) to 0 . 0 6 • (at 48h) in 5.5mM glucose; from 0.53• to 0.18• to 0.13• in 1 6 . 7 m M glucose; from 0 . 0 6 • to 0.04• to 0.03• in L - a r g i n i n e ; from 0.34• to 0.15• to 0.07• in tolbutamide. Islets were then per• islets c u l t u r e d in 5.5mM glucose were s e n s i t i v e to acute stimulation w i t h glucose, a r g i n i n e and tolbutamide, while sensitivity of islets cultured in 16.7mM g l u c o s e was lost for acute glucose, decreased for acute t o l b u t a m i d e and m a i n t a i n e d for acute arginine. Islets cultured in arginine, maximally desensitized after 6 hours of culture, showed d e c r e a s e d s e n s i t i v i t y to all stimuli during per• c u l t u r e in tolbu= tam• d e s e n s i t i z e d islets to all stimuli. In conclusion, 48 h o u r exposure to d i f f e r e n t seeretagogues i n d u c e d in h u m a n islets a timedependent desensitization and a loss of response to acute stimuli.

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INSULIN SECRETORY RESPONSE TO STIMULI BY ISLETS FROM RATS CHRONICALLY INFUSED WITH GLUCOSE.

GLUCOSE RESPONSIVENESS OF PANCREATIC B CELLS FOLLOWING CULTURE AT H][GH GLUCOSE.

F.J. B e d o y a and B. Jeanrenaud. Laboratoires de Recherches M~taboliques, Universit~ de Gen~ve, Switzerland. This w o r k was d e s i g n e d to study the islet secretory response to fuel and non fuel stimuli, following chronic glucose infusion to normal rats. Secretory function was a s s e s s e d in p e r i f u s e d islets. S e v e n days of g l u c o s e i n f u s i o n leads to a 46% d e c r e a s e in the ist p h a s e of the i n s u l i n r e s p o n s e to 16.7 mmol/l glucose, while the 2nd phase is decreased by 33%, compared to the saline i n f u s e d group. The Ist p h a s e of the r e s p o n s e to 20 m m o l / l leucine is d e c r e a s e d by 51%, w h i l e the 2nd p h a s e is d e c r e a s e d by 60%. W h e n 20 mmol/l ~-ketoisocaproate is tested, a d e c r e a s e d ist p h a s e (80%) and a lack of 2nd p h a s e are observed. The r e s p o n s e to 50 # m o l / l tolbutamide, 7 m m o l / l g l u c o s e is d i m i n i s h e d by 45%. By contrast, the response to 20 ~ m o l / l forskolin, 16.7 m m o l / l g l u c o s e is not affected and that to i00 nmol/l phorbol ester, 5 m m o l / l g l u c o s e is i n c r e a s e d 3-fold. Glucose utilisation through glycolysis is h i g h e r in islets from g l u c o s e i n f u s e d rats, w h i l e 14CO 2 p r o d u c t i o n from D-[UI4c] g l u c o s e is not altered. These f i n d i n g s s u g g e s t t h a t a l t e r a t i o n s in the m i t o c h o n d r i a l h a n d l i n g of fuel s t i m u l i are i n v o l v e d in the loss of the i n s u l i n r e s p o n s e seen in this m o d e l of islet desensitization.

P339 GENETIC ANALYSIS OF THE EXPRESSION OF DIABETES INDUCED BY THE DIABETES (db) MUTATION IN MICE K.Kaku, T.Kaneko, N.Hotta and M.A.Permutt Yamaguchi University, ~Nagoya University, Japan ~Washington University, USA Expression of obesity-induced diabetes associated with the diabetes (db) mutation in mice varies in inbred strains. This study utilized a genetic analysis to evaluate the number of genes responsible for the difference in diabetes responses between mice of the susceptible C57BL/KsJ (BL/Ks) and resistant 129/J inbred strains. BL/Ks (db/+) males and 129/J (+/+) females were bred to generate F1 hybrids, and the F1 females (db/+ and +/+) were backcrossed to BL/Ks (db/+) males. A total of 252 backcrossed (BC) males were obtained, of which 31 were db/db and obese. While the fed plasma glucoses of all BC mice were greater than 400 mg/dl, the expression of diabetes varied considerably, as measured by fasting plasma glucose, fed plasma insulin, and pancreatic insulin and proinsulin mRNA content. The proinsulin mRNA content was a good indicator of diabetes severity and islet dysfunction was seen in the inverse correlation between proinsulin mRNA content and fasting plasma glucose (r=0.69, p<0.O01), and a direct correlation between proinsulin mRNA and plasma insulin (r=0.61, p
Z. Ling, C. Van Schravendijk, P. In't Veld, F. Schuit*, D. Pipeleer,s. Dept. of Metabolism & Endocrinology and Dept. of Biochemisty*, Vrije Universiteit Brussel, Brussels, Belgium In vivo studies have suggested that chronically elevated glucose levels reduce the responsiveness of pancreatic B cells to glucose. We have investigated this possibility by comparing the effects of glucose on purified rat B cells which had been cultured for 10 days at 6, 10 or 20 mM glucose. In each cell preparation, insulin synthesis and release was dosedependently regulated by glucose. The concentrations at which glucose elicited (half)maximal effects varied with the pretreatment levels, the highest being required after culture at 6 mM glucose, the lowest after 20 mM. In 10 or 20 mM pre-exposed cells, the glucose-inducible hormone synthesis was 3-fold higher than after 6 mM exposure; the cells also exhibited a 10-fold higher glucose-inducible release of newly formed insulin. The 20 mM-pretreated cells contained less insulin (1.7_+0.3 ng/ ng DNA) than the 10 mM-group (4.5_-t-0.3ng/ng DNA) and presented a marked heterogeneity in cellular hormone content; they released 5-times less insulin in reponse to a maximal glucose stimulus. It is concluded that prolonged exposure of islet B cells to glucose levels above 6 mM increases their mean sensitivity to glucose. If the prevailing conditions favor the rate of hormone release over that of hormone synthesis, the total insulin store will progressively decline, and hence provide less hormone for subsequent secretory stimuli. The extent of the secretory deficit will depend on the cellular heterogeneity in glucose responsiveness.

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THE INFLUENCE OF LYSINE-ACETYL-SALICYLATE ON INSULIN RESPONSE TO GLUCOSE ANDARGININE IN MAN T.F. Veneman, T.W. van Haeften and E.A. van der Veen, Free University Hospital, Amsterdam.

EFFECTS OF THE ANOMERSOF SWEET TASTE INHIBITORS ON GLUCOSE-INDUCED INSULIN RELEASE AND ALLOXAN TOXICITY A. N i k i , H. Niki and T. Hashioka. Department of Internal Medicine, Aichigakuin U n i v e r s i t y , Nagoya, Japan

Salicylate (three day course) has been proposed to increase plasma insulin levels by inhibiting insulin metabolism and not by enhancing insulin release. We studied the acute effect of lysine-acetyl-salicylate (LAS) on glucose- and arginine-stimulated plasma insulin and C-peptide concentrations in 8 volunteers. Hyperglycemic clamps (17mMol/L; 60minutes) and arginine infusions (0.5g/kg; 30minutes) were performed after an infusion with LAS (0.5g salicylate; 60minutes)-or saline. First and second phase release were assessed as increment at 5 minutes and increment at end of infusion and as Areas Under Curve (AUC). Ar@inine-stimulated plasma insulin increments did not differ between saline and LAS (Mean_+SEM: 135+22 vs 130+22 and 175+37 vs 116+18pMol/L, p>0.2 (first and second phase~); plasma Cpeptide increments tended to be higher after LAS (0.37+0.07 vs 0.65+0.14, 0.10>p>0.05, and 0.55+0.14 vs 0.70+0.16 nMol/L, p>0.2). Glucose-stimulated plasma insulin increments were not different (388+80 vs 391+66 and 459+107 vs 444+69pMol/L (first and second phase)] nor were plasma C-peptide increments (1.18+0.20 vs 1.23+0.19 and 2.39+0.33 vs 2.08+0.22 nMol/L, both p>0.2). AUC's showed similar results. Conclusion: Acute inhibition of PG synthesis has no significant influence on glucose-, nor arginine-stimulated insulin release in man.

Glucose is believed to be metabolized in pancreatic B-cells f o r signal production for i n s u l i n release, while the sugar is considered to bind with a receptor in gustatory c e l l s f o r e l i c i t i n g sweet taste responses. To elucidate the s i m i l a r i t y between the glucose recogn i t i o n mechanisms of the two c e l l s , we studied effects of s p e c i f i c - c o m p e t i t i v e i n h i b i t o r s of the sweet taste receptor on glucose-induced i n s u l i n release and alloxan t o x i c i t y , using batch-incubated r a t pancreatic i s l e t s . The m-anomers of p-nitrophenyl-glucopyranoside (PNP-GIu) and p-nitrophenyl-mannopyranoside (0.1 - 5 mmol/l) dose-dependently i n h i b i t e d glucose(lO mmol/l)-induced i n s u l i n release, without a f f e c t i n g glucose oxidation in the i s l e t s , while the ~-anomers were i n e f f e c t i v e . p-Nitrophenyl-galactopyranoside had no e f f e c t . When the i s l e t s were pretreated for 5 min with alloxan (1.25 mmol/l) together with ~-PNP-GIu (15 mmol/l), subsequent i n s u l i n release by glucose (20 mmol/l) was higher than that from the i s l e t s pretreated with alloxan alone (p < 0.01). A greater protection was observed with the m-anomer than the B-anomer of PNP-Glu (p < 0.01). Unlike mannoheptulose, ~-PNP-GIu did not i n h i b i t the p r o t e c t i v e e f f e c t of glucose against a l l o x a n , but the e f f e c t of ePNP-GIu (15 mmol/l) was attenuated by mannoheptulose (15 mmol/l) (p < 0.01), a sweet substance and i t s e l f a week protector against a l l o x a n . These findings suggest a common, though not i d e n t i c a l , mechanism of glucose recognition by the gustatory c e l l and pancreatic B - c e l l .

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EFFECTS OF H E X A D E C Y L P H O S P H O C H O L I N E ON INSULIN S E C R E T I O N FROM P E R I F U S E D ISOLATED MOUSE ISLETS I. Rustenbeck, A. Schulz and S. Lenzen. Institute of P h a r m a c o l o g y and Toxicology, U n i v e r s i t y of G6ttingen, D 3400 G 6 t t i n g e n

IN INI"RACELITJIAR ~H DIRECTLY AFFECI~ THE INSULIN SECRETORY

An a c t i v a t i o n of a p h o s p h o l i p a s e A2 yields l y s o p h o s p h o l i p i d s (LPL) and free fatty acids and is r e p o r t e d to occur in g l u c o s e - i n d u c e d in sulin secretion. This h y p o t h e s i s is s u p p o r t e d by the finding that some LPL induce insulin release from i s o l a t e d p a n c r e a t i c islets, when p r o v i d e d exogenously. However, a d i s a d v a n t a g e of these n a t u r a l l y o c c u r r i n g LPL is that they are r a p i d l y r e e s t e r i f i e d and thus the additional p r e s e n c e of e s t e r i f i c a t i o n i n h i b i t o r s like p - h y d r o x y m e r c u r y b e n z o a t e or NaF is n e c e s s a r y to elicit a s u b s t a n t i a l insulin release. To cir cumvent this d i s a d v a n t a g e we used a newly synthesized LPL analogue, h e x a d e c y l p h o s p h o c h o ] i n e (HePC), which, can be d e g r a d e d slowly only by p h o s p h o l i p a s e s C and D. This c o m p o u n d was a far more potent s t i m u l a t o r of insulin s e c r e t i o n than l y s o p h o s p h a t i d y l c h o l i n e , the most potent natural LPL, in a p e r i f u s i o n system with collag e n a s e - i s o l a t e d albino mouse islets. In a medium c o n t a i n i n g 0.1% BSA, I00 pmol/l HePC had an i n s u l i n - r e l e a s i n g p o t e n c y c o m p a r a b l e to 30 mmol/l glucose. The effect was c o n c e n t r a t i o n d e p e n d e n t i.n the range of I0-i00 pmol/l. As with l y s o p h o s p h a t i d y l c h o l i n e , the s e c r e t i o n returned p r o m p t l y to basal rates after w i t h d r a w a l of the agent. This insulin r e l e a s i n g p r o p e r t y may be due to a c a l c i u m m o b i l i z a t i o n by HePC (5-100 pmol/l), which could be o b s e r v e d with a Ca 2§ ion s e n s i t i v e e l e c t r o d e in e x p e r i m e n t s with p e r m e a b i l i z e d p a n c r e a t i c islet cells.

Rorm~an and P-O. Berggren2, iDepartments of Medical Cell Biology, University of Uppsala, Uppsala~ 2Endocrinology, Karolinska Institute, Stmck/x)im and ~vedical Physics, G o ~ University, Gothenburg, Sweden A possible regulatory role of intraoellular pH (pHi) in insulin release was ~ t i g a t e d . ~-cells were isolated from obese hyperglycaemic mice (ob/ob). The intracellular H+ concentration was measzLred with the fluorescent indicators quene-i and BCECF. The free cytopla~nic Ca2+ oonmentration ([Ca2+]i) and membrar~ potential were measured with the f l u o r e s ~ t indicators quirt-2, fura-2 and bisoxonol, respectively. The glucose-Lnduoed increase in [Ca2+] ~ was ~ e d by intraoellular alkalinization. T h e - ~ in phi was observed both in the absence and presence of H~S-. A similar increase in pHi was not obtained by direct stimulation of protein kinase C with the phorbolester TPA. Addition of i0 mmol/l NH4CI to glucose-stimulated ~-oells rapidly ~ pHi, followed by a slow return towards the resting level. The alkalinization was a ~ e d by hyperpolarization, reduction in [Ca2+]i and inhibition of insulin release. At low glucose, i0 mmol/l NH4CI evoked a transient stimulation of insulin release ~ did not affect the stimulatory effect by high K +. Whereas i0 mmol/l TEA only had a small effect, 200 ~nol/l tolbutamide prevented most of the alkalinization-induced decrease in [Ca2+]i" Addition of i0 mmol/l NaOOoCHa markedly decreased pHi, with:~t char~ing the threst~id for gluoose-induoed increase in [CaZ+]{. Hence, pHi might modulate rather than directly regulate the insulin secretory process, an effect involving interaction with the ATP-regulated K +channels.

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A N E W R A P I D AND S H O R T - A C T I N G O R A L H Y P O G L Y C E M I C A G E N T OF D - A M I N O A C I D S.Mashiko,M.Fukuda* an d M . K i k u c h i * * . Institute for d i a b e t e s c a r e , A s a h i life f o u n d a t i o n , Second dept. of i n t e r n a l m e d i c i n e , G u m m a m e d i c a l s c h o o l ~ Dept. of endoer. & metab., Mitsui memorial h o s p i t a l ** T o k y o , J a p a n .

SOLUBILIZATION AND CHARACTERIZATION OF ACTIVE SOMATOSTATIN RECEPTORS FROM HAMSTER PANCREATIC BETA CELLS,

M. MaletLiI, J,C. Marie2, M, AnderssonI, G, Rosselin2 and V, Muttl IBiochemis~y II, KaroIinska Institute.S-I0401 Steckholm, Sweden ; 21NSERM U.55, H6piLalSaint-Antoine,F-75571 Paris Cedex 12, France. Our aim is to struclurallycharacterizesomaloslatin (SRIF) receptors

A derivative of D-phenylalanine(A4166,mol.wt 3 1 7 ) w a s f o u n d to s t i m u l a t e i n s u l i n r e l e a s e in m i c e and rats in v i v o and in v i t r o . T o c h a r a c t e r ize the s e c r e t a g o g u e , k i n e t i e changes in b l o o d g l u c o s e and h o r m o n e s by o r a l l y g i v e n A - 4 1 6 6 w e r e c o m p a r e d w i t h t h o s e by T o l b u t a m i d e in f a s t i n g c o n s c i o u s dogs p r e v i o u s l y o p e r a t e d for m e a s u r e m e n t of s p l a n c h n i c g l u c o s e d i s p o s a l . The a g e n t was f o u n d at 1 5 m i n . , m a x i m i z e d at 6 0 m i n . and dec r e a s e d r a p i d l y t h e r e a f t e r w h e n g i v e n at lOmg/kg, G l u c o s e l e v e l s w e r e d e c r e a s e d in d o s e - d e p e n d e n t manner at 2 . 5 - 2 0 m g / k g . The d e c r e a s e began at 45min. and a t t a i n e d a nadir of 34% b a s a l at 60min. w i t h l e v e l l i n g off at 3 0 0 m i n . a c c o m p a n i e d by s i m u l t a n e o u s s t e e p r i s e s in p o r t a l insulin w i t h a p e a k l e v e l of a v e r a g e d 53• 12P U / m l ( v s . basal 14• O.05,n:5)with a rapid decline to base-line at 150min. without significant changes in glucagon. Biphasic inhibition of h e p a t i c g l u c o s e o u t p u t was a c h i e v e d . C o m p a r a b l e d o s e s of T o l b u t a m i d e i n d u c e d g r a d u a l l y a p e r s i s tent maximal hypoglycemia of 37% basal at 120min.with monophasic insulin,glucagon release and i n h i b i t i o n of h e p a t i c glucose output. In conclusion, A-4166 showed differant characteri s t i c s from T o l b u t a m i d e in the r a p i d and s h o r t a c t i n g h y p o g l y c e m i c a c t i o n p r o b a b l y s u i t a b l e to ameliorate postprandial hyperglycemia without p r o t r a c t e d h y p o g l y c e m i a in type 2 diabetics.

P346 FANC~EATIC SEC~gETION OF P ~ T I N

from hamster insulinoma, In an initial important step we have found that SElf receptors may be solubilized in an aclive , unoccupied Form, using 0,3% CHAPS, The binding of 1251-SRIF-28 te the soluble fraction was lime-dependenl, saturable and reversible. The order of potency of lwo forms of SElf and an analogue of it for inhibiting binding was SElf-28 SElf-14 > SMS 201-995. Scalchard analysis of equilibrium binding dala indicated [he presence in the soluble extract of high (H) and low (L) affinity binding sites with the following characteristics : KdH= 0.17 nM and BmH = 3.16 fmol/mg protein ; KdL= 11.1 nM and BmL = 3.38 pmol/mg protein, GTP inhibited 1251-SRIF binding te soluble receptors and enhanced [he dissociation of soluble SElf-receptor complexes, suggesting that GTPbinding proteins are functionally associated

with SElf receptors in

solution. Soluble receptors were adsorbed on wheat germ aggtutinin,

confirming the glycoproleinnature of [he SRIF receptor. An apparent molecular weight of about 600,000 was found for solublereceptors by size exclusion gel chromatography. A specific protein of 37 kDa was identified after photoaffinity labeling of soluble receptors and SDS-PAGE. These results dearly indicate that we have soiubilized authentic and active SElf receptors,

P347 IN THE PIG

C.-G. Ostenson, B. AhrQn, S. Efendic, H. ~ s o n and M. Sund@n. Dept of Endocrinology, Karolinska Hospital, Stcckt~im (C.-G.0., S.E. and M.S.), Depts of Pharmacology and Surgery, Lund University, Lund (B.A.), and Dept of Surgery, Helsir~borg County Hospital, Helsingborg (H.M.) ~tatin-like ~reactivity (PLI) oocurs in Band D-oells of porcine islets, and porcine ~ t a t i n suppresses insulin secretion in rats and mice. We have ~ i z e d porc/ne pla~na PLI and examined whether PLI is released from the pig pancreas /n vivo. After gel-filtration, PLI in non-extracted pig plasma largely consisted of 2 high-molecular fractions ( M of 80-85,000 and 300-350,000, respectively), while a ~ i i ~ of PLI eluted at the ix]sition of synthetic porcine pancreastatin. After extraction on o c ~ i s i l y l s i l i c a , virtually all PLI disappeared except in the fraction co-eluting with porc/ne ~ t a t i n . Extracted PLI (pancreastatin) was measured by radioimmunoassay in pla~na samples from the carotide artery and superior pancreaticoduodenal ve/n of thiopental-anesthetized pigs. By multiplying the ~ - a r t e r i a l concentration differenoe of pancreastatin times the pancreatic versus plasma flow, a net pargzreatic pancreastatin output of 454 -+ 68 pmol/min was found. This output was markedly.reduced, in parallel to insulin, by electrical stimulation of ~ t h e t i c nerves along the superior artery (p<0.01). ~ r e , the pancreatic venous pancreastatin levels were elevated by a]/m~t i00 pmol/l during i.v. glucose infusion (p<0.01). In cc~clusicn, pig pla~na pancreastatin c~nn be measured by radio' y after extraction on octadecylsilylsilica. There is a net ~ t i c output of pancreastatin which is suppressed by sympathetic nerve stimulation and enhanmed during hyperglycemia.

O X Y N T O M O D U L I N A N D RELATED PEPTIDES CONTROL S O M A T O S T A T I N RELEASE IN RIN T3 CELLS IN CULTURE T. TANI, A. LEQUELLEC, C. JARROUSSE, F. SLADECZEK, J. MARTINEZ, A. ESTIVAL*, L. PRADAYROL* and D. BATAILLE. Centre CNRS-INSERM de PharmacologieEndocrinologie, Montpellier and * INSERM U151, Toulouse, France Rib/ T3 cells, originating from a rat pancreatic tumor, produce somatostatin. We describe in these cells a sensitivity towards the peptides of the glucagon-family similar to that present in the gastric mucosa: Oxyntomodulin, the 37oamino acid glucagon-containing p e p t i d e p r o c e s s e d from proglucagon in intestine and hypothalamus, was 10-20 times more potent than glucagon on somatostatin release and on cellular cyclic AMP levels. The C-terminal oxyntomodulin [19-37] fragment increased somatostatin release without modifying the cyclic AMP levels. In contrast to carbachol, none of the peptides modified significantly the inositol phosphate turnover or the intracellular free Ca 2+ measured by the fura-2 method. These data indicate that the peptides of the oxyntomodulin-glucagon family possess several receptorsecond messenger systems in RIN T3 cells, one linked to a d e n y l a t e cyclase and a n o t h e r i n d e p e n d e n t f r o m phospholipase C. The RIN T3 cells are a good model for analyzing the cellular mode of action of these peptides. It remains to be established whether the 'oxyntomodulin type' receptor discovered in RIN T3 cells is present in normal islet D cells. If so, this would open up a new avenue of research on the control of islet peptides by the proglucagon-deriving peptides.

A99

P348

P349

TRUNCATED GLP-I (PROGLUCAGON 78-107-AMIDE): A POSSIBLE INCRETIN FROM DISTAL GUT. B. Schjoldager, A. Wettergren, P,E. Mortensen J.Myhre, C. Zrskov, J. Christiansen, J.J. Holst. Institute of Medical Physiology C, and Department of Surgical Gastroenterology, Glostrup Hospital, U n i v e r s i t y of Copenhagen, Denmark. Truncated GLP-I is a hormone from the distal ileal mucosa which upon infusion in man inhibits postprandial gastric acid secretion, gastric emptying, and pancreatic secretion of trypsin and lipase. We studied the effects of intravenous infusion of synthetic truncated GLP-I in comparison with saline on fasting and postprandial pancreatic secretion of insulin and glucagon, blood glucose and enteroglucagon release in six normal volunteers receiving a low caloric content liquid meal. The infusion resulted in plasma concentrations of truncated GLP-I of 103 +/20 pmol/l (mean +/- SEM), similar to postprandial values. Plasma concentrations of the enteroglucagons oxyntomodulin and glicentin remained below i0 pmolA. Plasma concentrations of pancreatic glucagon decreased' during truncated GLP-I infusion, from 8.2 +/- 0.9 pmol/l (preprandial) to 6.3 +/- 0,9Vpmol/l (postprandial). There was no decrease during saline infusion. Plasma insulin increased after the meal from 30 +/~ 2 pmol/l to 59 +/- 8 pmol/l (saline) and from 36 +/4 pmol/l to 70 +/- 27 pmol/l (truncated GLP-I), but with no significant differences between the days. Blood glucose increased after the meal on both days, but not significantly. Postprandial values of blood glucose were inhibited by truncated GLP-I, from 4.7 +/0.2 (saline) to 3.8 +/- 0.3 mmol/l (truncated GLP-I), a significant inhibition of 19 %. We conclude that truncated GLP-I has significant effects on pancreatic g l u c o r e g u l a t o r y hormones and glucose metabolism.

The p r e s e n c e of specific receptors for g l u c a g o n like p e p t i d e - i [7-36]amide (tGLP-I) has been documented in different clones of tumoral i n s u l i n - p r o d u c i n g cells by two previous reports, yet i n f o r m a t i o n is lacking in normal islet cells. We have studied the specific b i n d i n g of [1251]tGLP-I in d i s p e r s e d normal rat islet cells and in tumoral cells (RINm5F). In normal and tumoral cells the S c a t c h a r d plot d e m o n s t r a t e d the p r e s e n c e of high- and l o w - a f f i n i t y b i n d i n g sites. The apparent Kd for the h i g h a f f i n i t y b i n d i n g sites was 1.8 nmol/l in normal and i.i nmol/l in tumoral cells; the Kd for the lowaffinity b i n d i n g sites was 0.46 ~mol/l in normal and 0.17 ~mol/l in tumoral cells. The 50% inhibition of the maximal b i n d i n g (400 pmol/l) was close to 3.0 nmol/l in both cell types. Both the b i n d i n g and the d i s s o c i a t i o n were time and t e m p e r a t u r e dependent, thus maximal b i n d i n g was achieved in 30 min at 37~ and 50% d i s s o c i a t i o n was attained in 7 min at 37~ Glucagon and GLP1 [l-36]amide at high c o n c e n t r a t i o n s (i0 ~mol/1) did not compete with the [IzbI]tGLP-I binding. In conclusion: the p r e s e n c e of specific r e c e p t o r s for tGLP-I in normal rat islet cells w i t h two affinity binding sites of similar c h a r a c t e r i s t i c s to that in tumoral islet cells, is documented.

P350

P351

OF ACTION OF NEUROPEPTIDE Y ON INSULIN SECRETION IN THE ISOLATED PERFUSEDRAT PANCREAS G.Skoglund, R.Gross, B.Ahr~n and M.M.Loubati~res-Mariani, Laboratory of Pharmacology, Faculty of Medicine, CNES, Montpellier, France and Depts. of Pharmacology and Surgery, Lund University, Lund, Sweden

INVOLVEMENT OF ALPHA-2 AND BETA-ADRENOCEPTORS IN INSULIN RELEASE INDUCED BY PANCREATICNERVE STIMULATON N. Duval, I. Angel, M.T. Eon, A. Oblin and S.Z. Langer Synth~labo Recherche (L.E.R.S.), 58 rue de la Glaci6re, 75013, Paris

MODE

Neuropeptide Y (NPY) has previously been demonstrated to inhibit insulin secretion and induce vasoconstriction in the perfused rat pancreas. To examine the mechanisms behind these actions, the interaction between NPY and prazosin (~x-adrenoceptor antagonist), yohimbine (~2-adrenoceptor antagonist), verapamil (Ca~ channel blocker) or forskolin (adenylate cyclase activator) was studied in the isolated rat pancreas perfused through the arterial system with KEB medium supplemented with 8.3 mM glucose at constant pressure. NPY (i.0 raM) reduced (by 50Z) insulin secretion (P<0.01) and flow (P<0.01). These effects by NPY were not affected by prazosin or yohimhine, not even at a high dose level (6.0 ~M). Verapamil (6.0 ~/4) reduced by itself insulin secretion (by 50Z; P<0.05). Verapamil and NPY together showed additive inhibitory effects on insulin secretion, which was inhibited by 75Z (P<0.01). In contrast, verapamil abolished the NPY-induced flow reduction. Forskolin potentiated insulin secretion markedly (by 500%; P
GLUCAGON-LIKE PEPTIDE-I [7-36]AMIDE NORMAL AND TUMORAL ISLET CELLS.

RECEPTORS

IN

I. Valverde, S. Garcia-Mateu, C. Ruiz-Grande, M. A. T r a p o t e and E. Furundarena. F u n d a c i 6 n Jim~nez Diaz, Centro A s o c i a d o C.S.I.C., Madrid, Spain.

The sympathetic nervous system plays an important role in the regulation of pancreatic insulin release. Ne have investigated the effects of pancreatic sympathetic nerve stimulation (PNS) in anaesthetized dogs, by measuring pancreatic insulin and norepinephrine (NE) outflow in the pancreatico-duodenal vein, under selective adrenergic receptors blockade. In chlorisondamin (I mg/kg iv) and atropine (I mglkg iv) pretreated beagle dogs, consecutive PNS at 0.3, l and 3 Hz <25 V, 2 ms for 3 min) potentiated pancreatic NE release, and p a r a l l e l y inhibited insulin release in a frequency dependent manner. The alpha-2 adrenoceptor antagonists idazo• or SL 84.0418 (at pancreatic infusion of 1,3 or I0 ug/kg/min ia or at 0.3, l and 3 mg/kg iv) markedly potentiated NE release, apparent l y through the interaction at pre-synaptic alpha-2 adrenoceptors, while concomitantly and p a r t i a l l y antagonizing the post-synaptic alpha-2 adrenoceptor mediated i n h i b i tion of insulin release. This antagonism was frequencydependent with larger antagonism at lower PNS-frequencies. Under blockade of beta-adrenoceptors with propranolol
AIO0 P352

P353

ACTIVATION OF ~-ADRENOCEPTORS IN RAT ISLETS OF LANGERHANS LEADS TO ENHANCED GLUCAGON BUT NOT INSULIN SECRETION

THE INFLUENCE OF GAMMA-AMINOBUTYRIC ACID (GABA) ON HORMONE RELEASE BY THE ENDOCRINE PANCREAS. P. Gilon, C. Remacle and J.C. Henquin. Unit6s de Diab6tologie et de Biologie Cellulaire, University of Louvain, Brussels and Louvainla-Neuve, Belgium.

R.J. Lacey, N.S. Berrow, J.H.B. Scarpello* and N.G. Morgan. Departments of Biological Sciences and Postgraduate Medicine*, University of Keele, Keele, Staffs ST5 5BG, U.K. ~-agonists are reported to increase islet hormone secretion in vivo, although it is unclear whether this effect is mediated via direct interaction with islet cell receptors, or whether it results from indirect mechanisms. To address this issue, we have investigated the effects of the selective ~-agonist clenbuterol on cAMP levels, insulin and glucagon secretion in isolated rat pancreatic islets. Clenbuterol (20~M) elicited a significant increase in cAMP levels within 5 minutes of incubation (control: 1.28 • 0.15; clenbuterol: 2.98 • 0.27 fmol cAMP/islet; p
The role of the high amounts of GABA contained in islets is unclear. The present study was an attempt to localize GABA by immunocytochemical methods (at the light and electron microscope levels) and to define its possible influence on hormone release by isolated mouse or rat islets. GABA and glutamate decarboxylase (the major enzyme of GABA synthesis) were only identified in B ceils. GABA was not co-localized with insulin in secretory granules. Exogenous GABA (1-100 ~tmol/1) or muscimol (10 ~tmol/1; agonist of GABAA-receptors) did not affect basal or stimulated insulin and somatostatin release. In contrast, GABA and muscimol slightly inhibited (20 %, P < 0.01) glucagon release induced by glucose omission or by 20 retool/1 arginine in 3 mmoVl glucose. On the other hand, bicuculline (10 ktmol/l; antagonist of GABAA-receptors) did not prevent the inhibition of glucagon release by 20 mmol/1 glucose. In conclusion, GABA does not seem to affect B and D cell function, but inhibits A cells by acting on GABAA-receptors. However, no evidence could be obtained to support the hypothesis that glucose-inhibition of glucagon release is mediated by GABA co-secreted with insulin.

P354

P355

GASTRIN-RELEASING PEPTIDE (GRP): BINDING AND FUNCTIONAL STUDIES IN MOUSE PANCREATIC ISLETS

GALANIN IS PRESENT IN HUMAN PANCREATIC NERVES AND INHIBITS INSULIN SECRETION FROM ISOLATED HUMAN ISLETS 8. Ahr@n, A. Ar'Rajab, G. BGttcher, F. Sundler and B.E. Dunning. Depts. of Pharmacology, Surgery, and Medical Cell Research, Lund Univ., Lund, Sweden, and Dept. of Medicine, Univ. of Washington, Seattle, Wa, USA

E.J. Verspohl, E.A. Landsbeck, R.J. Plehn, H.P.T. Ammon, M.A. Wahl. Depart. of Pharmacology, Inst. of Pharmaceut. Sciences, Tiibingen, FRG GRP is a pancreatic neuropeptide that increases insulin release, and increases net 45Ca2+ uptake by pancreatic islets. GRP binding sites and the involvement of inositol metabolism have not been investigated yet. Binding of 100 pM [nSI-TyrlS]GRP to collagenase-isolated mouse pancreatic islets at 24 ~ was time-dependent; maximal binding was 1.65 % of radioacitvity per 50 islets; half-maximal binding was achieved at approx. 15 rain; non-specific binding (presence of 1 #M unlabelled GRP) was less than 10 % of totally bound radioactivity. Tracer degradation was less than 13 %. The ICso of competition of binding by unlabelled GRP was 2.4 nM; iterative analysis of the data showed one binding site. Binding was specific with respect to the rank order of GRP analogues (N-terminus of GRP(1-16), NAC-GRP(20-27), and hormones not structurally related to GRP like VIP and galanin. The rank order of GRP analogues on glucose(16.7 mM)-induced insulin secretion (RIA) was similar to their affinity

rank

order.

GRP

augmented

inositol-l,4,5-trisphosphate

(determined by a mass assay; ECso = 0.8 nM). In a virtually Ca~+-free medium 5 nM GRP increased the 45Ca2+ release of preloaded islets. In conclusion: 1. Highly specific binding sites for GRP are present in mouse pancreatic islets. 2. GRP increases Insp 3 content of islets which possibly stimulates the observed Ca release from intracellular stores.

In several animal species, the neuropeptide galanin occurs in pancreatic nerves and inhibits insulin secretion. Galanin has also, in some species, been suggested to be the neurotransmitter mediating the inhibition of insulin secretion that accompanies adrenergic nerve activation. However, the presence and action of galanin in the human pancreas have remained unknown. Therefore, we determined the presence and nature of human pancreatic galanin-like immunereactivity (GLIR) and the effects of galanin on glucose-stimulated insulin secretion from human islets. Immunofluorescent staining of the human pancreas revealed GLIR in fine varicose nerve fibers in both islets and in the exocrine parenchyma. Furthermore, acid extracts of human pancreas (n=3) and isolated islets (n=3) contained 0.17• and 0.23• pmol GLIR/mg protein, respectively. The pancreatic GLIR coeluted with synthetic porcine galanin from Sephadex G-50. Moreover, synthetic porcine galanin inhibited glucose-stimulated insulin secretion from collagenase-isolated human islets in a dose-related manner with a minimum effective dose (MED)>I0 -s M. Thus, l)the human pancreas is innervated by galanin-containing nerves, 2)the human pancreatic GLIR is galanin-sized, and 3)porcine galanin inhibits glucosestimulated insulin secretion from human islets. Hence, we conclude tha~ galanin could be a local regulator of human insulin secretion.

A101

P356

P357

AMPLIFICATION O F G L U C O S E M E D I A T E D INSULINRELEASE BY

EXTRACELLULAR ATP STIMULATES INSULIN SECRETION BY CA2+ DEPENDENT AND INDEPENDENT MECHANISMS

GIP: A R E IONIC F L U X E S I N V O L V E D ? M.A. Wahl, E.A. Landsbeck, R.J. Plehn, H.P.T. A m m o n and E.J. Verspohl. Institute of Pharmaceutical Sciences, EberhardKarls

University, Tfibingen,

FRG.

Glucose-dependent insulinotropic peptide (GIP), a 42 aminoacid peptide w a s recently s h o w n to augment insulinsecretion induced by nutrients. It was the aim of our study to evaluate its mechanism of action, in pancreatic islets. W e therefore examined whether GIP affects 8SRb* efflux, 45Ca2* uptake and efflux and intracellular recorded electrical activity using isolated mouse islets. GIP neither inhibited eSRb§ efflux at 3 m M glucose nor altered S6Rb* efflux inhibited by 5.6 m M glucose or stimulated by ionophore A23187.4a CaZ, uptake in the presence of 16.7 m M glucose was augmented dosedependently with a maximal effect of 150% at about 10-s M (p<0.05). In the presence of 3 m M or 11 m M glucose, no effect of GIP w a s seen. Addition of GIP to a buffer containing 16.7 m M glucose elevated 4aCaS§ efflux, no alteration was seen in a Ca z* deprivated medium. Intraeellular recorded electrical activity challenged by 16.7 m M glucose showed a significant increase in the presence of 5"I0 -9 M GIP. From our data w e conclude that GIP m a y amplify glucose-induced insulinsecre-

tion by increasing Ca2. uptake.

P358 MECHANISMS OF THE AMPLIFICATION OF INSULIN RELEASE BY ARGININE-VASOPRESSIN. Z.Y. Gao, G. Drews, M. Nenquin, T.D. Plant and J.C. Henquin. Unit6 de Diabdtologie et Nutrition, University of Louvain, Brussels, Belgium; and I. Physiologisches Institut, University of Saarland, Homburg/Saar, Germany. The mechanisms by which arginine-vasopressin (AVP) affects pancreatic B cell function are still unclear. They were studied in normal mouse islets. AVP produced a dose-dependent (0.1-1000 nmol/t; ECho - 2 nmol/1) amplification of glucose-induced insulin release. This amplification was of slow onset and reversibility. AVP was ineffective when the concentration of glucose was _< 7 mmolB, but still very effective in 30 mmol/1 glucose. The increase in insulin release produced by AVP was accompanied by small accelerations of 86Rb and "~Ca effiux from islet cells. Omission of extracellular Ca accentuated the effect of AVP on 86Rb efflux, attenuated that on 45Ca efflux and abolished that on release. AVP had little effect on cAMP levels, but increased inositol phosphate levels in islet cells, even in the absence of Ca. AVP augmented glucose-induced electrical activity in B cells only slightly. This was not due to a direct action on ATP-sensitive K§ channels as revealed by patch-clamp recordings (whole-cell and outside-out patches). In conclusion, AVP potently amplifies glucose-induced insulin release in B cells. This effect involves a stimulation of phosphoinositide metabolism rather than a change in cAMP levels .or a direct control of the membrane potential.

G.-D. Li, D. Milani, W. Pralong and C.B. Wollheirn. Division de Biochimie Clinique, University of Geneva, Geneva, Switzerland ATP belongs to the Ca2+-mobilizing neurotransmitters which stimulate insulin secretion. Using RINm5F cells we examined whether the Ca 2+ mobilization is sufficient to explain the secretory response. ATP raised cytosolic Ca2+ ([Cae+]i) and depolarized the cells (ECso=5 uM) as measured with fura-2 and bisoxonol, respectively. Phorbol-myristate-acetate (5 min pretreatment), which uncouples receptors from phospholipase C, attenuated the [Ca2+]i rise and production of CaZ+-mobilizing inositol trisphosphate without affecting membrane depolarization by ATP. Diaz0xide (200 uM) that activates ATP-sensitive K § channels decreased ATP-induced depolarization by 50-70% and abolished Caz+ influx, monitored in single cells by the quenching of fura-2 with Mn 2+. However, blockade of L-type Ca 2+ channels did not attenuate [Ca2+]i rises. In perifusion, ATP (100 uM) elicited transient (>10-fold) insulin secretion. Ca2+ depletion with EGTA caused time-dependent abolition of [Ca2+]i responses, whereas insulin secretion was only partially suppresed. Simultaneous addition of EGTA or diazoxide with ATP did not inhibit secretion, but 5 min pretreatment with diazoxide, which blocks Ca 2+ influx, reduced secretion by 30%. Interference with Ca2+ mobilization by phorbol-myristate-acetate caused similar reduction of secretion. Epinephrine inhibited ATPinduced insulin release by 60%. Thus, insulin secretion by ATP comprises (i) phospholipase C activation and Ca 2+ mobilization, (ii) Ca 2+ influx not involving phospholipase C and L-channels, and (iii) Ca 2+ independent processes.

A102

PS 3 Islets: Ions and Ionic channels P359

P360

Externally applied ATP closes ATP-regulated potassium in insulin-secreting (RINm5F) cells causing a membrane depolarization.

INHIBITION OF CALCIUM UPTAKE IN ISLET CELL MICROSOMES BY A MONOCLONAL ANTIBODY TO HEART SARCOPLASMIC RETICULUM

By M.J. DUNNE and O.H. PETERSEN. The MRC Secretory Control Research Group, The Department of Physiology, The University of Liverpool, Liverpool, L69 3BX. Patch-clamp experiments have been carried out using whole-cell and excised outside-out membrane patches, to investigate the effects of externally applied ATP on the membrane potential and the gating of ATP-sensitive K+ channels. When added to the bathing solution at concentrations between 50 and 200 ~M, ATP caused a sharp depolarization of the membra,lr from around -70 to approximately -40 mV (n=6 separate cells). This depolarization was associated with the generation of Ca2+ spike potentials which were maintained for the duration of the stimulation. At higher concentrations (> 200 ~tM) the effects of ATP on the membrane potential were transient, consisting of a large initial depolarization, lasting between 2 and 10 seconds, which was then followed by a pronounced repolarization of the membrane (n=6). Only the early phase of-the depolarization was associated with the generation of spike potentials. Added to the external face of outside-out membrane patches, ATP (200 ~tM) caused a sharp decrease in the open-probability of KATP channel (n=5). ATP-evoked channel closure was not as pronounced as that evoked by externally applied tolbutamide (100 ~tM) (n=5). These data may indicate that the purinergic (P2-type) receptor, thought to be present in these cells, is either part of or is closely associated with the ATP-regulated potassium channel.

B. Manuel y Keenoy, D.O. L e v i t s k y , A. Sener and W.J. Malaisse. Laboratory of Experimental Medicine, Brussels Free U n i v e r s i t y , Brussels, Belgium and I n s t i t u t e of Experimental Cardiology, USSR Cardiology Research Center, Moscow, USSR. + The uptake of Caz by mcrosom% is thought to p a r t i c i p a t e in the control of c y t o s o l i c Ca2 a c t i v i t y in the i n s u l i n producing pancreatic B - c e l l . In the present study, a subc e l l u l a r microsomal f r a c t i o n was prepared from RINm5F c e l l s . In t h i s f r a c t i o n , the r a t i o o f microsomal/mitochond r i a l enzyme markers, as w~ll as t h a t of ruthenium redresistant/sensitive ~5Ca2' uptake,was twenty times h i gher than in the corresponding mitochondrial s u b c ~ l l u l a r fraction. The ATP-dependent net uptake of 45CaZ+ by RINmBF c e l l microsomes displayed an apparent K~ f o r ATP close to 1.5 mmol/l and an apparent Ka f o r Ca2 close to 0.2 pmol/l. I t was i n h i b i t e d at low temperature and by vanadate (apparent Ki : 50 p m o l / l ) . A monoclonal antibody d i r e c t e d against the Ca2--A~Pase of dog heart sarcoplasmic reticulum i n h i b i t e d , s C a 2 uptake by RINm5F c e l l ~ i c r o s o mes, w h i l s t f a i l i n g to a f f e c t the uptake of 45CaZ in the mitochondrial s u b c e l l u l a r f r a c t i o n , As judged from the q u a n t i t a t i v e aspect of these measurements, the uptake of Caz- by microsomes would account f o r only a modest f r a c t i o n of ATP consumption in i n t a c t i s l e t c e l l s , although being ~ e l l suited to play, at p h y s i o l o g i c a l concentrations of #a 2- and ATP, a major r o l e in the control of c y t o s o l i c Ca2 a c t i v i t y .

P361

P362

SUPPRESSION OF GLUCOSE-INDUCED OSCILLATIONS OF Ca2+ IN p-CELLS EXPOSED TO STREPTOZOTOCIN AND ALLOXAN.

EFFECTS OF AMINESAND AMINE PRECURSORSON MOUSE 8-CELL CALCIUM ACTIVITY. P Lindstr6m. Department of Histology and Cell Biology, Ume&, Sweden Dopamine and its precursor L-DOPA are potent inhibitors of insulin release in mouse islets. Serotonin is less potent than dopamine. L-5Hydroxytryptophan (L-5-HTP) has a small stimulatory effect but strongly potentiates the effect of glucose. I have now labelled mouse (Ume& ob/ob) 8-cells with fura-2/AM and measured intracellular calcium activity in single cells using dual wavelength (340/380 nm) fluorometry. Raising the glucose concentration from 1 mmol/I to 20 mmol/I induced an initial lowering of the calcium activity followed by an increase and slow irregular oscillations. The initial decrease often remained in perifusions with 0.1 mmol/I dopamine but the later increase was abolished. Serotonin (1 mmoi/I) had no effect on fura-2 fluorescence. L-DOPA (1 mmol/I) increased the frequency of oscillations. L-5-HTP (4 mmol/I) abolished the oscillations and the calcium activity remained increased. At 1 mmol/I glucose, L-5-HTP induced a slow rise in the fluorescence ratio reaching a maximum level after 10-15 min. This level was not less than that induced by 20 mmol/I glucose + 4 mmol/I L-5-HTP. The decarboxylase blocker ~z-monofluoromethyldopa (0.1 mmol/I) and verapamil (10 pmol/I), both inhibited the effect of L-5-HTP. In conclusion, dopamine may inhibit insulin release by inhibiting calcium inflow. L-5-HTP increases calcium inflow. There is no strict parallellism between effects on cytosolic calcium activity and insulin release.

E. Orapengiesser, E.Gylfe and B.Hellman. Department of Medical Cell Biology,University of Uppsala, Sweden Dual wavelength microfluorometry and the indicator fura2 were employed for studying bow diabetogenic agents affect the cytoplasmic Ca 2+ concentration (Ca2+i) in individual pancreatic B-cells from ob/ob-mice. In most B-cells a rise of the glucose concentration from 3 to 20 mmol/1 resulted in large amplitude oscillations of Ca2+i (0.2-0.5/rain) from a basal level of 60-90 nmol/l. Streptozotocin and alloxan (I4.4 mmol/1) rapidly abolished the glucose-induced oscillations of Ca2+i. The presence of a high glucose concentration during the exposure to the toxicants counteracted the action of alloxan but not that of streptozotocin. Loss of the cyclic variations of Ca2+i after streptozotocin treatment did not prevent a glucose-induced increase of Ca2+i in mildly affected cells. Most B-cells made insensitive to glucose by streptozotocin responded to 100 ~mol/l tolbutamide with an immediate rise of Ca2+i. The most advanced lesions obtained with exposure to the diabetogenic agents were manifested as uncontrolled and sustained increases of Ca2+i. The results indicate that experimentally induced lesions can break the oscillatory pattern with maintenance of a glucose response in terms of raised Ca2+i .

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TEMPERATURE DEPENDENT OSCILLATIONS OF CYTOPLASMIC CA2+ AND ACTION POTENTIALS IN SINGLE B-CELLS

MODULATION OF INTRACELLULAR FREE CALCIUM LEVELS BY THE SULFONYLUREA TOLBUTAMIDE IN SINGLE ISLET OF LANGERHANS B. Soria, A. Nadal, M. Valdeolmillos and L.M. Rosario. Department of Physiology, University of Alicante, Spain Sulfonylureas are thought to raise B-cell cytosolic Ca 2+ concentration ([Ca2+]~) primarily through blockade of ATP-sensitive ~ - c h a n n e l s , followed by membrane depolarization and stimulation of Ca ~ influx. Alternative mechanisms leading to [Ca2+]i rises have not been directly examined. We have now tested the effects of tolbutamide (50-100?~M) and diazoxide (100-400 uM) on islet [Ca = ];, which was monitored by I n ~ q l fluorescence.~Glucose (7-17 raM) induced [Ca ]; oscillations a~ previously reported. Tolbutaml~de increased [CaZ*]~ both in the presence of sub-stimulatory (3 ~mM) and stimulatory (ii and o~7 mM) glucose concentrations. This [Ca=--]~ rise occuKred rapidly and exceeded the pea~s of the [caZ*]~ oscillations. Moreover, the tolbutamide effe$~ was blocked by verapamil (i00 uM) and Ca-removal from the perfusion medium% Unlike tolbutamide, the ATP-blockable K -cha99el agonist diazoxide suppresse~ [Ca~-]~ oscillations and reduced average Ca "t levels. I~ is concluded that tolbutamide does not rise [Ca2§ by either of the follDwing previously suggested mechanisms: (i) Ca z* release from intracellular stores; (ii) voltage~insensitive ionophoretic translocation of Ca'-. Rather, tolbutamide is likely to increase [Ca2+]~ .by ultimately activating voltage-sensitive ~a ztchannels.

U.Panten, C.Schwanstecher, C.Heipel, E.Grapengiesser, E.Gylfe and B.Hellman. institute of Pharmacology and Toxicology, University of Gbttingen, FRG; Department of Medical Cell Biology, University of Uppsala, Sweden In single pancreatic B-cells glucose induces large amplitude oscillations of the cytoplasmic Ca 2+ concentration (Ca2+i) at 37~ Patch-clamp experiments at room temperature, however, have failed to demonstrate similar oscillations of action potentials. To analyze this discrepancy we examined how temperature influences these phenomena in single B-cells from mice. Ca2+i was monitored continuously using dual wavelength microfluorometry and action potentials were recorded using the cell-attached configuration of the patch-clamp technique. The glucose-induced oscillations of Ca2#i disappeared when the temperature was increased above 42~ and were reversibly inhibited below 30~ However, cooling did not prevent a glucose response in terms of the average rise of Ca2+i. When performing patch-clamp experiments at 37~ glucose-induced action potentials were formed to occur in bursts with durations similar to the large amplitude oscillations of Ca2+i. It is concluded that the oscillations of CaZ+i follow the action potentials which reflect alterations of the Ca 2+ permeability of the plasma membrane.

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CYTOPLASMIC Ca2+ RESPONSE TO QUININE IN PANCREATIC

LOOP DIURETIC-SENSITIVE CI'/CATION CO-TRANSPORT SYSTEMS IN THE PANCREATIC B-CELLS J. Sehlin and P.-E. Sandstr6m, Department of Histology and Cell Biology, University of Ume&, Ume&, Sweden. Previous studies have shown that loop diuretics inhibit B-cell function, probably by 'acting on CI-/cation co-transport. The role of Na + in such co-transport has now been examined in mouse islets by measuring 86Rb+ and 22Na+ fluxes. 22Na+ efflux was reduced by both ouabain (1 mmol/I) and furosemide (1 mmol/I) and the effects were additive. Extracellular Na + deficiency reduced the ouabain-resistant 86Rb+ influx and diminished the effect of furosemide but not that of D-glucose. The 86Rb+ efflux was rapidly and reversibly reduced by N a + deficiency. Furosemide decreased the 86Rb+ efflux and the effect of the combination of furosemide and Na + deficiency was not stronger than the effect of furosemide alone. However, part of the specific furosemide effect remained also in Na + deficiency. The data suggest that Na + participates in loop diuretic-sensitive Na+/K+/Ci - co-transport in the pancreatic B-cells. Since some of the furosemide effect on 86Rb+ efflux also persisted in Na + deficient medium, it is likely that also loop diuretic-sensitive but Na+-independent K+/CI - co-transport occurs in this cell type.

~-CELLS P-E Lund, E. Grapengiesser, E. Gylfe and B. Hellman. Department of Medical Cell Biology, University of Uppsala, Sweden Whereas it is established that closure of the ATP-sensitive K+-channels by glucose or sulfonylurea can induce large amplitude oscillations of the cytoplasmic Ca 2+ concentration (Ca2+i), little is known about the effects of suppressing also other K+ conductances. We therefore examined how quinine, an unspecific inhibitor of K+ conductance, influences Ca2+i and insulin release. Ca2+i was monitored in individual mouse B-cells using the indicator fura-2 and dual wavelength fluorometry, and insulin release was measured by perifusing cells mixed with polyacrylamide beads. At 3 retool/1 glucose, addition of I00 ]Jmol/1 quinine caused a rapid increase of Ca2+i from a basal level of 60-90 nmol/l to a peak value of 300-400 nmol/1. Ca2+i then remained elevated as long as quinine was present. Increase of glucose to 11 mmol/l resulted in large amplitude oscillations of Ca2+i replaced by a continuous elevation with addition of quinine. When glucose was raised from 3 to 20 mmol/1 in the presence of 100 pmol/l quinine there was a pronounced temporary lowering of Ca2+i associated with suppression of insulin release. The results indicate that quinine has similar effects to specific inhibitors of the ATP-sensitive K+-channels. However, its action is less finetuned, resulting in suppression rather than in generation of the large amplitude oscillationsof Ca2+i.

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CYCLIC AMP POTENTIATES L-TYPE CA-CHANNEL ACTIVITY IN MURINE PANCREATIC B-CELLS PA Smith, CMS Fewlxell and FM Ashcroft. University Laboratory of Physiology, Parks Rd., Oxford, OX1 3PT, UK. Agents that increase cyclic AMP (cAMP) levels potentiate B-cell electrical activity and insulin release. This is the mechanism by which glucagon is thought to increase insulin secretion. We have used cellattached membrane patches on mouse pancreatic g-cells to study the effects of cAMP on Ba-currents flowing through single L-type Ca channels at 20-24~ Cells were bathed in a high K-solution to control the B-cell membrane potential and 0.1gM BAY K 8644 was added to increase channel open times. The pipette contained 100raM Ba2+ and 10raM TEA +. Channel openings were induced by voltage pulses from -?0mV. Dibutryl cAMP (1raM), a membrane permeant analogue of cAMP, reversibly increased channel activity by up to 3-fold but did not affect the single channel current amplitude. Forskolin (10gM) had similar effects. These results support the idea that cAMP influences Bcell electrical activity by increasing L-type Ca-channel activity and raise the interesting possibility that glucagon stimulates insulin secretion by cAMP-dependent modulation of this Ca-channel. Since the effect of cAMP on Ca-channel activity resembles that of glucose, these agents may act via a common mechanism.

NA+/CA ++ EXCHANGE IN T H E B E T A C E L L IS ELECTROGENIC M.Hoenig, L.H.Culberson, C . A . W h e e l e r a n d D.C. Ferguson. Department of Physiology and Pharmacology, College of Veterinary Medicine, U n i v e r s i t y of G e o r g i a , A t h e n s , G A 30602 Plasma membrane vesicles from a glucoseresponsive insulinoma exhibit Na/Ca exchange. The electrogenicity of this exchange was evaluated with simultaneous accumulation of the potential-sensitive probe, tetraphenylphosphonium ([3H]TPP). Vesicles were R el o a d e d w i t h 160 ram NaCl. [3H]TPP and 4~Ca uptakes were assayed by adding the vesicles to t u b e s c o n t a i n i n g 160 m M K C l in 20 m M M o p s / T r i s (pH 7.4) a n d e i t h e r a) 1 ~ M [ 3 H ] T P P w i ~ 50 ~ S CaCl 2 o r w i t h 0.i m M E G T A or b) 50 # M C a C I 2. Uptake was terminated by rapid filtration. In the presence of EGTA, [3H]TPP uptake reached an equilibrium v a l u e of 10.9 • 0.5 n m o l e / m g p r o t e i n , w h i l e , in t h e p r e s e n c e o f Ca, u p t a k e i n c r e a s e d t r a n s i e n t l y t o a p e a k l e v e l of 2 9 . 6 + 4.1 n m o l e / m g p r o t e i n at i0 s e c (n=6), a t i m e W h e n t h e r a t e of 4 5 C a u p t a k e w a s a l s o m a x i m a l . A l s o c o n s i s t e n t w i t h a 3 N a + / l C a ++ s t o i c h i o metry, the initial velocity of 45Ca-efflux from Ca-loaded vesicles demonstrated a sigmoidal dependency on the external Na + conc e n t r a t i o n w i t h a H i l l c o e f f i c i e n t c l o s e to 3 (range 2 . 6 - 2 . 9 , n=3). In c o n c l u s i o n , Na/Ca e x c h a n g e in b e t a c e l l s r e g u l a t e s c y t o s o l i c Ca concentrations a n d its d i r e c t i o n m a y v a r y w i t h the membrane potential.

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LINOGLIRIDE, A NON-SULFONYLUREA HYPOGLYCEMIC DRUG, INHIBITS ATP-SENSITIVE POTASSIUM CHANNELS IN PANCREATIC B-CELLS P. Ronner and T.J. Higgins, Diabetes Research Center, University of Pennsylvania, Philadelphia, PA 19104-6015, USA.

EXPRESSION OF VOLTAGE-GATED POTASSIUM CHANNELS IN INSULIN-PRODUCING CELLS. Kenna S., Welsh M., Betsholtz C., Berggren P.-O., Rorsman P., Grupe A., Pongs O., Ashcroft F.M. and Ashcrofl S.J.H. Departments of Clinical Biochemistry & Physiology, Oxford, UK; Medical Cell Biology & Pathology, Uppsala, Endocrinology, Stockholm, & Medical Physics, Gothenburg, Sweden; Biochemistry, Bochum, F.R.G.

Tolbutamide and linogliride, a nou-sulfonylurea hypoglycemic drug, each transiently stimulate insulin release from the perfused rat pancreas. After pretreatment with tolbutam• linogliride no longer stimulates insulin release. We hypothesized that linogliride - like tolbutamide stimulates insulin release by inhibiting ATP-sensitive potassium channels. We studied single rat islet cells electrop~ysiologically in the whole-cell configuration at a potential of -70 mV and with 30 DM ATP in the electrode solution. Currents in response to i0 mV de- and hyperpolarizing voltage pulses were due to ATP-sensitive potassium channels, since they were inhibited 95-98% if the electrode contained 3 ~ ATP, or if the bath contained i ram tolbutamide. Within 3 miu, linogliride applied to cells by per• at concentrations of 30, i00, and 300 NMjinhibited the current by 59• 73• and 97• respectively (meaniS.E.M.; n=3, 5, and 2). Cells pretreated with linogliride for 20-40 min showed a K i for linogliride of 15-20 pM. Linogliride (3-30 pM) did not affect the rate of run-down of the current. The substituted guanidinium moiety of linogliride may well act homologously to the sulfonylurea moiety of sulfonylureas. Our data show that linogliride inhibits ATP-sensitive potassium channels and we propose that linogliride and sulfonylureas bind to the same site on the inside of the plasma membrane.

A previously cloned family of rat cortex voltage-gated Kchannels (RCK) functionally resemble the delayed rectifier Kchannels physiologically important in repolarisation of the 8-cell action potential. Our aim was to investigate the presence in insulin-producing cells of mRNA sequences similar to the RCK family. The polymerase chain reaction (PCR) was performed on cDNA from HIT-T15 and RIN m5F B-cells and from ob/ob mouse islets and also on DNA from a lgt11 HIT-T15 cDNA library, using primers to the $5 and $6 conserved regions of the RCK family. Eight different specific amplification products were obtained which sequence analysis suggested to derive from mRNAs coding for a family of voltage-gated potassium channels. Five of these (MK1-5) appear to be the mouse counterparts of the rat brain RCK family (RCK1-5), A sixth mouse sequence (MK6) is novel; a similar sequence was found in RIN cDNA (RK6) and DNA from the HIT-T15 l g t t l library (HaK6). Two further novel sequences were found, HaK7 from HIT-T15 and RK8 from RINm5F cDNA. Two PCR products (RK/MK/HaK1 and RK/MK/HaK6) were common to both 6-cell lines and islets.

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CALCIUM ACTIVATED K-CHANNELS FROM HIT T15 B-CE1.1~S INCORPORATED INTO PLANAR LIPID BILAYERS Y Oosawa, SJH Ashcroft and FM Ashcroft, University Laboratory of Physiology, Parks Rd, Oxford, OX1 3PT, UK Ca-activated K channels contribute to action potential repolarisation in B-cells. We have studied the properties of this channel after incorporation into planar lipid bilayers, which allows the solution on each side of the membrane to be controlled. Plasma membrane vesicles prepared from insulin-secreting HIT T15 B-cells were added to one side (cis) of a planar lipid bilayer and single channel currents recorded following vesicle fusion. We recorded single channel currents with a reversal potential of 0mV and a conductance of 233pS in symmetrical 140mM KC1 solutions. When [K] o was reduced to 5mM (by Na substitution), the reversal potential was more negative than -50mV, indicating the channel is K-selective. The relative permeability to chloride (PcI/PK) was 0.046. The channel open probability was both Ca and voltage-dependent: K-currents were activated by increasing the cis calcium concentration (K0. 5 -2[xM at 0mV) or by making the cis potential more positive (K0. 5 ~ -15mV at 13~tM Ca). These properties resemble those of Ca-activated Kcurrents recorded in patch clamp studies on B-ceils and suggest the intracellular end of the channel faces the cis side.

B-CELL RECEPTOR SPECIFIC FOR CYTOSOLIC MG2+ADP CONTROLS TOLBUTAMIDE-SENSITIVITY OF K + CHANNELS

C.Schwanstecher, C.Heipel and U.Panten. Institute of Pharmacology and Toxicology, University of G6ttingen, FRG The same type of K + channel (K+Axe channel) in the pancreatic B-ceil is inhibited by cytoplasmic ATP and tolbutamide and the half maximally inhibitory concentration (K~) of tolbutamide is decreased by exposure of excised inside-out membrane patches to ADP. Using mouse B-cells patch-clamp experiments were performed addressing the question whether ADP controls the tolbutamide-sensitivity of the K+nax channel by interaction with the same receptor site mediating ATP-induced channel closure. In reside-out" " membrane patches the ADP-inducedz+ decrease in KJ for tolbutamide required the presence of Mg and channel inhibition by ATP and nonmetabolizable nncleotides was not accompanied by alteration of the tolbutamide-sensitivity of the channel. At 37~ the K+AXe channel in cell-attached membrane ~hatches was half maximally inhibited by 2.2 ~amol/1 tolbutamide. is very low K~ value was observed in the presence of a glucose concentration (3 retool/l) which cannot maintain a low ADP level in B cells. It is concluded that the cytosolic MgZ+ADP complex controls totbutamide-sensitivity by interaction with a receptor site not identical with the site mediating channel closure and that this control is effective in the intact B-cell.

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THE RIDDLE OF GLUCOSE-6-PHOSPHATASE IN RAT ISLETS M.-A. Perales, A. Sener and W.J. Malaisse. Laboratory of Experimental Medicine, Brussels Free U n i v e r s i t y , Brussels, Belgium.

~ I V E GKIJOOSE ~ ~ Y C ~ C ~blob MICE

C o n f l i c t i n g data were r e c e n t l y reported concerning the presence of glucose-6--phosphatase in r a t i s l e t s , which were claimed e i t h e r to be v i r t u a l l y devoid of the enzyme or to d i s p l a y an a c t i v i t y 3-4 times higher than hepatocytes. This issue presents more than academic i n t e r e s t in view ~f the possible r o l e of glucose-6-phosphatase in both Ca2 storage by the endoplasmic reticulum and Dglucose f u t i l e c y c l i n g in i s l e t c e l l s . In the present study, glucose-6-phosphatase a c t i v i t y was measured in r a t l i v e r and i s l e t crude homogenates or microsomes. R e l a t i v e to p r o t e i n content, the y i e l d o f microsomal prot e i n was comparable in l i v e r and i s l e t s . As judged from the h y d r o l y s i s of D-[1-14C]glucose 6-phosphate in i n t a c t , deoxycholate- or histone I I A - t r e a t e d microsomes, the enzymic a c t i v i t y r e l a t i v e to p r o t e i n content represented in i s l e t s less than 2 % o f t h a t found in l i v e r . Moreover, as judged from the generation of hexose phosphate from D - [ 6 - ! ~ C ] g l u c o s e and carbamoyl phosphate, no phosphotransferase a c t i v i t y could be detected in pancreatic i s l e t s , whatever the s u b c e l l u l a r f r a c t i o n examined or microsomal treatment used. The i s l e t microsomal p e l l e t contained no i n h i b i t o r of l i v e r glucose-6-phosphatase a c t i vity. These f i n d i n g s impose r e s e r v a t i o n on the presence of glucose-6-phosphatase in r a t i s l e t s and i t s p a r t i c i p a t i o n to s t i m u l u s - s e c r e t i o n coupling.

IN ISLETS OF FED AND F~%'r~m

A. Khan, V. C~andramouli*, C-G. 0stenscn, H. LOW, B.R. ~u* and S. Efendic, Dept of Endocrinology, Karolinska Hospital, St(xzkholm, Sweden and *Depts of Medicine and B i ~ s t r y , Case Western Re2e_rv~ University, Cleveland, Ohio, USA. We have recently demcnstrated that gluoose cycle (gluoose --> glucose-6-P --> glucose) (GC) operates in the pancreatic islets of mioe. GC was markedly increased in the islets of fed obese hyperglycemic (ob/ob) mice oompared to their lean litter mates. Presently, we have studied the effect of nutriticr~l state of ob/ob mice on the activity of GC. Islets of both fed and fasted animals were incubated with 5.5 and 16.7 n~ [l-14C]glucose and 16.7 n~4 [l-14C]mannose. The yields of 1400_ and the amount of 14C from [I-14C] mannose inoorporat~ into glucose were determined. Glucose utilization was obtained frcm the yield of 3 ~ O from [5-3H]glucose. From these measur6m~nts the q~antity of he_xose utilized by the islets and the quantity of he_xose-6-phosphate dephosphorylation was estimated. Of the hexose phosphorylated by the islets of fed mice 43.3% was dephosphorylated at 5.5 n~4 and 42.3% at 16.7 n~4 gluoose. In fasted state the c o ~ ~ values were 31.2 and 37.1%. Inoorporation of~ from ~ 0 into glucose incubated with islets gave similar peroe~tages. In ccnclusice large peroentsges of gluoo~e ~ l a t e d by ob/cb mice islets are dephos~rylated. The extent of GC was not significantly altered by short-term fastir~. Thus irrespective of nutriticr~l state GC could have i~0ortant effects on the extent of insulin release by islets and oould be the basis for the diabetic syndrcme in mioe.

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ACTIVATION BY Ca2+ OF KEY MITOCHONDRIAL DEHYDROGENASES IN PANCREATIC ISLETS : PHYSIOLOGICAL AND PATHOLOGICAL ASPECTS J. Rasschaert, M.-H. G i r o i x , A. Sener and W.J. Malaisse. Laboratory of Experimental Medicine, Brussels Free Univers i t y , Brussels, Belgium and Laboratory of Developmental Physiology, U n i v e r s i t y of Paris 7, Paris, France

A ROLE FOR ACYL-COA ESTERS IN NUTRIENTSTIMULATED INSULIN SECRETION M. Prentki, D. Belin, C. Glennon, S. Vischer, K.-H. Kim, and B.E. Corkey. Division de Biochimie clinique, University of Geneva, Switzerland, Purdue University, Lafayette, IN and Boston University, MA, U.S.A.

N u t r i e n t secretagogues provoke Ca2+ accumulation in i s l e t c e l l mitochondria. The i n f l u e n c e of t h i s cation upon selected m i t o c h % d r i a l dehydrogenases was t h e r e f o r e i n v e s t i g a t e d . Ca= (8 pmol/l) lowered the Km of FAD-linked glycerophosphate dehydrogenase f o r i t s s u b s t r a t e , as judged from the generation of 3H20 from L-[2-3~ glycerol-3-phosphate by i n t a c t mitochondria. In i s l e t homogenates, NAD-linked i s o c i ~ r a t e dehydrogenase a c t i v i t y was also increased by Ca2- (Ka S 1.0 p m o l / l ) , at l e a s t in the presence of ADP, t h i s c o i n c i d i n g with an increased a f f i n i t y f o r the t r i c a r b o x y l i c acid. Likewise. in both i s l e t homogenates and i n t a c t mitochondria, Ca2§ increased 2 - k e t o g l u t a r a t e dehydrogenase a c t i v i t y with again an i n creased a f f i n i t y of the enzyme f o r the 2-keto acid but l i t t l e chan~e in maximal v e l o c i t y . Ruthenium red suppressed the CaZ--induced increase i n [ 1 - ~ " C ] 2 - k e t o g l u t a r a t e o x i d a t i o n by i s l e t mitochondria. Preincubation of the i s l e t with D-glucose increased the r a t e of [ l ~ ] 2 - k e t o g l u t a r a t e o x i d a t i o n by mitochondria. In i s l e t mitochond r i a from a d u l t r a t s i n j e c t e d with s t r e p t o z o t o c i n durina the neonatal p e r i o d , the d e t r i t i a t i o n of L - [ 2-3,H~!vcero~ 3-#hosphate was severely impaired, both in the absence ana presence of Caz*. This coincided with a decreased r a t i o o f aerobic to t o t a l g l y c o l y s i s in i n t a c t i s l e t s . This s i t e - s p e c i f i c defect could account, in p a r t at l e a s t , f o r the p r e f e r e n t i a l impairment of the f u n c t i o n a l response to D-glucose in t h i s experimental model of diabetes.

To gain insight into the coupling factors linking the metabolism of nutrients to insulin release we examined using HPLC the acylCoA profile of clonal pancreatic B cells (HIT) in response to various fuel stimuli. Malonyl CoA increased about 4-fold in response to glucose, pyruvate and glutamine plus leueine. The rise in malonyl CoA preceded insulin secretion. Only those nutrients promoting secretion caused elevations in malonyl CoA. Consistent with the situation in the liver, where malonyl CoA mediates the switch from fatty acid catabolism to lipid synthesis, glucose inhibited fatty acid oxidation, increased d e novo lipid synthesis and a rise in diacylglycerol mass in HIT cells. Succinyl-CoA levels which reflect anaplerotic input into the Krebs cycle were elevated by the nutrient agonists. The formation of malonyl CoA by acetyl CoA carboxylase is the rate-limiting step in lipid biosynthesis. Using an antisense RNA probe, HIT cells were shown to express high levels of acetyl CoA carboxylase mRNA. Exogenous long chain fatty acids (myristate and palmitate) were extremely potent stimulators of insulin release. The data lend strong support to the concept that lipid biosynthetic events and acyl CoA esters provide a crucial link between fuel metabolism and insulin release.

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P378

ADENINE NUCLEOTIDE PATTERN OF ISLETS FROM FED AND STARVED RATS= J. Tamarit-Rodriguez and E. Vara. Departamento de Bioquimica, Facultad de Medicina, Universidad Complutense. Madrid. Starvation increases islet palmitate oxidation and decreases the sensitivity of insulin secretion to glucose. We have now investigated whether these metabolic changes induce any variation of islet adenine nueleotides. They were measured with a fire-fly lueiferase assay after enzimatie conversion of ADP and AMP into ATP. Islet ATP reached its maximum level at 5 mmol/l glucose (6.7 ~ 0.5 pmol/i, n=9) whereas ADP was decreased by increasing sugar concentrations. AMP showed a clear dose-dependent stimulation by glucose: 2.9 ~ 0.5 (n=6), 4.5 ~ 0.3 (n=6) and 6.7 ~ O.6 pmol/i (n=6) at 0,5 and 20 F~nol/l, respectively. Starvation (24h) did not significantly modify ATP but it increased ADP (3-and 4-fold) and decreased AMP (5-and 9-fold at 5 and 20 mmol/l glucose, respectively). Fatty-acid oxidation inhibitors (0.25 m o l / l 2-bromostearate and lO umol/l 2-tetradecylglycidate) did not affect significantly ATP but they decreased ADP and increased AMP in starved islets to the levels recorded in fed controls at 5 and 20 mmol/l glucose. They also restored a normal insulin response to 20 n~nol/l glucose in starved islets. The results show that total islet AMP correlates positively with insulin release and therefore it might be implicated, as cause or effect, in the stimulus-secretion coupling mechanism of B-cells.

STIMULATION BY D-GLUCOSE OF POLYPHOSPHOINOSITtDE SYNTHESIS IN ISLET CELLS F. Blachier and W.J. Malaisse. Laboratory of Experiment a l Medicine, Brussels Free U n i v e r s i t y , Brussels, Belgium. N u t r i e n t secretagogues s t i m u l a t e the h y d r o l y s i s of p o l y phosphoinositides in i s l e t s , a phenomenon c u r r e n t l y ascribed to a c t i v a t i o n of phosphoinositidase C by c y t o s o l i c Ca2-. When i s l e t s p r e l a b e l l e d with m y o - [ 2 - 3 H ~ i n o i i t o l were incubated f o r 5 min in the absence of Ca2-, D-glucose indeed f a i l e d to a f f e c t the production of [ B H ] i n o s i t o l phosphates, but increased by 65 % the amount o f t r i t i a t e d p o l y p h o s p h o i n o s i t i d e s . Likewise, in homogenatew of p r e l a b e l l e d i s l e t c e l l s , ATP, in the absence of Caz-, increased [ 3 H ] p o l y p h o s p h o i n o s i t i d e s but n%t [ ~ H ~ i n o s i t o l phosphates. In the homogenates, Ca2 (~ 10 6 mmol/l) caused a c o n c e n t r a t i o n - r e l a t e d increase of [3H~ i n o s i t o l phosphate production, t h i s being matched by a comparable decrease o f . [ 3H ] p o l y p h o s p h o i n o s i t i d e s . In the presence of Ca2-, ATP f u r t h e r increased the production of [ 3H ] i n o s i t o l phosphates. The i n t e g r a t e d amount of t r i t i a t e d p o l y p h o s p h o i n o s i t i d e s and i n o s i t o l phosphates was increased by ATP to the same e x t e n t whet h e r in the presence or absence of Ca2+. The e f f e c t o f ATP in the a c e l l u l a r system was apparently not a t t r i butable to a p u r i n e r g i c mechanism, i t not being reproduced by m,~ -methylene-ATP. The glucose-induced stimul a t i o n of i n o s i t o l phosphate production in i n t a c t i s l e t c e l l s may thus i n v o l v e not ~ o l e l y the a c t i v a t i o n of phosphoinositidase C by Caz- but also an accelerated generation of p o l y p h o s p h o i n o s i t i d e s , p o s s i b l y due to a r i s e in c y t o s o l i c ATP.

A107

P379

P380

GLUCOSE-INDUCED INSULIN R~T.EASE IS NOT DEPENDENT ON ACTIVATION OF PROTEIN KINASE A S. J . P e r s a u d , P . M. Jones and S. L. Howell Biomedical Sciences Division, K i n g ' s C o l l e g e London, Campden H i l l Road, London N8 7AH, U.K.

CA2+-INDUCED LOSS OF CA2+-DEPENDENT PROTEIN KINASE ACTIVITY IN ELECTRICALLY P E R M E I L I S E D ISLETS. P.M. J o n e s , S . J . Persaud and S.L. H o w e l l , B i o m e d i c a l S c i e n c e s D i v i s i o n , K i n g ' s C o l l e g e London, K e n s i n g t o n , London, U.K.

The r o l e of cyclic AMP i n t h e e x o c y t o t i c r e l e a s e o f i n s u l i n i s not w e l l - d e f i n e d . We have i n v e s t i g a t e d the effects of the Rp isomer of adenosine cyclic 3',5'-phosphothioate (RpcAMPS) on insulin secretion from r a t i s l e t s o f Langerhans. C o l l a g e n a s e - i s o l a t e d i s l e t s were p r e i n c u b a t e d w i t h 500uM RpcAMPS f o r one h o u r a t room t e m p e r a t u r e , t h e n i n c u b a t e d f o r 30min a t 37~ i n t h e p r e s e n c e o f s t i m u l a t o r y c o n c e n t r a t i o n s of glucose + forskolin. Forskolin (I0uM) p o t e n t i a t e d i n s u l i n r e l e a s e a t 10mM g l u c o s e (10mM g l u c o s e 3 . 5 5 § 0.40 ng/islet/3Omin; + IOuM forskolin, 6.08 + 0.26~ mean + SEM, n=6, p < 0 . 0 l ) and this was significantly inhibited by 500uM RpcAMPS (10mM glucose § ]0uM forskolin § 500uM RpcAMPS, 3.37 + 0.36, n=6, p < 0.01 vs lOmM glucose + 10uH forskolin). RpcAMPS also inhibited cAMP-induced insulin secretion from electrically permeabilised islets without affecting 2§ Ca - i n d u c e d secretion. However, under similar conditions, treatment of intact i s l e t s w i t h RpcAMPS o v e r a r a n g e o f g l u c o s e c o n c e n t r a t i o n s (2-10mM) did n o t significantly impair insulin s e c r e t i o n (2mM g l u c o s e ~ 0.44 ~ 0 . l , + 500uMRpcAMPS, 0.55 ~ 0 . 0 6 ; 5mM glucose, 0.51 ~ 0 . 0 6 , § 500uM RpcJ~[PS, 0.38 ~ 0 . 0 7 ; 7.5mM glucose, 1.22 ~ 0 . 2 7 , + 500uM RpcAMPS, 1.00 + 0.32; 10mM glucose, 3.55 ~ 0.40, + 5 0 0 u M R p c A M P S ~ 2.56 0.68, n=5-6, p > 0. l for each comparison). These results s u g g e s t t h a t c y c l i c &liP-induced a c t i v a t i o n o f protein kinase A is not essential for either basal or glucose-stimulated insulin secretion.

Elevated intraeellular Ca 2+ s t i m u l a t e s i n s u l i n s e c r e t i o n fr~ electrically permeabilised islets, but Ca- -insensitivity develops within 20-30 mins. We have z+ . . . investigated the role of Ca -depen~nt kznases In thls process by measuring Ca- -induced protein phosphorylation in Ca- -responsive and Ca- -insensitive permeabilised islets. Insulin secretion was measured from permeahilised rat islets perifused w~h buffers containing v a r i o u s c o n c e n t r a t i o n s o f Ca . P r o t e i n phosphq~ylation was aw by measuring the transfer of ~ ' P from [ ~ ~'P]-AT~+ into proteins which mere separated by SDS-PAGE. Ca (10uM) produced rapid increases in insulin secretion (50nM Ca ~ , ]1.6+l.5 pg/islet/min; 10uM Ca 2 , 3 0 . 3 + 0 . 1 , mean+SEM, ~, p<0.0l), with s e c r e t i o n returning to b a s a l - - ( 5 0 n M Ca ~ ) within 20-30mins despite the continued presence of a s t i m u l a t o r y c o n c e n t r a t i o n o f Ca 2+ (]0uM Ca 2 + , ] I . 5 ~ 0 . 4 pg~slet/min). The l o s s o f s e c r e t o r y ~ s p o n s i v e n e s s to Ca-- was accompanied by a l o s s o f Ca- - i n d u c e d p r o t e i n p h o s p h o r y l a t i o n s . An i n i t i a l (] m i n ) e x p o s u r e to 10uM

Ca2+ stimulated 32p incorporation, notably into proteins of apparent M.W. 54-55kDa. However, no Ca-+-induced P incorporation was detected in 2 . . . . . Ca -insensxtlve Islets , although cyclic AMP (500uM) stimulated p r o t e i n phosphorylation in these islets (approx. M.W.25,47,60kDa). These results suggest that the ~ansient nature+of the insulin secret~y response to Ca-- reflects a Ca- -induced loss of Ca- -dependent protein kinase activity in e l e c t r i c a l l y permeabilised islets.

P381

P382

A L L O X A N M A R K E D L Y I N C R E A S E S THE B L O O D P E R F U S I O N OF THE P A N C R E A T I C ISLETS IN R A T S S. S a n d l e r a n d L. J a n s s o n . D e p a r t m e n t o f M e d i c a l C e l l Biology, Uppsala University, Uppsala, Sweden. It is y e t n o t fully u n d e r s t o o d w h i c h f a c t o r s c o n s t i t u t e t h e u n i q u e s u s c e p t i b i l i t y of t h e p a n c r e a t i c a - c e l l s t o a l l o x a n a n d s t r e p t o z o t o c i n (SZ). In t h i s s t u d y t h e a c u t e e f f e c t s of a l l o x a n a n d SZ on b o t h w h o l e p a n c r e a t i c b l o o d f l o w (PBF) a n d islet b l o o d f l o w (IBF) w e r e s t u d i e d in a n e s t h e t i z e d r a t s . A n i m a l s r e c e i v e d e i t h e r s a t i n e , a l l o x a n (75 m g / k g BW) or SZ (40 m g / k g BW) i n t r a v e n o u s l y a n d 3 rain a f t e r a d m i n i s t r a t i o n t h e PBF a n d IBF w e r e m e a s u r e d w i t h a m i c r o s p h e r e t e c h n i q u e . In s o m e a n i m a l s I ml of a 30 % (w/v) D - g l u c o s e s o l u t i o n w a s g i v e n i n t r a v e n o u s l y 5 min b e f o r e t h e t o x i n s . SZ h a d e s s e n t i a l l y no e f f e c t s on e i t h e r PBF or IBF c o m p a r e d t o s a l i n e - i n j e c t e d a n i m a l s , a n d did n o t c h a n g e t h e I B F - r e s p o n s e to g l u c o s e . On t h e o t h e r hand, a l l o x a n c a u s e d a 50 % r e d u c t i o n in PBF (0.31 + 0.03 vs 0.68 ,+ 0.04 m l / m i n x g p a n c r e a s ; P < 0 . 0 0 I ) , w h i l s t t h e IBF i n c r e a s e d s i g n i { i c a n t l y ( 1 6 4 ,+ 17 vs 66 _+ 3 ~ l / m i n x g p a n r e a s ; P<0.001). A c c o r d i n g l y t h e f r a c t i o n of t h e w h o l e p a n c r e a t i c b l o o d flow d i v e r t e d t h r o u g h t h e islets i n c r e a s e d f r o m 10.7 -+ 0.3 t o 51.9 ,+ 3.3 % in a l l o x a n - i n j e c t e d r a t s (P<0.-0Ol) A d m i n i s t r a t i o n of g l u c o s e b e f o r e a l l o x a n did n o t a l t e r t h i s b l o o d f l o w r e s p o n s e . It is possible t h a t t h e m a r k e d a c u t e i n c r e a s e in i s l e t b l o o d p e r f u s i o n i n d u c e d by a l l o x a n m a y c o n t r i b u t e t o t h e p r e f e r e n t i a l c y t o t o x i c i t y o f t h e G - c e l l s in vivo t o t h i s toxin.

ENZYMATIC ACTIVITY MEASUREMENTSAT A SINGLE ISLET CELL LEVEL D. Z~hner, A. Sener and W.J. Maiaisse. Laboratory o f Experimental Medicine, Brussels Free U n i v e r s i t y , Brussels, Belgium. The o v e r a l l metabolic and f u n c t i o n a l response of i s o l a ted pancreatic i s l e t s may mask an underlying heterogeneit y and recruitment of i n d i v i d u a l i s l e t c e l l s . The invest i g a t i o n of metabolic events and enzymatic a c t i v i t i e s in i n d i v i d u a l c e l l s i s l i m i t e d , however, by the s e n s i t i v i t y of c u r r e n t a n a l y t i c a l methods. Taking advantage of the p r e p a r a t i o n of [ 3 , 4 - 3 H ] 2 - k e t o g l u t a r a t e with high s p e c i f i c r a d i o a c t i v i t y (55 Ci/mmol), femtomolar amounts (1.0 fmol or more per sample) of NAD(P)H could be measured t~rough the conversion of the t r i t i a t e d 2-keto acid to L-glutamate, as catalyzed ~v glutamate dehydrogenase in the presence of excess N H 4 . The l a b e l l e d amino acid was then separated from i t s precursor by ion exchange chromatography. The a p p l i c a t i o n o f t h i s method to the assay of l a c t a t e dehydrogenase allowed i t s measurement in as l i t t l e as 0.0005 i s l e t / s a m p l e . Since each i s l e t contains about 2,000 c e l l s , the enzymatic a c t i v i t y was indeed measured on a s i n g l e c e l l basis. A comparable procedure was app l i e d to the measurement o f several metabolites (e.g. NAD(P)H, NAD(P) , s o r b i t o l and ~ - h y d r o x y b u t y r a t e ) and enzymic a c t i v i t i e s (e.g. polyol dehydrogenase, ~-hydroxybut y r a t e dehydrogenase, glutamate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase) in i s l e t c e l l s . In a d d i t i o n to i t s e x q u i s i t e s e n s i t i v i t y , t h i s novel r a d i o i s o t o p i c procedure also o f f e r s the advantages of f i a b i l i t y , r a p i d i t y , and s i m p l i c i t y .

A108

PS 5 Islets: Morphology-Differentiation P383

P384

COMPUTER-ASSISTED MORPHOSTRUCTURAL ANALYSIS OF PANCREATIC CELLS DURING NEONATAL REVERSIBLE DIABETES. A. Ast~sano, N. Daulriche, A. Anteunis and G. Rosselin. INSERM U.55, H6pital Saint-Antoine, F-75571 Paris C~dex 12, France; Structural changes involved in reversible diabetes were analysed 1day before the onset of pancrealic insulin recovery, i.e.72h poststreptozotocin injection to rat neonates. (Portha's model).Diabetic and control pancreatic samples were randomly selected and processed for EM. Comparative morphometrical analysis was performed using superimposition of a multiple square lattice system (41,5 and 8,59 pro). The relative islets volume (v/v) is twice as low in diabetics than in controls. Changes in the cellular islet compartments are considerable. 8 cell v/v which represents 83,5+1,32% of islet tissue in controls (n=4) drops to 14,1+7,8 in diabetics, A cell v/v increases from 1,74 to 5,12%, whereas ductal cell v/v nearly doubled. The abundance of a proliferating endocrine pancreatic subpopulation (PEPS) is remarkable in diabetics, accounting for 57% versus 9% in controls. PEPS are morphologically characterized by ribosomes in rosettes and spherical granules of different sizes and electron density, some resembling A and/or B granules, others being coated-like. Several types of endocrine-connected ductal cells were also identified by their H3-thymidine incorporation or the appearance of endocrine-like granules. Our findings suggest that PEPS cells are transitional forms, resist streptozotocin, increase their proliferation and are responsible for the subsequent reversion of diabetes (for further identification see abstract Dautriche et al).

EVIDENCE OF B CELL DYSFUNCTION IN PERSISTENT NEONATAL HYPOGLYCAEMIA WITH HYPERINSULINISM (PNHH).

J. RAHIER, R.M. GOEBBELS, K. CHANNAOUI Dept of Pathology. Univ. of Louvain, BRUSSELS BELGIUM The pathogeny of PNHH is still unclear. The disease does not result from an increase in the B cell mass, but probably from a functional disorder of B cells. This disorder is associated to nuclear abnormalities whose recognition permits distinction between focal and diffuse forms of the disease. We have used double immunolabelling techniques to determine the percentage of total B cell area that is recognized by an anti proinsulin antibody. We studied the islets of 24 PNHH infants (20 diffuse and 4 focal forms) and 24 control infants. The proinsulin antibody specifically labelled a cytoplasmic domain corresponding to the perinuclear Golgi region. The granular pole was not labelled. In control infants, the proinsulin/B cell area amounted to 5 • 1 % (SEM). It was much higher in the diffuse form of PNHH (19 • 3 %). Results were more variable in the focal form of the disease, but the proinsulin/B cell area was always higher in the lesion than outside. A correlation was found between the B cell nuclei abnormalities and the proinsulin/B cell area. In conclusion, these data suggest that the rate of proinsulin synthesis is markedly increased in PNHH and that the proinsulin to insulin processing is roughly normal. They reinforced the concept that PNHH is an heterogeneous disease.

P385

P386

SPECIFIC ANTISERA TO IAPP, PROINSULIN C-PEPTIDE l, AND 2: PROBES TO STUDY MURINE ISLET ~-CELL DIFFERENTIATION

EARLY MATURATION IN VITRO OF FETAL ISLET B-CELLS INDUCED BY GROWTH HORMONE C. Svensson and M. Welsh, Department of Medical Cell Biology, Box 571,751 23 UPPSALA The aim of this study was to investigate the effects of growth hormone (GH) on the early in vitro maturation of fetal rat islets. The pancreata of 21-day old fetuses were partially digested and cultured in RPMI 1640 supplemented with 10% fetal calf serum. After one day the medium was changed and supplemented with 1% fetal calf serum and GH (1 l i g / m l ) and cultured for another two days. The islets were then gently removed from the culture dishes and studied with regard to insulin release, insulin content, glucose oxidation, thymidin incorporation, the activity of low Km and high Km glucose phosphorylating enzymes and the contents of mRNA coding for insulin, the ADP/ATP carrier and cytochrome b. Islets treated with GH showed increased rates of insulin release (1.67 mmol/I glucose; 250_+50 vs 380+100 pg/islet x h (p<0.05), 16.7 mmol/I glucose; 1430+250 vs 1990+410 pg/islet x h (p<0.05)) and glucose oxidation rates (1.67 mmol/I glucose; 3.37+0.25 vs 4.95+0.32 pmol/islet x h (p<0.01), 16.7 mmol/I glucose; 10.0+2.0 vs 14.6+2.1 pmol/islet x h (p<0.05)). The thymidine incorporation was also significantly increased compared to controls 56+17% (p<0.05). The activities of high Km glucose phosphorylating enzymes were increased by GH (3789+412 vs 4689+681 DPM/200 islets (p<0.05)). There were no differences in the islet mRNA contents (p>0.05). It is concluded that GH induces early in vitro maturation of Bcells by a combined stimulatory effect on insulin release, Bcell replication and glucose metabolism.

O.D. Madsen, N. Blume, J.E. Petersen, H. Kofod and T. Dyrberg. Hagedorn Research Laboratory, Gentofte, Denmark. Rats and mice have two non-allelic preproinsulin genes which are both expressed in 6-cells in mature islet of Langerhans together with islet associated polypeptide (IAPP or amylln). Our aim was to produce antisera specific for each of these markers as probes to study islet ~-cell differentiation. By immunizing rabbits with synthetic peptides derived from the C-peptide sequences we have generated several antisera which in immunocytochemlcal analysis specifically bind to rat or mouse Cpeptide i, but not to C-peptide 2; and others which recognize an epltope unique to the rat and mouse C-2 peptides. In addition, highly potent antisera were raised against synthetic rat IAPPT_37. Analysis of ~-cell dedifferentiation in the NHI-6F rat insulinoma cells which carry an exogenous human insulin gene demonstrated progressive loss of expression of four 8-cell markers as follows: human insulin > rat insulin 2 > rat insulin 1 IAPP. We conclude that IAPP can be a pre-~-cell marker and that rat insulin 1 is more efficiently expressed in poorly differentiated ~-cells compared to rat insulin 2. In summary, our antisera are important immunocytochemical tools to analyse the expression of these individual 6cell markers during islet cell differentiation.

A109 P388

P387 ISOLATION AND PURIFICATION THE HUMAN PETAL PANCREAS

OF HUMAN FETAL BETA CELLS FROM

R.R. de Krijger, H.J. Aanstoot, A. Verkerk*, J.F. Jongkind* and G.J. Bruining, Dept. of Pediatrics, subdivision of Clinical Genetics, and Dept. of Cell Biology*, Erasmus University Rotterdam and Academic Hospital/Sophia Childrens Hospital Rotterdam, The Netherlands The aim of this study was to obtain single viable pancreatic beta cells from human fetal pancreases. The pancreases were obtained from legal abortions. Studies were carried out with approval of the ethical committee. A two-step collagenase digestion yielded 1.29 ! 0.17 x 105 cells per mg of pancreatic tissue (mean ~ SD, n=lO). This amounts to 7 to 30 million cells per pancreas. The viable cells were selectively recovered by Percoll gradient centrifugation. Thus the vitality increased from 55 • 8% to 81 • 4% (n=16, pK0.01). The percentage of beta cells in the viable fraction remained unaltered after this gradient centrifugation, varying from i% to 6% in an age-dependent way (n=17, r=0.74, p<0.01). These suspensions were sorted in a FACS based on the perpendicular light scattering of the cells and their staining with the antibody N1 (kindly donated by Dr. K.J. Lafferty, Barbara Davis Center, Denver, USA). This antibody stained 86 • 11% of all beta cells while the beta cells constituted 15.6 • 5.7% of the Nl-positive population (n=22). Combining the two sorting parameters an increase from 2.7 • 0.9% to 33.7 • 8.3% beta cells was achieved (n=5, ps In conclusion, human fetal beta cells can be purified from the human fetal pancreas by fluorescence activated cell sorting.

THE MAJOR ISLET HORMONES ARE CO-LOCALIZED WITHIN THE SECRETORY GRANULAE IN THE FETAL PORCINE PANCREAS. O. Korsgren, A. Lukinius, R. Schneil, A. Andersson and L Grimelius. D e p a r t m e n t of Medical Cell Biology and Department of Pathology, Uppsala University, Uppsala, Sweden. The early ontogenic development of the porcine endocrine pancreas was investigated at the ultrastructural level. Pancreases from 50 and 70 days otd fetuses (term is 115 days) were stained with a double immuno-gold labelling technique using two different sizes of gold particles. This enabled us to localize two antigens in the same section. A monoclonal antibody against insulin in combination with a polyclonal rabbit antibody against either glucagon or somatostatin were used. All endocrine cell types were present in the pancreatic primordia of both age groups, either organized into small nests or as separate cells. In general the endocrine cells contained predominantly one hormone with granulae similar to those seen in adult cells. However, in a majority of the insulin-containing cells, small amounts of either glucagon or somatostatin were found within the insulin granulae. Likewise, a small amount of insulin was found in the granulae of every second glucagon or somatostatin producing cell. These findings suggest that some endocrine cells are able to synthesize at least two different hormones, and that these peptides are co-stored in the same granulae. Altogether these data support the concept of a common precursor for the hormone-producing cells within the pancreas.

P389

P390

SUBCELLULAR DISTRIBUTIONOF ISLETTHYROTROPINRELEASINGHORMONE E. Vara, L.-A. Idahl, P. LindstrSm, J. Sehlin and J. TamaritRodriguez. Departamento de Bioquimica, Universidad Complutense, Madrid, Spain, and Department of Histology and Cell Biology, University of UmetL, Ume&, Sweden We have previously shown that islet secretion of thyrotropinreleasing hormone (TRH) is inhibited by glucose. This is at variance with recent evidence indicating that TRH is contained within insulin granules. Therefore, we have measured the contents of TRH, insulin, glucagon and somatestatin radioimmunilogically in subcellular fractions (nuclei, rnitochondria, secretory granules, microsomes, supernatant) from collagenase-isolated islets of normal mice, ob/ob-mice, rats and rat fetuses (21.5 days). Whereas insulin, glucagon and somatostatin were mainly confined to the secretory granule fraction, the bulk of TRH occurred in the supernatant, thus indicating no co-sedimentation between TRH and any of the other hormones. Rat fetus islets showed a 5-fold higher supernatant content of TRH (5 pg/g.g protein) than adult islets of all species. ob/ob-Mouse islets contained a much higher insulin content (> 100%) and a much lower somatostatin (<20%) and glucagon (<40%) content in their granule fraction, as compared with other adult islets. The results do not preclude the possibility that islet TRH is localized to B-cells but they indicate that the bulk content of TRH is not stored in granules. This opens the possibility that insulin and TRH are secreted by different mechanisms.

IDENTIFICATION OF GAP JUNCTION AND TIGHT JUNCTION PROTEINS OF PANCREATIC B-CELLS

P. Meda, Departmentof Morphology,Universityof Geneva Geneva, Switzerland

Pancreatic B-cells are connected by gap and tight junctions, the membrane structures for cell-to-cell communication and separation of membrane regions, respectively.We have investigatedthe proteins comprising these structures in the rat pancreas and in a transplantable rat insulinoma. Immunolabelling of cryosections and crude membrane preparations 0Nestern blots) with antibodiesagainst three gap junction proteins, showed that both normal and tumoral B-cells expressthe 43 kDa connexin (CX) 43 but not CX32 and CX26, the gap junction proteins of exocrine pancreas. Northern blots of total islet and tumor ARN, hybridisedwith cDNA clones with CX specific sequences,also detected only a ARNm for CX43. The amount of this ARNm increased two fold in islets from glibenclamide-treated (1 mg daily, for 2 days) rats showing enhanced B-cell communications. Since the existenceof B-cell tight junctions has been questioned,we also searchedfor the major protein (ZO-1) of these structures. Immunolabellingwith antibodies against ZO-1 showed this 225 kDa peptide in the B-cells of both in situ and isolated islets and insulinoma. This study shows that 1) CX43 and ZO-1 are the major components of B-ceUgap and tight junctions, respectively;2) CX expressionincreaseswith B-cell communication during sustained stimulation of insulin secretion by glibenclamide.

Al10 P391 DISTRIBUTION

P392 OF GALANIN

IN THE MOUSE AND RAT PANCREAS

S.Lindskog, B.Ahr~n, B.E.Dunning, and F.Sundler. Depts. of Pharmacology, Surgery, and Medical Cell Research, Lund University, Lund, Sweden, and Medicine, University of Washington, Seattle, Wa, USA. The neuropeptide galanin is a sympathetic post-ganglionic neurotransmitter in the canine endocrine pancreas and inhibits insulin secretion in the mouse and the rat. However, the distribution of galanin in the mouse and rat pancreas has not been established. We therefore examined the intrapancreatic localization of galanin-like immunoreactivity (GLIb) in these species, both under normal conditions and 2 days after i.v. injection of 6-hydroxydopamine, which induces chemical sympathectcmy by destroying the adrenergic nerve terminals. The completeness of this sympathectomy was verified by tyrosine hydroxylase immunostaining which was absent. Immunofluorescent stainin Z revealed, in both species, that GLIR occurred in fine, varicose intrapancreatic nerve fibers. The fibers innervated the islets as well as the exocrine parenchyma and the blood vessels. GLIR nerve fibers were more numerous in the mouse than in the rat. After chemical sympathectomy, OLIR fibers were almost absent in the rat pancreas, whereas in the mouse, virtually no difference was observed regarding the density of the fibers when compared to controls. We conclude that l)the pancreas in both the mouse and the rat is innervated by galanincontaining nerve fibers and 2)in the rat, but not in the mouse, most of the intrapancreatic galanin-containing nerves are adrenergic.

P393 I N F L U E N C E O F I N T E R M I T T E N T F E E D I N G ON I N S U L I N SEC R E T I O N OF I S L E T S I S O L A T E D F R O M M A T U R E RATS. P. M a s i e l l o , M. N o v e l l i , V. F i e r a b r a c c i , M. B o m b a r a a n d E. B e r g a m i n i . C I R I N , S e z i o n e di P a t o l o g i a G e n e r a l e , U n i v e r s i t y of P i s a , P i s a , Italy. T o i n v e s t i g a t e the p o s s i b l e p r o t e c t i v e e f f e c t of a d i e t a r y r e s t r i c t i o n , i.e. i n t e r m i t t e n t f e e d i n g , on the a g e - r e l a ~ e d d e c l i n e of i n s u l i n s e c r e t o r y responsiveness, islets were isolated from 12-moo l d S p r a g u e - D a w l e y rats, e i t h e r fed ad l i b i t u m (controls) or fed e v e r y o t h e r d a y (IF) o v e r 10 months. During a 60-min static incubation of i s l e t s t a k e n f r o m b o t h fed a n d 2 4 - h f a s t i n g r a t s of e a c h g r o u p , i n s u l i n r e l e a s e at 2.8 m m o l / l g l u cose was higher in c o n t r o l i s l e t s t h a n in IF i s l e t s (1.8 + 0.2 v e r s u s 0.24 ~ 0 . 0 5 n g / i s l e t , p ~ 0.01, in T e d rats; 1.4 + 0.1 v e r s u s 0.56 0 . 0 8 n g / i s l e t , p ~ 0.01, in f a s t i n g rats). Upon s t i m u l a t i o n w i t h 16.7 m m o l / l g l u c o s e and o t h e r s e c r e t a g o g u e s , the i n c r e m e n t a l i n s u l i n r e l e a s e o v e r b a s a l w a s m u c h l a r g e r in IF t h a n in c o n t r o l i s l e t s . I n t e r e s t i n g l y , IF p r e v e n t e d t h e i n c r e a s e in i s l e t i n s u l i n c o n t e n t , k n o w n to o c c u r in a g i n g rats, p a r t i c u l a r l y in fed a n i m a l s (fed IF a n d c o n t r o l s : 86 + 5 a n d 225 + 6 n g / i s l e t , respectively, p<0.O01; f a s t i n g - I F and c o n t r o l s : 155 + 6 a n d 184 + 6 n g / i s l e t , r e s p e c t i v e l y , p < 0.01). I n d e e d , the n u t r i t i o n a l s t a t u s of t h e a n i m a l s inf l u e n c e d t h e h o r m o n a l c o n t e n t of the i s l e t s o f c o n t r o l and IF r a t s d i f f e r e D t l y , w i t h o u t a f f e c t i n g i n s u l i n r e l e a s e . In c o n c l u s i o n , IF i m p r o v e s t h e e f f i c i e n c y o f i n s u l i n s e c r e t o r y r e s p o n s e in v i t r o a n d m o d i f i e s i s l e t a d a p t a t i o D to f a s t i n g .

PERSISTENT REDUCTION OF PANCREATIC B-CELL MASS AFTER TEMPORARY PROTEIN-CALORIE MALNUTRITION IN THE RAT I. Swenne, L.A.H. Borg, C.J. Crace and A.H. Schnell LandstrSm. Department of Medical Cell Biology, University of Uppsala, Uppsala and Department of Paediatrics, University of Sheffield, Sheffield, UK. Protein deficiency has been implicated in the etiology of malnutrition-related diabetic syndromes in the third world. To test the long-term effects of temporary protein-calorie malnutrition on pancreatic B-cells 3-week-old rats were weaned onto either low protein diet (5% protein; LP rats) or control diet (18% protein; C rats). From six weeks of age all rats were refed the control diet up to twelve weeks of age. In 6-week-old LP rats the glucose tolerance and insulin secretory response to glucose were severely impaired. Glucose tolerance but not insulin secretion recovered during the refeeding period up to twelve weeks of age. Upon histological examination the pancreas of 6-week-old LP rats appeared atrophic, the pancreatic islets were small but without signs of degeneration. Morphometric analysis of immunostainable B-cells showed a reduction of total B-cell mass. Electron microscopy confirmed atrophy of the islets without degenerative changes. Morphometry at the ultrastructural level showed a diminution of individual B-cell size. T h e s e morphological changes were not rectified by nutritional rehabilitation but partly persisted at twelve weeks of age. Temporary protein-calorie malnutrition thus causes permanent structural and functional alterations of the pancreatic B-cell, which in turn may predispose for diabetes.

Alll

PS 6

Amylin/IAPP/CGRP P394

P395

ISLET A M Y L O I D AND I M M U N O R E A C T I V E ISLET A M Y L O I D P O L y P E P T I D E IN T Y P E 2 D I A B E T E S M E L L I T U S M.G. N i e u w e n h u i s , M.J.H. Berends and C.J.M.Lips, U n i v e r s i t y Hospital, U t r e c h t and W e s t e i n d e Hospital, The Hague, The N e t h e r l a n d s . In post m o r t e m p a n c r e a t i c tissue sections from 150 p a t i e n t s islet a m y l o i d (IA) d e p o s i t i o n s and i n t r a c e l l u l a r islet i m m u n o r e a c t i v i t y for islet a m y l o i d p o l y p e p t i d e ( I R - I A P P ) w e r e s t u d i e d and r e t r o s p e c t i v e l y c o r r e l a t e d w i t h the c l i n i c a l data. The p r e s e n c e of IA was s c r e e n e d for w i t h Sulfated A l c i a n Blue. I m m u n o h i s t o c h e m i e a l i n v e s d g a tion was p e r f o r m e d w i t h rabbit a n t i - h u m a n IAPP ( a m y l i n , P e n i n s u l a ) a n d a n t i - i n s u l i n (Dako).Of 41 I A - p o s i t i v e sections 25 w e r e from overt type-2diabetics. W i t h i n the limits of the sampling m e t h o d the p o s i t i v e p r e d i c t i v e value of IA-posit i v i t y for overt type-2 d i a b e t e s was 61%. O n l y in d i a b e t e s of m o r e than 20 years d u r a t i o n 100% I A - p o s i t i v e islets were found. In i n s u l i n - t r e a ted t y p e - 2 - d i a b e t i c s I A - p o s i t i v e islets r a n g e d from 1.5-i00%(n=5), in n o n - d i a b e t i c s (n=7) from 0-16%, w i t h one e x e e p t i o n ( 6 5 % ) . C o m p a r e d to freshIy fixed p a n c r e a t i c tissue from o p e r a t i v e material in n o n - d i a b e t i c s I A P P - I R in islet B - c e l l s was found to be s e n s i t i v e to the state of tissue p r e s e r v a t i o n ; this in c o n t r a s t to I R - i n s u l i n and IR-IAPP in IA. D e p o s i t i o n of IA in the islets was a s s o c i a t e d w i t h w e a k IAPP B - c e l l immunoreactivity. In c o n c l u s i o n , m o d i f i c a t i o n in production, storage and/or release of IAPP m a y preceed overt type 2 diabetes.

A M Y L O I D FORMS I N T R A - C E L L U L A R L Y IN B - C E L L S OF H U M A N I N S U L I N O M A S AND M O N K E Y ISLETS. A Clark I, JF M o r r i s I, LA Scott I, A M c L a y 2, A Foulis 2, N B o d k i n 3, BC H a n s e n 3. iDept of H u m a n Anatomy, Oxford, UK. 2Pathology Dept, G l a s g o w Royal Infirmary, UK. 3Dept of Physiology, Baltimore, USA. A m y l o i d formed from islet a m y l o i d p o l y p e p t i d e (IAPP) is c h a r a c t e r i s t i c of type 2 d i a b e t e s in m a n and other p r i m a t e s and of some insulinomas. I A P P - i m m u n o r e a c t i v i t y (IAPP-IR) is p r e s e n t in i n s u l i n g r a n u l e s and l y s o s o m e s of n o r m a l Bcells, but the factors w h i c h cause d e p o s i t i o n of a m y l o i d a r e unknown. To d e t e r m i n e the a n a t o m i c a l sites of fibril formation, 12 surgical s p e c i m e n s of i n s u l i n o m a and postm o r t e m p a n c r e a s from 3 n o n - d i a b e t i c and 2 pred i a b e t i c m o n k e y s w e r e p r e p a r e d for e l e c t r o n microscopy. I A P P - I R was d e t e c t e d w i t h a p o l y c l o n a l a n t i b o d y r a i s e d to IAPP19_37 and p r o t e i n - A gold. In insulinomas, e x t r a c e l l u l a r a m y l o i d i m m u n o r e a c t i v e for IAPP was d e t e c t e d in 7/12 cases and i n t r a c e l l u l a r I A P P - I R fibrils w e r e i d e n t i f i e d in 5/12 specimens. In t i s s u e from n o n - d i a b e t i c and p r e - d i a b e t i c m o n k e y s I A P P - I R was p r e s e n t in i n s u l i n g r a n u l e s and l y s o s o m e s and in the p r e - d i a b e t i c a n i m a l s i n t r a c e l l u l a r a m o r p h o u s / f i b r i l l a r m a s s e s of IAPP p o s i t i v e m a t e r i a l w e r e also found. T h e s e findings s u g g e s t that a m y l o i d fibrils are i n i t i a l l y formed intracellularly. Extracellular amyloid deposits could result e i t h e r from e x t r u s i o n of fibrils from the cells or from d e a t h of B - c e l l s c o n t a i n i n g amyloid. A b n o r m a l t r a n s c r i p t i o n and or p r o c e s s i n g of pro IAPP could lead to a c c u m u l a t i o n of i n s o l u b l e p e p t i d e amd fibril formation.

P396

P397

INCREASED A M Y L I N LEVELS IN P A T I E N T S ON C H R O N I C HEMODIALYSIS A. B e r z l a n o v i c h , B. Ludvik, E. Hartter, B. Lell, H. Graf and R. Prager, 2 ~d D e p a r t m e n t of Medicine, U n i v e r s i t y of Vienna, A u s t r i a Amylin, a new p e p t i d e i s o l a t e d from a m y l o i d deposits of type II d i a b e t i c islets, is t h o u g h t to play a p o t e n t i a l role in the p a t h o g e n e s i s of type II diabetes. I n c r e a s e d a m y l i n levels are found in states of i n s u l i n r e s i s t a n c e such as o b e s i t y and d i a b e t e s mellitus. E l e v a t e d a m y l i n levels in p a t i e n t s w i t h renal f a i l u r e could c o n t r i b u t e to the i n s u l i n r e s i s t a n c e in uremic p a t i e n t s and, a d d i t i o n a l l y , give e v i d e n c e for renal e l i m i n a t i o n of amylin. We t h e r e f o r e i n v e s t i g a t e d amylin, i n s u l i n and C - p e p t i d e levels in 20 p a t i e n t s on h e m o d i a l y s i s . The p a t i e n t s r a n g e d in age from 40 to 73 years (mean 53o9• SEM), b o d y mass index was 22.8• kg/m=, fasting b l o o d g l u c o s e levels were i10• mg/dl. F a s t i n g a m y l i n levels were s i g n i f i c a n t l y e l e v a t e d c o m p a r e d to h e a l t h y controls (38.4• vs. 12.6• pg/ml, p<0.001) and fell to 2 2 o 6 • pg/ml (p=0.002) at the end of hemodialysis. Insulin (19.4• vs. 16.9• uU/ml, ns.) and C - p e p t i d e levels (21.4• vs.17.1• ng/mle ns.), b o t h e l e v a t e d for the fasting state, showed no s i g n i f i c a n t d e c r e a s e during hemodialysis. In c o n c l u s i o n a m y l i n levels are i n c r e a s e d in p a t i e n t s w i t h renal failure s u g g e s t i n g renal e l i m i n a t i o n of amylin. The c h r o n i c e l e v a t i o n of serum a m y l i n in these p a t i e n t s m a y c o n t r i b u t e to the insulin r e s i s t a n c e seen in p a t i e n t s w i t h renal failure.

C O R R E L A T I O N OF ISLET A M Y L O I D & R E D U C E D B - C E L L MASS W I T H B - C E L L F U N C T I O N IN D I A B E T I C MONKEYS. EJP de Koning, A Clark, N B o d k i n * Oxford, UK & *Baltimore, MD, USA.

& BC Hansen*.

P a n c r e a t i c islet a m y l o i d o s i s is a c h a r a c t e r i s t i c f e a t u r e of type 2 d i a b e t e s in m a n but its r o l e in the a e t i o l o g y of the d i s e a s e is unknown. S p o n t a n e o u s d i a b e t e s in m a c a c a m u l a t t a is c h a r a c t e r i s e d by s e q u e n t i a l p h a s e s of obesity, h y p e r i n s u l i n a e m i a (fasting i n s u l i n >80~U/ml) & a b n o r m a l g l u c o s e t o l e r a n c e l e a d i n g to d i a b e t e s (fasting p l a s m a g l u c o s e >8.0mM). To r e l a t e the d e g r e e of a m y l o i d o s i s to p h y s i o l o g i c a l parameters, p o s t - m o r t e m p a n c r e a t i c t i s s u e from 16 m o n k e y s was s t a i n e d w i t h i m m u n o p e r o x i d a s e for i n s u l i n (B-cells) and islet a m y l o i d p o l y p e p t i d e (IAPP = e x t r a c e l l u l a r amyloid). Morphometric a n a l y s e s w e r e d o n e w i t h o u t k n o w l e d g e of the animals' m e t a b o l i c status. B - c e l l area % endocrine area (~%endo) in the d i a b e t i c s (n=5) was r e d u c e d to 20% of that in n o n - d l a b e t i c c o n t r o l s (n=3), p<0.001. Islet a m y l o i d (IA) was absent in controls, was p r e s e n t in 2/5 o b e s e (x IA%endo=l.6), 1/3 h y p e r i n s u l i n a e m i c (x I A % e n d o = 0 . 3 ) and in 5/5 d i a b e t i c a n i m a l s (x IA%endo=68). I n c r e a s e d IA c o r r e l a t e d w i t h a r e d u c t i o n in the B-cell area (r=-0.95, p<0.001). G e o m e t r i c m e a n islet area i n c r e a s e d w i t h p r o g r e s s i o n to diabetes. C o n c l u s i o n s : islet a m y l o i d d e p o s i t i o n can p r e c e e d the o n s e t of s p o n t a n e o u s d i a b e t e s in monkeys. E x t e n s i v e islet d a m a g e i m p l i c a t e s a m y l o i d d e p o s i t i o n in the d e t e r i o r a t i o n of B-cell f u n c t i o n in type 2 diabetes.

Al12

P398

P399

NON-LINKAGE OF THE ISLET AMYLOID POLYPEPTIDE GENE WITH TYPE 2 DIABETES.

DIRECT INTERACTION OF ISLET AMYLOID POLYPEPTIDE (lAPP) WITH CALCITONINEGENE RELATEDNPEPTIDE (CGRP) RECEPTOR PRESENT IN 8 CELL MEMBRANE

Cook JTE I, Patel pl, Clark A I, H~p~ener JWM 2, Lips CJM 3, Mosselman S2, O'Rahilly S I, Page RC ~, Wainscoat JS 4, and Turner RC I. i. Diabetes Research Labs, Radcliffe Infirmary, Oxford. 2. Institute of Molecular Biology & 3. Dept of Internal Medicine, University of Utrecht 4. Dept of Haematology, John Radcliffe Hospital, Oxford.

A. BARAKAT,J-C. MARIE and G. ROSSELiN. INSERMU.55, H6pital Saint-Antoine, 184 rue du Fg Saint-Antoine, Paris 12~, France

Type 2 diabetes is associated with the deposition of islet amyloid. The major formative peptide, islet amyloid polypeptide (IAPP), has recently been characterised and an abnormality of the structure or expression of this gene is a possible candidate for the inherited component of type 2 diabetes. A restriction-fragment-length-polymorphism (RFLP) of the gene has been identified with restriction endonuclease PvuII. To study the relationship between the IAPP gene and type 2 diabetes, two distinct genetic approaches have been undertaken. Firstly, non-linkage has been demonstrated in four pedigrees, with four normoglycaemic first degree relatives having an allele associated with diabetes in other family members, and one affected relative not having the putatively associated allele. The LOD score taking age-related penetrance into account of -1.68 made linkage unlikely (p=0.02). Secondly, in a population-based RFLP survey, no linkage disequilibrium of the alleles was found between 88 unrelated Caucasian type 2 diabetic subjects, 15 normoglycaemic Caucasian control subjects (age 59-94 years) and 52 randomly-selected blood donors. (X2=2.72 with 4 degrees of freedom; not significant). A mutation in or near the islet amyloid polypeptide gene is thus unlikely to be a common cause of type 2 diabetes.

Islet Amyloid Polypeptide (lAPP), secreted from pancreatic islets in response to glucose, is a diabetes-associated 37 amino-acid peptide that is 50% identical to calcitonin gene-related peptide (CGRP). We have previously described a receptor for CGRP in hamster pancreatic 8 cell tumor membranes. No specific binding of lAPP is observed in this system. However when 1251-CGRP (10 -1 2M) is used as a tracer, lAPP (0.39-200 nM) inhibited competitively this binding, half inhibition being observed at 200nM. A synthetic antagonist,838 hCGRP, is also able to inhibit the binding with the following potency: CGRP>8-38 CGRP>IAPP. Cross-linking studies using dithiobis succinimidyl propionate (DSP)revealed that, in non reducing conditions, three major bands of 38, 21.3 and 17.7 KD of bound 1 251.CGRP are present on autoradiograms. CGRP (1.2x106M) and lAPP(10 6 M) stimulated the accumulation of cyclic AMP in cultured Rin m5F pancreatic cells.These findings indicate that lAPP might directly act on 8 cells through CGRP receptors. The paradoxical relationship between the effect of CGRP in rising cellular cyclic AMP and its inhibitory action on insulin release remains to be elucidated.

P400

P401

REDUCES THE CONTENT OF CYCLIC AMP IN ISOLATED RAT ISLETS M. Pettersson, S. Karlsson, S. Lindskog, M. Schwartz, B. Ahr~n. Dept. Pharmacology, Lund University, Lund, Sweden

AMYLIN AND CGRP DECREASE INSULIN-MEDIATED GLYCOGENESIS~RAT D I A P H R A G M IN V I V O

CGRP

Calcitonin gene-related peptide (OGRP) is an intrapanereatic neuropeptide inhibiting glucose-stimulated insulin secretion from isolated rat islets. However, its mechanism of action is not established. We examined whether CGRP affects the islet content of cyclic AMP. Batches of 20 isolated, overnight cultured, rat islets were incubated for 30 minutes in KEB medium supplemented with IBMX (0.i mM) and glucose (3.3 or 16.7 mM) without or with addition of CGRP at a concentration which inhibits insulin secretion (i N/4). Cyclic AMP was extracted by TCA and, after sonication, measured with radioinm%unoassay. The islet cAMP content was 19.1• fmol/islet after incubation at 3.3 mM glucose (n=5) and raised to 36.0• fmol/islet at 16.7 mM glucose (n=5; P<0.05). CGRP abolished this increase (19.2• fmol/islet; n=14; P<0.01). After including pertussis toxin in the overnight culture medium (i00 ng/ml), the islet cAMP content was 28.4• fmol/islet after incubation at 3.3 mM glucose (n=2), and 60.4• fmol/islet at 16.7 mM glucose (n=2; P<0.01). CGRP did not affect the cAMP content in these islets (50.7• fmol/islet; n=5). We conclude that CGRP reduces glucose-stimulated production of cAMP in normal rat islets by a pertussis toxin-sensitive mechanism, inferring an action on an inhibitory G protein.

E.A. Foot, C.DaCosta and Department of Biochemistry, O x f o r d , O x f o r d O X I 3QU, U K

B. Leighton University of

A m y l i n is a H - c e l l h o r m o n e t h a t c a u s e s i n s u l i n r e s i s t a n c e in s k e l e t a l m u s c l e in v i v o a n d in vitro. It has been suggested that an abnormality in a m y l i n h o m e o s t a s i s plays an i m p o r t a n t r o l e in t h e d e v e l o p m e n t of t y p e 2 (non-insulin-dependent) diabetes mellitus. A m y l i n h a s a h i g h d e g r e e of h o m o l o g y w i t h t h e neuropeptide calcitonin gene-related peptide (CGRP) w h i c h a l s o c a u s e s i n s u l i n r e s i s t a n c e in m u s c l e in v i v o a n d in vitro. We established t h e d o s e - r e l a t e d e f f e c t s of C G R P a n d a m y l i n on i n s u l i n - s t i m u l a t e d r a t e s of g l y c o g e n s y n t h e s i s in t h e r a t d i a p h r a g m in vivo. Various doses of i n s u l i n (0.01, 0.i, i, I0, 100 ~ g / k g ) a n d [U-14C]glucose were administered i n t r a p e r i t o n e a l l y i n t o 1 8 - h f a s t e d r a t s (70 g) a n d a f t e r 2 h t h e i n c o r p o r a t i o n of [U-14C]glucose into diaphragm glycogen was measured. Intraperitoneal administration of insulin did not decrease plasma glucose levels. Basal and maximally stimulated rates of g l y c o g e n s y n t h e s i s w e r e 5.5 • 0.5 a n d 407 f 29 D P M / 2 h / g , r e s p e c t i v e l y . T h e e f f e c t s of i n s u l i n w e r e d o s e r e l a t e d (ECs0 v a l u e w a s a b o u t 0.7 ~ g / k g ) . C o - a d m i n i s t r a t i o n of a m y l i n (4, 40 o r 400 ~ g / k g ) o r C G R P (2, 20 o r 200 ~ g / k g ) resulted in significant dose-related i n h i b i t i o n in t h e r a t e s of g l y c o g e n s y n t h e s i s in s k e l e t a l m u s c l e in vivo.

Al13

P402 EFFECTS OF CALCITONIN GENE-RELATED PEPTIDE ON IN VIVO INSULIN ACTION S.B. Choi, S. Frontoni, L. Sloan, M. Katz and L. Rossetti. University of Texas Health Science Center at San Antonio, U.S.A. CGRP is a peptide with structural homology to amylin which is present in nerve terminals of skeletal muscle and intestine. The effect of this peptide on in vivo insulin action was studied in conscious rats. All rats received 200 rain euglycemic (5.7 raM) insulin (28 pmol/kg.min) clamp with [3-3H]-glucose. CGRP was infused at 100 (low) or 500 (high) pmol/kg.min during the last 100 rain. Hepatic glucose production (16.1_+3.3 ilmol/kg.min during infusion of insulin alone) was stimulated to 25.6_+2.8 and 73.9___8.3 p.mol/kg.min at low and high CGRP infusions, respectively. Glucose uptake (133.3_+7.8 i~mol/kg-min) decreased to 102.2_+3.9 and 93.9_+1.7 tlmol/kg.min with CGRP at 100 and 500 pmol/kg-min, respectively. Whole body rates of glycolysis (3H20 generation) were similar during insulin alone (80.0_+5.6 p.mol/kg.min), low (82.2_+4.4 limol/kg-min), and high CGRP (73.3_+7.8 i~mol/kg.min) infusions. In contrast, CGRP determined a s e v e r e impairment in muscle glycogen synthesis (8.8_+3.3 and 9.4-+2.8 ~mol/kg-min during the low and high CGRP, respectively, vs 35.0-+3.3 ~mol/kg-min). Conclusions: (1) In conscious rats CGRP determines severe hepatic and peripheral insulin resistance (2) CGRP induces insulin resistance primarily through inhibition of glycogen synthesis at low and (3) through stimulation of hepatic glucose production at high concentrations. These results indicate that CGRP exerts potent effects on glucose metabolism and may contribute to the pathogenesis of insulin resistance.

PS 7 Insulin Therapy and Devices P403

P404

T R E A T M E N T OF S U B C U T A N E O U S INSULIN RESISTANCE - PROBLEMS AND SOLUTIONS. K. Wiefels, A. Hfibinger and F.A.Ories. Diabetes-Research Institute at the University of D~sseldorf, F.R.G. Two female patients, who were resistant to subcutaneously (se.) administered insulin, were first treated intraperitoneally (ip.) with an external pump (H-TRON, Hoeehst), then intravenously (iv.) with a constant rate insulin infusion pump (infusaid Mod.420, filled with diluted H-TRONIN ). Patient one, B.M., (pat. two, B.I.) age 39 (57) years, duration of diabetes 25 (20) years, c-peptid < 0,3 ng/ml, (7,3 ng/ml) experienced constant ketotie high bloodglucose values, despite of 500 (410) units of insulin, administered sc. Both patients were treated by continuous ip. insulin infusion over 16 (59) months. Catheter occlusions occured every 8 (5,9) months (mean), range 1-17, mean Hbal-values were 8,9 and 8,1%. After pump implantation in 1988, malfunction occured after 3 and 4 months respectively. In the first patient a second pump of the same model was reimplanted and is functioning well since 21 months. The insulin need and HbAl-values remained constant and hospital admissions could be reduced, compared to ip.treatment. We conclude that iv. treatment of sc. insulin resistance by this constant rate infusion pump can improve the life-style of the patient if pump or catheter related problems are eliminated.

DECREASEDFREE INSULIN LEVELS DURINGEUGLYCEMICHYPERINSULINEMIC CLAMPIN NON-INSULIN DEPENDENTDIABETICSON INSULIN THERAPY D. Babi(, B. Ro~i6, M. Granid and Z. ~krabalo, Institute for Diabetes, Zagreb, Dugi DGI 4a, Yugoslavia Euglycemic hyperinsulinemic clamp technique is considered as useful test for assessing peripheral insulin resistance. The aim of this study was to investigate a possible effect of insulin antibodies on blood insulin concentration during euglycemic hyperinsulinemic clamp. We have compared free insulin levels in the group of 16 non-insulin dependent diabetics (mean age 51.7• years, body mass index 28.5• kg/m2) on insulin therapy (average 7.6 months) with a control group consisted of 16 age-weight matched non-insulin dependent diabetics (mean age 52.5• years, body mass index 27.7• kg/m2) treated with oral antidiabetic drugs. Using the euglycemic hyperinsulinemic clamp technique at the constant low-dose insulin infusion rate (0.303 pM/min/m2) free insulin levels were 300.2• pM/l in the insulin treated patients. This was significant l y lower than in controls (599.8_+81.4 pM/l, p
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P405

P406

HYPERGLYCEMIA, HYPERKETONEMIA, BASAL INSULIN SECRETION WERE PERFECTLY NORMALIZED BY PRE-MEAL REGULAR INSULIN INJECTIONS WITH OR WITHOUT BEDTIME SULFONYLUREA IN TYPE 2 DIABETES MELLITUS R.Kawamori, M.Kubota, T.Watarai, S.Ishida, M.Ikeda, and T.Kamada ist Dep.of Med., Osaka Univ. Med. Sch., Japan

H O R M O N E AND S U B S T R A T E P R O F I L E S A F T E R L O N G T E R M CONTINUOS INTRAFERITONEAL INSULIN INFUSION(CIPII) R.Bauersachs, K.D.Hepp, B.Ruhland, K.Piwernetz, R.Renner, P.Bottermann(1), P.Dieterle(2) and R.Landgraf(3). Diabetes Center M~nchenBogenhausen, i: II.Med.Klinik, Klinikum Rechts der Isar, Technical University Munich, 2: 3.Med.Klinik, Krankenhaus MHnchen-Neuperlach, 3: Klinik Innenstadt, University Munich Aim: The intraperitoneal route of insulin delivery is thought to be more physiological than the peripheral one. This study evaluates the quality of metabolic control with respect to hormone and substrate levels after 6 and 12 months of CIPII as compared with intensified conventional insulin therapy (ICT). Method: 6 patients (IDDM) on CIPII (5 implanted pumps, one external pump) were matched with 6 patients on ICT. Profiles of insulin, OH, cortisol, prolactin, glucagon and triglycerides, cholesterol, FFA, glycerol, ~-HOB, lactate, alanine were measured at 0, 6 and 12 months. Results: At study entry all parameters were not significantly different between the two groups. After 12 months BG control was comparable: MBG [mmol/l]: 9.0• 7.9• (CIPII); HbAlc[%]: 6.7• (ICT), 6.8• (CIPII). Both, 24h insulin requirements and insulin levels were not different. All other measured values remained in the range of a healthy control group, with the exception of lactate [~mol/l] (1002• (ICT), 928• (CIPII)) and eortisol [nmol/l] (661• (ICT), 716• (CIPII)). Conclusion: Even though BG control could not be fully normalized, most hormone and substrate parameters were within normal limits. After elimination of technical pitfalls with the implanted pump, further improvement of metabolic control under CIPII is likely.

The substitution of prandial insulin by injections of regular insulin was performed in nonobese type 2 diabetics with secondary failure on sulfonylureas (n=37). Patients were given regular insulin 30 min preprandia!ly for 3 meals. Doses were initiated with 10U, 8U and 6U for breakfast, lunch and dinner, respectively. Then, according to the daily profile of plasma glucose taken every 3-4 days, doses were adjusted to obtain normal preand 2-h post-prandial glycemias. 4 weeks later, in 30 patients, perfect normalization of both meal-related and pre-breakfast glyeemia was established with insulin doses of 9.6i2.9, 7.5!2.5, and 7.0!2.5 U for breakfast, lunch, and dinner, respectively. Urinary C-peptide excretion rate/plasma glucose, from i0 PM to 7 AM, was increased markedly after 4 weeks insulin treatment, showing recovery in basal insulin secretion. 7 patients with high pre-breakfast plasma glucose (160illmg/dl) in spite of normal bedtime glycemia (113i20mg/dl) were given bedtime glibenelamide (1.25-5 mg) in addition to pre-meal regular insulin. Glibenclamide increased urinary C-peptide excretion rate from i0 PM to 7 AM up to 1.50!0.64 ug/h, (without glibenelamide, 0.56• ug/h), then not only prebreakfast glycemia but also otherwise elevated plasma concentration of ketone bodies and FFA were normalized. It was demonstrated that in most of nonobese type 2 diabetics with secondary failure on sulfonylurea, sufficient regular insulin before each meal could restore basal insulin secretion enough to regulate the hepatic glucose production during night time, and that in type 2 diabetics with markedly decreased basal insul~n secretion, sulfonylurea could stimulate basal insulin secretion but not stimulate secretion enough to regulate prandial glyeemia.

P407

P408

FASTING AND POST PRANDIAL SUBSTRATE OXIDATION IN TYPE I DIABETICS DURING SUBCUTANEOUS AND INTRAPERITONEAL INSULIN TREATMENT.

PERIOPERATIVE MANAGEMENT OF TYPE 2DIABETES MELLITUS: A RANDOMIZED CLINICAL STUDY: J.J.Diez, C. Grande and M.T. Ibars. Hospital General de Guadalajara. 19002 Guadalajara.Spain Perioperative metabolic control of 14 type 2 diabetic patients undergoing elective surgery under general anesthesia and treated with two different techniques of iv insulin infusion has been evaluated. After random assignment, 7 patients were included in the standar (I) and 7 in the intensified treatment (II) group. There were no differences in sex, age, weigth, dibetes duration or hemoglobin AIC levels. In group I a mixture of 5% glucose (125 ml/h), potassium (3.8 mmol/h) and insulin (2 U/h) was given. In group II insulin was administered by using an iv infusion pump, and glucose determinations to control infusion rate were performed at least 12 times a day. On the operation day blood glucose concentrations of group II patients were between 6 and 10 mmol/l and at lower levels than those of the group I patients (8-13 mmol/l). However, these differences did not reach statistically significant values, At 2, 3 and 4 days after operation glycemic profiles were very similar in both groups. Incidence of hypoglycemia was I episode/28 patient-days in group I vs. I episode/9.3 patient-days in group II (p< 0.05) Our results suggest that intensified treatment does not significantly improve perioperative metabolic control in type 2 diabetic patients and might increase the risk of developing hypoglycemia.

M.C. Librenti, G. Galimberti,G. Petrella, E. Orsi, P. Micossi and G. Pozza. H.S. Raffaele Scientific Institute, Milano, Italy. To estimate ~ metabolic rate and substrate oxidation, indirect calorimetry was performed by means of MMC HORIZON (Sensor Medics, Anaheim, Ca) in 8 type I diabetic patients, (age 41.4• 4 males and 4 females) treated with subcutaneous (SC) insulin therapy and 6 months after intraperitoneal (IP) by an implantable device (INFUSAID Model 1000, Norwood, MA, USA). Calorimetry was carried out at fasting and after a standard meal (700 Kcal: CHO 55%, lipid 23%, protein 22%). HbAlc improved from 8.24~0.4 SC to 7.22• IP, p<.005 while daily insulin requirement and body weight unchanged. Energy expenditure at fasting was: SC 24+_4, IP 23+_.3; post prandial was: SC 27~3, IP 25+--2 Kcal/Kg/die. Respiratory quotient at fasting was: SC 0.76+0.04, IP 0.80• post prandial was: SC 0.92+_0.09, IP o.93• Carbohydrate oxidation at fasting was: SC 16+_9, IP 27+16; post prandial was: SC 59+__19, IP 61~11%. Lipid oxidation at fasting was: SC 66+15, IP 53+,17%; post prandial was: SC 25&18, IP 20+_19%. Protein oxidation at fasting was: SC 18+_6, IP 20• post prandial was: SC 16+_3, IF 18~4%. Fasting and post prandial evaluations showed highly significant differences for each parameter both during subcutaneous and intraperitoneal treatment, while no statistical differences between SC and IP calorimetric evaluations were observed.

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P409

P410

C L I N I C A L E F F I C A C Y OF M U L T I P L E INSULIN I N J E C T I O N R E G I M E N IN P A N C R E A T O G E N I C D I A B E T E S J.Yokoyama, J.Miura, A.Mimura, Y.Mori, K.Ito, M . O h n o and Y.Ikeda, Tokyo, J a p a n P a n c r e a t o g e n i c d i a b e t i c s (PD), s e c o n d a r y either to c h r o n i c c a l c i f i e d p a n c r e a t i t i s or to pancreatectomy become unpredictably hypoglycemic d u r i n g c o n v e n t i o n a l i n s u l i n t h e r a p y and are often in the s o - c a l l e d b r i t t l e diabetes. We i n v e s t i g a t e d tissue s e n s i t i v i t y to i n s u l i n (SI) , e v a l u a t e d by means of the g l u c o s e - i n s u l i n clamp technique, and i n s u l i n r e q u i r e m e n t s in feedback control p e r f o r m e d w i t h B i o s t a t o r ~ in 16 PD. Based on these data, m u l t i p l e insulin i n j e c t i o n r e g i m e n was d e t e r m i n e d and was e v a l u a t e d in comparison with previous conventional insulin regimen. SI was s i g n i f i c a n t l y i n c r e a s e d in PD c o m p a r e d w i t h that in Type I d i a b e t i c s (6.6+2.3 vs 3.5• m g / k g / m i n glucose, p<0.01). PD r e q u i r i n g n o c t u r n a l i n s u l i n i n f u s i o n m o r e than 1.0 m U / h r in f e e d b a c k c o n t r o l w e r e t r e a t e d w i t h p r a n d i a l a c t r a p i d i n s u l i n (Bolus) using a pen injector and once u l t r a t a r d insulin i n j e c t i o n (Basal) while PD r e q u i r i n g n o c t u r n a l i n s u l i n i n f u s i o n less than 1.0 mU were t r e a t e d only w i t h Bolus. HbAI was s i g n i f i c a n t l y (p<0.01) decreased after Blous or B a s a l - B o l u s r e g i m e n (9.9• vs 8.5• 10.3+2.2 vs 9.4• respectively). Furthermore, large swings of blood glucose and f r e q u e n c y of h y p o g l y c e m i c events w e r e r e m a r k a b l y improved. These r e s u l t s suggested that m u l t i p l e i n s u l i n i n j e c t i o n r e g i m e n should be c o n s i d e r e d in PD a l t h o u g h PD are c o n t r o l l e d with r e l a t i v e l y small dose of i n s u l i n b e c a u s e of an i n c r e s e d i n s u l i n sensitivity.

F U N C T I O N A L INSULIN S U B S T I T U T I O N THERAPY: R E S U L T S FROM 294 U N S E L E C T E D TYPE ! (INSULIN-DEPENDENT) DIABETIC PATIENTS. C. Bali, H. Abrahamian, H. Hoffmann, B. Maierhofer, and K. Irsigler. 3rd M e d i c a l D e p a r t m e n t and L. B o l t z m a n n R e s e a r c h - I n s t i t u t e , City H o s p i t a l V i e n n a - L a i n z . 294 type 1 patients w e r e t r a i n e d in f u n c t i o n a l insulin s u b s t i t u t i o n (a b a s i s - b o l u s regimen) from 1987 to 1990 by i n p a t i e n t group t e a c h i n g and followed in a special o u t p a t i e n t clinic. They were e v a l u a t e d w i t h r e s p e c t to m e t a b o l i c control (HbAlc) , bodyweight, daily i n s u l i n dose, m e t a b o l i c d e r a n g e m e n t s leading to h o s p i t a l admission, a c c e p t a n c e of the t h e r a p e u t i c r e g i m e n (discontinuation on p a t i e n t ' s request, q u e s t i o n naire), and c o m p l i a n c e (analysis of patients' records). M e a n age and diabetes d u r a t i o n w e r e 36 and 18 years, r e s p e c t i v e l y , the m e a n treatment period was 19 months (total 442 p a t i e n t years). M e t a b o l i c control was s i g n i f i c a n t l y improved (HbAIe: 8,6 vs. 8,2%, p < 0,001), b o d y w e i g h t did not change, daily i n s u l i n dose was reduced s i g n i f i c a n t l y (65 vs 43 IU/day; p < 0,01). H o s p i t a l a d m i s s i o n rate for h y p o g l y c emia was 0 , 0 0 4 / p a t i e n t year, for h y p e r g l y c e m i c d e r a n g e m e n t 0 , 0 2 / p a t i e n t year. 1,6% of users changed the t h e r a p e u t i c s t r a t e g y on their own request; rating by q u e s t i o n n a i r e was positive. Data on c o m p l i a n c e and t r e a t m e n t failure w i l l be given. We conclude that by f u n c t i o n a l insulin s u b s t i t u t i o n therapy in an u n s e l e c t e d group of type 1 p a t i e n t s a s i g n i f i c a n t and lasting i m p r o v e m e n t of m e t a b o l i c c o n t r o l can be achieved w i t h o u t the side effect of w e i g h t gain, t h o r o u g h t r a i n i n g and r e g u l a r f o l l o w - u p provided. Patients' a c c e p t a n c e is enthusiastic, regardless of m e t a b o l i c control.

P411

P412

E V A L U A T I O N OF A M U L T I P L E INJECTION R E G I M E W I T H H I G H F A S T / L O W INSULIN RATIO IN TYPE 1 DIABETES. Th. K a r d o y i a n n i s , N . Z a c h a r i o u , A . K o f i n i s , K . P e t r o u , and B.Karamanos. D i a b e t e s Center. Athens Greece To assess the e f f e c t i v e n e s s and a c c e p t a b i l i t y of an intensified regimen (IT), c o n s i s t i n g of three pre-meal bolus injections of r e g u l a r insulin (Actrapid-HM, Novopen) and one i n t e r m e d i a t e (Monotard-HM) at bed-time,(being less than 25% of total insulin dose), we studied 20 Type 1 patients, during two, 2-month periods, treated with IT and two daily injections of M o n o t a r d - H M (CT), in a crossover pattern. Daily glycosuria, two 4-point blood g l u c o s e (BG) profiles per study period and HbAI were measured. BG was lower with IT at each time point, at 0 8 . 0 0 = 8 . 3 ~ . 5 vs I0.2~.7, 12.00=7.4~.4 vs 9.3~.8 ,17.00=7.8~.4 vs 10.3• 2 2 . 0 0 = 8 . 2 ! . 4 vs 10.0+__.8 mmol/l, p<.01 for all. HbA1 was also lower with IT, 6.60~.I vs 7.38• p<.001 .Glycosuria e x p r e s s e d as % positive tests was less with IT, at 08.00=34.4• vs 53.5~6.2, at 12.00=30.6• vs 48.7~5.4, at 1 7 . 0 0 = 3 4 . 2 ~ 5 . 1 vs 53.5~5.9, at 2 2 . 0 0 = 3 3 . 0 ~ 5 . 6 vs 52.2~6.0%, p<.01 for all. H y p o g l y c e m i c episodes were less with IT, 4.7~.8 vs 6.9• The d a i l y insulin dose was not d i f f e r e n t b e t w e e n the two regimens, 49.2~2.1 vs 50.8• units p>.05. D u r i n g IT, Monotard-HM c o n s t i t u t e d only 24.2% of the total insulin dose. In the end of %he study , 90% of the patients preferred to continue with IT. Conclusions: In Type 1 diabetics, IT w i t h high f a s t / s l o w insulin ratio 3:1, in c o m p a r i s o n to CT a) r e s u l t s in b e t t e r m e t a b o l i c control b) less h y p o g l y c e m i c r e a c t i o n s and c) is p r e f e r r e d by the m a j o r i t y of the patients.

D O U B L E BLIND C R O S S - O V E R S T U D Y U S I N G H U M A N L E N T E AND ULTRALENTE INSULINS ADMINISTERED AT BEDTIME A. GROSS J. P. DIGY E. M. L E U T E N E G G E R , H 6 p i t a l R. Debr6,

RAYNAUD and Reims, F r a n c e

O u r a i m was to c o m p a r e f r e e i n s u l i n levels, their clinical and metabolic consequences using lente (L, Monotard HM ) or ultralente (UL, U l t r a t a r d HM) i n s u l i n in a b a s a l - b o l u s regimen. Ten type I (insulin-dependent) diabetic p a t i e n t s (M + SD age, 41,8 + 8 years, d u r a t i o n of disease, 14,2 + 7,3 years, BMI, 24,7) t r a i n e d to h o m e b l o o d g l u c o s e m o n i t o r i n g w e r e i n c l u d e d in a d o u b l e b l i n d study. P r e v i o u s l y treated by various multiple injections regimens, t h e y w e r e r a n d o m l y a l l o c a t e d to L or U L i n j e c t e d at b e d t i m e p l u s 3 i n j e c t i o n s of r a p i d i n s u l i n b e f o r e meals, w i t h a c r o s s - o v e r after 8 weeks. Diet was n o t changed. Free i n s u l i n and g l y c a e m i c p r o f i l e s u s i n g 7 d a i l y determinations were obtained at inclusion, a f t e r 8 and 16 weeks. G l y c a e m i c control w e r e a l s o a s s e s s e d by HbAlc, f r u c t o s a m i n e , i n s u l i n daily dosage, frequency and severity of h y p o g l y c a e m i c attacks. A t t h e e n d of the study, w e d i d n o t o b s e r v e a m o n g all t h e s e c r i t e r i a a n y s t a t i s t i c a l d i f f e r e n c e s r e l a t e d to the use of L or U L (M + S.E.M., HbAIc, 6,78 + 0,13 vs 6,93 + 0,12 %, f r u c t o s a m i n e , 2,6 + 0,i vs 2,7 + 0,I %, free insulin daily profiles, basal insulin d a i l y doses, 15 + 1 vs 16 + 1 IU, n u m b e r of h y p o g l y c a e m i c r e a c t i o n s 7,9 + 1,6 vs 7,5 _+ 1,8 p e r month). L or U L c a n be b o t h s u c c e s s f u l l y i n j e c t e d at b e d t i m e in a b a s a l - b o l u s regimen.

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P413 TIMING OF PRE-MEAL INSULINS IN DIABETIC P ~ ON A MULTIPLE DAILY INJECTION R E G - - . A QUESTIONNAIRE SiIJDY. L.N. JCrgensen, Novo N o r d i s k A/S, D K - 2 8 0 0 Bags v a e r d and F.S. Nielsen, H v i d C r e Hospital, DK2930 Klampenborg, Denmark. D i a b e t i c p a t i e n t s on a m u l t i p l e d a i l y i n j e c t i o n r e g i m e n are r e c o m m e n d e d to inject rapid a c t i n g i n s u l i n 30 min. b e f o r e mealtime. It is a w i d e s p r e a d impression, however, t h a t m o s t p a t i e n t s a c t u a l l y inject t h e m s e l v e s i m m e d i a t e l y b e f o r e meals. The aim of this study was to e v a l u a t e the t i m i n g of i n s u l i n i n j e c t i o n s in d i a b e t i c p a t i ents on m u l t i p l e daily injections, i00 consec u t i v e p a t i e n t s a n s w e r e d an a n o n y m o u s q u e s t i o n naire on t h e i r t i m i n g of injections. (All the p a t i e n t s h a d been r e c o m m e n d e d to inject i n s u l i n 30 min. b e f o r e the meals.) The r e s u l t s w e r e as f o l l o w s : ( n = 1 0 0 ) 0 - 1 0 min. b e f o r e m e a l s : 4 5 % ; ii20 min. b e f o r e m e a l s : 2 5 % ; >20 min. b e f o r e meals: 30%. These figures w e r e identical for b r e a k fast, lunch, and d i n n e r insulin. The r e a s o n s for not i n j e c t i n g as r e c o m m e n d e d were: "it is e a s i e r to remember": 74%; "it is m o r e convenient": 40%; "fear of h y p o g l y c a e m i a " : 10%; "it gives b e t t e r control": 8%. It is n o t e w o r t h y that t h e s e p a t i ents w e r e h i g h l y m o t i v a t e d and educated. In conclusion, these r e s u l t s show that: i) o n l y a m i n o r i t y of the p a t i e n t s c o m p l y w i t h the c o r r e c t t i m i n g of rapid a c t i n g insulin; 2) the p o o r c o m p l i a n c e is due to c o m p l e x i t y and i n c o n v e n i e n ce of the therapy, m o r e t h a n " m e t a b o l i c considerations".

P414 C O M P A R I S O N OF THREE D I F F E R E N T R E G I M E S W I T H ONE DAILY INSULIN I N J E C T I O N IN TYPE 2 D I A B E T E S Ch. T o u n d a s , A . K o f i n i s , D . R o u s s i , B . P a v l o p o u l o s , a n d B.Karamanos. Diabetes Center,Athens,Greece. To compare the e f f e c t i v e n e s s of three regimes comprising one daily insulin i n j e c t i o n and isocaloric but differently distributed diets, in NIDDM, we studied 20 patients for 3 c o n s e c u t i v e 6-week periods (A,B,C). Insulin was given: D u r i n g A: L e n t e - M C before breakfast, B: A c t r a p h a n e - H M before breakfast, C: A c t r a p h a n e HM before lunch. Calories were d i s t r i b u t e d 2/10 breakfast, 4/10 lunch, 4/10 dinner for A and B, and I/I0, 5/10, 4/10, for C. Diabetes control was assessed by blood glucose (BG) selfm o n i t o r i n g pre- and 2 hours p o s t - p r a n d i a l l y and HbAI at the end of each period. M e a n daily BG mmol/l(six measurements/day) was 9.8 • vs. 8.0~.5 vs 7.2 • for A, B and C respectively, A-B p>.05, A-C p<.01, B-C p >.05. HbAI % was i0.6~.3 vs i0.2~.2 vs 9.4X.2, A-B p>.05, A-C p<.001, B-C p<.001. BG during periods A, B and C was: P r e - b r e a k f a s t 8.9 • vs 7.2 ~ .5 vs 6.0 ~.4, A-B p<.05, A-C p<.001, B-C p<.01, Postb r e a k f a s t 10.8~.5vs 8.6~.5vs8.0~.4, A-B p<.O01, A-C p<. 001, B-C p>.05, Pre-lunch 8.0• 6.7• 6.7• A-B p<.05, A-C p<.05, B-C p>.05, P 0 s t - l u n c h 11.7• vs9.0•177 A-B p<.O01, A-C p<.001, B-C p>.05. Pre-dinner 8.S• 6.9~.4vs 5.8• p<.O01, A-C p< 001, B-C p< 01, P o s t - d i n n e r ll.5• 9.6• 8.0• A-B p<.001, A-C p< .001, B-C p<.001. Conclusion: In Type 2 d i a b e t i c s t r e a t e d with one insulin dose/day, administration of A c t r a p h a n e - H M before lunch with less calories at breakfast, compared to p r e - b r e a k f a s t Lente or A c t r a p h a n e with normal diet distribution, results in better control and significantly smaller p o s t p r a n d i a l glycemic excursions.

P415

P416

PRESERVATION OF INSULIN SECRETION IN INSULINDEPENDENT DIABETICS BY INTENSIVE INSULIN THERAPY.

A d j u s t m e n t of insulin dosage d u r i n g long haul flights. T.A. Sane, V.A. Koivisto, P. Nikkanen, and R. Pelokonen; Third and Second D e p a r t m e n t s of Medicine, Helsinki U n i v e r s i t y Hospital, F i n l a n d

I. Hulinsky, IKEM, Prague, Czechoslovakia We enroled 42 newly diagnosed IDDM patients, mean age 23.8 yrs (range 17-31), and treated them by multiple dose (basal/bolus) insulin administration by pump and/or pens from disease onset (intensive insulin therapy group - liT), as well as 12 patients previously treated by conventional means from IDDM onset, mean age 29.0 yrs (range 18-41), mean duration of diabetes 2.65 yrs. Both groups were followed for a mean of 2.2 yrs, in order to study the effect of liT on metabolic parameters in these patients. The pvalue of difference in c-peptide secretion at time 0' and ( 6' after lmg Glncagon i.v.) between both groups was 0.445 (0.942) at the time of enrolment, 0.129 (0.084) after one month, and 0.018 (0.012) after one year. The mean glucose control index, as well as average glycaemias in BG profiles, and HBAI~ was in the normal range as of implementation of IIT. The mean insulin dose (+SD) was 0.53 (_+0.19) IU/kg, but no correlation was found between this dose and c-peptide secretion. We conclude that I ~ has a beneficial effect on prolonging the period of autologous insulin secretion when implemented from disease onset, stabilizing the metabolic profile and thereby possibly retarding the progression of organ complications.

We e x a m i n e d the effect of long haul flight with time zone shift of 6-10 hours on the insulin r e q u i r e m e n t and g l y c a e m i c control in 27 type I d i a b e t i c p a t i e n t s aged 38+-14 yrs., insulin dose 35+-11 U/d, HbAI 9.3+-1.4 % (MEAN+-SD). Glycaemic control was a s s e s s e d by selfm o n i t o r i n g of blood glucose. On w e s t w a r d travel the mean insulin dose increased by 1.1+-0.5 U or 3.1+-1.9 % of total daily dose per time shift hour, given as I-2 extra injections of shorta c t i n g insulin before meals during the flight. On e a s t w a r d travel the insulin dose decreased by 1.0+-1.1 U or 2.6+-2.7 % of total daily dose per time shift hour. An extra dose of shortacting insulin was given before l a t e - e v e n i n g meal on the plane, w h e r e a s the s u b s e q u e n t m o r n i n g dose of i n t e r m e d i a t e - a c t i n g i n s u l i n was d e c r e a s e d by 29+-18 %. The a v e r a g e blood g l u c o s e c o n c e n t r a t i o n on w e s t w a r d (8.6+-2.3 mmol/l) and e a s t w a r d travel (9.4+-2.5 mmol/l) were slightly h i g h e r (p<0.01) than on the n o n t r a v e l control day (7.1+-1.4 mmol/l). The number of low (<3 mmol/l) or high (>15 mmol/l) blood g l u c o s e values were not d i f f e r e n t from control day. In conclusion, good g l y c a e m i c control can be m a i n t a i n e d d u r i n g w e s t w a r d and e a s t w a r d flight by i n c r e a s i n g or d e c r e a s i n g daily insulin dose 2-4 % of total daily dose per time shift hour.

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COMPARISON OF ACTIVITY OF MAGNESIUM AND ACTRAPID HM INSULINS S Colagiuri, J Bryson, S Keating, L Tan, J King and C Eigenmann. Prince of Wales Hospital, Sydney, Australia

NEW INJECTION PREPARATIONS WITH PROTRACTED P R O P E R T I E S C O N T A I N I N G AMIDATED H U M A N M O N O ARGININE INSULIN. P. Balschmidt, K.H. Wolffbrandt, B. Dath, P. Nilsson and F.B. H a n s e n , Novo Nordisk A/S, Gentofte, D e m n a r k . New insulin preparations with protracted properties have been formulated using [ArgA0 l-human insulin-(B30-amide) of biosynthetic origin. This insulin derivative shows low solubility in the physiological pH range, and full potency in the mouse blood glucose test. The prolonged effects of different pharmaceutical formulations were evaluated in a pharmacokinetic rat model. External g a m m a counting of 12 s I-labelled derivative was used to measure the rate of disappearance (T 50 ~ ) from the site of injection. VelosulinTr~ showed a Tso ~ value of approximately 0.5 h, whereas the T s 0 value for a suspension preparation of the derivative was > 12 tl. Preparations containing dissolved derivative at pH 4 were tested in a factorial design with Zn 2+ (3 Zn 2§ or 30 Zn 2* per hexamer), with or without buffer 0 5 mM acetate) and with glycerol or sodium chloride as isotonic agent. The obse~wations showed that a high Ts0 ~ value was dependent on high Zn 2* content or on the combination of low buffer capacity and low ion strength. The prolonged blood glucose lowering effect of a dissolved preparation was confirmed in streptozotocin diabetic rats and was shown to be of longer duration than the effect of Ultratard T M HM. Antigenicity test of a suspension preparation in rabbits showed approximately the same low response as Ultratard TM HM.

This study compared magnesium insulin (Mgl),a zinc free insulin containing Mg salts, and Actrapid HM (ACT) insulin. Pharmacokinetic comparison was made in 8 healthy nondiabetic subjects using the euglycaemic clamp. Three separate 6 hour studies were randomly performed in each subject using 0.2 units/kg insulin injected subcutaneously in abdomen. Day I - ACT U]00 conventional needle; Day 2 ACT U50 sprinkler needle; Day 3 - Mgl U50 sprinkler needle. Compared with ACT UIO0, plasma insulin levels were higher after Mgl at 15 min (p<0.05), similar at peak and lower from 120 to 270 min (p<0.02). ACT U50 showed an intermediate profile. Glucose clamp results reflected these profiles. Total glucose infused (mean • was 135.4 • 15.1 g for ACT UIO0 compared to 90.2 • 13.4 g for Mgl (p~O.02) and 116.9 • 1].9 g for ACT U50 (NS). Hourly glucose infused was significantly lower for Mgl v ACT UIO0 between 3 - 6 hours and v ACT U50 between 3 - 4 hours. Mgl and ACT UIO0 (0.2 U/kg) given 30 min before a test breakfast was examined in Type 2 (non-insulin-dependent) subjects. Mgl resulted in ]ower BG levels at all times up to 120 mins which reached significance at 15 and 60 min (p
P419 IN VITRO AND IN VIVO D E M O N S T R A T I O N OF THE I N T E R F E R I N G E F F E C T OF A C E T A M I N O P H E N ON GLUCOSE DETECTION BY A SENSOR D. Moatti, G. Velho and G. Reach. Service de Diabdtologie, H6tel-Dieu Hospital, 1 Place du Parvis Notre Dame, 75004 Paris We assessed in vitro and in vivo the interfering effect of acetaminophen on glucose measurement by a needle type amperometric/enzymatic glucose sensor. The sensor output at 5 mmol/1 glucose concentration in phosphate buffer was 14.4 +_ 2.0 nA (n=7). It was 25.3 _+_2.1 nA in the presence of 5 mmol/1 glucose and 200 gmolB acetaminophen (usual pharmacological plasma concentration). Without acetaminophen such output current would be observed in response to about 12 retool/1 glucose. The sensor was implanted subcutaneously in anaesthetized rats (n=3). Increasing plasma glucose, by intravenous glucose infusion, from a 6.8 + 0.8 to a 11.0 + 0.7 mmo/1 plateau induced an increase in the sensor output from 6.6 + 0.9 to 9.0 + 1.2 nA. During an intravenous acetaminophen infusion (plasma plateau around 200 gmol/1) plasma glucose did not change, remaining below 7.4 mmol/1. Sensor output increased from 6.8 + 1.2 to 10.4 + 1.0 nA at 15 rain and remained stable at that level throughout the infusion. Using this output current for glucose monitoring would result in an overestimation of about 6 mmol/1 on subcutaneous glucose concentration. This study demonstrates that acetaminophen, a common antalgic agent, diffuses into the subcutaneous tissue and produces a major bias in the estimation of subcutaneous glucose by the sensor.

P420 "GLUCOSENSOR UNITEC ULM': A NEW' DEVICE FOR CONTINUOUS BLOOD GLUCOSE MONITORING, H. Zier, W. Kerner, F.S. Keck, J. Br~ckel, and E.F. Pfeiffer, Abteilung Innere Medizin I, Medizinische Klinik und Poliklinik der Universit~t UIm

The "Glucasensor Unitec Ulm" is a newly developed portable system for long-term control of blood sugar, preferentially in diabetic patients. It consists from an enzymatic glucose sensor, a roller-pump and a digitalmonitor and allows continuous monitoring of venous blood glucose levels, minute by minute, 24 hours a day. The enzymatic glucose sensor is placed in an acryl flow chamber and connects the platinum/silver electrode with the various electronic components. Roller-pump and digital monitor are controlled by a microprocessor. The results of 24 hour glucose monitoring demonstrate good correlation with a reference method. Dilution of blood is held constant, demonstrating a perfect function of the roller-pump. The dilution of blood with saline/heparin amounts to 1:3 at minimum pump speed, to1:4 at maximum pump speed. The capacity of battery operation is guaranteed for 6-8 hours. The electronic drift of the amplifier is negligible (< 1%). The drift at 100 mg/dl glucose is less than 16% per 24 hours, without glucose the drift is 1% per 24 hours.

Al18 P421 AMINIATURIZED SENSOR FORGLUCOSE MEASUREMENTIN SUBCUTANEOUSTISSUE OF HUMAN SUBJECTS W. Kerner., F.S. Keck, J. Brfickel, H. Zier, and E.F. Pfeiffer, Abteilung fOr Innere Medizin I, Medizinische Klinik und Polildinik, Universit&'t UIm, Germany A miniaturized amperometric sensor for glucose measurement in subcutaneous tissue was developed. The platinum anode (isolated wire; diameter 0.125 ram) was placed into the lumen of a eannula (stainless steel; bevelled tip; G 27; length 24 mm). A layer of glucose oxidase was covalentiy bound to the tip of the wire and covered with medicalgrede polyurethane. The wire did not extend into the area of the bevel of the needle. In vitro testing revealed that the sensor response to glucose was stable for several days, linear over a wide range of concentrations (0 - 15 mMol/1) and largely independent from oxygen availability. After preincubation with plasma for several hours, the sensors were placed directly into the subcutaneous tissue of 5 type I diabetic subjects. Amperometric measurements were performed against an epicutaneous Ag/AgCI reference electrode. Plasma glucose concentrations of the patients were regulated throughout the experiment by intravenous infusion of glucose and/or basal amounts of insulin. Thus concentrations of 5, 10, 15 mMoM were attained for successive 120 rain. periods. Rasma glucose and sensor output correlated fairlywell in each patient (range of r values: 0.88 - 0.92). The regression Unas, however, did not meet the origin, pointing at the significance of interfering non - glucose substances. Moreover there was a drift of the sensor current during the experiment resulting in a 10 to 20 % loss of output Beside this drift, in some patients a sudden decrease of the sensor signal was observed, which was interpreted as the result of rnicrobleeding at the implantation site. in conclusion: (1) Subcutaneous glucose can be measured in diabetic patients with a miniaturized sensor. (2) Influence of interfering substances should be eliminated by addition of another membrane. (3) Biocompatibility of the outer membrane must be improved before practical application of the sensor is possible.

P422 TECHNICAL AND CLINICAL EVALUATION OF THE EXACTECH BLOOD GLUCOSE METER K. Dannehl, T. Koschinsky, Diabetes Research Institute, Diisseldorf, Germany The ExacTech blood glucose meter has been introduced for self-momtoring of blood ~lucose (SMBG) and for clinical emergency use. There'fore, 7 p.en-sized devices which use electrochemistry to ~ive a digital ~lucose readout 30 seconds after wfiole blood application, and 6 batches of disposable strips have been evaluated by 2 technicians accordin~ to Diabetes Care 11: 619-29, I988, and then compared'to 13 Type 1 diabetic patients. For the 7 devices examined by the equally performing technicians the maximal total deviation within ttie clinically relevant blood glucose range (1.7-20mM) equalled deviations from the reference value between 13% and 21%. The variability between the 6 stripbatches increased this deviation up to 34%. Examineffbvthe diabetic patients these values equalled deviations between 23% and 57%. 11 out of 13 patients underestimated hypoglvcemic values. Applving the acceptance analysis, fhe device handling bq tile technicians resulted in 5 out of 7 devices classified as zood and in 2 as acceptable, while handlin~ by alFdiabetic patients resulted m all devices classifieH as acceptable. While clinically acceptable for SMBG and emergency use in climcal practice the ExacTech device as any other device for SMBG still differs from the performance of a standard laboratory device.

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Hypoglycaemia P423 PIVOTAL ROLE OF CATECHOLAMINES IN GLUCOSE COUNTERREGULATION TO HYPOGLYCAEMIA. C. Fanelli, P. De Feo, G. Perriello, E. Torlone, F. Santeusanio, P. Brunetti and G.B. Bolli. University of Perugia, Italy. It is presently believed that catecholamines are not important in hypoglycaemic counterregulation if glucagon secretion is normal. To reassess the issue, 6 studies (S1-6) were performed in 8 normal subjects. In $1 (only infusion of insulin, rate 15 mU/m2/min for 12h)plasma glucose decreased from 4.94 +__0.11 to 2.77 + 0.05 romp1/1 at 12h. In $2 (insulin as in $1 + alpha-, beta-blockade and variable glucose infusion to reproduce the same plasma glucose concentration of Sl~resulted in N30% lower hepatic glucose production (HGP,3-3H-glucose) after 11/2h, - 1 5 % greater glucose utilization (GU) after 5h,but --30% greater C-peptide (C-P) and - 8 0 % greater growth hormone. To quantitate the effects of catecholamines independently of changes in C-Peptide and other counterregulatory hormones, the PAPclamp (AJP 252:E565,1987) was performed as control study ($3) and compared with $4 ($3 + alpha-, beta-blockade + variable 1ucose infusion to reproduce plasma glucose of $3), and with $5 4 without glucose infusion). In $4, HGP was - 2 5 % lower and - 1 0 % greater than in $3. In $5, plasma glucose after 3h was "1 retool/1 lower vs $3 (p<0.001) despite identical glucagon and other counterregulatory hormone responses. Finally, in $6 ($2 without glucose infusion), plasma glucose nadir at 6h was "0.9 mmol/1 lower than in $1 (p<0.001) despite N30% increased responses of glucagon and other counterregulatory hormones. Conclusions. In contrast to the preserit view, catecholamines play an essential role in counterregulation even when glucagon secretion is normal, because lack of catecholamines effects is not compensated by responses of other counterregulatory hormones.

P424 NO RELEVANT POST-HYPOGLYOAEMIC HYPERGLYCAEMIA UNDER FUNCTIONAL INSULIN TREATMENT K.Howorka, H.Grillmayr, H.Thoma, and Ch. Schlusche, Institute of Biomedical Engineering, University of Vienna, Vienna, Austria. The purpose of this study was to investigate the relevance of the Somogyi effect (posthypoglycaemic~ counterregulatory elevation of blood glucose) under functional insulin therapy, which separates basal, prandial and correctional insulin use. Subjects were I0 Type I (insulindependent) diabetic patients (age: 30• /x!SD/; diabetes duration: 11• years). During an observation period of 207~iI days per patient, glycaemic control was determined on the basis of mean blood glucose (MBG) measured by glucose meter with memory capacity, insulin required for correction of hyperglycaemia, and fasting blood glucose. Mean glycaemic control for all 24h periods after hypoglycaemic episodes was compared to mean glycaemic control for all hypoglyeaemia-free days. (Frequency of hypoglycaemia defined as blood glucose<50mgdl was 0.9• episodes/week/patient.) Results for hypoglycaemia-free days vs. periods following hypoglycaemia were: MBG (mmol/l) 7.8~i.I vs. 6.7• (p<0.0001); fasting blood glucose 7.5• vs. 7.1• after night-time hypoglycaemia; n.s.); amount of regular insulin required for correction of hyperglycaemia (IE/24h)3.9• vs. 1.6• (p<0.0001). These results confirm the clinical observation that hypoglycaemiais more likely to be followed by a period of relatively low blood glucose rather than hyperglycaemia. Post-hypoglycaemie counterregulatory blood glucose elevation is not clinically relevant under functional insulin treatment.

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ACTIVATION OF PANCREATIC NORADRENERGIC NERVES DURING EXTREMEHYPOGLYCEMIAIN HALOTHANE-ANESTHETIZEDDOGS. P.J. Havel, B.E. Dunning, T.O. Mundinger, R.C. Veith, and G.J. Taborsky Jr., Depts. of Medicine and Psychiatry, Univ. of Wash. and VA Medical Center, Seattle, WA U.S.A.

ROLE OF DECREASED INSULIN SECRETION IN THE CORRECTION OF HYPOGLYCEMIA. S.R. Heller and P.E. Cryer. Washington University School of Medicine, St. Louis, MO 63110, U.S.A.

To obtain evidence that the sympathetic innervation of the pancreas

We tested the hypothesis that decreased insulin secretion is the most important factor in the correction of hypoglycemia. Nine normal subjects were studied on 6 occasions: ~) Saline 0-240 min. 2-6) Insulin 0.6 mU'kg- "min.- 0-80 min. (to produce a plasma glucose of 3.2 m~ol/L), then O, O.l, 0.2, 0.4 or 0.6 mU'kg-l'min. 80-180 mln. Glucose steady state was achieved at 140-180 min. when glucose (mmol/L), insulin (pmol/L) and C-peptide (nmol/L) levels were: l) 4.9• 32m4 and 0.27• 2) 5.0• 29• and 0.14• 3) 4.8• 39• and 0.06• 4) 4.3• 53• and <0.02; 5) 3.6• 128• and <0.02; and 6) 3.2• 195• and <0.02. Glucagon and epinephrine remained elevated only in studies 5 and 6. Thus, insulin infused in a dose that prevented substantial reductions in portal insulin (study 4) attenuated recovery but did not prevent glucose increments to levels above those that activate counterregulatory systems. We conclude that decrements in insulin, coupled with increments in glucagon, are important in promoting complete recovery from hypoglycemia. However, recovery to glucose levels above those required to activate counterregulatory systems, and well above those that produce neuroglycopeMa, occurs even when portal insulin is not suppressed completely.

contributes to the pancreatic response to systemic stress, we measured the spillover (output) of norepinephrine (NE) into pancreatic venous plasma during insulin-induced hypoglycemia (IIH) (5.0-2-_1.4U/kg) in halothane-anesthetized, laparotomized dogs (n=9). We found that marked hypoglycemia (plasma glucose = 1.9_+0.2 mM) did not activate pancreatic noradrenergic nerves (A pancreatic NE-output = +0.12_+0.78 pmol/min). Therefore, we produced a more extreme IIH, by infusing somatostatin (SS) 2.5

gg/min IV ( n = l l )

to prevent the

counterregulatory glucagon response. In these animals, plasma glucose fell to 0.8+0.1 mM and NE-output increased from 2.46_+0.36 pmol/min to 5.28+0=90 pmol/min, (A = +2.82+0.90 pmol/min, p<0.005).

In

contrast, in dogs with spinal cord transection (n=6), pancreatic NE-output did not increase (A = +0.6+_2.04 pmol/min), despite extreme IIH (plasma glucose = 0.8+0.2 mM), suggesting that IIH increased pancreatic NE-output by centrally activating pancreatic noradrenergic nerves, rather than by a direct effect of low glucose. When SS was discontinued, NE-output increased by an additional 2.94+_1.20 pmol/min (p<0.025), despite recovering glucose levels. Conclusions: 1) Exogenous SS restrains NE release from pancreatic sympathetic nerves. 2) Pancreatic noradrenergic nerves are activated by a central mechanism during extreme hypoglycemia. 3) Such activation should stimulate glucagon secretion to help counteract the life-threatening hypoglycemia.

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INCREASED EFFICIENCY OF GLUCOSE COUNTERREGULATORY R E S P O N S E IN H Y P E R T H Y R O I D I S M . P.Moghetti, R.Castello, F.Tosi, L.Perobelli, G./ardini, A.Bolner, C . M a g n a n i , G . P i e m o n t e and M.Muggeo. Chair of Metabolic Diseases, U ~ i v e r s i t y of V e r o n a , Italy. To evaluate the i m p a c t of hyperthyroidism on co~nterregulatory r e s p o n s e to h y p o g l y c e m i a , in 6 h;gperthyroid and 8 a g e - and B M I - m a t c h e d normal females i.v. i n s u l i n (0.I U / K g b . w . ) w a s g i v e n as a b e l u s and b l o o d w a s d r a w n f r o m 0-120 min for glucose, epinephrine, norepinephrine, g l u c a g e n , A C T H , c o r t i s o l a n d GH m e a s u r e m e n t s . In the basal state plasma glucose levels were similar in the two g r o u p s and ACTH and GH c o n c e n t r a t i o n s w e r e not s i g n i f i c a n t l y d i f f e r e n t . Plasma catecholamines were significantly lower in hyperthyroid patients (epinephrine 2223 vs. 68• rig/l, p<0.Ol; norepinephrine 62210 vs. 238221 rig/l, p
EFFECTS OF 3 HOURS EXERCISE IN TEE EVENING Pfohl M., Schm~lling R.M., Renn W., Schnauder Jakober B. and Eggstein M. Med. Univ.-Klinik, Abt. IV, D-7400 T~bingen

G.,

Exercise of long duration may be expected to induce hypoglycaemia or alterations of insulin requirement in the following hours. In order to quantify this effect we investigated 8 type I diabetic patients during and after bicycle ergometry for 3 hours. They exercised in the evening at a work load of 30% maximum corresponding to 53• Watt. Blood glucose was continously monitored during exercise and in the following night. For comparison, the same parameters were determined in the same individuals without exercise. Basal NPH insulin remained unchanged on both occasions. During the 3 hour exercise period blood glucose declined by 58 mg/dl to 81 mg/dl. After exercise blood glucose rose to 137 mg/dl compared to 90 mg/dl without exercise and remained higher throughout the night and until the following morning (121 mg/dl versus 103 mg/dl). There were no nocturnal hypoglycaemic episodes. After exercising ACTH rose from 2,3 to 3,5 pmol/l compared to 2,6 and 1,8 pmol/l at rest, cortisole rose from 6,5 to 13,3 ug/dl compared to 5,8 and 2,5 ug/dl. Growth Hormone rose from 42 to 298 pmol/l after exercise, at rest it remained unchanged at 78 and 70 pmol/l. Epinephrine and norepinephrine rose only slightly during exercise and were not different in the following nights. Thus exercise for 3 hours in the evening does not reduce insulin requirement in the following night, blood glucose is even higher than without. This effect can be explained by a significant rise of ACTH, cortisole and Growth Hormone during and after exercise.

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Thermogenesis and Sweat P r o d u c t i o n During Sustained Hypoglycaemia in Man. D.G. MAGGS, A.R. SCOTT AND I.A. MACDONALD. Nottingham, UK.

EFFECTS OF INSULIN-INDUCED HYPOGLYCEMIA ON CEREBROVASCULAR PERMEABILITY IN DIABETIC RAT

Acute hypoglycaemia is associated with an adrenal medullary response and increased thermogenesis, but a fall in body temperature. The purpose of this study was to monitor thermogenesis and body temperature during sustained hypoglycaemia. Ten healthy men (age 20-26 years) were studied on three occasions (resting supine O at 30 C) with th~ infusion of saline (placebo) or insulin (60 mU/m /min) and glucose to maintain euglycaemia (81 mg/dl) or hypoglycaemia (45 mg/dl) for 60 minutes. Thermogenesis (gas exchange), sweat rate, body temperature and plasma catecholamines ~ere measured throughout. Sweat rate (8 to 38 g/hr/m~; P=0.003) and thermogenesis (5.2 to 5.6 kJ/min; P=O.002) increased within 10 minutes of the onset of hypoglycaemia and reached a plateau by 20 minutes, which was then maintained. Skin temperature began to fall after 20 minutes and core temperature after 30 minutes, both were still falling after 60 minutes of 9 O hypoglycaemla (skin -2.4 C, core -0.36~ P
H.Tanaka, K.Ishikawa, S.Higa, K.Murakami and G.Mimura Second Depertment of Internal Medicine, School of Medicine, University of the Ryukyus, Okinawa, Japan The effects of hypoglycemia on cerebrovascular permeability to Evans blue-albumin complex were studied in both streptozotocin-induced diabetic rats and control rats. After 3 months of diabetic state, 200units/kg of insulin was given intraperitoneally and then O.Ig/kg of Evans blue was injected 30 minutes prior to sacrifice intravenously. The extraction of extravasated Evans blue was performed by homogenizing the whole brain in 60% trichloroacetic acid, and Evans blue in the supernatant was measured. The results were expressed by Evans blue/g wet tissue: Group I, control-normoglycemia (n=4), 0.02• g/g; Group II, control-hypoglycemia (n=4), 0.89• Group III, diabetic-normoglycemia (n=4), 2.18• Group IV, diabetic-hypoglycemia (n=4), 3.94• In basal condition, diabetic rats showed increased permeability compared with the controls (p
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I N F L U E N C E OF DURATION OF DISEASE AND AGE ON SEVERE HYPOGLYCEMIA IN DIABETIC CHILDREN A. Verrotti, A. Blasetti, L. Pavone, S. Tumini, P. Rocchi, F. Chiarelli and G. Morgese. University D e p a r t m e n t of Pediatrics, Chieti, Italy. In a 3-years prospective study, we evaluated a group of 47 (19 male) diabetic children, age ( m e a n + SD) 138.8 + 102 months, duration of disease 78.4 + 42.7 months, to evaluate the frequency of severe hypoglycemia (defined as glycemia < 50 mg/dl plus an episode of loss of consciousness, seizures or both). During the study, 15 (31%) children had severe hypoglycemic episodes; a m o n g these children, 7 patients experienced one episode, 6 patients two episodes and 2 patients three episodes of severe hypoglycemia. No significant difference was found between the hypoglycemic and non-hypoglycemic children regarding metabolic control (HbAI: 11 + 2.3% vs 10.9 + 1.7, respectively) and insulin requirement (0.93 + 0.1 U/Kg/die vs 0.98 + 0.2). T h e duration of disease was longer in children who experienced two or three episodes of severe hypoglycemia compared with those who experienced one episode: 96.5 + 33.9 m o n t h s vs 72.0 + 20.7; p <0.05. Patients with two or more severe hypoglycemias were younger than children who had one episode (152.1 + 56.0 m o n t h s vs 199.8 + 59.0; p < 0.05). Our data show that duration of disease and age may influence the frequency of severe hypoglycemia in diabetic children. In our experience diabetic children with a long duration of disease are at risk of severe hypoglycemia and need a special care to try to avoid this harmful complication.

FACTORS ASSOCIATED WITH THE INCIDENCE OF SEVERE HYPOGLYCEMIA IN TYPE 1 (INSULIN-DEPENDENT) DIABETIC PATIENTS M. GNiBer, V. JSrgens, U. Bott, V. Scholz and M. Berger. HeinriehHeine-University Diisseldoff, Germany. A diabetes treatment and teaching programme (DTTP) was instituted in 10 hospitals. 784 consecutive diabetic patients were recruited for follow up after 1, 2 and 3 years to evaluate the long term effect of the DTTP. Exclusion criteria: blindness, renal insufficiency. To date 744 patients have been reevaluated 1 year, 698 2 years and 546 3 years after the DTTP. In order to identify patients with an increased risk for severe hypoglycemia we analyzed the data of 546 patients (injecting insulin at least 1 year before the DTTP) for the year preceeding the DTTP and for the two years following the DTTP. 441 patients (group A) had no severe hypoglycemia after the DTTP, 105 patients (group B) had at least one severe hypoglycemia after the DTTP. No significant difference between the groups A and B was observed concerning the following items: Sex, diabetes knowledge test, duration of diabetes (11_+7 versus 11_+7 years), body mass index (23.6_+2.9 versus 23.5_+3.2 kg/mZ), insulin dosage (47_+15 versus 47_+17 U/day) frequency of blood glucose self monitoring, social status, sports activity, alcohol drinking habits. The most important factor to predict severe hypoglycemia after the DTTP was a positive history for severe hypoglycemia before the DTTP (incidence/patient/year 0.16 group A, 0.84 group B, p=0.000). Special teaching and treatment interventions for patients with a positive history of severe hypoglycemia should be developed and evaluated.

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N O N - I N V A S I V E DETECTION OF N O C T U R N A L H Y P O G L Y C E M I A IN T Y P E 1 D I A B E T I C P A T I E N T S .

ASSAYS OF COUNTERACT

I.Bendtson,J.Gade*,G.Wildschir and C.Binder. Steno M e m o r i a l H o s p i t a l , G e n t o f t e and *Dept.of Medical Informatics and Image Analysis, A a l b o r g U n i v e r s i t y , Denmark. Asymptomatic, nocturnal hypoglycemia occurs with a f r e q u e n c y of 29-56% in i n s u l i n t r e a t e d d i a b e t i c patients. In d a y t i m e h y p o g l y c e m i a has shown electroencephalographic c h a n g e s s i m i l a r to n o r m a l sleep. We e v a l u a t e d eight t y p e 1 d i a b e t i c p a t i e n ts on two c o n s e c u t i v e nights, w i t h o u t any c h a n g e s in t h e r a p y (2-4 inj./day). M e a n age 31 (range 2246) yr, H b A l c 8.2 (6.5-10.1)% no n e u r o p a t h y or n e u r o l o g i c d i s t u r b a n c e s was present. S p o n t a n e o u s hypoglycemia (bloodglucose (BG) < 3.0mmol/l) o c c u r e d in 6 n i g h t s (38)%, in four n i g h t s the BG was < 2.0 mmol/l. N o n e of the p a t i e n t s w o k e up d u r i n g h y p o g l y c e m i a , none c l a i m e d h y p o g l y c e m i a the f o l l o w i n g morning. D u r i n g sleep we r e g i s t e r e d E E G , E M G and AOG c o n t i n u o u s l y . The t r a c e s was u s e d in the N i g h t i n g a l e (R) a p o l y g r a p h i c sleep analyser. In the h y p o g l y c e m i c n i g h t s the times for the first and s e c o n d sleep cycles w a s 30% l o n g e r and the total R E M (rapid eye movements) was s l i g h t l y lower t h a n the v a l u e s in normal nights. The sleep p a t t e r n s d u r i n g h y p o g l y c e m i a c o u l d not be c l a s s i fied according to t r a d i t i o n a l sleep stages. E v a l u a t i o n of EEG a m p l i t u d e and total f r e q u e n c y c o n t e n t was t h e n u s e d for h i e r a c h i c a l c l u s t e r i n g analysis. The c l a s s e s of EEG i n f o r m a t i o n s h o w e d specific frequency spectra for each p a t i e n t during hypoglycemia. In conclusion: C o m m e r c i a l sleep a n a l y s e r s c a n n o t be u s e d for e v a l u a t i o n of hypoglycemia. EEG a n a l y s e s m i g h t be u s e d as a n o n - i n v a s i v e m e t h o d to d e t e c t s e v e r e h y p o g l y cemia.

NASAL GLUCAGON PREPARATIONS INSULIN INDUCED HYPOGLYCEMIA.

A.R.S~rensen and L.L.Kimer. A/S, Bagsv~rd, D e n m a r k

Nordisk

P o w d e r b a s e d g l u c a g o n p r e p a r a t i o n s for nasal delivery to counteract insulin induced h y p o g l y c e m i a , h a v e b e e n a s s a y e d in a r a b b i t model. Hypoglycemia is induced by sc. i n j e c t i o n of 2 iu insulin/rabbit. One h o u r later, the g l u c a g o n p r e p a r a t i o n is b l o w n into the nose, and the change in b l o o d g l u c o s e (BG) is monitored. U s i n g this model glucagon (0.i-i mg) p r e p a r a t i o n c o n t a i n i n g 0-2 mg didecanoyl-~-phosphatidylcholine (DDPC) as e n h a n c i n g agent and p o w d e r b a s e to make 20 mg h a v e b e e n assayed. An index (I) is c a l c u l a t e d for each preparation, w h e r e I is the area u n d e r the BG curve (scaled: a v e r a g e BG from t = -i0 to t = 0 equals 100%) from t = 0"to t = 60 min. after the nasal g l u c a g o n a d m i n i s t r a t i o n r e l a t i v e to the area o b t a i n e d after a sc. i n j e c t i o n of the same dose. The usual p o w d e r base for freeze d r i e d i n j e c t a b l e g l u c a g o n p r e p a r a t i o n s , lactose, y i e l d e d an I = 0.55, w h e r e a s a - c y c l o d e x t r i n p r e p a r a t i o n w i t h o u t DDPC gave an I = 0.50. The b e s t p r e p a r a t i o n a s s a y e d was a g r a n u l a t e of ~c y c l o d e x t r i n w i t h 10% DDPC,' w h i c h g a v e an I = 1.12. The r e s u l t s g e n e r a t e d in this m o d e l i n d i c a t e s a p o t e n t i a l for an easy a d m i n i s t r a b l e g l u c a g o n p r e p a r a t i o n w h i c h can be u s e d to c o u n t e r a c t i n s u l i n i n d u c e d h y p o g l y c e mia in d i a b e t i c patients.

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COMPUTERISED MONITORING OF REACTION-TIME, A POTENTIAL SAFEGUARD AGAINST HYPOGLYCAEMIA-INDUCED MISMANAGEMENTOF MACHINES. G.Druck,E.Chantelau,t. Heinemann,and E . S a r t o r i u s . H e i n r i c h - H e i n e - U n i v e r s i t y , OOsseldorf (FRG)

INSULIN-LIKE HISTIOCYTOMA

Hypoglycaemia prolongs the r e a c t i o n time in man, thus compromising the a b i l i t y t o operate machines.Hence, monitoring o f a prolonged r e a c t i o n time(PRT) could be used t o prevent e.g. s t a r t i n g a car during unnoticed hypoglycaemia. A PRT-monitor was developed by E.S. from a pocketcomputer(SHARP Ltd.,Osaka,Japan) d i s p l a y i n g complex random numbers t o be reproduced by a t e s t person w i t h i n < i second.After intense t r a i n i n g , t h e device was t e s t e d i n 8 h e a l t h y c o n t r o l subjects , and i n 15 t y p e - 1 ( i n s u l i n dependent) d i a b e t i c p a t i e n t s , e i t h e r in good(n=7) or in poor (n=8) c o n t r o l . A l l subjects were clamped at normoglycaemia(5.O and 3.6 mmol/1),at hypoglycaemia(2.5mmol/1), and post-hypoglycaemic at 5.0 mmol/1 by means of a Bios t a t o r . During each l e v e l o f glycaemia, the subjects had t o t r y the PRT-monitor 5 times, and each t r i a l could be repeated once( n: 10 t r i a l s par person).Results: the monitor i n d i c a t e d c o r r e c t l y non-PRT at 5.0 mmal/1 in 83Z of the p a t i e n t s t r i a l s , and in 79Z o f the c o n t r o l t r i a l s . At 3.6 mmol/1, non-PRT was i n d i c a t e d i n 7 1 Z ( p a t i e n t s ) , a n d in 68%(controls) of the t r i a l s . A t 2.5 mmol/1, PRT was c o r r e c t l y i n d i c a t e d i n 74%(patients), and in 55%(controls) o f the t r i a l s . Conclusions: monitoring o f hypoglycaemiai n d i t e d PRT i s f e a s i b l e by means of r e l a t i v e l y simple computer devices. Provided the s p e c i f i c i t y o f 74% as associated with our PRT-monitor can be enhanced by f u r t h e r s o p h i s t i c a t i o n s o f t h i s technique, such PRT-monitors might be attached t o e.g. cars, in order t o prevent hypaglycaemic p a t i e n t s from s t a r t i n g t o d r i v e .

Nova

TO

GROWTH FACTOR (IGF) II P R O D U C I N G A S S O C I A T E D WITH H Y P O G L Y C E M I A

T.Wasada, N.Hizuka, M~ K.Haruki, K.Ikejiri, and Y.Hirata. Diabetes Center, Dept. of E n d o c r i n o l o g y , Tokyo W o m e n ' s Med. College, Tokyo 162; Dept. of B i o c h e m i s t r y , Med. Institute of Bioregulation, Kyushu University, Fukuoka 812, Japan A 5 3 - y r - o l d woman d e v e l o p e d a large retrop e r i t o n e a l h i s t i o c y t o m a and episodes of fasting hypoglycemia. The tumor r e s e c t i o n resulted in complete r e s o l u t i o n of hypoglycemia. Following an 8-yr remission, h y p o g l y c e m i a returned w i t h local r e c u r r e n c e of a large tumor. Plasma i m m u n o r e a c t i v e insulin (IRI) was low independently of glycemia. Plasma IRI responses during iv and oral glucose tolerance tests were b l u n t e d and glucose curve was diabetic, while these were n o r m a l i z e d after the second r e m o v a l of the tumor. Low p l a s m a IGF-I (<35 ng/ml) and m o d e s t l y high IGF-II(843 ng/ml) m e a s u r e d by each specific RIA were raised and lowered, respectively, after surgery. The tumor contained large amounts of IGF-II (12.9 ~g/g tissue). Under acid gel chromatography, free IGF-II from p l a s m a emerged at the fractions c o r r e s p o n d i n g to 125 I-IGF-II as a single component. The HPLC r e c o n f i r m e d that a tumor IGF-II was identical to a r e c o m b i n a n t human IGF-II. The tumor c o n t a i n e d 100-times m u c h IGF-II m R N A c o m p a r e d w i t h normal human liver. Among the leader exons of IGF-II gene, a novel exon, a c o u n t e r p a r t of r a t exon i, was found to be p r e d o m i n a n t l y expressed. This case provides a further u n d e r s t a n d i n g of the p a t h o p h y s i o l o g y of t u m o r - i n d u c e d h y p o g l y c e m i a and a new e v i d e n c e of u n u s u a l IGF-II m R N A e x p r e s s i o n in human tumor.

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GLUCOSE COUNTERREGULATION IN TYPE I DIABETIC PATIENTS WITH DECREASED SYMPTOMS OF HYPOGLYCAEMIA AFTER INSULIN PUMP TREATMENT A.H~binger, K.Wiefels, D.Ziegler and F . A . G r i e s Diabetes Research Institute, DUsseldorf, Auf'm Hennekamp 65 In o r d e r t o s t u d y w h e t h e r i m p a i r e d h y p o g l y c a e m i c awareness (IHA) after intensified insulin treatment w i t h i n s u l i n pumps ( C S I I ) i s a s s o c i a t e d with impaired glucose counterregulation, glucose counterregulatory hormones (GCH) were m e a s u r e d in 7 t y p e I d i a b e t i c patients with impaired symptoms a f t e r 6 months C S I I ( g r o u p I ) and i n 9 patients w i t h u n c h a n g e d symptoms o f h y p o g l y caemia a f t e r 6 months o f C S l l ( g r o u p 2 ) . GCH r e s p o n s e t o an i n s u l i n i n d u c e d h y p o g l y c e m i a was measured before (1.test) and a f t e r 6 months (2.test) of CSII. Intravenous insulin infusion induced glucose nadirs of approximately 30 m g / d l . G l u c o s e r e c o v e r y showed no d i f f e r e n c e s b e t w e e n t h e g r o u p s in b o t h t e s t s . The r e s p o n s e s of g]ucagono cortisol and g r o w t h hormone showed a l s o no d i f f e r e n c e s b e t w e e n t h e g r o u p s in b o t h tests. The e p i n e p h r i n e r e s p o n s e was u n s i g n i f i c a n t l y h i g h e r in t h e ~econd t e s t : areas under c u r v e (AUC) (pg x m l x 90 min - I ) g r o u p I : 2 0 , 9 4 5 and 2 6 , 6 7 5 , p = 0 . 3 9 3 ; g r o u p 2 : 2 0 , 7 4 5 and 2 7 , 0 8 9 , p = 0 . 3 0 2 . E p i n e p h r i n e r e s p o n s e s were unaltered in g r o u p 2, w h i l e t h e r e s p o n s e i n creased un~ignificant~y in g r o u p I : AUC (pg x ml x 90 m i n - ) 1 1 , 9 7 7 and 1 6 , 3 4 5 , p = 0 . 3 6 2 . The r e s u l t s demonstrate that impaired a w a r e n e s s o f h y p o g l y c a e m i c symptoms a f t e r C S l l is not associated with impaired glucose recovery o f h y p o g l y c a e m i a o r i m p a i r e d r e s s o n s e o f GCH.

DOES THE SPECIES OF INSULIN MODIFY RY RESPONSES TO HYPOGLYC~4IA IN HEALTHY ADULTS? T.W. Jones, S. Caprio, M.P. Diamond, L. Hallarman, R.S. Sherwin and W.V. Tamborlane. Yale University School of Medicine, New Haven, CT, USA It has been suggested that pork and human insulin induoe different oounterregulatory responses to hypoglycemia, although this resmins controversial. To examine this issue, hormonal responses to a mild hypoglycemic stimulus were determined in 35 healthy adults (26+2 yrs) using the glucose clamp techr/que to ensure standardization of glucose and insulin levels. With a continu~as infusion of either pork (P, n=15) or human (H, n=20) regular insulin (0.8 mU/kg/min, NovoNordisk), plasma glucose was reduced over 40 minutes frc~ 4.7_+0.07 to 3.3+0.04 n~nol/l (both groups) and maintained at that level (with variable rate glucose infusion) for an additional 60 minutes. Steady state insulin levels were similar (44_+7 vs 39_+4 ~U/ml, P vs H) as were basal counterregulatory hormone levels. When plasma glucose was lowered, hormonal response curves were similar with no significant differences in peak values of epir~phrine (324_+74 vs 324+57 pg/ml, P vs H), norepine~hrine (274+39 vs 312_+34 pg/ml, P vs H), qlucagon (163_+29 vs 175_+20 pg/ml, P vs H), growth hormone (14_+3 vs 17+3 ng/ml, P vs H) or cortisol (19.7_+3 vs 16.6+1 ug/dl, P vs H). Our data suggest that pork and human insulin produce a comparable and robust hormonal response in healthy adults under conditions of controlled hypoglycemia. Reports of altered responses to hypoglycemia in diabetic patients transferred from pork to hlal~n insulin may result frc~ factors other than the effect of insulin species per se on counterregulation.

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HYPOGLYCAEMIC WARNING SYMPTOMS AND INCIDENCE

HYPOGLYCEMIA UNAWARENESS AND HUMAN INSULIN J.H.Anderson, J.H.Holcombe, J.A.Grimes and J.A.Galloway; Lilly Research Laboratories, Indianapolis, IN, USA. Recent anecdotal and scientific reports have suggested that human insulin induces less counter-regulatory hormonal response than animal insulin and that patients may experience less warning symptoms when treated with human insulin. We assessed the risk of severe outcomes of hypoglycemia and hypoglycemia unawareness by an analysis of six multicenter, prospective, double-blind studies involving 1514 patients. New patients (n=286) were randomized to receive human insulin or animal insulin, while 1228 patients previously treated with animal insulin were randomized to human insulin or to continue their current animal insulin. Fourteen of 795 (2%) animal insulin treated and 13 of 719 (2%) human insulin treated patients reported severe hypoglycemia during the study. Hospitalizations for hypoglycemia were less than 1% in both groups; there were no deaths in either group associated with hypoglycemia. Hypoglycemia was more common in insulin-dependent patients, but there was no difference between human and animal groups. Hypoglycemia symptom awareness was reported as the "same" (74%, 70%), "greater" (11%, 18%), or "less" (15%, 12%) in the animal and human treated groups respectively. In conclusion, we found no differences between human and animal insulin in respect to incidence of hypoglycemia, severe outcomes or awareness of symptoms.

OF SEVERE HYPOGLYCAEMIA DURING TREATMENT WITH HUMAN AND ANIMAL INSULIN. h M0hlhauser, bl. Berger, L. Heinemann, E. Fritsche and K. Lennep. Heinrich-Heine Universtiy of D(]sseldorf, FRG. It has been claimed~[W. Berger, Lancet igBg] that treatment with

human insulin [HI) reduces autonomic hypoglycaemic

warning symptoms which might increase the risk of severe hypoglycaemia [SH]. We have assessed hypoglycaemic symptoms and

incidence

of

SH

by

an

interviewer

administered

questionnaire in 297 patients treated with I-II (age 38+18 yrs, diabetes duration animal

13+10 yrs) and 25q patients treated with

insulin [AI; age 38+18 yrs, diabetes duration 18+11

yrs] in a multicenter cross sectional study. When asked which symptom usually occurs first during hypoglycaemia 2q% of HI and 20% of TI patients answered "sweating", 20% and lg%. resp., answered "trembling" and 12% and lq%, resp., answered "unrest".

According

to

the patients' judgement,

their "most reliable symptoms of hypoglycaemia" were in both groups "trembling" (16%

versus

12%].

[23% versus 26%] and "sweating"

The

incidence

of

SH

was

0.41

cases/patient/year in HI and 0.q8 in TI patients. None of the differences was statisticallysignificant. This study shows that hypoglycaemie

warning

symptoms

and

the

incidence

of severe hypoglyeaemia are comparable between patients treated with human and animal insulinpreparations,

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INSULIN SPECIES HAS NO ~J#'F~Cf ON GLUCOSE ~ RDGUiATION 93 H Y P O G L Y ~ IN NORMAL VOLU~.'a~S S. A ~ i e l , C . M i l l , I . M a c D o n a l d , I.HaN~r,D.Forsterj J. Childs. Guy's Hospital, LONDON and Nottingham University, ~ G L A N D .

CHANGES OF MOOD AND PSYCHOMOTOR FUNCTION DURING HUMAN AND PORCINE INSULIN INDUCED HYPOGLYCAEMIA T. L i n g e n f e l s e r , M. M a a s s e n , D. O v e r k a m p , H. G l ~ c k , M. E g g s t e i n a n d B. J a k o b e r . University of Tuebingen, Department of Medicine, Western Germany R e c e n t s t u d i e s h a v e r e p o r t e d a d i f f e r e n c e in symptoms during hypoglycaemia induced by human (H) o r p o r c i n e (P) i n s u l i n . O b j e c t i v e a s s e s s ment of the patients mood and psychomotor function has not been performed. We have conducted a controlled study to compare the effect of either H or P insulin induced hypoglycaemia on mood changes and psychomotor performance. Nine insulin-dependent diabetics were studied after Biostator~-controlled normoglycaemia over night. Falling blood glucose levels (100-65-50-40 mg/dl) were reached within the same time. At each of the aformentioned levels tests of psychomotor ability were performed and mood was assessed using a standard scaled questionnaire. The dose of insulin needed to achieve hypoglycaemia and free hormone levels were not different at any time. Mood and psychomotor function deteriorated significantly during hypoglycaemia, but no differences were found between H and P insulin, apart from a more pronounced slowering of optical reaction time under P insulin (at 5 0 m g / d l : p = 0 . 0 2 3 , a t 40 m g / d l : p = 0 . 0 3 6 ) . We conclude that H and P insulin induced hypog l y c a e m i a c a u s e s s i m i l a r c h a n g e s of m o o d a n d p s y c h o m o t o r f u n c t i o n . H o w e v e r t h e r e is a m o r e pronounced prolongation of optical reaction time under P insulin.

Reduced awareness of hypoglycaemia in insulin dependent diabetics has been attributed to conversion to human insulin (huI). Diminished adrenergic responses to uncontrolled hypoglycaemia have been reported with huI. Further to investigate the effects of insulin species on hypoglycaemia, an hyperinsulinaemic clamp (1.5 mU/kg.min) was used to lower blood glucose (BG) reproducibly and slowly in 5 normal subjects, twice, using huI or porcine insulin (porl) in random order and double-blind. BG was lowered step-wise from 5 to 2.6 ~mDl/l over 3 hours and restored. Catecholamine responses were similar. (adrenaline 5.25 • 0.47 nmol/l huI vs 5.5 • 0.89 porl~ p NS; noradrenaline 2.20 • 0.15 nmol/l huI vs 2.13 +_ 0.35 porI) and began at BG 3.2 • 0.I mmol/l huI vs 3.1 • 0.i porI, p NS and 3.0 • 0.i vs 2.9 _+ 0.3, p NS respectively). Autonc~ic occurred at BG 2.9 • 0.I amDl/l (huI) and 2.8 • 0.2 ~mol/l (pocI), p NS, neuroglyoopenia at 3.0 • 0.i and 2.8 • 0.I n~ol/l, p NS and psyc/xm~tor deterioration (4 point-reaction time) at BG 2.8 • 0.2 (huI) and 2.7 _+ 0.2 ~ o i / i (porI), p NS. We conclude that insulin species has no effect upon the magnitude of a d r ~ i c responses; glucose threshold for counterregulation nor presentation of hypoglycaem~ia in normal volunteers, provided the hypoglycaemic stilm/lus is reproducible. We suggest that, if similar results are found in diabetics, change in ir~ulin species may not be responsible for loss of awareness of hypoglycaemia.

PS 9 Oral Therapeutic Agents P443

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SL 8#.0418 : HYPOGLYCAEMIC EFFECTS AND GOOD CARDIOVASCULAR TOLERANCE IN HEALTHY VOLUNTEERS OF A NEW 2-ANTAGONIST. P.Rosenzweig*, E.Fuseau*, M.C.Delauche*, P.L.Morselli*, I.Berlin** - * Syntheiabo Recherche, Dept of Clinical Research, 58 rue de la Glaei@re, 75013 Paris, France 9* Therapharm Recherche, 92100 Boulogne Billancourt,France

SL 84.0418 : A NEH ALPHA-2 ANTAGONIST NITH ANTI-HYPERGLYCAEMIC PROPERTIES. STUDIES IN RAT MODELSOF DIABETES I, Angel, H. Schoemaker, N. Duval, A. Ob]in and S.Z. Langer, Synth~labo Recherche (L.E.R.S.), 58 rue de ]a Glaciate, 75013 Paris

SL 84.0418, a novel pyrrolo-indole derivative, is a potent and selective alpha-2 adrenoceptor antagonist of preferential pers activity. SL 84.0418 has antihyperglycaemie properties in different animal models and modes of administration. The aim of the study was to assess the clinical, cardiovascular and biological tolerance and effect on glycaemia of SL 84.0418 in 12 healthy male young volunteers in a double blind randomised, placebo controlled, study according to a latin square design. Single doses from 1 up to i00 mg were given orally to 6 fasted subjects per dose with one week wash-out between doses. Haemodynamic parameters and glycaemic levels were evaluated during 24 hours after dosing. Th~ overall clinical and cardiovascular tolerance was good : no significant effect was observed on supine nor standing heart rate and blood pressure. No effect on glycaemia was seen up to the dose of 20 mg. Moderate hypoglycaemic effects were observed mainly at 1 hour after dosing : mean (sem) gZycaemia fall from baseline were -0.34 (0.ii) mmol/l after 40 mg, -0.69 (0.23) mmol/l after 60 mg, -0.80 (0.i0) mmol/l after 80 mg and -0.96 (0.16) mmol/l after i00 mg. These data suggest that SL 84.0418 may be an interesting new well tolerated antihyperglycaemic drug for the treatment of type II (non insulin dependent) diabetus mellitus.

Ne have investigated the potential use of alpha-2 adrenoceptor antagonists as ant]hyperglycaemic drugs for the treatment of t y p e - i [ diabetes. SL 84.0418 (2-(4,5- dihydro-lh-imidazol-2-yl)-l,2,4,5-tetrahydro-2-propylpyrrolo[3 2,1-HI-]indole hydrochloride) is a new, potent, selective and o r a l l y active alpha-2 adrenoceptor antagonist with preferential a c t i v i t y in the periphery. Nhile not affecting glucose-induced insulin release in v i t r o , i t potent l y antagonizes the i n h i b i t o r y effect of the alpha-2 adrenoceptor agonist UKl4304 on this parameter. In v]vo, SL 84.0418 fa]]s to s i g n i f i c a n t ] y affect basal glycaemic leve]s. However, SL 84.0418 potentiates glucose-induced ]nsulin release and reverses hyperglycaemia induced by glucose, epinephrine or UK14304. SL 84.0418 is further devoid of hypoglycaemic a c t i v i t y in animal models of type-I diabetes. In the neonatal-streptozotocin model of type-I[ diabetes, SL 84.0418 is however more effective than the a]pha-2 antagonist idazoxan in reversing UK14304-induced hyperglycaemia. SL 84.0418 (3 and lO mg/kg po) reduces basal hyperglycaemia in type-If diabet i c rats and f u l l y normalizes t h e i r impaired glucosetolerance. In this animal model of t y p e - I f diabetes, SL 84.0418 is more e f f e c t i v e tha# glibenclamide or idazoxan. I t is suggested that SL 84.0418 may be a potent and c l ] n i c a l l y e f f e c t i v e oral anti-hyperglycaemic drug f o r the treatment o f type-II diabetes.

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IMPROVEMENT OF GLYCEMIC CONTROL AND WEIGHT LOSS IN TYPE 2 (NON-INSULIN-DEPENDENT) DIABETICS AFTER ONE YEAR OF DEXFENFLURAMINE TREATMENT. E. Tanher-Lassen, P. Damsbo, J.E. Henriksen, B. Palmvig and H. Beck-Nielsen. Hvid6re Hospital, Copenhagen, Denmark.

BENFLUOREX AMELIORATES DIET-INDUCED "SYNDROME X" IN RATS

Type 2 diabetes (non-insulin-dependent) is frequently associated with obesity, and weight loss in these patients is known to improve glycemic control. The aim of this study was to investigate the effect of dexfenfluramine treatment for one year on weight loss and glycemic control in 40 obese type 2 (non-insulin-dependent) patients, BMI: 34.0 _+1.3 kg/m 2. The study was performed as a randomized, donhle-blind, placebo-controlled, parallel study with respect to body weight, fasting blood glucose (fBG) and HbAlc. All patients had a diabetes diet of 5-6000 kJ and were followed at five week intervals by a doctor and a dietitian, alternately. Results: 8 patients (20%) dropped out. Both groups lost weight: Dexfenfluramine (n=15) treated (98.6 _+3.9 kg to 92.9 +.4.1 kg p<0.01), Placebo (n=17) treated (94.9 +3.5 kg to 92.2 -+3.7 kg p<0.01). The weight loss was significantly greater in the dexfenfluramine treated group (6.3 _+1.4% vs 2.9 _+0.8% p<0.05). The glycemic control was improved in the dexfenfluramine group(fBG: 11.6+_0.9 to 9.4_+0.6 mmol/l p<0.05; HbAlc: 8.5-+0.5 to 7.6.+0.4% p <0.02), whereas no significant improvement was seen in the placebo group (fBG: 11.8_+0.9 to 11.1.+0.8 mmol/l NS; HbAI~: 8.3+_0.5% to 8.3-+0.4% NS). Conclusion: Treatment with dexfenfluramine for one year accelerates weight loss and improves glycemic control in obese type 2 (non-insulin-dependent) diabetics.

Insulin resistance is central to the cluster of diseases, termed "syndrome X". High-fat and high-fructose fed rats demonstrate insulin resistance associated with elevated plasma (highfructose) and tissue (high-fat) triglycerides and increased blood pressure. Our aim was to evaluate the effects of benfluorex (Servier) on insulin levels, insulin action, serum and tissue triglyceddes and blood pressure in adult, male rats. Both fructose- (35% of calories) and fat-feeding (59% of calories) impaired insulin action (euglycemic clamp plus 2,6[3H]deoxyglucose, insulin ~700 pM) in averaged skeletal muscle (to 60 and 72% of starch control respectively, both p<0.01). Fructose-feeding elevated mean blood pressure (13+3%, p<0.01) and circulating (from 0.97+0.09 to 1.73+0.20 mmol/L, p<0.01), but not skeletal muscle, triglycerides. In contrast, fat-feeding increased triglyceride measured in red quadriceps muscle (5.81 vs 9.22 mg/gm wet weight, p<0.01). Benfluorex (50 mg/kg/day p.o.) lowered basal insulin levels (by 38+9%) and prevented skeletal muscle insulin resistance in both diet groups; prevented fructose-induced hypertriglyceridemia (1.13+0.16 mmol/L, ns) and hypertension; and normalized red quadriceps triglycerides (6.22 mg/gm, ns) in the fat-fed group. We conclude that high-fat or high-fructose fed rats have several characteristics of "syndrome X" and that benfluorex effectively counters the adverse effects, potentially through its hypoinsulinemic and/or lipid lowering actions.

P447

P448

FLUOXETINE

INCREASES

INSULIN ACTION

B.J. Potter van Loon, J.K. Radder, M. Fr61ich, H.M.J. Krans, A.E. Meinders, Dept. of Internal Medicine, University Hospital, P.O. Box 9600, 2300 RC, Leiden, The Netherlands Insulin resistance is one of the defects in obese persons with type II diabetes mellitus. It has been demonstrated that the anorectic agents fenfluramine and dexfenfluramine diminish insulin resistance in type II diabetic subjects, irrespective of the weight-reducing effect. In a double-blind, placebo controlled, crossover study we examined whether fluoxetine, a serotonin-reuptake inhibiting anorectic agent, improves insulin action on the liver and on the peripheral tissues. Suppression of hepatic glucose production (HGP) and stimulation of peripheral glucose uptake (PGU) were measured in 8 obese type II diabetic subjects (D) (age 53(SEM:2)yrs, body mass index(BMI) 34(l.l)kg/m 2) and in 8 obese control subjects (C) (age 47(3)yrs, BMI 32.9(l.l)kg/m 2) by means of the sequential, hyperinsulinaemic, euglycaemic glucose clamp technique with continuous infusion of 3-3H glucose. Studies were performed after 2 weeks of fluoxetine- (F) and after 2 weeks of placebo- (P) treatment. Patient weight remained stable during the study. In D half-maximal PGU was achieved at an insulin level of 180 mU/l during F, compared to 225 mU/l during P (19<0.05). Maximal PGU was similar. HGP tended to be lower during F (9.45 vs. 10.37 umol/kg/min, O.10
LH Storlien, ND Oakes, AB Jenkins, EW Kraegen and DJ Chisholm. Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney 2010, Australia

LONG TERM EFFECT OF TETRAHYDROLIPSTATIN, A PANCREAS LIPASE INHIBITOR, ON GLUCOSE TOLERANCE. M.L. Drent, K.T. de Bruin and E.A. van der Veen. Department of Endocrinology, Free University Hospital, Amsterdam, The Netherlands. In acute studies ingestion of fat with carbohydrate results in lower blood glucose levels, possibly due to slower gastric emptying or stimulation of gastric inhibitory polypeptide, a known stimulator of insulin release. We studied the long term effect of decreased fat absorption by a lipase inhibitor on glucose tolerance. Twenty-two healthy obese patients (mean weight 83.2+9.82 kg) entered a 16 week weight reduction tYeatment period on a 500 Kcal-reduced diet. Eight of these patients additionally used Tetrahydrolipstatin (THL) 50 mg t.i.d., a pancreas lipase inhibitor, which reduces fat absorption by inhibiting triglyceride hydrolysis. An oral glucose tolerance test (75 gram glucose) was performed before and after the treatment period. Mean weight loss on placebo was 5.29+3.94 kg (p<0.001); on THL 6.06Z2.76 kg (p<0.001); no-significant difference was found between both groups (p=0.6). Mean decrease in fasting plasma glucose on placebo was 0.39+0.43 mmol/l (p=0.005); on THL 0.70~0.44 ~mol/l-(p=0.003). Mean decrease in 2 hour plasma glucose on placebo was 1.14+1.75 mmol/l (p=0.03); on THL 1.16+0.86 mmol/l (p=07006); no significant differences were found between both groups, p=0.1 and p=0.9 respectively. In conclusion, the presence of unabsorbed fat in the gastrointestinal tract by the use of THL had no significant influence on glucose tolerance.

A125

P449

P450

SECONDARY FAILURE IN TYPE 2 DIABETES MELLITUS

METABOLIC EFFECTS OF SULPHONYLUREA-ME~FORMIN ASSOCIATION IN TYPE 2 DIABETES. S.Marena,V.Tagliaferro,@.Montegnosso,O.Bozzo and G.Pagano Institute of Internal Medicine-University of Turin-Italy The effectiveness of sulphonylurea-metformin association has not been clearly defined. We investigated the metabolic effects of metformin (500mgx2) or placebo addition for 4 weeks to sulphanylurea (glibenclamide 5mgxS) treated type 2 diabetics, evaluating glycaemic and lipaemie profile and HbAIc level. Euglycaemio clamp (plasma insulin concentration of lOO!2uU/ml) by artificial B-cell (EsaoteItaly) was carried out together with the study of insulin binding to circulating monocytes. Six patients (age 50!3yr BMI=21!0.7) partecipated in the study according to a double-blind cross-over design. Fasting glucose did not varied while mean diurnal blood glucose showed a reduction after metformin (164~18 vs 209~33mg/dl, P=0.OO31) together with HbAlc amelioration (9.5~i vs i0.6~1%, P=O.OO25).LIpa~ mic profile showed a reduction of total cholesterol (209! !8 vs 244z29mg/dl , P=0.035) and triglycerides (I13Z25 vs 201~46mg/dl, P=0.006) and an improvement in HDL cholesterol (52Z3 vs 47+_4mg/dl, P=0.022) after metformin. Glucose requirement assessed by euglyeaemic clamp increased after ~aetformin (6.4+0.8 vs 4.1zO.4mg/ml/min , P=O.O02), hepatic glucose production decreased (I.5z0.6 vs 4.2Z0.1mg/kg/min , P=0.O08), while residual glucose production during insulin infusion did not significantly varied. Insulin binding to circulating monoeytes was significantly higher after metformin (6.3~1.2 vs 3.8~0.8 %, P=0.0016). These results confirm the metabolic amelioration of combined treatment suggesting its probable mechanism of action.

M- Granid. D. Babid. I. Pavli~--Renar and Z. Skrabalo. Institute for Diabetes. Zagreb. Dugi Dol 4a. Yugoslavia By using euglycemic hyperinsulinemic clamp we examined the impact of the peripheral insulin resistance on the occurrence of the secondary failure of oral hypoglycaemic agents in type 2 diabetes. The examination was carried out in the group of patients with secondary failure who have previously undergone a 3 month education program regarding the adequate dietary measures and physical activity (7 men and 3 wom~n~ mean age 57.535-6 yrs; body mass index 22-7 32.9 k g ; m % . These patients had HbA1c~9%. fasting blood glucose ~I0 mmol;1 and positive C-peptide with the maximal daily glibenclamide dose prior to and after a 3 month education program, representing thus I/3 of the total number of patients included at the beginning of educationThe results were compared with the group of patients with no previous education :11 men and 3 wom~n: mean age 57.2 -6.6 yrs; body mass zndex 26.933.0 kg/mL)- As a parameter for the resistance estimation, a metabolic glucose clearance was used- The comparison showed lower sensitivity in the group wlth no previous educatlon ~2.2--I 2 ml/kg/mln versus 4 5T2.5 ml/kg/min: p
.

.

.

.

+

,

.

P451 FREE

P452 RADICAL

SCAVENGING AND ANTI-PLATELET GLICLAZIDE.

EFFECT

OF

N Scott, A Saniabadi, J Brown, JJF Belch and PE Jennings. Department of Medicine, Ninewells Hospital and Medical School, Dundee, Scotland. Free radical mechanisms and increased platelet aggregation have been implicated in the pathogenesis of diabetic mic~oangiopathy. Therapeutic intervention with a sulphonylurea capable of scavenging free radicals and reducing platelet aggregation may be beneficial. We therefore assessed these abilities fm-uZtro for two Sulphonylureas, Gliclazide and Glibenclamide. The free radical scavenging assay distinguishes between superoxide scavengers and general free radical scavengers. The drugs were dissolved in buffer in a final concentration of 0.5, 1.0, 2.5, and 5.0 ug/ml. The percentage inhibition corresponding to the amount of general scavenging (mean i SD) of the assay for each concentration of Gliclazide respectively was ii.0 2.5%, 20.8 • 2.9%. 31.4 • 2.2%, 47.2 + 0.8%. Glibenclamide had no scavenging effects at concentrations up to 25.0 ug/ml. Using a technique of whole blood platelet aggregation we also investigated the effects of these agents on blood from type 1 diabetic patients and normal controls. In normal controls, using 0.6 ug/ml collagen as the aggregating agent the percentage of single platelets remaining increased from 52%, median (range 8-70) to 60% (17-80) p<0.O04 after incubation with 5 and i0 ug/ml Gliclazide. In the diabetics the corresponding values were a rise in percentage count from 41% (17-65) to 45% (17-76) and 53% (17-77) p<0.004 with 5 and lO ug/ml Gliclazide. With Glibenclamide the counts were unaffected. The results demonstrate that Gliclazide, unlike Glibenclamide, is a general free radical scavenger and will also reduce platelet aggregation.

'rH]~O,APy REVERSES HYP]~NSVLIN'J~LLA I N I',,~N-

DIABETIC I R O N ~ E D THAIASS~4IA MAJOR PATIENTS. W.V. Tamborlane, S. Caprio, S. Boulware, H.A. Pearson, P. Merkel, R.S. Sherwin and T.W. Jones. Yale Univ. School of Mad., New Haven, cr 06510 U.S.A. Hyperinsulinemia and insulin resistance precede the develo~ of insulin-deficient diabetes in thalassemia major (qlM). To examine whether sulphonylurea therapy reverses these abnormalities, glyburide (1.253.75rag/day) was administered to 7 non-diabetic thalassemic patients (17.5~_3yr, ferritin 3578_+938ng/ml). Glucose responses to oral glucose (1.75 g/kg) were normal before and after glyburide. Before therapy, insulin responses were markedly increased (area under insulin response curve 16.0-+2.7 vs 6.3_+0.6 mU/ral.rain in normals, p <0.01). In contrast, insulin responses fell after 3 months (to 8.6-+1.2, p<0.Ol) and were normalized after 12 months (6.8_+I.I,p=NS vs controls) of glyburide. Peak insulin levels were also normalized, falling f ~ 236-+37 uU/ml to 117_+24 after 3 months, and 84_+15 after 12 months (vs 83_+8 uU/ml in normals). In keeping with these findings, euglycemic insulin clamp studies (40mU/m2.min, n=4) showed increased insulin-stimulated glucose metabolism (frc~ 162_+18 to 215-+45 mg/m2.min), an effect due to enhanced peripheral glucose uptake since hepatic glucose production (3H3 glucose) was fully suppressed before and after glyburide. Adverse effects of therapy were limited to infrequent and mild hypoglycemic s y ~ . Conclusion: In nondiabetic, hyperinsulinemic qM patients, glyburide normalizes insulin responses to oral glucose and i~proves insulin sensitivity. If insulin resistance and compensatory hyperinsulinemia contribute to the development of diabetes, glyburide may be useful in postponing this complication of iron overload.

A126

P454

P453 EFFECTS OF G E M F I B R O Z I L IN PATIENTS WITK HYPERLIPIDEHIA.

TYPE

2

DIABETIC

F.Fallucca, F.Troili, M.Pitaro, M.Testa, S.Balducci, E.Fratieelli, M.Ceci and A.Maldonato. Diabetes Unit, Inst. C 1 . H e d i c a 2, La Sapienza University, Roma, Italy. We s t u d i e d the e f f e c t s of g e m f i b r o z i l (GFZ) on g l u c o s e m e t a b o l i s m and B - c e l l s e c r e t i o n in 22 hyp e r l i p e m i c type 2 d i a b e t i c patients, p r e v i o u s l y t r e a t e d w i t h diet alone (5 pts) or w i t h s u l f o n y l ureas. P l a c e b o (PL) or GFZ were s e q u e n t i a l l y added to p r e v i o u s t r e a t m e n t in r a n d o m o r d e r for two c o n s e c u t i v e S - m o n t h p e r i o d s (crossover). On start and e v e r y 3rd m o n t h a s t a n d a r d b r e a k f a s t was given (BTT) and b l o o d d r a w n at 0", 60" min. A f t e r GFZ treatment plasma triglycerides and total c h o l e s t e r o l were d e c r e a s e d and HDL c h o l e s t e r o l increased. All o t h e r m e t a b o l i c v a r i a b l e s w e r e decreased: plasma glucose (PG-0": 3m, 160• vs 194• p < 0 . 0 5 ; 6m, 159• v s 186• p < 0 . 0 2 5 . PG60": 3m, 219• v s 244• p<0.05; 6m, 209• vs 242• mg/dl p<0.01), i n s u l i n (6m: IRI-0", 18.8• 1.9 vs 2 6 . 0 • p<0.01; IRI-60", 39.9• vs 52.6• pU/ml p<0.01), % H b A l c (3m, 7 . 1 • vs 7.6• p<0.05; 6m, 7.1• vs 7 . 4 • p<0.05) and u r i n a r y C p e p t i d e (3m, 59• vs 96• p<0.001; 6m, 59• vs 95• ~ g / 2 4 h p<0.01). All e f f e c t s were more e v i d e n t in those p a t i e n t s s t a r t i n g from higher plasma triglyceride levels (above 376 mg/dl, m e a n v a l u e of the group), in w h o m the dec r e a s e i n d u c e d by GFZ was greater. These d a t a suggest t h a t GFZ t r e a t m e n t may be u s e f u l in hypertriglyceridemic NIDDM since it combines p o s i t i v e e f f e c t s on lipid and g l u c o s e metabolism.

THE DIRECT ANTIHYPERGLYCAEMIC MECHANISMS OF METFORMIN IN TYPE 2 DIABETES MELLITUS. G. Perriello, P. Misericordia, F. Santeusanio, P. Brunetti, G.B. Bolli. University of Perugia, Italy. To assess the mechanism(s) by which metformin (MF) decreases plasma glucose (PG) in Type 2 diabetes directly, i.e. independently of long-term improvement of PG control, 13 subjects with NIDDM (fasting PG 9.7 -- 1 mmol/l;HbAlc 8.2 _+ 0.3%; BMI 25.3 -- 1.1, mean -- SEM) on diet only, were studied after administration of MF (500 mg at -5h and at -lh) or placebo, with the hyperglycemic-hyperinsulinemicglucose clamp + glucose tracer (3-~H) and indirect calorimetry. Glucose (G) was infused at variable rate from -3b to Oh (step 1, no insulin), from 0 to 2h (step 2, insulin infusion rate 0.1 mU/kg/min), and from 2 to 4h (step 3, insulin infusion rate 1 mU/kg/min) to clamp PG at the fasting value of -3h (9.5 _+ 0.5 mmol/l). Results. At low insulin, MF suppressed hepatic glucose production (HGO) by 45% in step 1 (plasma insulin 93 _+ 14 pmol/I), by 93% in step 2 (plasma insulin 145 + 29 pmol/1), but had no effects on glucose utilization (GU). At high insulin (660 + 50 pmol/l, step 3), MF increased GU by 13% (from 7.78 -+ 0.65 to 8.89 -+ 0.67 mU/kg/min), a result primarily of 65% increase in G storage. Free fatty acids were more suppressed after MF vs placebo in step 1 (0.44 _+ 0.04 vs 0.54 --- 0.04 mM, p<0.05) and step 2 (0.18 -+ 0.02 vs 0.25 _+ 0.03 mM, p<0.05), whereas they were maximally suppressed in step 3 at similar levels on both occasions. Conclusions. MF enhances the suppressive effects of both hyperglycaemia and hyperinsulinaemia on HGP, and lypolisis, as well as their stimulatory effects on peripheral GU (primarily storage). Since the effects of MF on the liver were more than five times greater than those on the muscle (the major site of GU), it is concluded that MF decreases PG in NIDDM primarily by suppressing HGP rather than by increasing GU.

P455

P456

REVERSIBIUTY OF THE DEFECT IN GLYCOGEN SYNTHASE ACTIVITY IN SKELETAL MUSCLE FROM OBESE TYPE 2 (NON-INSUUN-DEPENDENT) DIABETICS TREATED WITH DIET AND METEORMIN. P. Damsbo, A. Handberg, and H. Beck-Nielsen. Hvidore Hospital, Klampenborg, Denmark

METFORMIN TREATMENT DOES NOT AFFECT AMINO ACID METABOLISM IN TYPE 2 DIABETES. P. Tessari, G. Biolo, D. Bruttomesso, S. Inchiostro, G. Panebianco, C. Fongher, L. Sabadin, M. Vettore, M. Carlini, A. Tiengo. University of Padova, Italy. Biguanides are widely used in the therapy of type 2 diabetes but little is known about the effects of these drugs on amino acid metabolism. Therefore, we have studied phenylalanine turnover and oxidation in 6 controls and in I0 diabetic patients before and following one-month treatment w i t h metformin (850 mg b.i.d.), both in the posl-absorptive state and during a continuous oral administration of a synthetic mixed meal. A d u a l isotope technique was employed, i.e. L-12,6-3HI -phenylalanine was infused i.v., L-II-14CI -phenylalanine was administered with the meal (~Ii Kcal/kg). Absorption of dietary phenylalanine was virtually I0()% in both studies. Before metformin, post-absorptive plasma phenylalanine concentration (56_+4 vmol/l) and rate of appearance (Ra) (0.74+-0.10 unol/kg.min) were comparable to normal values (44 +-4 ~mol/l and 0.73~0.05 ~mol/kg.min). Post-meal total phenyla~anine Ra (0.77 +-0.10 u mol/kg.min), endogenous Ra (0.61 +-0.09 umol/kg.min), dietary phenylalanine oxidation (0.098+-0.010 ~mol/kg.min) and initial splanchnic uptake (iSU)(48+-3%) were comparable to controls (0.80+_0.07 ~mol/kg.min; 0.65_+0.06 ~nol/kg.min; 0.085+-0.009 umol/kg.min; 58+-4%, respectively). After metformin, post-absorptive (144 *_22 mg/dl) and post-meal plasma glucose (166+-21 ~ng/dl) were improved (p<0.01) with respect to pre-treatment values (167_+17 and 219+-21 mg/dl, respectively). Post-absorptive phenylalanine concentration (53 +-4 ~mol/1) and Ra (0.73+-0.09 U~nol/kg.min), post-meal total Ra (0.77_+ 0.09 ~mol/kg.min), endogenous Ra (0.57 +-0.02 umol/kg.min), dietary phenylalanine oxidation (0.082+-0.009 ~mol/kg.min), and iSU (54+4%) were comparable to pre-treatment. Therefore, our data show that, at ]east on the basis of phenylaIanine metabolism, metformin therapy is without deleterious effects on amino acid metabolism.

It was our aim to study if the defect in insulin stimulated Glycogen Synthase(GS) activity in type 2 diabetics, is secondary to hyperglycemia and reversibile to diet and metformin treatment. In a placebo controlled, double-blind, randomized trial we studied the effect of 3 months' treatment on GS activity in skeletal muscle from obese patients with type 2 diabetes.Nine received metformin BMI: 32-+1 kg/m2; HbA,c: 9.5-+0.6%; fBG: 10.8-+1.2 mM. Nine received placebo: BM1:32-+2 kg/m 2, HbA~c: 9.9+1.3%; fBG: 12.7-+1.4 mM. All had a 6000 KJ diet. Euglycemic hyperinsulinemic clamp was performed before and after treatment. Muscle biopsies were taken from m. vastus lateralis at basal, 20 and 80 mU/m2.min insulin level. GS was assayed(0.0 and 10 mM G6P) to calculate fractional velocity % (FV). Results: In both groups BMI and HbA,c was reduced equally and fBG was reduced to 8.1 mM. Only in the metformin treated group insulin was able to stimulate GS FV after 3 months: Metformin before: 4.2+_1.2%, 5.2_+1.5%, 6.2_+1.3% (NS); after : 3.3-+0.4%, 6.0-+ 1.4%, 10.1-+3.0% (p<0.05). Placebo before: 6.4-+ 1.9%, 6.3-+ 2.1%, 9.3-+ 2.4% (NS); after: 3.1 _+0.7%,3.8-+ 0.8%, 7.8-+2.2% (NS). Conclusion: The defect in GS activity in skeletal muscle from obese type 2 (non-insulin-dependent) diabetics, is not secondary to hyperglycemia, and metformin seems to improve insulin sensitivity of this enzyme specifically.

A127

P458

P457 METFORMIN IMPROVES NORMOGLYCEMIC INSULIN

INSULIN RESISTANT

SENSITIVITY INDIVIDUALS

IN

E . I . M . W i d 4 n . J.G. E r i k s s o n , L.C. G r o o p . F o u r t h Department of Medicine, Helsinki University Hospital, Helsinki, Finland. Can metformin (MF) i m p r o v e i n s u l i n s e n s i t i v i t y in first degree relatives (REL) of t y p e 2 diabetic patients? To address this question MF and placebo (PL) w e r e in a r a n d o m i z e d manner g i v e n t o 9 i n s u l i n r e s i s t a n t R E L (age 50 • 3 yrs, BMI 26.2 • 0.9, insulin 8 • 2 ~U/ml). Glucose and lipid metabolism were measured before and after treatment with a +80 ~U/ml euglycemic c l a m p in c o m b i n a t i o n with indirect calorimetry and infusion of [3-3H]glucose. Isotopic glycolysis was assessed by measuring a p p e a r a n c e of t r i t i a t e d w a t e r . P L or 500 m g M F were given twice the day before the clamp and o n c e o n e h o u r b e f o r e t h e clamp. F a s t i n g p l a s m a g l u c o s e d i d n o t d i f f e r b e t w e e n P L a n d M F (5.1 • 0.2 vs 5.3 • 0.i mmol/l). Basal glucose oxidation (GOXB) rates were similar after PL and MF (1.31 • 0.17 vs 1.26 • 0.13 mg/kg.min). Isotopic glycolysis was similar a f t e r P L a n d M F a n d a c c o u n t e d f o r a b o u t 75% of GOXB. IG c o r r e l a t e d with GOXB (r=0.91;p<0.01) after PL but not after MF (r=0.18;p=NS). MF resulted in a 23% increase in insulin stimulated glucose metabolism (4.78 • 0.52 v s 3.88 • 0.28 m g / k g . m i n ; p < 0 . 0 5 , v s PL), w h i c h w a s predominantly due to an increase in nonoxidative glucose metabolism (2.76 • 0.48 v s 1.57 • 0.23 m g / k g . m i n ; p < 0 . 0 1 ) . CONCLUSIONS: MF improves insulin sensitivity in insulin resistant normoglycemic individuals, mainly by enhancing non-oxidative glucose metabolism. MF also seems to interact with the contribution of IG to GOXB.

METFORMIN DOES NOT IMPROVE INSULIN SENSITIVITY IN NONDIABETIC OBESE SUBJECTS X. DEBUSSCH~ H. PUY, S. FENDRI, J.M. MARCELLI, S. ARLOT, J.D. LALAU and J. QUICHAUD, Service d'Endocrinologie, HSpital Sud, Amiens, France. Metformin improves peripheral insulin sensitivity in type 2 (non-insulin-dependent) diabetes, but its action on insulin resistance in non diabetic obese subjects has not yet been defined. Peripheral insulin sensitivity has been assessed using a 3-step hyperinsulinaemic (40,100,350 mU/m2/min) euglycaemic clamp in ~0 non-diabetic obese patients (BMI : 29.3 3.5 kg/m--), before and after a month's metformin treatment (1700 mg/d), either on a weight-maintaining (n = 5) or on a weight-reducing diet (n = 5 ; 6.9 + 1.0 Z weight-loss). Before treatment, maximal rat~ of gluc?se disappearance (Rd max) was 9.7 ~ 0.8 mg.kg- . min--, and insulinaemia for halfima~imal glucose utilisation (Kd) was 172 + 21 ~U.ml . Both these parameters are frankly impaired e o ~ a r e d # o S non-obese controls (Rd max 15.5 ~ 0.6 mg.kg .min ; Kd 60 + 13 ~U.ml- ), but are much less impaired than in 13 obese (BMI 3,4.4 +,2.4) type 2 diabetics (Rd max 6.0 + 0.4 mg.kg-• -~ ; Kd 226 + 22 ~U.ml-l). Under metformin treatment, insulin sensitivity improves in weightlos~ng subjects (Rd max i0.0 ~ 1.0 vs 9.0 j 1.0 _~. kg -.min ~ ; Kd 121 + 19 vs 175 + 30 ~U.ml_~). In contrast, Rd max (10.4 + 1.2 vs 10.7 ~ 1.3 mg2~g min--) and Kd (175 ~ 38 vs 167 + 32 ~U.ml -) remain unchanged using metformin without attempting weightloss. In conclusion, metformin in weight-stable obese patients, has no effect on peripheral insulin resistance as assessed by this hyperinsulinaemic euglycaemic clamp technique.

PS 10 Management of Diabetes P459

P460

AN INTEGRATED CCMPUTERISED SYSTEM FOR DIABETES MANAGEMEN~ M.J. Caden, A.G. Shannon, S. Colagiuri and P. Gallagher University of Technology Sydney, Prince of Wales Hospital Randwick, TCG Systems Automation Marketing Chippendale.

TELEMATIC EXPERT SYSTEM DIABETO : EVALUATION ON 100 DIABETICS.

A computerised system comprising an intelligent handheld computer, comprehensive dietary information and a barcode scanner allows quick, easy and precise coIlection of the information required for the management of diabetes. In addition to food intake, exercise pattern, episodes of hypoglycaemia, insulin dosage and periods of illness, blood glucose data may be collected by connecting the handheld unit to a standard commercial glucometer. The data stored in the handheld unit can be transferred to the physician's computer prior to the patient's visit. A preliminary scan of the data may indicate warning signals for the patient or doctor. It is also possible to transmit treatment advice at this stage - an advantage for patients in remote areas. Software at the practice can generate a wide range of reports including dietary analysis, blood sugar levels and relevant correlations. Availability of these reports before a patient visit makes for an informed and efficient clinical session. Long term storage of data allow a patient's progress with various management strategies to be monitored. Results of trials indicate that this system provides an easy and effective way of evaluating and implementing diabetes management schemes.

M.C. TURNIN*, R. BEDDOK*, J. CLOTFES*, R. ABADIE*, J.C. BUISSON**, C. SOULE-DUPUY**, F. BAYARD* and J.P.

TAUBER*.*Service d'Endocrinologie-Diab6tologie, CHU Rangueil, 31054 Toulouse Cedex, France.**Institut de Recherche en Informatique de Toulouse, 31400 Toulouse, France. DIABETO is a computer assisted educational system on dietetics based on free access through minitel, the french public videotex network. It contributes to allow diabetics to monitor their diet themselves and to balance their meals with personalized counselling. The system has been evaluated on a 100 patients sample, randomized and split into two groups. For six months the first group was using the system and the other kept as control. DIABETO led to a significant improvement of the knowledge of dietetics in the f'trst group (p<0.0005). DIABETO allowed an improvement of the dietary habits: decrease of calorie intake on patients initially overeating (p<0.05), increase of glucids in diet from 39.7+3.7% to 42.9+5.0% on patients eating less than 45% of glucids initially, decrease of lipids in diet from 41.94_-4.7% to 37.4_+5.7% on patients eating more than 35% of lipids initially (p<0.0005). For the next six months the control group was using the system, additionally to similar improvements shown on the first group it was possible to see a significant improvement of glycosylated hemoglobin from 11.0+2.9% to 9.9+2.6% (p<0.005) and fructosamin from 5.00-20.91% to 4.57_+0.92% (p<0.001). DIABETO appears to be an effective therapeutic tool in the control of metabolic diseases.

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P462

QUALITY

OF

BLOOD

GLUCOSE

SELF

MONITORING

IN

Karinganamattam,

L,

ELDERLY INSULIN TREATED PATIENTS, D,

Hemmann,

Heinemann

I.

and

bl0hlhauser,

IV], Berger,

J.

Heinrich-Heine

University

of

DOsseldorf, FRB, L i t t l e information is available on the quality of blood glucose self monitoring (BQSfvl] in elderly patients, We investigated the quality

of BGSlVI in

131 patients older than 60 years

[mean+SD age 67+5 years, duration of diabetes lq+8 years) consecutively admitted for participation at a structured 5-day inpatient treatment and teaching programme for insulin treated elderly

patients,

Relative

differences

glucose

measurements

laboratory

blood

day

the programme

1 of

introduction

into

BGSM

(after using

between patient were

theoretical the

visual

assessed

and at

and practical test

strips

H~moglukotest 20-B00 R) and on day q [after 3 days of practical training of BGSM], On day 1, the mean relative difference between patient and laboratory measurements was 12% and on clay q

g% [p<0,05), The results of a subgroup of 20 patients

who preferred to use reflectance meters were ]8% and 20% [NS),

respectively,

This

study shows that

elderly

patients

can achieve a high quality of blood glucose self monitoring using visual

test strips,

do not achieve better

Patients using reflectance

meters

results than those using visual test

strips.

KNOWLEDGE ON DIABETES AND PERFORMANCES AMONG HEALTH PROFESSIONALS OF NONDIABETOLOGICAL DEPARTMENTS

A. Piaggesi, L. Bini, E. Castro Lopez, R. Miccoli, O. Giampietro, and R. Navalesi. Cattedra di Malattie del Metabolismo, Istituto di Cfinica Medica 1I, University of Pisa, Italy. We tested the level of knowledge on diabetes and professional skills in a group of 32 non-diabetological (Neurology, Surgery, Otoiatric, Gynecology, Dermato-logy, Oculistic, Ortopaedics) health care professionals of the Policlinic of Pisa on recognition and treatment of hypoglycemias (HYP), storage, mixing and administration of insulin (INS), blood-glucose sticks monitoring (BGM), prevention and treatment of diabetic foot (DF). The evaluation was carried out through a multiple-choice questionnaire and the observation of the performance of subjects, by means of pre-defined observation schedules, during preparation and administration of a blend of rapid-intermediate action insulins, the execution of blood-glucose sticks and the management of 9simulated hypoglycemia. As to HYP, 81.25% of subjects gave no correct definition, 85.38% did not know asymptomatic hypoglycemias, 96.87% answered no exaustively as to treatment of hypoglycemias. For INS, 65.62% did not know: the standards for a correct storage of insulin, 84.37% the difference between "clear" and "opaque" insulins, 93.75% the technique for a correct injection of insulin; 87.5% neglected the interval between administration of rapid insulin and meals. For DF, 75.43% admitted that the foot of diabetics was infrequently controlled, 53.15% did not know the rules for a correct hygiene of feet. Assessment of performance of professionals through schedules, evidentiated incorrect execution of sticks (78.84% dropped no sufficient blood on reactive pad, 64.31% did not respect the timing, 82.66% did not give a precise result) and administration of insulin (80.2% mixed and 92.43% injected improperly). Due to inadequate specific preparations, educational interventions are needed for non-diabetological health professionals, shared by themselves (93.75% wished to partecipate to a riqualification course on diabetes).

P463

P464

I N D I C A I I O N 5 DF PSYCItOLOGICAL SIRESS AS A RISK FACTOR FOR IYPE 1 D I A B E I E S IN CttILDHOOD B.ft~ggl6f, L.B[om, G.LSnnberg, 8.Sahlin, G.Dahlquist. The D e p a r t m e n t s of Child Psychiatry and EpidemJo]ogy, Universily o r Ume~, Ume~, Sweden. This study is p a r t o f a n a t i o n w i d e case r e f e r e n t study c a r r i e d out in Sweden September 1985 to August 1986. A l l r e c e n t onset type 1 ( i n s u l i n - d e p e n d e n t ) d i a b e t i c cases aged 0-14 years in Sweden were i n v i t e d to p a r t i c i p a t e in the study t o g e t h e r w i t h age-, sex- and g e o g r a p h i c a l l y matched r e f e r e n t s . L i f e events the l a s t year b e f o r e the c l i n i c a l onset o f type 1 diabetes were recorded. A l l t o g e t h e r 338 cases and 528 r e f e r e n t s answered a q u e s t i o n n a i r e . When using d i f f e r e n t q u a l i t a t i v e measures on the r e p o r t e d events t h e r e was a tendency (p=0.08) to an increase in the frequency o f severe ] i f e events among the cases. When looking s p e c i f i c a l l y at events r e l a t e d to actual or t h r e a t e n e d losses w i t h i n the f a m i l y t h a t may affect children differently in d i f f e r e n t age groups, a s i g n i f i c a n t l y higher frequency o f such events were r e corded among cases 5-9 y e a r s . When analysing such events as a r i s k f a c t o r f o r type I d i a b e t e s the r e l a t i v e r i s k was 1.82 (95% c . l . 1.09, 3 . 0 3 ) . I t is concluded t h a t s t r e s s f u l l i f e events r e l a t e d to actual or threatened losses w i t h i n the f a m i l y occuring in the v u l n e r a b l e age group 5-9 years are a s s o c i a t e d w i t h the onset o f c h i l d hood type I d i a b e t e s .

IMPACT OF PUBERTAL ALTERATIONS AND PSYCHOSOCIAL FACTORS IN TYPE 1 DIABETES. N.S. Fineberg, A.W. Helmy, S.E. Fineberg, R.G. Gibson, and D. Orr, and M. Golden, Indiana University School of Medicine, Indianapolis, IN Pubertal insulin insensitivity may contribute to metabolic instability in adolescence. Ten intrapubertal and i0 postpubertal individuals were selected on basis of their metabolic control over 6-9 months (good vs. poor HbAIJ9.6% or 210.3%, 12-20 yrs, ii males and 9 females). A 24 hour glycemic profile, noctural and fasting hormones studies, and psychosecial data were obtained. After overnight euglycemia, a 2 hour hyperinsulinemic euglycemic glucose clamp (40 mU/min/m 2) was performed. Premeal and overnight fasting glucose correlated with HbA 1 (r=.54 and .64, p<.02), and were higher in the poor control groups, but didn't differ by pubertal status, Body image was better within puberty (p=.002), and in those with good control (p=.0Ol). A better body image correlated with HbA 1 and age (r=-.60 and -.56, both p<.01). Family functioning was better in postpubertal patients (p=.035). Insulin dosage and insulin sensitivity determined by clamp didn't differ between intra or postpubertal groups or by glycemic control. Quality of metabolic control in adolescence is associated with psychosocial factors rather than measures of insulin sensitivity.

A129 P465

P466

~ 1 DTABWrI~ ~ YEARS OF THE DI.q;R/Lq~. RRI.AI'IOBS wrrH T]}~ EV{~JII"IO~ ABD HETABOLIC (I]NTBEr A. Godz~, J . ~ 1 o , L. l A ~ r o , JN. G(i~ale~z-Clel~ute, J. Guareh, N. Riesc~, M. Valdes ~ E. Vilardell.

QUALITY OF LIFE IN TYPE I INSULIN-DEPENDENT DIABETIC PATIENTS M. Costea, C. Ionescu-Tirgoviste, I. Mincu, Clinic of Diabetes, Nutrition and Metabolic Diseases, Bucharest, Romania

FSY(H]E~GICAL I~,~5"T (IF

~ o c r i n o l o g y arid Diabetes, @ H o s p i t a l Clinic. B a r c e l o n a . ~ A I N .

AIMS: To a s s e s s p s y e h i a t r i o m o r b i l i t y ,

Psychiatry

attitudes

Unit.

towards

the disease and their relation with metabolic control, 69 type 1 diabetic patients were evaluated (age 25.3+_6.9 y.(17-48), man/woman 58.0/42.0%, evolution time 25~_16 months (0.1-72), HbAlc 7.3~_1.9%). METHODS." I0 analogic scales (referred to attitudes towards diabetes) and 4 standard psychometric tests of psychopathology and personality were administered: Beck Depression Inventory (BDI), General Health Questionnaire (GHQ), State Trait Anxiety Inventory and Eysenck Personality Questionnaire (STAI), all validated in our population. ~ . " 20.9% patients showed psychiatric morbility (GHQ>7) (14% in general population). This was related with time evolution (r=-0.21, p
This study aims to evaluate the level of psychosocia] adaptation of type l diabetic patients related to the intensity of professional, familial and social stress. Two groups were studied: A, included 250 type l diabetics ( l l 8 M, 132 F, mean age: ~ SD 37~15 yrs., duration of diabetes: 12~ 5 yrs.); and B included lO0 healthy subjects (43 M, 57 F, mean age: 35 ~ 12 yrs.). Methodological procedure u t i l i z e a standardized questionnaire with 28 items adapted according to PULSESand AFFLECK scales. No variation were noted in normals regarding the type of stress, and the degree of intensity. The most significant statistical correlations were found between the level of education and the occurence of psychosocial stress: rprof.= -.884; rfam. = -.981; rsoc.=-.7Ol; the negative value of r shows a reverse-proportional dependency of the correlated variables. The high rate of psychosocial inadaptation in diabetics versus nondiabetics is determined by: professional stress (3],2% vs 12%; p
P467

P468

INFLUENCE OF FUNCTIONAL INSULIN SUBSTITUTION ON HEALTH LOCUS OF CONTROL - A CONTROLLED STUDY H.Grillmayr, K.Howorka, K.Nelson, C.Schlusche and H.Thoma, Institute for Biomedical Engineering & Physics, U n i v e r s i t y of Vienna, Vienna, Austria. Training in functional, n e a r - n o r m o g l y c e m i c insulin substitution is a clearly defined education program differentiating basal, prandial and c o r r e c t i o n a l d o s a g e s (with m u l t i p l e injections or continuous delivery) and emphasizing patient self-responsibility. This study i n v e s t i g a t e d the effect of such training on p e r c e i v e d locus of control of diabetes and related h e a l t h b e l i e f s in 26 Type 1 (insulindependent) diabetic p a t i e n t s (x+SD - age: 34• d i a b e t e s duration: 15• yrs, HbAlc: 6.5• treated w i t h m u l t i p l e daily injections after a general diabetes education programme. Questionnaires developed by Bradley and c o l l e a g u e s were a d m i n i s t e r e d to the i n t e r v e n t i o n group (n:16) before and after training in functional insulin s u b s t i t u t i o n and to a control group (n=10) after the general diabetes e d u c a t i o n and 4 weeks later. R e s u l t s (x• of health locus of control scales showed s i g n i f i c a n t changes in the i n t e r v e n t i o n group on Internality (before: 4.5+0.6 / after: 5.0• p<0.05;) and Total P e r s o n a l Score: 3.9• / 4.3• p<0.03. Other scales Treatment: 2.7• / 2.6• p<0.7; Externality: 2.3• / 2.0• p<0.6; Chance: 2.0• / 1.4• p<0.1; Personal Control: 3.9• / 4.4• p<0.1; Medical Control: 2.2• / 2.7• p<0.4; F o r e s e e a b i l i t y : 3.5• / 3.8+1.0 p<0.4; Total M e d i c a l Score: 2.4• / 2.8• p<0.8. - showed no s i g n i f i c a n t changes, nor were changes seen in the control group. A training program in functional insulin s u b s t i t u t i o n improves perceived a b i l i t y to control diabetes.

S E L F - A S S E S S E D C O M P L I A N C E AND I N S U L I N - D E P E N D E N T DIABETES MELLITUS. Hanestad, B. R. H a u k e l a n d Hospital, Bergen, Norway. The aim of the study was to a n a l y s e the connection b e t w e e n s e l f - a s s e s s e d c o m p l i a n c e and sex, age, duration, p e r c e i v e d impact of diabetes and m e t a b o l i c control (HbAI). 247 persons who c o n s e c u t i v e l y v i s i t e d a Diabetic Clinic p a r t i c i p a t e d in the study. The subjects s e l f - r a t e d the impact of diabetes in daily life and c o m p l i a n c e with i n j e c t i o n treatment, s e l f - m o n i t o r i n g of b l o o d - g l u c o s e and urine, foot-care, diet, weight regulation, exercise, smoking and a l c o h o l habits. Most d i f f i c u l t to comply with were smoking habits (27.0%), weight (23.7%), a c t i v i t y (22.3%) and diet (i9.5%). C o m p l i a n c e score was not related to sex (p = .127) or duration of diabetes (p = .188). The age group of 50-60 years has s i g n i f i c a n t l y h i g h e r c o m p l i a n c e score c o m p a r e d to the lower age groups. No s i g n i f i c a n t difference in c o m p l i a n c e score was found b e t w e e n persons in poor and good m e t a b o l i c control (p = .191). Persons who p e r c e i v e d great impact of diabetes in daily life have a lower compliance score than persons who report lower impact of diabetes in daily life (p = .079). These results indicate: i) Diabetic r e g i m e n r e l a t e d to lifestyle is most d i f f i c u l t to comply with, 2) sex, m e t a b o l i c control, duration and impact of diabetes is not s i g n i f i c a n t l y c o n n e c t e d to c o m p l i a n c e score and 3) older people report h i g h e r score than y o u n g e r people.

A130

P470

P469

INSULIN PATIENTS

Assessment of Well-Being in Diabetic Patients P.Y.Benhamou *, F.Borgel **, B.Pasquier **, J.L.Bosson * and S.Halimi *. *Service Endocrinologie DiabEtologie, ** Unite de Rdadaptation Neurologique, C H R U , Grenoble, France. The need for assessment of quality of life and psychosocial aspects in diabetes care is emphasised.We designed a prospective study,aiming to evaluate the validity of quantitative scales.The study concerned 111 consecutive admitted diabetic patients .The Barthel index (BI, N = 100) evaluated functional abilities in daily living and the Frenchay index (FI, N = 60) social activities. Twelve analogic visual scales (range 0 - 100 ram) were used for the assessment of health care burden (HCB) and quality of life (QL). 52 men and 59 women were included, mean age 51 + 17 years, mean duration of disease 12 _+9 years, with type 1 (n=56) or type 2 (n=55) diabetes, 73 were insulin - treated.BI was 97.4 _+7.5 and FI was 44.7 _+ 7.5. Anova showed signicant differences between type 1 and type 2 diabetics for HCB ( 58 + 32 vs 44 + 35, p = 0.04) and time-dependency (54 + 35 vs 18 _+ 33, p = 0.0001), and between insulin-treated and oral agent-treated patients for HCB (59 + 32 vs 41 + 33, p = 0.01) and medicalisation (47 + 35 vs 25 _+ 33, p = 0.01). HCB was analysed by stepwise multiple regression among 5 selections of patients: group A (all patients), group B (type 1), group C (all type 2), group D (insulin-treated type 2), group E (oral agenttreated type 2). The most significant factors were respectively: diet,drug-taking,medicalisation (A); fear for complications, injection, diet, time-dependency (B); diet, drug-taking (C); fear for complications, injection, diet (D); diet, medicalisation (E). QL in group B correlated with time-dependency (r = - 0.31, p = 0.02), as time-dependency with age (r = - 0.41, p = 0.0001). These simple tools could be useful for evaluation of diabetes health care delivery and educational programs.

IMPACT TO DIABETIC

H. T i n d a l l 1, 5. P a y n t e [ 2 and M. Henderson 2. Departments of Medicine and C l i n i c a l Psychology2~ The General I n f i r m a r ' y , Leeds. U.K. Poor` d i a b e t i c control because of f a i l u r e to adhere to diet is a common problem in type 2 d i a b e t i c p a t i e n t s . The aim of the study was to i n v e s t i g a t e whether` i n s u l i n t h e r a p y gi ven once d a i l y could improve contr'ol and whether` it affected ' q u a l i t y of l i f e ' (QOL). With ethics committee a p p r o v a l 30 d i e t a r y noncompliant p a t i e n t s median ( r a n g e ) age 69 (50-79) y e a r s were r andoml y a l l o c a t e d to receive f o r 6 months either` Humulin M3 i n s u l i n once d a l l y or cont i nu e with oral hypoglycaemlc therapy (controls). Blood glucose series (6 p o i n t . BSS) were performed at t o r day 5 and 6 months (t6). Blood samples were taken f o r HbA 1 ( i s o e l e c t r i c f o c u s i n g ) and p a t i e n t s completed QOL ques~:ionnaires at t ~ 3 and 6 months. Statistical a n a l y s i s was perforr~ by 2-way a n a l y s i s of v a r i a n c e with r e p l i c a t i o n s . At 6 months~ BSS (all points p <0.05) and HbAlc (12.1 [ 9 . 9 - 1 6 . 2 ] ~ t vs 10.6 [6.7-13.4] ~ t61 p < 0.01) s i g n i f i c a n t l y i~ in the i n s u l i n - t r , e a t e d group~ but not in the c o n t r o l s . I n s u l i n had a p o s i t i v e effect on 5 of 6 subscales of mood which were measured; p a t i e n t s became s i g n i f i cant ly more c o n f i d e n t (p < 0 . 0 1 ) , more energetic ( p < 0.01), more elated ( p < 0.05)~ clearheaded (p < 0.05) a n d less depressed (p < 0.05) on insulin, whereas controls remained u n c h a n g e d . This study shows that diabetic control a n d QOL can be improved with insulin given once daily to older patients not adhering to diet. Further work is needed to establish whether the improved QOL is secondary to insulin therapy~ better diabetic control or both.

P472

P471 THE

IMPROVES QUALITY OF LIFE IN OLDER WITH POORLY-CONTROLLED TYPE 2 DIABETES

OF A M~ILTI-DISCIPLINARY FOOT PROBLEM~

APPROACH

FI T h o m s o n , EA Masson, AVeves, DES Fernando, IA Gem and AIM Boulton. Manchester Royal Infimary, Manchester, UK. The mnagement of diabetic foot ulceration requires the coordination of m a n y services. We report our experience of a multidiscipllnary approach incorporating physicians, surgeons, diabetic foot nurses, chiropodists and an orthotist. Since February 1987, 2 6 0 r e f e r r a l s h a v e b e e n seen; m e a n a g e 61 years (66% male). 77% had either neuropathy or a c o m b i n a t i o n of n e u r o p a t h y and peripheral vascular disease. 153 (59%) presented with foot ulceration. The commonest site os u l c e r a t i o n was the toes suggesting inappropriate footwear as an important aetiolosical factor. 8 1 % of u l c e r s were treated without formal surgery. Local surgery was required in 8% and major amputation in II%. No major amputations were required In p a t i e n t s with purely neuropathic ulcers. O v e r h a l f t h e a t t e n d e r s identified as being at risk of foot ulceratlon were supplied with either extra depth or specially made shoes. Foot ulceration recurred in only 12% of cases, less than in previously reported series. The clinic represents a reor~anisation of e x i s t i n g r e s o u r c e s , without additional expenditure. Major amputation, costing approximately Z7OO0 per case, was reduced b y 39% a f t e r establishment of the clinic. An effective redistribution of resources may improve the identification and education of 'at risk' patients end reduce treatable foot disorders and amputation.

EFFECTS OF LENGTH OF INITIAL HOSPITALIZATION OF C H I L D R E N WITH NEWLY DIAGNOSED DIABETES MELLITUS T.Simell, E.A.Kaprio, J.M~ienp~i~, A.Hakulinen, K.N~int6-Salonen and O.Simell. Children's Hospital and Cardiorespiratory Re-

search Unit, University of Turku, Turku; Children's Hospital, University of Helsinki, Helsinki; Peijas City Hospital, Vantaa; Aurora City Hospital, Helsinki; Children's Hospital, University of Kuopio, Kuopio, Finland We studied in a randomized clinical trial the effects of length of initial hospitalization of children with newly diagnosed diabetes on: 1) metabolic balance of the child; 2) psychosocial adjustment of the child and family; 3) costs of care; and on 4) personnel's work load. Half of the newly diagnosed diabetic children were hospitalized for one week (study group, N =31), the other half (control group, N =30) for four weeks according to our traditional practice of care. Follow-up was two years. We now report the outcome of the metabolic balance. In both groups, near-normoglycemia was achieved in two months after the diagnosis and was maintained over the study period. At the end of two years, HbA1 values were slightly higher in the study group (means 9.0 vs. 8.7%, n.s.) while H b A l c and fructosamine values were lower (means 7.7 vs. 7.9%, and 2.6 vs. 2.8 mmol/I, respectively, n.s.). In the study group, 12 children had C-peptide values > 0 . 3 nmol/I and 8 children insulin dose --<0.5 units/kg/24 hours while in the control group 6 children had C-peptide values > 0 . 3 nmol/I and 2 children insulin dose < 0 . 5 units/kg/24 hours. We conclude that the length of initial hospitalization has no significant effects on the metabolic balance of children with newly diagnosed diabetes mellitus.

A131

P473

P474

THE INTRODUCTION OF DIABETIC NURSES SAVES MONEY L. Waller, S. Ehnberg and L. Welin, Department of Medicine, ~stra Hospital, S-416 85 Gothenburg, Sweden The organization of specialized diabetic out-patient clinics has probably improved the management of d~abetes in terms of better quality-of-liFe, improved metabolic centre] etc, all of which is difficult to measure. The objective of this study was to find out whether an out-patient clinic a]so saves hospital beds and thus money. There are 203 beds in the Department oF Medicine with a catchment area of 240 000 people. Our First diabetic nurse started in the autumn of 1984 and an out-patient diabetic clinic has been in operation since March 1985. No extra resources besides a diabetic nurse have been added. All hospital records From 1981-1988 For patients with diabetes mellitus as the main diagnosis were retrospectiveiy examined. We found s decrease in elective admissions for adjustment off diabetes treatment during 1985-88, i.e. after the opening of the out-patient clinic. The decrease was on average 336 days/year. The cost saved for these hospital days was 336 x 1609 Skr = 540.624 Skr (= 52.498 s The cost for a diabetic nurse is 198.315 Skr (: 19.256 s Thus a diabetic nurse together with an organized outpatient clinic saves 342.309 Skr (: 33.239 s at our hospital.

SCREENING FOR DIABETES: A SUCCESSFUL AND WORTHWHILE SYSTEM M.J. Davies and J.L. Day Diabetes Centre, Ipswich Hospital, Ipswich, Suffolk

P475 A NATIONWIDE SURVEY OF AMBULATORY TREATMENT OF DIABETICS IN FINLAND Kangas, T.T., National Public Health Institute of Finland, Health Services Research Unit. A nationwide survey on the current mode of treatment of diabetes in Finland was carried out in 1989 using a selfadministered questionnaire given to all diabetics who got their drugs through pharmacies during a seven week period. Altogether 33.205 diabetics were reached, comprising 82% of the estimated maximal group of drug treated diabetics who could have been reached during the time of the survey. Only 3.8% refused to answer the questionnaire and additional 0.3% of the answers were disqualified, - The results presented deal with diabetics aged 35 years and over (N: 18.838), In the age group of 35-64 years (38% of patients) insulin therapy alone was used by 22%, oral therapy alone by 70% and combination therapy by 8% of the patients. 73% attended primary health care centers, 15% specialist-run hospital out-patient departments and 5% occupational health care units. 8% were treated by private practitioners. In the age group 65 years or older insulin therapy alone was used by 12%, oral therapy alone by 81% and combination therapy by 7% of the patients. In the older age group 86% attended primary health care centers, 7% specialist-run hospital out-patient departments and 7% were treated by private practitioners. 69% of younger age group patients and 65% of older age group patients reported to visit their doctor at least 3 times a year. 80% of the patients treated with insulin alone visited their doctor at least 3 times a year. The results comprise the first part of a comprehensive nationwide survey of the delivery and structure of health services for diabetics.

A simple method of screening for asymptomatic diabetes in the community was developed using a postal request with self-testing of urine for glycosuria. A carefully designed instruction letter was posted to all 2984 subjects aged 45-70 years identified by the local Health Service register, from one practice. Known cases were excluded (n = 73, 2.38%). All were requested to test with enclosed Clinistix either once, (after main meal) or twice, (pre- and post-breakfast), record the result and return stamped addressed cards. The return rate was remarkable at 79.25% (n : 2365), with no difference between those requested to perform one or two tests (p = 0.25), and increased with age (r = 0.97). 73 subjects (3%) recorded glycosuria of whom 68 attended for a 75 gm. oral glucose tolerance test. Of these 17 (0.8%) had diabetes and 11 (0.46%) impaired glucose tolerance. Similar tests in 50 randomly selected negative responders revealed no cases of diabetes. The new prevalence of diabetes is 3-7%. Of those with diabetes 45% were asymptomatic. 66% had not attended their General Practitioner in the previous three months. Cost per subject screened was s The high return rate and diagnostic yield of this simple, cheap procedure suggests it is both necessary and worthwhile.

A132

PS 11 Nutrition P477

P476 EVALUATION OF A NEW BAR CODES OUESTIONNAIRE TO PROVIDE INFORMATION ABOUT USUAL INTAKE IN DIABETIC SUBJECTS.

NUTRITIONAL ABILITY AND DIET REALITY IN DIABETIC

M.M. V e n t u r a , Santeusanio U n i v e r s i t ~ di

PATIENTS UNDER INSULIN THERAPY.

To o b t a i n the

V. M o r i c o n i , M.T. S u l p i z i , P.G. F a b i e t t i , F. and P. Brunetti. Ist. Patologia NedLca, Perugia, Italy.

the

accuracy

best of

possible

the

dietary

diet,

the

results

expert

and i n c r e a s e

needs to knew the

h a b i t s of the s u b j e c t . U n F o r t u n a t e l y , assessment 0f individual dietary intake is often l e f t out because of the l e n g t h of time r e q u i r e d . This study was u n d e r t a k e n to e v a l u a t e a new bar codes s e l f - a d m i n i s t e r e d questionnaire dietary

elaborated

by means of

Miles)

compared

1985).

Sixty

33

aged M• SO)

regarding

eating

by means

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a conventional

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femal e;

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diabetic

34.8 ~ 13.8

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asked

subjects

Roblin and M.C. Tumin. French Study Group on Nutritional Education in Diabetes. France. In order to compare the ability of diabetic patients to realise an adapted diet and the real application of this diet, 54 patients (age: 48 + 3 yr; duration of diabetes 14 + 4 yr) from 5 centers, treated with insulin twice

(Willett,

a day, were studied. Patients were under regular supervision in the

(27 male,

center for at least one year, diet prescription (1650 + 53 kCal; 202 + 9 g

33.6 ~ 13.6 provide

(frequency

Meter

J.P. Le Floch, F. B6cot, J.P. Digy, A. Murat, L. Perlemuter, X.

years

information

and amounts

food

of

carbohydrates) including 3 meals and 3 snacks. Centralized HbAlc (HPLC, N=3.5-4.5%) ranged from 5.0 to 8.5 %. Nutritional data were

year.

recorded using a standardized computer software under the supervision

Food

of a dietician and included 1) a 24h diet record (R) of the last weekday;

into energy and nutrients was automatically obtained in approximately fifteen minutes.

2) a 24h simulated (S) ideal diet. Observed results (mean + SE) were

With

the

computerized

consumption whereas,

the

conventional The

results

no s i g n i f i c a n t

bar

codes

computerized saving

model)

method

over

the

the

last

conversion

of

data

conversion

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questionnaire of

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two methods were i d e n t i c a l ; differences.

questionnaire

elaborated

method r e p r e s e n t s

expressed as an indice depending on individually prescribed diet: I(%)=[(observed-prescribed)xl00/prescribed]. Comparisons were

there

were

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by

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diabetics

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the

of

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patterns

of

made with paired t-test. Indices of calorie intakes differed significantly

(R:

16 _+ 3%, S: 6 _+ 3%; p<0.001). Indices did not differ for

carbohydrate (R: 1 + 3%, S: 2 + 3%; NS) and protein (R: 16 + 3%, S: 19 + 4%; NS) intakes, whereas they did for fat intakes (R: 29 + 9%, S" 8 + 1%; p<0.001). Daily distributions of calories differed especially at dinner and 10 p.m. snack (p<0.01). No significant center effect or individual confounding factor was noted. These results suggest that there is a difference between the nutritional ability of diabetic patients and the reality of diet, especially conceming fats.

P478

P479

SIX MONTHS RESULTS OF A WEIGHT REDUCTION PROGRAM IN OBESE DIABETIC PATIENTS. M.C. Blonk, M.A.J.M. Jacobs and R.J. Heine. D e p a r t m e n t of Endocrinology, Free U n i v e r s i t y Hospital, Amsterdam.

FAVOURABLE GLYCEMIC EFFECTS OF A NEW FORMULA DIET IN 30 TYPE II DIABETIC PATIENTS W. St~irme~:, P. Bartramj E. Kramer, H. Kasper, J.

The aim of this study was to investigate the l o n g - t e r m efficacy of an intensive weight reduction p r o g r a m (IP) in obese type 2 (noni n s u l i n - d e p e n d e n t ) diabetic patients. Fourtyfour patients (age: 29 to 70 yr; BMI: 27.4 to 45.2 kg/mi), were randomized to either IP or to a c o n v e n t i o n a l p r o g r a m (CP) consisting of an energy restricted dietary advice only. IP included in addition behavioral m o d i f i c a t i o n and exercise training. The changes in median (range) total body weight (BW) and in percentage fat mass (bioelectrical impedance) after six months were for IP and CP: -2.4 (-8.0 to 0) kg vs -0.9 (-15.4 to 4.9) kg (p<0.10) and -i.i (-1.9 to 4.3)% vs -0.i (-6.5 to 3.7)% (NS), respectively. Mean (SEM) HbAlc values declined similarly for IP and CP: 7.7• to 7.1• and 7.6• to 7.1• Body weight loss correlated with the change in HbAlc: r=0.65, p<0.001. No changes were observed in the w a i s t / h i p ratio, blood pressure, triglyceride levels and in VLDL-, LDL-, IDL- and H D L - c h o l e s t e r o l concentrations. In conclusion, the IF tended to achieve a greater, albeit modest, reduction in body weight than the CP. The observed improvement of glycaemic control c o r r e l a t e d with the achieved weight loss.

We developed a fibre enriched (15 g/l soy polysaccharide) liquid formula diet (A) (53% carbohydrates (CHO), 32% fat, 15% protein). 77% of total CHO consist of physically and acid modificated starch, 23% of fructose. In 30 type II diabetics with different therapeutic regimens we compared the glycemic response of diet A with that of an isoenergetic maltodextrins and saecharose containing liquid diet (B) and a continental breakfast (C). Each test-meal contained 48g CHO and equal amounts of protein and fat, The sequence of the meals was randomized. Over a 4 hour postprandial (p.p.) observation period ingestion of diet A was followed by a markedly flattened p.p. rise in blood

Schrezenmeir'. Departments Universities of Wfirzburg and

of *Mainz.

Internal FRG.

Medicine,

glucose (BG). Maximal rise above f a s t i n g BG(mmol/1) - A: 2.6• B: 4.3+_1.4, C: 4.0+_1.3. (p < 0,001 A v s B;. p < 0,001 A v s C) The a r e a s u n d e r the p.p. BG c u r v e (mmol/lx4h) were also significantly r e d u c e d - A: 251_+229, B: 569+273, C. 506+222 (p < 0,001 A vs B, p < 0,001 A v s C). The d e s c r i b e d d i f f e r e n c e s in p.p. BG were more p r o n o u n c e d in insulin t r e a t e d p a t i e n t s t h a n in diabetics with a d i e t a r y regimen only, or p a t i e n t s with s u l f o n y l u r e a s . Similar r e s u l t s we obtained by evMuation of the c o r r e s p o n d i n g p.p. serum insulin profiles in the g r o u p of non insulin t r e a t e d patients. We conclude t h a t the p r e s e n t e d new formula diet might be helpful to avoid excessive h y p e r g l y c e m i a in the management of diabetic p a t i e n t s receiving an artificial e n t e r a l nutrition.

A133

P480

P481

ALCOHOL WiTH DINNER REDUCES BLOOD GLUCOSE NEXT MORNING IN TYPE 2 DIABETIC PATIENTS S. Tulokas, R. Pelkonen, E Haapa. L Kosldnen and T. Koivula and V.A. KoMsto, Diabetes Center and Univ~oity Hospital, Tampere, l-lelsinki University Hospilal, Heisinld, Finland We studied the effect of alcohol with dinner on the blood glucose and insulin response and fasting blood glucose next morning in 16 type 2 diabetic patients, aged 57+2 yrs, BMI 27-+1 kg/m 2, HbAlo 7.7*-'0.2% (mean-+SEM). The same test meal (30% of daily calories) was given without or with alcohol (1 g/kg) in a randomized order at 7 p.m., 2 days apart. Premeal glucose and insulin concentrations, respectively, were similar before control (8.8-+0.4 mM, 12.8-+1.8 mU/I) and alcohol (8.4-+0.4 mM, 15.0-+1.8 mU/I) meal. One hour after the meal, insulin concentration was higher after alcohol (51-+4 mU/I) than control meal (40-+4 mU/I, p<0.01), whereas serum glucose concentrations were identical (10.4+0.7 mM in both). NeXt morning, fasting blood glucose concentration was lower (7.4-+0.5 mM vs 8.1-+0.5 mM, p<0.02) and serum triglyceride level was higher (2.0-+0.2 mM vs 1.7-+0.2 mM, p<0.01) after alcohol than control meal, respectively, whereas serum insulin concentrations were similar. Conclusions: In type 2 diabetic patients alcohol with dinner 1) augments meal-induced insulin secretion, and 2) reduces fasting blood glucose and increases serum triglyceride concentration next morning. Thus, alcohol with dinner has a hypoglycemic effect next morning in type 2 diabetes.

I N F L U E N C E OF D I E T A R Y C A R B O H Y D R A T E TYPES ON I N S U L I N S E N S I T I V I T Y IN MAN.

B.Kiens, H. Lithell and E.A.Richter. August Krogh Institute, University of Copenhagen, Denmark; and Department of Geriatrics, Uppsala University, Sweden A two-step sequential euglycemic, hyperinsulinemic clamp was used to a s s e s s dietary influence on insulin sensitivity in humans. 8 healthy male subjects consumed a controlled, isocaloric diet for 2x4 weeks with 4 weeks pause in between. In both periods the diet contained 45E~ carbohydrate, 40-43E~ fat and 13-15E~ protein and differed only in type of carbohydrates (mainly complex vs mainly simple). At a plasma insulin concentration of 60mU/l the glucose infusion rate was significantly lower after 4 weeks on the complex diet (7.5 mg/kg/min) than on the simple (8.1 mg/kg/ min) At a plasma concentration of 350 mU/l the glucose infusion rate was 11.7 mg/kg/min on the complex and 13.0 mg/kg/min on the simple diet (p
P482

P483

CAN VARYING CONTENT OF CARBOHYDRATE IN DIET MODIFY INSULIN SECRETION, PERIPHERAL INSULIN SENSITIVITY AND LIPID LEVELS IN OBESE SUBJECTS? M. Nonato, P.M. Piatti, A. Saibene, E. Contrino, S. Costa, F. Zilli, A.E. Pontiroli, G. Pozza. Istituto San Raffaele, Clinica Medica VII, Universita' di Milano, Milano, Italy.

FIBRE SUPPLEMENTATION DOES NOT CHANGE THE GLYCEHIC RESPONSE OF ENTERAL FEEDINGS IN ELDERLY PATIENTS WITH TYPE ]] DIABETES OR IMPAIRED GLUCOSETOLERANCE, M. Vandewoude, I. De Leeuw, F. Coucke and M. Govaerts. Dept. of Endocrinology, Metabolism and Clinical Nutrition, University of Antwerp (UIA), Antwerp, Belgium. Although enteral feedings are used more and more to support the n u t r i t i o n a l status of elderly people and impaired glucose tolerance is common in this population, no data are available about the glycemic responses of this form of c l i n i c a l n u t r i t i o n in this specific population. Theoret i c a l l y the addition of soluble fibre could f l a t t e n the glycemic response. For this reason the glycemic index (as defined by Jenkins) was calculated after performing three tolerance tests on three consecutive days comparing 50 g of glucose in 365 ml water with the same volume containing the same amount of carbohydrates of a reference feeding and a test feeding containing 6.9 g of soluble fibres. Fat and protein content were not d i f f e r e n t in both preparations (resp. ]2.4 and 11.6 g; 13.4 and 12.4 g). Two groups of 6 g e r i a t r i c patients were submitted to this experience: group A with impaired glucose tolerance (80 y SD 5) and group B with non-insulin-dependent diabetes (79 y, SD 6). In group A no difference in glycemic index was disclosed between the feeding with and without soluble fibre (52, SD 16 versus 56, SD 18). In group B no significant differences could be observed in regard of group A or in between the 2 experimental feeding solutions (56, SD 22 versus 65, SD 24). Conclusion: the addition of fibre does not influence the glycemic response of enteral feedings in g e r i a t r i c patients with glucose intolerance.

The aim of this study was to compare the short term effects of two hypocaloric (800 Kcal) diets: high-complex-carbohydrate (60%) -low fat (20%) (group I) and low-complex-carbohydrate {20%)-high fat (60%) (group II) on insulin release, insulin sensitivity (M/I index) and lipid levels in 12 non diabetic obese women (age:40.6+3.0 yrs; BMI 37.5+2.5 kg/m2) matched for age and BMI. A hyperglycaemic (+90 mg/dl) clamp was performed before and after 21 days of diet. The percentage of weight reduction was similar in both groups (5.5+0.7 and 5.5~0.6 %), basal blood glucose and insulin levels, first and second phase insulin release were unchanged after diet in both groups. M/I index and FFA levels were similar before diet in the 2 groups; after diet they were unchanged in group II, and decreased 22% (p<0.02) and 48% (p<0.02) respectively in group I; 3-OH-butyrate levels increased 389% in group I and 161% in group II (p<0.01 vs basal for both groups). Cholesterol and triglyceride levels did not significantly change after both diets. In conclusion, a) a short period of highcarbohydrate diet increases FFA levels and induces insulin insensitivity probably mediated by glucose-fatty acids cycle, b) neither diet modifies insulin release and lipid levels.

A134

P484 EFFECTS OF D I E T A R Y N-3 FATTY ACIDS ON G L U C O S E H O M E O S T A S I S IN H Y P E R T R I G L Y C E R I D E M I A A . V r ~ n a and L. Kazdov~, I n s t i t u t e for C l i n i c a l and E x p e r i m e n t a l Medicine, Prague, C z e c h o s l o v a k i a H y p e r t r i g l y c e r i d e m i a (HTG) is f r e q u e n t l y associated w i t h impaired g l u c o s e t o l e r a n c e (GT) and insulin resistance. We t h e r e f o r e studied the effects of fish oil (FO) rich in n-3 fatty acids (FA), a h y p o t r i g l y c e r i d e m i c preparation, on glucose tolerance, insulinemia, insulin s e c r e t i o n and insulin action in an animal m o d e l of HTG; in rats made h y p e r t r i g l y c e r i d e m i c b ~ feeding a high sucrose (70 cal%) diet. The diet was fed for 2-3 weeks; one group of rats was given i m l / r a t / d a y of olive oil (controls), and the second the same amount of FO (Activepa 30 TG, M a r t e n s a.s., N o r way). As expected, F0 a d m i n i s t r a t i o n m a r k e d l y d e p r e s s e d t r i g l y c e r i d e m i a . S i m u l t a n e o u s l y , we found m i l d i m p r o v e m e n t of GT m e a s u r e d after oral glucose load (3g/kg) in the F O - t r e a t ~ d group, s u r p r i s i n g l y showing a m i l d e r increase in p o s t load i n s u l i n e m i a as c o m p a r e d w i t h olive o i l - t r e a ted controls (0.39 ~ 0.03 and 0.51 ~ 0.05 nmol/l, r e s p e c t i v e l y , p < 0.05, 60 mins after the load). Insulin release from isolated islets w~s also lowere~ by fish oil a d m i n i s t r a t i o n (3.6 ~ 0.I and 5.7 ~ 0.8 p m o l / 1 0 islets/60 mins, r e s p e c t i v e l y , p ~ 0.05). On the other hand, i n s u l i n action on adipose tissue (synthesis of lipids and glycogen) was m a r k e d l y i n c r e a s e d by d i e t a r y FO (150-200 % of controls). It can be c o n c l u d e d that d i e t a r y n-3 FA may affect g l u c o s e h o m e o s t a s i s by c o m p l e x m e c h a n i s m s i n v o l v i n g changes in insulin s e c r e t i o n and action as well as changes in lipid fuel availability.

PS 12 Clinical Diabetes P485

P486

Dehydroepiandrosterone reduces fasting plasma glucose but not prandial glucose profile or peripheral insulin resistance in type II diabetes

ABNORMALITIES OF CARBOHYDRATEMETABOLISM IN CYSTIC FIBROSIS: PATHOGENETIC MECHANISMS. D.Cucinotta,F.De Luca,A.Costantino,A.Di Benedetto,T.Arrigo, E.Di C e s a r e , L . F r i t t i t t a , S . C o n t i Nibali and G.Squadrito. I n s t i t u t e of Internal Medicine,University of Messina,ltaly. To f u r t h e r elucidate the pathogenesis of glucose metabolism abnormalities,often observed in c y s t i c f i b r o s i s (CF),in a selected population of 30 CF patients (16 males aged 13.3~ 4.9 years,BMl 17.6~2.2),with normal glucose tolerance acco~ ding to WHO c r i t e r i a , w e i n v e s t i g a t e d : B - c e l l function (insul i n and C-peptide responses to both oral and i . v . glucose), i n s u l i n s e n s i t i v i t y (euglycaemic clamp) and receptors ( c i r c u l a t i n g monocytes),specific autoimmunity ( i s l e t c e l l a n t i bodies,ICA).When compared to a control group (16 healthy su bjects aged 10.5+1.5 years,BMl 18+2.3)during OGTT CF patien ts showed not d i f f e r e n t i n s u l i n area (124+57 vs 104+48 ~U/ ml)but i n s u l i n peak was always delayed (between 90 and 120 min);moreover i n s u l i n response to i . v . g l u c o s e was blunted (0-I0 min area=137+32 vs 308+82 ~U/ml,2p
J.C. Levy, M. Burnett, S. Manley and R.C. Turner. Diabetes Research Laboratories, Radcliffe Infirmary, Oxford, U.K. Dehydroepiandrosterone (DHEA) reduces glycaemia and prevents pancreatic failure by reducing insulin sensitivity in animal models of type II diabetes. We assessed clinical effectiveness and mode of action of DHEA for 10 days (1.6g/day) in 7 obese type II diabetic females (BM1 32.9 _+SD4.9kg.m-2, fasting plasma glucose 11.2_+SD3.1mmolA) in a double-blind placebo-controlled randomised crossover study9 Clinical response was assessedby twenty-four hour glucose, insulin and non-esterified fatty acid (NEFA) profiles, and beta-cell function and insulin sensitivity were assessed by a constant 5mg.kg IBW-tmin -1 glucose infusion, followed by 50 and 900mU/I insulin euglycaemic clamps. Fasting plasma glucose was 1.8_+1.6mmol/I lower on DHEA compared with placebo, p < 0.05. There was no change in fasting plasma insulin: geometric mean 13(SD range 7-24)mU/I and 14(8-25) on placebo and DHEA, respectively, NEFA: 0.64_+0.12 and 0.57+_.0.14mmol/I, two hour post-prandial glucose: 6.2-+1.1 and 6.1_+.1.2mmol/I, or insulin: 18(11-29) and 18(11-31)mU/I, beta-cell function (90minute incremental insulin to incremental glucose ratio during the glucose infusion): 1.2-+l.0mU/mmol and 0.9+_0.9,or total body glucose uptake at either physiological or maximal insulin clamp concentrations9 Plasma HDL on placebo, however, (l.0_+0.2mmol/I) was lower on DHEA (0.75_+0.3mmol/I), p < 0.01. Conclusion: DHEA reduces fasting plasma glucose in obese female type II diabetic subjects, possibly by increasing hepatic insulin sensitivity.

A135 P487

P488

Measurement of Pancreas size in Diabetes Mellitus by Real-time

CELIAC DISEASE IN DIABETIC CHILDREN AND ADOLESCENTS.

Ultrasonography.

C.Johansson,P-O.Elfstrand,N.Sigure,H.Viander and A.Lanner,Departement of Pediatrics J~nk~ping,Sk~vde, Bor~s,Sweden,Departement of Hedical Hicrobiology,]urku, Finland and Phsrmacia Diagnostics,Uppsata,Sweden.

T.Nakayama, I.Ueta, K.Hisatake, K.Yoshida and F. Ohno. 2nd Dept, of Internal Medicine, Koehi Medical School, JAPAN. We performed an ultrasound scan on 25 Type-1 (Insulin
The main aim of this study was to de~ermine a minimum prevalence o f c e l i a c disease among d i a b e t i c ch ild re n and adolescents.442 out o f 459 p a t i e n t s with Type 1 diabetes m e l l i t u a st three p e d i a t r i c departements accepted to p a r t i c i p a t e in the i n v e s t i g a t i o n and were tested f o r g l i s d i n IgA/IgG-antibodies with a commercial k i t (Pharmacis Gluten IgA E I A ) . L a t e r a l l sara were also tested f o r r e t i c u l i n IgA/IgG-antibodtes.22 p a t i e n t s had a g l i a d i n I g A - l e v e l o f 25 AU or h i g h e r . ] o f these were already known as having c e l i a c disease and 1 was not w i l l i n g to be biopsied.18 p a t i e n t s underwent j e j u n a l biopsy.B o f these had t o t a l and 2 had s u b t o t a l v i l l o u s atrophy. These p a t i e n t s were judged as having c e l i a c disease and were recommended gluten free d i e t . W i t h i n two months g t i a d i n I g A - t e v e l s went back to normal in p a t i e n t s adhering to the d i e t . 5 p a t i e n t s has so far gone through a second j e j u n a l biopsy with normal h i s t o l o g y in a l l . The ten newly diagnosed p a t i e n t s with c e l i a c disease plus 6 p a t i e n t s already known give a minimum prevalence o f c e l i a c disease in d i a b e t i c children end adolescents of 3,6 %.

correlation was found between Q value, serum immunoreactive trypsin and urinary C-peptide secretion dose. (~' =0.6& p<0.01 ; ~' =0.48, !3<0.05)

These results suggested that Q value is a good

marker far endocrine and exocrine pancreatic function, and may be a good indicator for remission in Type-1 diabetic patients.

P489 Plasma peroxidisability and the total peroxyl radical trapping antioxidant activity of plasma from diabetic and non-diabetic patients. "ME Britton, *DG Wickens, "8K Macfarlane, "JS Yudkin and "TL Dorrnandy. *Department of Chemical Pathology and the "Academic Unit of Diabetes and Endocrinology, Whittington Hospital, London N19 5NF.

Increased oxidative stress in diabetes may result from either over-production of oxygen free radicals or inadequate antioxidant mechanisms. Conflicting literature evidence on the antioxidant status of diabetic patients has led us to explore two novel approaches to this problem, ill. Plasma total [peroxyl] radical antioxidant parameter [TRAP] based on the inhibition by plasma of the peroxidation of aqueous dispersions of linoleic acid induced by peroxyl radicals generated by thermal decomposition of 2,2' azo-bis [2 amidinopropane hydrochloride] [ii]. Plasma peroxidisability assessed by measuring the increase in U.V.-absorption at 234nm of plasma following incubation for 24 hours at pH 5.5. The TRAP in 25 diabetic patients [679 + 155gruel/IT is significantly [0.05 >p>O.01] lower than in eight normal controls [817 + 111gmol/I]. In our series the TRAP did not correlate with plasma peroxidisability, type of diabetes, glucose control [HbAI~] or with presence of complications. These results support the hypothesis that diabetic patients are mere susceptible to oxygen free radicals due to impaired antioxidant defence mechanisms. The lack of correlation between plasma peroxidisability and TRAP may result from the TRAP reflecting aqueous soluble antioxidants whilst plasma peroxidisability is dependent upon PUFA substrate as well as antioxidant status.

P490 Anionic charges on e r y t h r o c y t e s are reduced in non diabetic a t h e r o s c l e r o t i c patients and in non diabetic offspring of type 2 diabetic patients. Bruno A; Gruden G; Estivi P; Cavallo Perin P; Pagano G. Istituto di M e d i c i n a Interna. Torino A r e d u c t i o n of anionic charges of e r y t h r o c y t e s has been reported in IDDM. The m e c h a n i s m of this association could depend on genetic factors and or on acquired w i d e s p r e a d alterations of m e m b r a ne charges. Present study evaluates anionic charges on e r y t h r o c y t e s in relation to a l b u m i n u r i a in a t h e r o s c l e r o t i c and h e a l t h y subjects with family history of diabetes. We m e a s u r e d the b i n d i n g of a cationic dye Alcian Blue (AB) to red cell m e m b r a nes and AER (RIA Kit Sclavo) on overnight urine collections in: A)I0 patients with clinical and echodoppler evidence of leg lesions (BMI=23.56~ 0.58 age=53.44~3.11), non diabetic and with negative family h i s t o r y for diabetes; B)I0 non diabetic o f f s p r i n g of type 2 diabetic patients (BMI= 19.89• age=40~3.53); C)II h e a l t h y v o l u n t e e r s m a t c h e d for sex, age, BMI. All subjects (A,B~C) had normal blood pressure, renal function and negative family h i s t o r y for h y p e r t e n s i o n ; B and C had no a t h e r o s c l e r o t i c lesions. AB was s i g n i f i c a n tly lower (p<0.0001) in group A (87.33• SEM n g / 1 0 6 r e d cells) and in group B (87.5812.76) than in controls (99.55~0.73). All AER values were~20 ug/min. These p r e l i m i n a r y results suggest that the r e d u c t i o n of AB on e r y t h r o c y t e s is present in genetic and a c q u i r e d c o n d i t i o n s not c h a r a c t e r i z e d by i n c r e a s e d AER.

A136

P491

P492

Alteration of relationship between random 91ucose and HbAlc by type of diabetes and mode of treatment.

E F F E C T OF L O N G - T E R M M O N I T O R I N G OF G L Y C A T E D H A E M O G L O B I N IN I N S U L I N - D E P E N D E N T D I A B E T E S M E L L I T U S M L y t k e n Larsen, M H e r d e r , EF M o g e n s e n . D e p a r t m e n t s of C l i n i c a l C h e m i s t r y a n d E n d o c r i n o l o g y M, Odense University Hospital, Denmark.

J P Hosker, D McAughey and S Tomlinson. Manchester Diabetes Centre, U.K. Simultaneous measurementsof fingerprick random blood glucose (RnG), measured by Glucostix read on Glucometer II, and venous HbAlc were obtained in 209 Type 1 and 419 Type 2 diabetic subjects. There was greater correlation between RnG and HBAlc in Type 2 subjects (r=0.52, p
B e f o r e i n t r o d u c i n g r o u t i n e m e a s u r e m e n t s of g l y c a ted h a e m o g l o b i n (HbAlc) w e w a n t e d to c l a r i f y , whether knowledge about HbAlc-values would result in c h a n g e s in t h e r a p y and i m p r o v e m e n t of m e t a b o lic r e g u l a t i o n . 240 type 1 (insulin-dependent) d i a b e t i c s w e r e r a n d o m i s e d into two c o m p a r a b l e g r o u p s r e g a r d i n g age, sex, d u r a t i o n of d i a b e t e s and t h e i r f i r s t H b A l c - v a l u e . H b A l c w a s m e a s u r e d by a h i g h l y p r e c i s e i s o e l e c t r i c f o c u s i n g m e t h o d . D u r i n g the f o l l o w i n g y e a r all m e a s u r e m e n t s of H b A l c (including r a n d o m i s a t i o n values) w e r e b l i n d e d in the c o n t r o l group, b u t u s e d in the a s s e s s m e n t of m e t a b o l i c c o n t r o l in the o p e n group. A f ter o n e y e a r 222 p a t i e n t s w e r e still f o l l o w e d in the clinic. M e a n H b A l c w a s s i g n i f i c a n t l y l o w e r in the o p e n g r o u p (p
PS 13 Pregnancy P493

P494

RACIAL SUSCEPTIBILITY TO GESTATIONAL DIABETES

REASSESSMENT OF THE TIMING FOR THE GLUCOSE C H A L L E N G E T E S T TO S C R E E N G E S T A T I O N A L DIABETES. E.Kyo,N.Toyoda,K.Yoshimura,T.Ichio,Y.Sugiyama Department of O b s t e t r i c s and Gynecology, Mie U n i v e r s i t y S c h o o l of M e d i c i n e , Tsu, Mie, J a p a n The aim of the s t u d y is to reassess the timing to d e t e c t g e s t a t i o n a l diabetes recommended in the S e c o n d International WorkshopConference in C h i c a g o (1984), n a m e l y between 23 and 27 w e e k s of pregnancy. Pregnant women who v i s i t e d our U n i v e r s i t y and the affiliated clinics r e c e i v e d oral 50g glucose challenge test (GCT), and 1-hour blood samples were transported to our laboratory for assaying plasma glucose and s e r u m insulin. Pregnant women w h o s e l-hour g l u c o s e v a l u e was 7.8mM or m o r e were r e c o m m e n d e d to r e c e i v e 75g OGTT. The data w e r e c l a s s i f i e d to 3 g r o u p s a c c o r d i n g to the t i m i n g of the test, A: 8 - 2 2 w e e k s (n=352), B: 2 3 - 2 7 w e e k s (n=406), C: 2 8 - 4 0 w e e k s (n=lg0). The mean~SD of plasma glucose (mM) was

A. Dornhorst, J.S.D. Nicholls*, E Probst, K. Patterson*, R.S. Elkeles, R.W. Beard* D e p a r t m e n t s ' of Clinical E n d o c r i n o l o g y , a n d O b s t e t r i c s & Gynaecology*, St. Mary's Hospital hospital Medical School, Norfolk Place, London. W2, England. Of 2375 women a t t e n d i n g the a n t e n a t a l clinic at St. M a r y ' s Hospital, L o n d o n between J a n u a r y 1988 a n d September 1989 who delivered within this period, 43 were diagnosed as having gestational diabetes by criteria published previously by the St. Mary's group. T h e gestational diabetics were older t h a n the general a n t e n a t a l clinic women (33.4 + 5.2 vs 28.9 + 5.4 yrs, m e a n + SD, p < 0.001), a n d also h a d a higher body mass index at booking (28.7 +5.1 vs. 24.1 + 4.1, p < 0.001). A h i g h e r p r o p o r t i o n of the gestational diabetics were n o n - C a u c a s i a n (39/43 vs. 9 9 5 / 2 3 3 2 , p< 0 . 0 0 0 0 1 ) . T h e f r e q u e n c y of gestational diabetes was 0.3% amongst the Caucasians a n d 3.8 % in the n o n - C a u c a s i a n women. G e s t a t i o n a l d i a b e t e s was equally common in the Asian, A f r o - C a r i b b e a n , a n d Oriental races (3.4% vs. 3 . 3 % vs. 3.0% respectively ). H o w e v e r it was m o r e common in the Middle E a s t e r n women t h a n in the n o n - C a u c a s i a n group as a whole (6.0% vs. 3 . 8 % , Z2=7.04, p< 0.01). These results d e m o n s t r a t e an i n c r e a s e d f r e q u e n c y of gestational diabetes in n o n - C a u c a s i a n women in a large U.K. city. M i d d l e E a s t e r n w o m e n m a y h a v e a p a r t i c u l a r susceptibility to develop this disorder.

5.98• 6.15• 6.70• and serum i n s u l i n ( m i c r o unit/ml) 63.3139.0, 67.0~42.8, 89.0• r e s p e c t i v e l y . The glucose and insul i n values in g r o u p C were significantly (P<0.01) higher than t h o s e in g r o u p A or B, but t h e r e was no s i g n i f i c a n t d i f f e r e n c e b e t w e e n groups A and B. D e t e c t i o n rate of impaired glucose tolerance (IGT) is 3 . 3 % , 2.5%, 9.8% r e s p e c t i v e l y . T h e s e d a t a s u g g e s t that pregnanc y - i n d u c e d c h a n g e s in g l u c o s e m e t a b o l i s m do not b e c o m e o b v i o u s u n t i l 28 w e e k s of p r e g n a n c y , and the recommended period, 23 to 27 weeks of pregnancy, is too e a r l y to s c r e e n gestational diabetes.

A137

P496

P495 PREDICTIVE FACTORS OF INSULIN THERAPYIN DIABETES R. Corcoy, M. Ba]sells, D. Mauricio, M. Puig, J.M. Pou and A. de Leiva

GESTATIONAL

Codina,

M.

AIM: To i d e n t i f y predictive factors f o r insulin therapy in gestational diabetics (GD). 159 GD (National Diabetes Data Group c r i t e r i a ) were f i r s t treated with d i e t and insulin was added according to the recomendations of Second International Workshop-Conference on Gestational Diabetes. In insulin-requirlng and non-insulin-requiring GD we investigated: a g e , body mass index (BMI), weight increment (WI), week o f diagnosis, oral glucose tolerance test (OGTT), family history o f diabetes (FH), previous glucose intolerance (PGI) and i s l e t - c e l l antibodies (ICA). STATISTICAL ANALYSIS: T-test, chi-square. RESULTS: Insulin-requiring GD were s i g n i f i c a n t l y older (32.0 • 5.1 vs 29.7 • 4.0 years), had higher BMI (24,01 • 4.0 vs 22.8 • 3.2 kg/m2), fasting glycemia (5.3 • 0.8 vs 4.9 • 0.6 mmol/l), higher rate o f PGI (15 % vs 0%) and were diagnosed e a r l i e r (28.0 • 6.2 vs 33.4 • 5.0 weeks) than t h e i r non-insulin-requiring counterparts. Groups did not d i f f e r in WI (9.9 • 3.8 vs 10.6 • 4.0 kg) , OGTT glycemia at lh (12.2 • 1.6 vs 12.1 • 1.3 mmol/1), 2h (10.6 • 1.7 vs 10.6 • 1.2 mmol/1), and 3h (7.8 • 2.0 vs 8.6 • 9.3 mmol/1), FH (46 vs 50%), and presence o f ICA (11.3 vs 4.5%). In conclusion, the

GESTATIONAL DIABETES~ HIGH RISK FOR DIABETES 12MONTHS POST PARTUM p. Hallgren and A. Frid dept of Medicine, hospital of Falun and dept of Medicine, hospital of Lund, Sweden. The aim of this ongoing study is to evaluate glucose tolerance 12 months post partum in women with gestational diabetes. 50 women entered the study consecutively july 1985 - may 1987. Diagnosis was established using 3h oral 100 g glucose load in 33 cases, 2h 75g glucose load in 9 cases and a combination of fasting and postprandial glucose values in 8 cases. All women had a 75 g oral glucose tolerance test (OGTT) 12 months post partum. 19 women (38%) had impaired glucose tolerance (IGT), 7 women (14%) had diabetes, according to WHO criteria. There was no difference between the women with normal OGTT compared to those with IGT regarding age, body mass index, parity, time of diagnosis, birth weight or frequency of insulin treatment. The women with diabetes after ~2 months had a higher parity, 2.7• vs 1.450.2 (p<0.01), and an earlier diagnosis, week 22• vs 28 • (p<0.05), than the normals. They had more often been insulin treated than the normal women, 57% vs ]6% (p<0.801). The study shows that a large percentage of women with gestational diabetes have diabetes and more than half of the women have abnormal glucose tolerance test already 12 months post partum.

following predictive factors f o r insulin therapy in GD were i d e n t i f i e d : Previous history of glucose intolerance ( Odds r a t i o (OR) 1.45), onset before 30 weeks o f pregnancy (OR 1.43), fasting 91ycemia >= 5.8 mmol/l (OR 1.19) and age >= 30 years (OR 1.24).

P497

P498

THREE MONTH~ GESTATIONAL DIABETES MELLITUS ARE SUFFICIENT TO PROVOKE ALTERATIONS IN CELL PLASY~ MEMBRANES P. Borboni, * L. Y~uzzanti, *A.M. Cugini, *R.A. Rabini, ~ Cester, w Faloia, w Morosini, ~ Romanini, w De Pirro. 9 Istituto di Biochimica, w di Endoerinologia, ~ Ginecologica, UniversitA di Ancona; Cattedra di Endocrinologia, II UniversitA di R o m a - Italy Both insulin-dependent and non-insulin-dependent diabetes mellitus provoke alterations in plasma membrane properties. The aim of the study was to characterize alterations properties induced by gestational diabetes mellitus (GDM). Twelve GDM patients (aged 29@_. 4 years) presenting gestati~al diabetes at the 6th month of pregnancy, in good metabolic contr~l (insulin therapy) and fourteen healthy pregnant women (N) (aged 28 • 6 years) were studied at the time of term delivery (89-41 weeks). Na*K§ (NA), Acetyleholinesterase (AchE), membrane fluidity (P) and cholesterol/phospholipid ratio (C/P) were studied on placenta membranes and red blood cell (RBC) plasma membranes fr~n both mothers and umbelieal cord. In comparison to N, GDM patients showed: i) decreased NA ( p < O.O1), P (p< 0.01) and AchE (p < 0.01) in RBCs from mothers and n e w b o m s as well as in placenta; ii) decreased C/P (p< 0.05) in RBCs from mothers, but increased C/P (p~.O.Ol) in RBCs from newborns; moreover, increased C/P (p< 0.01) in placenta. These data point out that: i) t~hree months of diabetes mellitus and/or insulin therapy, in the absence of important hyperglycaemia, are sufficient to induce alterations of plasma membrane properties both in the mother and in the newborn; ii) in GDM NA, but not AchE P and C/P, changes as previously reported in diabetic patients.

PERINATAL MORTALITY AND CONGENITAL MALFORMATIONS DIABETIC PREGNANCIES IN NORWAY DURING 1967- 86.

IN

Jak Jervell, Johan Halse, Per Magnus, Narve Moe, Leiv Bakketeig. Rikshospitalet and National Institute of Public Health, Oslo, Norway Have the changes in care of diabetes generally and diabetic pregnancy specifically improved the prognosis of diabetic pregnancies in a whole country? Our National Medical Registry of Births gives detailed medical information regarding pregnancies outcome and also mothers' prepregnancy health. During 1967-86 a total of 1989 offspring of mothers diabetic prior to pregnancy was reported. Perinatal mortality (28th week of pregnancy to 7 days postpartum) was reduced from 188 per thousand births (152-224) (95% confidence interval) in the first five year period to 30.4 (16-44) during the last. The relative risk compared to the total births had fallen from 9.8 to 3.5. The relative risk of cardiovascular and neurological malformations had fallen from 5.6 during the first 15 years to 1,8 during the last five years. There was an increase in cardiovascular and neurological malformations in the general population from 2.5 to 4.5 per thousand, and a fall in malformations in children of diabetic mothers from 17.7 to 8.3(n.s.). Perinatal mortality and possibly malformation rates have improved during the 20-year period. The results were best at University clinics indicating that centralization of these complicated pregnancies could be beneficial.

A138 P499

P500

MALFORM~ATIONS IN 238 D I A B E T I C P R E G N A N C I E S , NO CORRELLATION BETWEEN HbAICAND MALFORMATIONS GL N i e l s e n and P Hostrup, Dept. of G y n e c o l o gy, A a l b o r g H o s p i t a l , DK - 9000 A a l b o r g The s t u d y was d e s i g n e d p a r t l y to e v a l u a t e our p r o g r a m for p r e g n a n t d i a b e t i c w o m e n and p a r t l y to i d e n t i f y r i s k f a c t o r s for fetal m a l f o r m a t i o n . F r o m 1976 to 1988 we h a v e t r e a ted 210 i n s u l i n - d e p e n d e n t d i a b e t i c p a t i e n t s who r e a c h e d >28 w e e k s of g e s t a t i o n and w e r e d e l i v e r e d of 205 live b a b i e s w i t h b i r t h w e i g h t >l.000g. A d d i t i o n a l l y t h e r e w e r e 23 s p o n t a n e o u s and 5 i n d u c e d a b o r t i o n s for m e d i c a l reasons. M e a n m a t e r n a l H b a l c in the i. t r i m e ster was 7,6% and in the 3. t r i m e s t e r 6,6% (reference 4,2-5,6%). The total n u m b e r of m a l f o r m a t i o n s was 16; c o n s i s t i n g of I b a b y w i t h m u l t i p l e lethal, t h r e e w i t h m a j o r card i a c ( r e q u i r i n g o p e r a t i o n ) , t h r e e m i n o r card i a c (no surgery) and 9 m i n o r m i s c e l l a n e o u s malformations. Based upon 4 Hbalc measurem e n t s t h r o u g h o u t p r e g n a n c y we found no d i f f e r e n c e s in g l y c a e m i c c o n t r o l b e t w e e n the two groups, m e a n H b a l c w e r e 6,9% a m o n g m o t h e r s w i t h m a l f o r m e d o f f s p r i n g and 7,1% in the g r o up w i t h h e a l t h y babies. B a s e d u p o n t h e s e d a t a we c o n c l u d e t h a t m a t e r n a l H b a l c d u r i n g p r e g n a n c y c a n n o t p r e d i c t fetal m a l f o r m a t i o n in insulin-dependent diabetic patients.

E f f e c t s of M a t e r n a l Weight V a r i a t i o n s and Gesta t i o n a l D i a b e t e s M e l l i t u s on F o e t a l Weight A. P e z z a r o s s a , ~]. O r l a n d i , *F. C o p p o l a , *E. R i c ciarelli & A. Gnudi Chair of Endocrinology & Institute of Obstetrics & Gynecology, University o f Parma, Parma, I t a l y Aim of t h i s study was to evaluate the effects of maternal weight (MM) and of gestatienal weight gain (WG) on foetal weight in women with (GDM) and without (N) gestational di n betes m e l l i t u s . 125 pregnant women: 89 (N) and 37 (GDM) we re divided into: normal weight and normal weight gain (N) (NN= 41; GDMN= 14): obese (0) (NO= 29; GDMO= 15), and exce! sive weight gain (> 12 kgs) (E) (HE= 19; GDME= 8). We evalu ated: (A) the number of newborns with BW 90th p e r c e n t i l e ; (B) the difference in grams from the 50th p e r c e n t i l e value of a large population from our country; and (C) the correla tions between MW, WG and birthweight. Results: (A): NN:7.3% NO: 13.3%; NE:39.1%;'GDMN: 28.5%; GDMO: 31.2%; GDME: 75%. (B): NN: -18~53; NO: 215• NE: 352!I02; GDMN: 205~I03; GDMO: 325~I08; GDME: 895~150 g. Birthweights were s i g n i f i c a n t l y higher in HO and HE vsNN, GOMEv s GDMNand GDMO, GDMN vs NN and GDME vs NE. (C): a p o s i t i v e c o r r e l a t i o n (p< 0.05) was found between WG above I0 kgs and BW in NE and GDME. Thus, MW and WG influence birthweight at a d i f f e rent extent in normal pregnancy and GDM. The heavier babies were found in groups with excessive weight gain. Thus,women with excessive weight gain, e i t h e r with GDM or not, are at increased risk f o r developing f o e t a l macrosomia.

P501

P502

A L T E R A T I O N S IN FETAL LUNG M A T U R A T I O N A S S O C I A T E D WITH M A T E R N A L DIABETES. A. Zapata, C. Grande, J.M. H e r n ~ n d e z - G a r c f a . H o s pitales:"12 de Octubre", Ctra. de Andalucfa Km-5.4,28041 Madrid and "La Paz", p o de la Castellana,261 .Madrid.SPAIN. The effects of maternal diabetes on the pulmonary surfactant phospholipids were studied in 66 diabetic and 52 normal pregnancies. The diabetic patients were subdivided according to the quality of diabetic control. Nineteen patients had strict metabolic control (White's class :9-B; 7-D; 3-F) and 47 patients were poorly controlled (~Tnite's class: 19-B; 8-C; 14-D; 6-F). Lipids extraction of amniotic s was quantitative and individual phospholipids were isolated by two-dimensional chromatography. There was no difference in evolution of the lecithin/ sphingomyelin (L/S) and phosphatidylinositol/sphingomyelin (PI/S) ratios of the pregnant diabetics with strict control metabolic compared to the control group. The incidence of mature surfactant (L/Sa2.7 and presence of phesphatidylglycerol) at 37 weeks were no different to those in normal pregnancies. In diabetics poorly controlled, at 37 weeks only 25% had mature surfactant and there was a si~_ifieant difference with respect to the control group (X =6.45;p<0.05) . PI/S ratios in these diabetics were higher than in controls. These differences were statistically significant (t=3.048;p<0.01) at 34-36 weeks: I .3+0.25 {n=46) and I .I-+0.3(n=28) , respectively. There was an acceleration of fetal puln~nary maturation in 6 women with poor metaholic control and it is of interest that these pregnant women suffered episodes of hypoglycae/nia.

INCREASED EXPRESSIONOF LAMININ-B1 IN EMBRYOSOF DIABETIC RATS R. Sala, E. Cagliero, M. Lorenzi and U. J. Eriksson. Eye Research Institute and Departments of Ophthalmology and Medicine, Harvard Medical School, Boston, USA, and Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden The mechanisms underlying the teratogenicity of poorly controlled

diabetes have not been defined. Since disturbed regulation of the extracellular matrix (ECM) is a prevalent feature of the vascular complications, and since the ECM exerts fundamental roles in organogenesis, we have investigated whether altered synthesis of ECM components occurs in embryos of diabetic rats. Embryos were dissected from the uterus of control and streptozotocin-diabetic rats (Sprague-Dawley, U-strain) on gestational day 11. Total RNA was extracted from 10 malformed and 5 non-malformed embryos from diabetic rats and from 17 controls; RNA pools were tested by Northern analysis. Irrespective of malformations, in the embryos from diabetic rats the levels of 8-actin mRNA were not changed (and were thus used as internal control), the fibronectin transcript was 91 + 10 % of control, while the laminin-B1 mRNA levels were increased to 154 +16 % of control (p < 0.02). Of note, the laminin transcript was not increased in a malformed control embryo (107 % of the control group), nor did it change at gestational day 12 versus 11 (92 % of day 11). Thus, embryos of poorly controlled diabetic rats exhibit ECM abnormalities that do not appear to be simply a consequence of malformations or altered growth rate. Disturbed ECM regulation may represent a central problem in the sequelae of diabetes.

A139 P503

P504

FREE RADICAL SCAVENGING ENZYMES PROTECT AGAINST GLUCOSE-INDUCED EMBRYONIC MALFORMATIONS IN VITRO. U. J. Eriksson and L. A. H. Borg. Department of Medical Cell Biology, University of Uppsala, Uppsala, Sweden. The pathogenesis of congenital malformations in diabetic pregnancy is unknown. Previous studies in vitro have shown that elevated D-glucose concentrations in the culture medium induce embryonic malformations and growth retardation. The aim of the present investigation was to evaluate if generation of free oxygen radicals is involved in these disturbances of embryonic development. Rat embryos of a gestational age of 9 days were cultured for 48 hours in 10 or 50 mmol/I D-glucose. The effects on embryonic development of addition to the culture medium of catalase, glutathione peroxidase, superoxide dismutase and citiolone were determined. All these agents caused a significant protection against growth retardation and embryonic malformations induced by 50 mmol/I D-glucose. Superoxide dismutase was the most efficient compound and normalized almost all embryonic parameters, e.g. number of somites, crown rump length, as well as DNA and protein contents. Separate measurements showed that the enzymes added to the culture media were taken up by both the yolk sac and the embryo proper and that citiolone induced an increased activity of superoxide dismutase in the embryonic tissues. It is concluded that D-glucose, at least partly, causes embryonic dysmorphogenesis in vitro by generation of free oxygen radicals.

THE USE OF FRUCTOSAMINE ASSAY IN THE EVALUATION OF GLYCAEMIC CONTROL DURING PREGNANCY. L.J. Morgan, W.J. Jeffcoate, A.R. Scott and B. Kirk. City Hospital, Nottingham. NG5 IPB. U.K. In order to assess the value of fructosamine assay in pregnancy, the records were reviewed of 44 pregnancies in women attending a joint diabetic antenatal clinic between September 1986 and December 1989. Seven were controlled with diet alone, and the remainder received insulin. All were encouraged to perform seven point blood glucose profiles on two days of each week, using reflectance meters. Mean fasting blood glucose and mean seven point profile glucose were calculated from measurements made in the two weeks preceding each fructosamine measurement. In addition, the percentages of glucose results greater than target values (<6 mmol/l pre-prandial; <8 mmol/l ix hours post prandial) were calculated. The median values from 247 fructosamine assays were plotted against gestational age. There was a significant negative correlation between gestational age and fructosamine (r2 = .17, p = .007). Mean fasting blood glucose also fell during pregnancy (r2 = .23, p = .OO1), but there was no significant correlation between fructosamine and mean Tasting blood glucose, blood glucose profile, or the percentage of observations greater than target values. These data suggest that fructosamine does not reflect closely the results of glucose measurement during pregnancy, and low observations may be the result of factors such as falling albumin concentration.

P505

P506

UK DIABETIC PREGNANCY MANAGEMENT SURVEY 1989 H.J. Bodansky, F. Brice, S. Redmond and P. Buchan Dept. of Medicine, The General Infirmary, Dept. of Obstetrics and Gynaecology, St. James's University Hospital, Leeds, British Diabetic Association, London.

SHORT TERM N O R M A L AND

This study attempted to assess the current care and management of diabetic pregnancy in the UK. Questionnaires were sent to 376 relevant Consultant Physicians in 239 districts; 54% responded providing information from 185 (78%) districts. There was a combined diabetes/obstetric clinic in 66 districts with annul median case loads of 13 (I-200). Preconception counselling was offered in 22 clinics. Diagnostic criteria for diabetes arising during pregnancy were WHO 153; O'Sullivan and Mahan 4; others 24. In those using WHO criteria; 149 treated impaired glucose tolerance similarly to diabetes. Blood glucose meters were not used in 67 centres. Target blood glucose values were < 6 mmol/l (3-12 mmol/l, n = 172) pre-meal and 8 mmol/l (5-10.5 mmol/l, n = 139) post-meal. Insulin was introduced at values of 7 mmol/l (5-12 mmol/l, n = 162) pre-meal or 9 mmol/l (5.5-15 mmol/l, n = 129) post meal. Few respondents could provide fetal loss/perinatal mortality rates for diabetic cases, due to lack of recorded data. Where data were available, fetal loss was almost always intra-uterine. Only 78 centres allowed pregnancy to progress to term. Caesarean section rates for diabetic women were 35% (890%, n = 111), but only 12% (7-20%, n = 102) for nondiabetic women. Routine admission towards term was advocated in 23 centres. It is concluded that care and management strategies for diabetic pregnancy are highly variable throughout the UK.

C H A N G E S OF hPL AND DIABETIC PREGNANCY

hCG

SECRETION

IN

Cz. W 6 j c i k o w s k i and O. W o j c i e c h o w s k a . Laboratory of E n d o c r i n o l o g y , I n s t i t u t e of O b s t e t r i c s and G y n e c o l o g y , M e d i c a l A c a d e m y in G d a ~ s k , P o l a n d The s e c r e t i o n of s o m e h o r m o n e s has a p u l s a t i l e c h a r a c t e r . The a i m of s t u d y was to i n v e s t i g a t e a s h o r t time c h a n g e s of h u m a n p l a c e n t a l l a c t o g e n (hPL) and c h o r i o n i c g o n a d a t r o p i n (hOG) in t h i r d t r i m e s t e r of n o r m a l p r e g n a n t w o m e n (n=lg) and p r e g n a n c y c o m p l i c a t e d by d i a b e t e s t y p e I (n=8). B l o o d was s a m p l e d e v e r y 15 min for 5h p e r i o d . P l a s m a hPL and hCG w e r e d e t e r m i n e d q u a d r u p l y by the RIA m e t h o d s . I n t r a a s s a y c o e f f i c i e n t s of v a r i a t i o n of hPL and hCG w e r e 1.5% and 1.7%, r e s p e c t i v e l y . S i g n i f i c a n t p e r i o d i c o s c i l a t i o n s of p l a s m a hPL c o n c e n t r a t i o n in n o r m a l p r e g n a n t w o m e n w e r e found. The o s c i l a t i o n s v a r i e d f r o m 3 0 - t o 60 m i n w i t h a m e a n of 51 min. The m e a n a m p l i t u d e of the o s c i l a t i o n s was 1 . 6 6 ~ 0 . 2 u g / m l ( 0 . 4 - 3 . 5 u g / m l w h i c h is 2 1 . 6 % of 3rd h o u r s m e a n value. In d i a b e tic g r o u p hPl p l a s m a c h a n g e s h a v e i r r e g u l a r c h a r a c t e r and do not c o r r e l a t e w i t h cos f u n c t i o n . H o w e v e r , the a m p l i t u d e of c h a n g e s was h i g h e r t h a n in n o r m a l p r e g n a n c y . D u r i n g the s t u d y a m e a n blood glucose concentration was 9 . 9 ~ 1 . 2 m m o l / 1 . S h o r t t e r m f l u c t u a t i o n s of hCG p l a s m a c o n c e n r a t i o n s in n o r m a l and d i a b e t e s c o m p l i c a t e d p r e g n a n c y h a v e not a c h a r a c t e r of p e r i o d i c o s c i l a t i o n s . T h e s e r e s u l t s i n d i c a t e t h a t in t h i r d t r i m e s t e r of p r e g n a n c y the s e c r e t i o n of hPL but not of a hCG has p u l s a t i l e p a t t e r n . D i a b e t e s d i s t u r b e d the p u l s a t i l e s e c r e t i o n of hPL.

A140 P507 P U B E R T Y AiTD ~ E R T I L I T Y Iff YOUI,~ T Y P E I (II%ULII~DEJPEifDEZT) D I A B E T I C ;lOl,.~,i~~. K.P. R a t z m a n n , E. S t e i n d e l and S. Gurr. C e n ~ e r of D i a b e t e s and L~etabolic Disorders, B e r l i n / G.D.R. The a i m of the p ~ e s e n ~ study was to analyze the somatic matumaOion, the a p p e a r a n c e and course of ~he m e n s t r u a l cycle, and the f e r t i l i t y in q0q y o u n g i n s u l i n - d e p e n d e n t (~ype q) d i a b e t i c w o m e n (age: 16.2 yrs.; fl'7-20 yrs.) as c o m p a r e d t o q05 a g e - m a t c h e d n o n - d i a b e t i c controls. A9 9he time of the study, m e n a r c h e h a d no9 yet occurred in 3~ of ~he d i a b e ~ i c girls as opposed ~o q# of 9he n o n - d i a b e t i c girls (33.9 ~ vs q3.3 ~; p < 0 . 0 ~ ) . The a v e r a g e age of m e n a r c h e of diabetic girls was s i g n i f i c a n t l y h i ~ h e r ~ h a n tha~ of n o n - d i a b e O i c con?rols (J3.# + ft.2 vs q2.9 + 1.2 yrs.; o<0.0fl), bhu.s show~ng a d e l a y e d p u b e r t a l d e v e i o p m e n ~ . There w a s a close r e l a t i o n s h i p b e t w e e n ~he q u a l i t y of m e ? a b o l i c control and the occurrence of m e n s t r u a l d i s o r ders (polymenorrhea, olii~omenorrhea, s e c o n d a r y a m e n o r r h e a ) . ~he latter occurred five ?imes more often in p a ~ i e n ? s w i ~ h poo~ metabolic control than in diabetic w o m e n w i t h ~i~ht m e t a b o lic control (43.3 ~ vs 8.S ,$; p < 0 . O ~ ) . [e failed ~o d e m o n s ? r a t e any c o r r e l a t i o n b e t w e e n per i p h e r a l h o r m o n e c o n e e n 0 r a 0 i o n (es~radiol, progesterone, prolac~in, FS?{ and L~2) and 9he quality of m e ? a b o l i c control. In the to?al study groups, n e i o h e r ~he 52 diabetic w o m e n nor bhe ~2 n o n - d i a b e t i c w o m e n t o o k any f o r m of contraception. 2# p r e g n a n c i e s occurred in the diabebic w o m e n as opposed to #fl p r e g n a n c i e s in ohe n o n - d i a b e t i c controls (#6 ;5 vs ~8.8 .3; p < 0 . O q ) . In S u m m a r y the study d e m o n s t r a t e s a d e l a y e d pubertal development, h i g h e r f r e q u e n c y of m e n s t r u a l d i s o r d e r s and less f e r t i l i t y in y o u n g diabetic women.

PS 14 Clinical Nephropathy P508

P509

MICROALBUMINURIA IN OIABETIC CHILDREN

INCIPIENTDIABETICNEPHROPATHYIN A SWEDISHPROVINCE- IMPACT

H. Oziatkowiak, O. Nazim, K. Sztefko and M. Korecka

OF PAST GLYCEMIC CONTROL, BLOOD PRESSURE AND SMOKING HABITS

Department of Endocrinology and Clinical Biochemistry, Institute of Pediatrics, University School of Medicine, Wielicka Sir. 265, 30-663 Krakdw, Poland Microalbuminuria is the only marker of early diabetic nephropathy. The aim of our study was to determine the prevalence of elevated urinary albumin excretion rate in diabetic children related to age, sex, diabetes duration and metabolic control as well as to compare different urine collection periods in the assesment of microalbuminutia. Urinary albumin excretion was determined in 92 children aged 2 - 18 years with insulin-dependent diabetes mellitus. All children collected three 24-hour urine samples divided in four fractions during a sixth-monthslong period of time for albumin analysis by radioimmunoassay. 28% of the diabetic children had urinary albumin excretion rate values exceeding the upper limit for healthy controls which was 15mg/24 hours. Microalbuminuria more than 30mg/24 hours was present in 19,5% of patients. No significant association was observed between microalbuminuria and age, sex ' hemooiobin A.I but the prevalanY. ce of elevated albumln excretion rose with increasing diabetes duration. The strong positive correlation was found between albumin concentration in overnight, morning and %-hour urine collection /p
J. Berglund and K. Newberg. Department of Medicine, Sundsvall Hospital, Sweden. The prevalence of microalbuminuria, a sign of incipient diabetic nephropathy, was systematically assessed in a defined population of Type 1 (insulin-dependent) diabetics in a Northern Swedish province.The relation of past, as well as present, glyeemic control, blood pressure level and smoking habits to the appearance of microalbuminuria was analyzed. Based on three over-night urine samples, a 22% prevalence of microalbuminuria (n=42) was found. A similar number of matched diabetics with normal urinary albumin excretion were selected. In comparison, microalbuminuric subjects had significantly higher blood pressure at present (mean 133/79 vs 123/75, p<0.01), as well as during the seven years prior to the diagnosis of microalbuminuria (140/82 vs 134/79, p<0.05). Past and present glycemic control (assessed by glycosylated h;emoglobin, HbA lc) was similar in both groups. Glomerular filtration rate (51Crom-EDTA-clearance) was slightly lower in

mieroalbuminurics (102+_27vs 115+_27, p<0.05). An analysis of life time tobacco consumption (assessed by "pack-year") did not reveal significant differences between the two groups, although microalbuminurics had slightly higher consumption. In the present study arterial blood pressure level was more strongly

associated with the subsequent development of mieroalbuminuria in diabetics, than either glycemic control or tobacco consumption.

A141

P510

P511

MICROALBUMINURIA IN AN UNSELECTED POPULATION OF TYPE 1 (INSULIN-DEPENDENT) DIABETICS. 1 2 1 O. Eshoj , B.Feldt-Rgsmussen , Henning Beck-Nielsen and S.H. Sandee-Petersen . From Medical Department M, Odense University Hospital (i) and Steno Memorial Hospital, Gentofte (2), Denmark. Urinary albumin excretion rate (AER) have never been investigated in a population-based material. In smaller groups of selected patients the prevalence of microalbuminutia have been estimated to around 10% and AER found to correlate positively to blood pressure and HbAIc. In the actual croessectional, population-based sudy of 640 type 1 (insulin-dependent) diabetics (282 women and 358 men, mean age 35 ~ 12 years, mean duration of diabetes 19 • 12 years (• AER, blood pressure and HbA]c were measured. We found the prevalences of normoalbumlnuria (<15 ~g/min) to be 76%, of microalbuminuria (15-200 ~g/min) to be 15% and of macroalbuminuria (>200 ;g/~in) to be 9%. Further we showed a strong positive correlation between AER and HbAIc (r=0.27), systololic blood pressure (r=0.35), diastolic blood pressure (r=0.29), diabetes duration (r= 0.29) and age (r=0.24) respectively (p-values < 0.0001) (Spearmanns Rho). We conclude that the prevalence of microalbuminuria has been underestimated in selected materials, whereas the prevalence of macroalbuminuria was overestimated. Furthermore our unselected material confirms the former findings of correlation between AER and poor metabo%~!% ~:%ntrol, blood pressure and further diabetes duration and age.

EFFECTS OF PUBERTY ON THE DEVELOPMENT OF MICROALBUMINURIA IN ADOLESCENTS WITH ONSET OF DIABETES DURING CHILDHOOD. F. Chiarelli, G. LaPenna, M.T. Petitti and G. Morgese. University Department of Pediatrics, Chieti, Italy. Fifty prepubertal type 1 (insulin-dependent) diabetic children were followed prospectively for 7 years. At the beginning of the study the patients had to fulfil the following inclusion criteria: onset of diabetes before 7 years of age, duration of disease > 3 years, urinary albumin excretion rate (UAE) < 15 j~g/min/1.7S m 2 on at least two occasions, normal blood pressure, HbAlc < i0%, age < ii years for boys and < i0 years for girls and no signs of puberty. Patients were aged 8.7-10.9 years with a duration of diabetes of 3.4-10.1 years. UAE and supine blood pressure were measured every a months. Seven years later, all the children were pubertal and 5 of them had developed persistent microalbuminuria (UAE > 30 /ug/min/ 1.7S m 2 on at least three occasions): at the end of the study the microalbuminuric adolescents were aged 15.S17.9 years with a du~'ation of diabetes of 12.2-16.8 years; their metabolic control worsened throughout puberty (HbAIe: 8 . 4 + 0 . 8 % at the beginning vs 10.6• at the end of the study; p < 0.01). Puberty is a risk factor for worsening of metabolic control and development of microalbuminuria in diabetic adolescents with onset of disease during early childhood.

P512

P513

Early onset and chronic hyperglycaemia - risk factors for microalbuminuria in T y p e I d i a b e t e s .

RISK FACTORS ASSOCIATED WITH URINARY ALBUMIN EXCRETION IN TYPE 1 (INSULIN-DEPENDENT) DIABETES : A STUDY OF 306 PATIENTS. B.M. B i l l a u l t and Ph. L. Passa. Department o f Endocrino- l o g y . HOpital S a i n t - L o u i s - 1, avenue Claude V e l l e f a u x 75010, P a r i s , France. This s t u d y was designed t o i n v e s t i g a t e r i s k f a c t o r s associated with Urinary Albumin Excretion (UAE) in type 1 (insulin-dependent) diabetic patients. In 308 consecutive patients the following parameters were recorded : age, sex, duration of diabetes, body mass index, arterial hypertension (blood pressure values>t160 and/or 95 mmhg or presence of an antihypertensive treatment), HbAI, smoking status, plasma triglycerides, total cholesterol, daily insulin dosage and UAE (mean of two 24 hour urine collection determinations). Patients with UAE > 30 mg/24 hour (n = 64) compared to those with normal UAE (n = 242) had significantly higher values for prevalence of hyper-tension (p <0.002), HbAI (p <0.001), triglycerides (p< 0.01) and daily insulin dosage (p <0.02). Dealing with other parameters, the two groups were comparable. Using a stepwise linear regression analysis, the signi-ficant (p < 0.01) and independent variables associated with UAE were in the whole population : hypertension, HbA1, triglycerides and daily insulin dosage ; in patients with increased UAE : hypertension and age ; in patients with normal UAE : daily insulin dosage. Specific health care delivery is mandatory in type i diabetic patients with increased UAE as this condition is associated with other cardio-vascular risk factors.

W J Kalk, C Osler, D Taylor, V R Panz and S G Reinach. Division of Endocrinology and Metabolism, Medical School, Johannesburg, South Africa. T h e i n f l u e n c e o f age o f o n s e t of d i a b e t e s a n d p r i o r l o n g - t e r m (mean 44 m o n t h s ) g l y c a e m i c c o n t r o l o n the p r e v a l e n c e of m i c r o a l b u m i n u r i a w a s i n v e s t i g a t e d in 90 s u b j e c t s w i t h i n s u l i n d e p e n d e n t d i a b e t e s (age 1 3 - 3 0 yr; d u r a t i o n 3-16 yr). 34 p a t i e n t s h a d an e l e v a t e d a l b u m i n e x c r e t i o n r a t e (AER - m e d i a n 38.5, r a n g e 22192/ug/min). C o m p a r e d to t h o s e w i t h n o r m a l A E R ( ~ e d i a n 9.• (n=56) m i c r o a l b u m i n u r i c s u b j e c t s w e r e y o u n g e r at d i a g n o s i s ( 1 1 . 4 • vs 8.4• yr, p = 0 . O O 4 5 ) , h a d l o n g e r p r e p u b e r t a l d u r a t i o n ( 2 . 2 ~ 3 . 0 vs 4 . 0 • yr, p = 0 . 0 1 4 ) b u t similar postpubertal duration of diabetes (5.0• vs 5.2• yr). Previous glycaemic c o n t r o l w a s p o o r e r in m i c r o a l b u m i n u r i c patients (mean H b A i 1 2 . 9 • vs 1 0 . 7 • p < 0 . 0 0 0 0 1 ) . 30 o f t h e 34 (88%) o f t h e m i e r o a l b u m i n u r i c patients had a mean HbAi>10.8% (normal 8.0%). The prevalence of microalbuminuria decreased with i n c r e a s i n g age o f o n s e t o f d i a b e t e s (x 2 for trend =8.60, p=0.DO34) and increased with increasing mean HbAi (x2=21.46, p=0.0026). Age of onset of diabetes and mean HbAi were negatively correlated (r=-0.40, p < 0 . O O l ) . We conclude that poor glycaemic control and young age o f o n s e t o f d i a b e t e s a r e r i s k f a c t o r s f o r the d e v e l o p m e n t of m i c r o a l b u m i n u r i a . Unlike d i a b e t i c r e t i n o p a t h y , t h e p r e p u b e r t a l y e a r s of d i a b e t e s a p p e a r to i n f l u e n c e t h e d e v e l o p m e n t o f microalbuminuria.

A142

P514

P515

ASIAN PATIENTS WITH DIABETES-RELATIONSHIP TO COMPLICATIONS. P. MARTIN~ D. NAGI, M. STICKLAND a n d H. T I N D A L L . U n i v e r s i t y D e p a r t m e n t of M e d i c i n e , The General I n f i r m a r y , Leeds. U.K. MICROALBUMINURIA

IN

A s i a n p a t i e n t s h a v e m a i n l y t y p e 2 d i a b e t e s a n d an i n c r e a s e d i n c i d e n c e of m i c r o a l b u m i n u r i a (AER; p r e d i c t s f u t u r e n e p h r o p a t h y in t y p e 1, b u t may p r e d i c t v a s c u l a r c o m p l i c a t i o n s in t y p e 2 d i a b e t e s ) . We t h e r e f o r e i n v e s t i g a t e d the r e l a t i o n s h i p between m i c r o proteinuria a n d the p r e s e n c e of c o m p l i c a t i o n s in 60 Asian diabetics compared with 60 E u r o p e a n d i a b e t i c s matched f o r age, s e x , d u r a t i o n ot: d i a b e t e s a n d t y p e of t h e r a p y . Patients provided 2 timed o v e r n i g h t u r i n e s f o r measurement of g l o m e r u l a r (AER; t r a n s f e r r i n ) a n d t u b u l a r ( o~/ M, NAG) ind ces S t a t i s t i c s were performed by Mann-Whitney U test. Fifteen Asians and 9 Europeans had hypertension. Thirty-four A s i a n s (57~; 26 m a c r o - , 8 m i c r o - v a s c u l a r ) and 22 E u r o p e a n s (37~; 12 macro-~ 10 m i c r o - v a s c u l a r ) had complications. F o u r t e e n A s i a n s had m i c r o a l b u m i n u r i a (10 m a c r o . v a s c u l a r c o m p l i c a t i o n s , 2" n e u r ' o p a t h y , 2 no complications) but only 5 E u r o p e a n s (3 m a c r o - , 2 micro-vascular) (AER A s i a n s v s E u r o p e a n s 10 E1-189~ ug/min vs 5.0 []-123] ug/min; p <0.04). Asians w i t h c o m p l i c a t i o n s h a d r e s i d e d l o n g e r in OK (p < 0 . 0 3 ) , had h i g h e r AER (13 [ 1 - 3 1 9 0 u g / m i n vs 5 []]l-360--_~ug/min; p < 0.03), systolic (143 L~95-180~ mmHg vs 124 C ] 0 5 160"] mmHg; p <0.02) and d i a s t o l i c ( 80 ~" 60-100] mm Hg vs 75 E60-900 mmHg; p < 0.05) BP than Asians without complications, whilst tubular protein handling was s i m i l a r ( p > 0 . 6 ; NS). In c o n c f u s i o n , m i c r o p r o teinumia in A s i a n s was a s s o c i a t e d w i t h c o m p l i c a t i o n s , particularly m a c r o v a s c u t a r , a l t h o u g h two had no complications. Therefore, longitudinal studies are r e q u i r e d in A s i a n s to e s t a b l i s h w h e t h e r m i c r o a l b u m i n u r i a is a s s o c i a t e d w i t h c o m p l i c a t i o n s o r p r e d i c t s f u t u r e n e p h r o p a t h y or b o t h .

NO PROGRESSION TO CLINICAL NEPHROPATHY DESPITE 20 YEARS OF TYPE-I(INSULIN-DEPENDENT) DIABETES :A LONG-TERM FOLLOW-UP. E.Chantelau,P.Wichmann,F.Beat,H.Eehterhoff,P.T.Sawicki,and U.Didjurgait. Heinrich-Heine-University,DOsseldorf(FRG) After >20 years of traditional therapy for type-I diabetes mellitus, about 40% of patients being referred to our department present with clinical proteinuria(CP; >5O0 mg/1) It is unknown whether intensive treatment with insulin pumps(CSII), and antihypertensive drugs(AHD) in cases of established hypertension(blood pressure >140/90 mm Hg) has an effect on the incidence of CP on the long run.Therefore, 20 type-i diabetic patients, who had volunteered for CSII in 1980, were followed until 1990; glycohaemoglobin GHB (normal mean=lOO%),microprotainuria in random urine samples (normal < 50mg/1), and blood pressure(BP) were monitored at least 3 times/year. On entering the study in 1980, the patients' mean(SD) age was 27(12)years, and duration of diabetes 12(5)years, and none of the patients had CP. Results of the follow-up(i980 vs.19go data):GHB 158(18)% vs 155(26)%; microproteinuria 40(56) mg/1 vs. 63(91) mg/1; mean BP 127/84 mm Hg vs. 125/79 mm Hg; hr. of patients on CSII 20 vs. 17;nr. of patients on AHD 1 vs.4. From 1980 to 1990, the annual mean GHB of all patients was 137(17)%, and the annual man BP was 134/84 mm Hg.Oonclusions: none of the patients progressed to CP after a mean of 22 years of type-I diabetes mellitua, and there was virtually no change in microproteinuria for 10 years. This favourable result may be ascribed to keeping the annual mean GHB well below150% of the normal mean, and BP <140/90 mmHg during a decade of intensive therapy. Thus, our study lends support to the hypothesis that good control of glycaemia and blood pressure can prevent diabetic nephropathy.

P516

P517

CARDIOVASCULAR RISK FACTORS IN TYPE I DIABETIC PATIENTS WITH CLINICAL NEPHROPATHY G. Passariello,G.Marrazzo,G.Pizza,L.Carbone, S. Sgambato and F;D'Onofrio -Institute of General Medecine - Universi ~ ty of Naples - Naples - Italy

Early glomerular and urological dysfunctions in insulindependent diabetes: evidence for an incipient uropathy. JY Pottier, J Le Cloirec, A Moisan, J Yaouanq, N Lemoullec, H Allannic and JY Harry. Service des Maladies MGtaboliques, Hopital-Sud; Rennes. Service de MGdecine NuclGaire, Centre Eugene Marquis; Rennes. France.

In diabetes mellitus cardiovascular mortality among patten ts with increased urinary albumin excretion (UAE) seems to be higher than in patients with normal UAE. Therefore, we investigated blood pressure,concentrations of total cholesterol, lipoproteins, fibrinogen and platelet adhesion in 40 insulin dependent diabetic with UAE greater than 30 ug/ min and in 20 individually matched insulin dependent diabe tics with normal UAE (<30 ug/min). Two groups had similar age, sex, diabetes duration,and glucose control. Patients with UAE (>30 ug/min) had significantly higher concentration of total cholesterol (6.1+0.7 vs 4.q+0.3 mmol/l p<0.01),LDL-cholesterol (3.47+0.22 vs 2.8+0.14 mmol/l p< 0.O05),VLDL-eholesterol (0.34+0.07 vs 0.15+0.03 ~nol/l p< 0.001)than controls but significantly lower concentration of high density lipoprotein 2 subfraction cholesterol(0.29 +0.03 vs 0.63+0.04 mmol/l p<0.001).Concentrations of total triglycerides (1.14+0.11 vs 0.68+0.06 mmol/l p30 ug/min). The mortality of cardiovascular diseas~ during 5 yera follow up was significantly higher among patients with poteinuria (p<0.0Ol)indicating that these risk factors contribute to the increased mortality in patients with clinical nephropathy.

Increased glomerular t-titration rate (GFR) is common in short term diabetic patients just as urinary stasis in long-term patients with overt nephropathy and autonomic neuropathy. To evaluate glomerular and urinary functions, renal scintigraphy was performed in 115 type I diabetic patients with albumin excretion rate (AER) less than 300 rng]24h, and 45 controls. Results: 1~ GFR (99mTc-DTPA) was increased in the diabetic group (p <10 -9) and correlated with renal plasma flow (RPF, 131I-Hippuran) (r =0.5; p <10-9). Fifty patients were h y p e r f i h e r i n g with RPF and Filtration Fraction (FF=GFR/RPF) higher than normoffitering patients. 2 ~ Slow intrarenal or pyeloureteral DTPA excretion, unilateral or bilateral, was observed in 3 controls and 60 patients (p<10-7): 24 hypelffltering, 36 normof'fltering (NS). Mean GFR, RPF, FF, duration of diabetes, blood glucose, HbAl.c, frequency of peripheral neuropathy did not differ from those of patients with normal scintigraphy. 3 ~ Asymmetrical 99mTc-DTPA renal uptake with asymmetrical GFR was observed in thirteen patients (7 hyperfiltering) and often associated with slower elimination. No evidence for stenotic renal atheroma or p a r e n c h y m a t o u s d i s e a s e was f o u n d on the angiopyeloureterography. Conclusion: incipient uropathy is a very common and early phenomenom wich occurs irrespective of incipient nephropathy.

A148

P519

P518 IMPAIRED

CARDIOVASCULAR

YOUNG MICROALBUMINUEIC

RESPONSE

TO COLD

PRESSER

TEST

IN

DIABETIC PATIENTS.

E.Bognetti, F~Meschi, M.Rota, D.Cofano, A.Palermo, G.Chiumello Scientific Institute H.San Raffaele,Pediatrin Department, Universit~j of Milan Cardiovascular and hormonal responses to cold presser test were measured in ii Type 1 diabetic patients with microalbuminuria (median 21.3 mcg/min,range 16-233.5),11 Type 1 diabetic patients with normoalbuminuria (median 5 mcg/min, range 1.9-10.6) matched for age (mean 19.8 yrs • 3.1 vs 19.2 yrs • 3.2),duration of diabetes (mean 12.5 yrs + 4.5 vs 12.8 yrs • 5.2), metabolic control (mean HbAIo 9.8 % ! 1.8 vs 8.9 % ~ 2.1), and in 9 normal control subjects.No differences were evident between baseline values of heart rate,plasma renin,aldosterone and eateeholamines concentrations of diabetic patients,but higher mean blood pressure was evident in microalbuminurie (mean 89.2 mmHg • 7.2) than normoalbaminuric (mean 80.5 mmHg • 4.5,p < 0.01) patients and controls (mean 81.9 mmHg • 7,p 0.05).Blood pressure increase to cold presser test was significantly lower in microalbuminurie (mean 21.4 mmHg • 7.8,p < 0.005) than normoalhuminuric patients (mean 32 mmHg • 7.8) without differences in catecholamines release. Heart rate increase at stress resulted reduced in microalbuminurie patients (mean 9 beats/min + 12.8) compared with controls (mean 2S.8 beats/min • 6.l,p ~ 0.005) with no difference between diabetic patients.No difference was evident in renin aldosterone release during the test between the groups. These findings suggest that in microalbuminuric patients the mechanisms involved in blood pressure regulation during a standard cold test are impaired. Defects in renal vasocostriction may be involved.

TRANSCUTANEOUS OXIHETRY IN TYPE I (INSULIN-DEPENDENT) DIABETIC PATIENTS WITH PERSISTENT MICROALBUHINURIA G.Psttin~,g. Breschi,F.Inneeanti,G.C.Bartelome$, and G. 5sghisri. Diabetes Unit, Spsdeli Riunits Transeutaneous oximetry (Te02) has been widely ussd in the evaluation of peripheral vascular dissass of Type I (insulin-dependent) diabetic patients. To verify whether also microangiopathy (retinopethy or persistent microalbums i.e. an urinary albumin sxcretlen between 20 and 200pg/min) would be related with an altered skin oxygen supply we monitored To02 by a Clsrk-typs electrode from the dorsal foot skin of 27 patients without clinical~instrumental (C4 dappler) evidence of peripheral vascular disasse and in 11 age and sex matched controls. Besides To02 we measured the time necessary to obtain a standardized postacclusive isehasmia (IT) and to reach the half value of basal Te02 after release of ischaemia (T/2). Either Tc02 or IT of diabetics were similar to those of controls while T/2 was significantly higher in the diabetic than in non diabetic individuals(37.BL14.1 (SD)sec vs. 26.9+10.4sec; p=0.026). While none of the considersd parameters was modified in presence of retinopathy, both T/2(49.6+22ssc) and IT(167+49sec) were significantly longer in patients with persistent microalbuminuria (n=5), than in the normoalbuminuric group(n=22;T/2=35+11sec, IT=99+28sec) and in controls (T/2=26.9310.4sec ~ F=5.62, p=O[O07, IT=78+35sec;F= 3.78, P=O.05~. In summary, skin oxygen supply seems eltared in diabetic patients with persistent mieroalbuminutia, even in absence of peripheral vascular discass.

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EFFECT OF SHORT TERM STRICT CONTROL OF BLOOD GLUCOSE ON URINARY EXCRETION OF ALBUMIN AND Bg-MICROGLOBULIN IN NIDDM Moridera K., Kurahati H. and Igarashi T. Kobe City General Hospital, Kobe Japan We studied the effect of short term strict blood glucose control on the early stage of diabetic nephropathy. 19 of poorly controled NIDDM with microalbuminuria (9 males, i0 females, age 57.5• yr, duration of DM 9.4• yr) were studied. Before and after one month of strict blood glucose control by hypoglyemic agent or/and insulin, albumin, $2 microglobulin, NAG and creatinin levels in 24 h urine specimens were determined. Before treatment FBG, HbAI, and 24 h glucosurie were 236• mg/dl, 13.3• and 41.5• g/24h. Urinary albumin excretion rate significantly improved from 79• mg/24h to 56• mg/24h (p
Effects of a two months reduced-protein diet on renal hemodynamie function in Type-I diabetic patients without nephropathy. A Guivarch, JY Poirier, A Moisan, C Le Cloirec, C Siemen, JP Hespel, JY Herry and H Allannic. Service des Maladies M6taboliques, Hopital-Sud, Rennes. Service de M6decine Nuclgaire, Centre Eugene Marquis, Rennes; France. In order to assess the influence of a low protein diet on renal function and to control the patients' adhesion to this diet, 22 well-educated Type I diabetic patients without microalbuminuria were randomly divided into two groups. Their usual daily protein intake (PI) was estimated during one week using a computer program. In group I, PI remained unchanged (1.68 g.kg-l.d-1; 11 patients). In group II a 30% PI restriction from 1.7 + 0.5 g.kg-l.d -1 to 1.1 - 0.3 was provided during two months. Glomerular filtration rate (GFR, 99raTcDTPA), renal plasma flow (RPF, 131I-Hippuran), filtration fraction (FF= GFR/RPF) were measured in all patients at baseline and day 4th (inpatients period) then two months later in group II. Results: 1~ In group I the renal functions at baseline compared with those at day 4th were unchanged: GFR= 149 + 19 ml/mn.l.73m2 vs 150 + 14 and FF = 26.9 + 3% vs 26 + 2.6. In group ]I both GFR and FF decreased at day 4th: GFR= 149 _+18 vs 140 + 28 !0< 0.05; FF= 25.9 + 3.5 vs 22.7 + 3.9 p< 0.01 (Wilcoxon test). 2 ~ Two months later mean GFR and FF increased slightly (142.5 + 24 ml/mn.l.73m2 and 24.1 + 3.6%) just as mean daily PI (1.6 + 0.36 g.kg-]). Conclusion: a moderate protein restriction is effective for lowering f'dtration fraction in diabetic patients; however the inconstant long-term adhesion to this diet restricts its routine application.

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R E S U L T S OF A T R A I N I N G P R O G R A M FOR R E D U C I N G BLOOD P R E S S U R E IN H Y P E R T E N S I V E D I A B E T I C P A T I E N T S D. H e y d a r i a n , K.Howorka, H.Grillmayr, H.Thoma, Ch. Schlusche, Institute for Biomedical Engineering and Physics, U n i v e r s i t y of Vienna, Vienna, Austria. In order to increase t h e r a p e u t i c efficacy and l o n g - t e r m c o m p l i a n c e in the t r e a t m e n t of secondary hypertension in diabetes, an outpatient education model was developed consisting of three modules: (I) Basic i n f o r m a t i o n on pathophysiology, training in self-measurement of blood pressure (BP), and non-pharmacological techniques for reducing BP (weight loss, reducing salt intake); (2) BP m e d i c a t i o n and adjustment of dosage a c c o r d i n g to self-measurements of BP; (3) summary and information on c a r d i o v a s c u l a r and renal risk factor reduction. A pilot study was p e r f o r m e d on 21 patients (/x• 44• d u r a t i o n of diabetes: 22• yrs). A follow-up examination (ambulatory assessment and w r i t t e n q u e s t i o n n a i r e ) was conducted 6• months after training ended and showed a change in number of BP self-measurements per week (median/Qi-Q3/) p r e - t r a i n i n g : 0.5 /0-7/; follow-up: 7 /3-13/; p<0.009), in BP (mmHG-systolic: 160 /140-180/; 125 /140-115/, p
ACUTE RENAL RESPONSE TO AN ACE-INHIBITOR AND A S E L E C T I V E B E T A - B L O C K E R IN INCIPIENT N E P H R O P A T H Y . M. M a u Pedersen, K.W. Hansen, C.K. Christensen, J.S.

C h r i s t i a n s e n and C . E . Mogensen. Medical D e p a r t m e n t M (Diabetes & E n d o c r i n o l o g y ) , A a r h u s Kemmunehospital, A a r h u s , Denmark. Angiotensin converting-enzyme inhibitors may be r e n o p r o t e c t i v e b e y o n d t h e i r a n t i h y p e r t e n s i v e action. However, it is unclarified w h e t h e r A C E - i n h i b i t o r s are p r e f e r a b l e to selective b e t a - b l o c k e r s in o r d e r to slow p r o g r e s s i o n of incipient a n d o v e r t diabetic n e p h r o p a t h y . I n 8 T y p e 1 ( i n s u l i n - d e p e n d e n t ) diabetic p a t i e n t s , k i d n e y f u n c t i o n was a s s e s s e d b e f o r e a n d a f t e r i n t r a v e n o u s injection of r e s p e c t i v e l y metoprolol 10 mg a n d enalaprilat 10 mg to elucidate p o s s i b l e d i f f e r e n c e s in a c u t e r e n a l haemodynamic r e s p o n s e . Glomerular filtration r a t e (clearance of ~ I iothalamate) was u n c h a n g e d with b o t h d r u g s . Metoprolol i n d u c e d r e d u c t i o n in r e n a l plasma flow (RPF, clearance of ~ q - h i p p u r a n ) (564 + 126 v s 528 + ]00 m l / m i n ] l . 7 3 m ~, 2p<0.03) and a r i s e in filtration f r a c t i o n (FF) and total r e n a l r e s i s t a n c e (TRR) (0.260 + 0.026 v s 0.273 _+ 0.026, 2p<0.04 and 0.178 + 0.038 v s 0.192 + 0.039 mmHg/ml/min, 2p<0.05). Urinary albumin excretion rate (UAE, RIA) was u n i n f l u e n c e d . As o p p o s e d to metoprolol, enalapril t e n d e d to i n c r e a s e RPF (576 + 97 v s 605 + 95 ml/min/1.73m ~, 2p=0.07), while FF a n d TRR were s i g n i f i c a n t l y r e d u c e d (0. 270 + 0.030 v s 0.251 + 0.032, 2p<0.05 a n d 0.174 + 0.027 vs 0.158 + 0.030 mmHg/ml/min, 2p<0.03). F u r t h e r m o r e UAE d e c r e a s e d (42.9 x]-: 1:8 v s 34.5 x / § 2.0 ~tg]min, 2p=0.05). Systemic blood p r e s s u r e was moderately r e d u c e d with enalapril, b u t u n c h a n g e d with metoprolol. T h u s in incipient n e p h r o p a t h y ACE-inhibition a c u t e l y b r o u g h t a b o u t s i g n s of e f f e r e n t r e n a l vasodilation a n d haemodynamic c h a n g e s compatible with a r e d u c t i o n in i n t r a g l o m e r u l a r p r e s s u r e . Metoprolol c a u s e d r e n a l v a s o c o n s t r i c t i o n with an i n c r e a s e in FF.

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DISCREPANT RENAL SHORT-TERM RESPONSIVENESS TO FELODIPINE, A CALCIUM ANTAGONIST, IN HYPERTENSIVE, INSULIN-TREATED PATIENTS WITH TYPE 1 AND TYPE 2 DIABETES MELLITUS

ACE-INHIBITION VERSUS CALCIUM CHANNEL BLOCKADE IN M~CRALBUMINURIC TYPE I (INSULIN-DEPENDENT) DIABETIC PATIENTS. H.J.G.Bilo, E.van Ballegooie, R.O.B.Gans, B.J.Potter van Loon, K.Bakker, E.P.J.Michels and A.J.M.Donker. Nijmegen, Amsterdam, Zwolle, Haarlem and Leiden, The Netherlands. In order to investigate possible differential effects of various antihypertensive drugs in type I (insulin-dependent) diabetes, we studied the acute effects of ACEinhibition (ACE, 25 mg captopril, n=7), calcium channel blockade (CCB, 20 mg nifedipine, n=8) and placebo (P, n=9) in normotensive, microalbuminuric patients. GFR (ml/min, 1251-iothalamate), ERPF (ml/min, 131I-hippuran), filtration fraction (FF=GFR/ERPF) and blood pressure (mmHg, BP) were measured at baseline (HL) and 1,2, and 3 hours after tablet ingestion. None of the variables changed in the placebo group. In the ACE-group, GFR remained stable (BL=140~18, 2h=140~20, mean+SD), ERPF rose (BL=547+96, 2h:592+110, +7.9~7.9~, p(O.O5, Wilcoxon) and FF feTl (BL=O.~6+O.03, 2h=0.24+O.02, p
E.

Jungmann,

Zeuzem, E.-H. Department of

P.-M.

Schumm-Draeger,

Scheuermann, Endocrinology,

T.

and K. Center

Nickelsen,

S.

Sch~ffling. of Internal Medicine, Johann Wolfgang G o e t h e - U n i v e r s i t y , T h e o d o r S t e r n - K a i 7, D-6000 F r a n k f u r t am Main 70, FRG Long-term e f f e c t s o f f e l o d i p i n e (Hoechst AG; 10 mg o n c e d i a l y ) were examined in a o n e - y e a r s t u d y in 8 p a t i e n t s w i t h t y p e ] d i a b e t e s [DM] ( g r o u p 1, a g e , 55 • 3 y e a r s , HbAI, 8 . 4 • 0 . 2 Z) and 9 p a t i e n t s w i t h t y p e 2 DM ( g r o u p 2, a g e , 65 § 2 y e a r s , HbAI, 9 . 0 § 0 . 6 %). F e I o d i p i n e consistently d e c r e a s e d b l o o d p r e s s u r e (group 1, 1 3 8 / 7 4 v s . 160/92 mmHg, group 2, 1 4 7 / 8 0 v s . 1 7 0 / 9 5 mmHg, both p < 0 . 0 5 ) . A f t e r 4 weeks t r e a t m e n t , u r i n a r y albumin e x c r e t i o n [UAE] was i n c r e a s e d by 143 % and f r a c t i o n a l a l b u m i n u r i a [UAEfract] by 83 % (both p < 0.05) in group 1. In group 2, UAE was d e c r e a s e d by 25 % and UA~fract by 48% (p < 0 . 0 5 v s . group 1 ) . After long-term t r e a t m e n t , UAE and UAEfract were d e c r e a s e d by 27 % (p ( 0 . 0 5 v s . 4 w e e k s ) i n group I and by 37 % in group 2. N e i t h e r r e n i n , a I d o s t e r o n e o r human a t r i a l n a t r i u r e t i c p e p t i d e l e v e l s n o r serum l i p i d s o r m e t a b o l i c c o n t r o l were s i g n i f i c a n t l y i n f l u e n c e d by f e l o d i p i n e t r e a t m e n t . Thus, t h e r e were p o t e n t i a l l y untoward r e n a l s h o r t - t e r m r e s p o n s e s t o f e l o d i p i n e i n type 1 DM d e s p i t e e f f e c t i v e b l o o d p r e s s u r e r e d u c t i o n . N e i t h e r in type I n o r i n t y p e 2 DM, h o w e v e r , was kidney function unfavourably i n f I u e n c e d by f e I o d i p i n e l o n g - t e r m t r e a t m e n t .

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EFFECTS OF ANGIOTENSIN CONVERTING ENZYME (ACE) INHIBITION ON CLINICAL COURSE OF ADVANCEDOIABETIC NEPHROPATHY. B. Schulz, E. Zander, J. Strese, G. Zander and G. Kirsch Central Institute of Diabetes "Gerhardt Katsch"~ Karlsburg, GOR This study aimed at assessing the effect of long-term ACE inhibition on the deterioration rate of kidney function in patients with renal failure due to diabetic nephropathy. Twenty Type i (insulin-dependent) diabetics with a mean diabetes duration of 26.5+2.5 years and serum creatinine concentrations of 197.2+1"2.8 pmol/l underwent ACE inhibiting treatment (captoprT1) during a prospective investigation period (B) of 15.1+l.l months. Results from this period were compared with those from a retrospective investigation period (A) of 26.1+7.9 months before captopril (i.e. furosemide, nifedipine, methyldopa) treatment in each patient. Dietary regimen and insulin treatment schedule were not changed durin 8 either period. After initiation of additional ACE inhibition we observed an improvement in blood pressure control from 157+4 to 150+5 mm Hg (p < O.Ol) after 18 months and a better metabolic--control (decrease of mean blood glucose levels and HbAI). The mean decline rate of glomerular filtration rate improved from -0.59 ml/min.month (A) to -0.007 ml/min.month (B) reaching a stabilization of kidney function. Serum creatinine concentrations exhibited marked increase (p
THE E F F E C T OF R A M I P R I L ON G L U C O S E T O L E R A N C E AND INSULINSENSITIVITY IN P A T I E N T S W I T H H Y P E R T E N S I O N E.K~enburg, M.Brunnbauer, B.Ludvik, G . S c h e r n t h a n e r and R. Prager; 2.Medical Department, U n i v e r s i t y of V i e n n a We t e s t e d the effect of Ramipril, a new ACEinhibitor on g l u c o s e tolerance and insulin s e n s i t i v i t y in 8 patients with b o r d e r l i n e essential h y p e r t e n s i o n (mean age 53 years, mean b l o o d p r e s s u r e 160/DSmmHg). A f t e r two weeks of p l a c e b o t r e a t m e n t we p e r f o r m e d an oral glucose t o l e r a n c e test w i t h 75mg glucose and an e u g l y c e m i c clamp test (insulin infusion 40mU/m2/min) w i t h m e a s u r e m e n t of glucose turnover. A f t e r two weeks of 5mg Ramipril d a i l y the p r o c e d u r e s were repeated. During the OGTT g l u c o s e was not s i g n i f i c a n t l y different b e f o r e and after Ramipril (0min: 86,gmg/dl• v.s. 95,7mg/dl• 60min: 126,4mg/dl• v.s. 160,3mg/dl• 120min: 106,8mg/dl• v.s. 109,1mg/dl• also insulin levels were identical b e t w e e n the two m e a s u r e m e n t s (0min: 12,2UE/mI• v.s. 9UE/mI• 60min: 69,6UE/mI• v.s. 67,7UE/mI• 120min: 53UE/mI• v.s. 41,1UE/mI• as well as Cpeptide levels (Omin: 3ng/ml~l,i v.s. 2,7ng/ml• 60min: 16,9ng/ml• v.s. 14,Dng/ml• 120min: 13,2ng/ml• v.s. 12,2ng/ml• During the euglycemic clamps there was almost no d i f f e r e n c e b e t w e e n the g l u c o s e infusion rate before and after Ramipril t h e r a p y (191,6mg/m2/min~60,6 v.s. 204,7mg/m2/min• In c o n c l u s i o n 2 w e e k s of R a m i p r i l - t r e a t m e n t had no significant effect on glucose tolerance and insulin sensitivity in patients w i t h essential hypertension. The clinical b e n e f i t or improvement of glucose t o l e r a n c e during treatment with A C E - i n h i b i t o r s seems to be of m i n o r clinical relevance in b o r d e r l i n e h y p e r t e n s i v e patients.

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METABOLIC EFFECTS OF FIVE DIFFERENT ANTIHYPERTENSIVE AGENTS IN NON-INSULIN DEPENDENT DIABEFES MELLITUS JV Anderson,SlruthersAD, BallJA, Bun/n JM, Kohner EM, HealyMJR and Bloom SR. R.P.M.S.,H.mmersmith Hospital,London WI2 01LS,U.K. The~ has~ o u s l y b ~ no comparativestudyofthemetaboliceffectsof thiazides,betablockers,and thenewer classesof . . t i h ~ v e s inthesame gro~ ofdiabetic1~iems.

PROGNOSIS OF D I A B E T I C PATIENTS W I T H E N D - S T A G E RENAL DISEASE Derfler Kurt, P.Balcke, M.Hirschl, A.C.Hauser, W. Waldh~usl. I.Med.Dep.,Univ.of Vienna,Austria.

Aft~ a nm-in~od r~kioginertmedicmon toconr~m the~sence of hy~on, 19 patients[aged54.4 • 7.I(SD) years;bodyweight 86.4 • 14.2(SD)kg] enteredthe study. Dinbetictherapycontinuedunchanged. Patients we~ treatedwitheach of sixdifferentdeags(bendrofluazide2.5 rag,atenolo150 rag,amilurMe5 rag,nifedipinedow release10rag,c a ~ 12.5mg and placebo)inarandomiseddoubleblindplacebocontrolledfashion,eachgiven twicedailyfer6 weeks. Pulserate,bloodpt~sure, standardbiochemistry, fastingbloodglucose,glycosylatedhaemoglobin, averagegluco~ teststripscore, 24hoururineglucose,plasmalipidsandintermediarymetabclitesweremeasured. Resultswere analysedusinganalysisofvariance All medicatio~nslowered blood i z e , ~ . Catalinal with placebo b~ a n z i d e raised mean fasting glucose (9. 8 cf 7.4 mmol/l), glycosylated hacmoglob/n (8. 8 d 7.4% ),24 hoururineglucose(30 d 13 mmcl]24 h )and causedhypokalaemia, alkalosis,hyperuricaemiaandhypokalaemia(P<0.05throughout).Nifedil~ine significantlywooJenedglycosylatedhaemoglobinand24 h urineglucosebutnot fastingglucose.Effectsonlil~dandintermediurymetaboliteswereminur. Despketheircontinuedrecommendation,we feelthiazidesshouldno longerbe used. Nifedipinemayworsen glucosetolerance.The otherdrugsstudiedshould be considered a~fir~line M t e ~ v e s .

During the last 15 years diabetic patients with end-stage renal disease (M=102; 35 IDDM, 67 NIDDM) were treated by chronic hemodialysis (HD). In 37 of these renal t r a n s p l a n t a t i o n (RT) was performed.This retrospective study evaluates the outcome and factors implicated in the previously described poor prognosis of such patients. Patients With VC Without V C HD-patients:{N) 35 30 Age (years) 56• 54• Years / diabetes 17.5• 16.5• HD-treatment/month 5.2• 15• Died N (%) 34 (97%) 25 (83%) RT-patients:(N) 7 30 Age (years) 51• 48• Years / diabetes 20.1• 18.8• Months after RT 14• 24.1• Died N (%) 4 (57%)* 8 (27%)* *vs.*p<0.001; V C = v a s c u l a r complications present prior to the start of HD treatment (myocardial infarction 29%; stroke 26%; peripheral gangrene / amputation 50%). 85% of these severe VC occurred at a serum-creatinine > 6 mg/dl. Decreased survival was related to age and duration of diabetes, but not to its type. However, vascular complications present prior to the onset of HD treatment /RT,demonstrated to be decisive for the worse prognosis of these patients. This fact may be partially improved by early start of HD treatment or RT.

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DIABETIC END-STAGE RENAL DISEASE IN TYPE I (INSULIN-DEPENDENT) DIABETES MELLITUS. N.E. FRANDSEN, A. GREEN AND A. K. SJOLIE. DEPARTMENT OF ENDOCRINOLOGY ODENSE UNIVERSITY HOSPITAL AND INSTITUTE OF CLINICAL GENETICS ODENSE UNIVERSITY, DK-5000 ODENSE.

SEVERE NEPHROPATHY IN TYPE 2 DIABETES. J.J. Altman, R. Hakim, C.A. Dupuy, F. Daniel, R. Patte, N. Simon and H. KUntziger ~. Centre H o s p i t a l i e r PasteurVal]ery-Radot 26, rue des Peupliers 75013 Paris, France.

All i n s u l i n - t r e a t e d patients (n=413 male and 314 female) mith diagnosis and living in the Fyn County, Denmark, as of July i. 1973 were studied regarding the p r e v a l e n c e and s u b s e q u e n t d e v e l o p m e n t of e n d - s t a g e renal disease (ESRD) during the period July 1. 1973 to June 1. 1981. At p r e v a l e n c e date, 5 p a t i e n t s ( p r e v a l e n c e p r o p o r t i o n = 0,7%) had ESRD. During the followup period all these p a t i e n t s died, a d d i t i o n a l l y 30 p a t i e n t s d e v e l o p e d ESRD of ~hich 26 died. As of June i. 1981 4 p a t i e n t s out of 627 r e m a i n i n g living p a t i e n t s had ESRD ( p r e v a l e n c e p r o p o r t i o n = o,6%). The average annual risk of d e v e l o p i n g ESRD in this p o p u l a t i o n ~as e s t i m a t e d at 0,88% and 0,45% for male and female patients, r e s p e c t i v e l y . E s t i m a t e d c u m u l a t i v e life time risks of d e v e l o p i n g ESRD for p a t i e n t s from age 20 years ~ere 35% and 20~ for male and female patients, r e s p e c t i v e l y . This e p i d e m i o l o g i c a l survey c o n f i r m s that ESRD r e p r e s e n t s a serious burden to p a t i e n t s with Type I (insulindependent) diabetes, the a p p a r e n t lo~er p r e v a l e n c e of ESRD is e x p l a i n e d by high m o r t a l i t y a s s o c i a t e d with ESRD.

We have p r o s p e c t i v e l y studied d e c l i n e of glomerular f i l t r a t i o n r a t e (GFR) of 40 uremic d i a b e t i c out p a t i e n t s dur i n g 3 years and e s t a b l i s h e d the epidemiology of diabetes in 286 p a t i e n t s on d i a l y s i s (D) in our h o s p i t a l . 30 % of the out p a t i e n t s were type 2 d i a b e t i c s (M • SD) : age = 59 • 9 years, d u r a t i o n o f disease = 17 • 7 years, of high blood pressure (HBP) = 76 • 64 months, and of renal i n s u f f i c i e n c y (RI) = 35 • 18 months. I n i t i a l c r e a t i n i n e c l e a rance was 52 • 7 m l / m i n / l . 7 3 m2 and 27 • 23 a f t e r 3 years. Degradation of GFR was 0.00015 • 0.00008 I/umol/month. Type l d i a b e t i c s had the same dur at i on of HBP and RI, but decline of GFR was slower (O.O0010 • 0.00008, p < O.Ol). Among a l l our p a t i e n t s on D, 26 % are d i a b e t i c s with 45 % o f type 2. D was s t a r t e d a f t e r 70 years in 27 % of them. In 1989, incidence of type 2 d i a b e t i c s o l d e r than 70 years s t a r t i n g D was 20 %. With the same l evel of blood pressure and glucose c o n t r o l , d i a b e t i c nephropathy is more r a p i d l y progressive in type 2 where c o n s e r v a t i v e treatment is needed. RI leading to D has a growing incidence in type 2 p a t i e n t s . The s t r a t e g y of renal replacement therapy f o r those old p a t i e n t s who are not going to be g r a f t e d w i l l be a major concern the next decade.

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THREE YEARS FOLLOW-UP OF TYPE 1 DIABETIC PATIENTS AFTER KIDNEY TRANSPLANTATION A. Ekstrand, E. P e t t e r s s o n , C. G r 6 n h a g e n - R i s k a , L. L a a t i k a i n e n , E. Laasonen, E. M a t i k a i n e n , J. Ahonen, L. Groop, H e l s i n k i U n i v e r s i t y H o s p i t a l , Helsinki, Finland Twenty-nine type 1 diabetic patients (DM) (13F/16M, age 39+1 yrs, d u r a t i o n of d i a b e t e s 26+1 yrs), w h o r e c e i v e d a k i d n e y g r a f t d u e to endstage renal failure were followed for 3 y e a r s a f t e r k i d n e y t r a n s p l a n t a t i o n (Tx) . T e n non-diabetic kidney-transplanted patients s e r v e d as c o n t r o l s (4F/6M; age 41+5 yrs). F i v e DM patients died during follow-up (3 cardiov a s c u l a r events, 2 infections) . H b A I d i d not significantly change during follow-up (from i0.6+0.5% to 9.5+0.6%) . Subcutaneous fat m e a s u r e d f r o m c a l f CT s c a n i n c r e a s e d in DM f r o m 5.0+--0.6 to 6.1+_0.9 m m (p<0.05). P r e v a l e n c e of neuropathy remained unchanged during follow-up (41% vs 38%). M o t o r b u t n o t s e n s o r y c o n d u c t i o n velocity measured from nervus medianus i n c r e a s e d in DM (MCV: 4 6 . 9 + 1 . 0 vs 5 0 . 3 + 1 . 4 m/s, p < 0 . 0 5 ; SCV: 4 1 + 1 . 8 v s 3 8 . 7 + 3 . 0 m/s, p=NS) . Autonomic neuropathy was uninfluenced by Tx (expiratory/inspiratory ratio i. 0 6 + 0 . 0 1 vs 1.06+-0.01). B e f o r e Tx p r o l i f e r a t i v e r e t i n o p a t h y w a s o b s e r v e d in 69% of DM p a t i e n t s ; 20% of t h e m improved, 70% remained unch.a n g e d while 10% w o r s e n e d a f t e r Tx. P r o g r e s s i o n of c o m p l i c a t i o n s was not related to glycemic control. In conclusion, progression of microand m a c r o v a s c u l a r c o m p l i c a t i o n s in t y p e 1 d i a b e t i c patients is not influenced by Tx . N o r m a l i z a t i o n of k i d n e y f u n c t i o n r a t h e r t h a n improved glycemic control enhanced motor conduction velocity during the first 3 years a f t e r Tx.

COMPARATIVE STUDY OF E.COLI ADHERENCE ON UROEPITHELIAL CELLS OF DIABETIC AND NON-DIABETIC WOMEN N.Galanakis,Ph. Philippides,S.Pentea,A.Tzonou,Th. Kountouris Mavroudis,E.Hatzimichelakis and H.Giamarellou.lst Dept.Propedeutic Medicine,Athens University School of Medicine, Laiko General Hospital,Athens,Greece. The role of E.coli adherence on Uroepithelial Cells (UC) regarding the promotion of Urinary Tract Infections (UTIs) in diabetic subjects is still unknown. Therefore the adherence ability of an E.coli(0:75,H:12) strain on UC of 45 diabetic women(23 type 1 and 22 type 2 with mean age 37.5 • SE 2,13 ys) without any evidence of previous UTIss was studied in vitro by a method described by C.SwangorgEden(Infect Immun 1977,18:767-774) in comparison with an age-matched control group of 25 healthy women. The degree of glycosuria was measured in urine samples by a glycose specific enzymatic method. Statistical analysis of the results was performed by discriminant analysis and multiple linear regression method,E.coli adherence on UC was sigDificantly increased in the diabetic group [1202 (959-15"03) vs 428(283"646) bacteria per 50 UC,p<0.00~ .A statistically significant correlation(p = 0.04) betweeen E.coli adherence and degree of glycosuria was observed,while no correlation was found as regards age,duration and type of diabetes. It is concluded that diabetic glycosuria is positively associated with increased E.coli adherence on UC, thus, contributing to a higher incidence of UTIs in diabetic women.

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PS 15 Experimental Nephropathyand Diagnosis P535

P534 STEREOLOGICAL

STUDIES

OF

EARLY

PHASES

OF

DIABETIC GLONERULOPATHY R. ~ s t e r b y , H.J. Bangstad, K.F. Hanssen, S.M. Mauer, M.W. Steffes, G.C. Viberti and J. V~alker. Institute of Pathology, Aarhus Kommunehospital, DK-8000 Aarhus C, Denmark, University of Minnesota, USA, Aker Hospital, Oslo, Norway and Guy's Hospital, London, England. A detailed s t u d y of individual glomeruli from Type 1 (insulin-dependent) diabetics was done with the aim of pinning down the source of variation in mesangial volume fraction, to improve the reliability of this structural estimate, and to s t u d y the correlation between glomerular volume and expression of glomerulopathy. 3 or 4 glomeruli per biopsy were sampled b y a I0 ~t-stepwise sectioning into the glomernlus, p r e p a r i n g sections for light microscopy with 10b intervals exhaustively t h r o u g h the glomerulus. Sections for electron microscopy were cut with 30 or 60 ~ intervals, the set of 3-7 levels located with a random position. The distance between levels was estimated, enabling calculation of individual glomerular volumes. At each electron microscopy-level photomontages of the profile were produced for the estimate of mesangial volume fraction. Micrographs were sampled from the largest profile for determination of basement membrane thickness. This protocol is p r e s e n t l y being applied to diabetics with or without incipient nephropathy. The results showed that the intra-glomerular variation in mesangial volume fraction constitutes a major part of the variation in this parameter, so that estimates at more than one level p e r glomerulus improves the reliability of the parameter. No correlation obtained between c u r r e n t glomerular volume and the s e v e r i t y of glomerulopathy.

MAGNETIC RESONANCE IMAGING OF THE KIDNEY IN TYPE i (INSULIN-DEPENDENT) DIABETIC PATIENTS WITH NEPHROPATHY. G.Rankel, R.Mangili, A.Sironi, M.Makarovic, G.Zerbini, L.Gianolli, A.Del Maschio and G.Pozza. Istituto Scientifico San Raffaele, Milan, Italy. Magnetic resonance scans of the human kidney are characterised by higher signal intensity in the cortical than in the medullary area. Such difference can be quantified by calculating the cortical/medullary signal intensity ratio (CMR), which may be abnormally low in some chronic non-diabetic glomerular diseases. We investigated whether this may be an early phenomenon in diabetic nephropathy by measuring the CMR in 9 patients with Type 1 (insulin-dependent) diabetes mellitus and overt nephropathy, 9 patients with microalbuminuria, 8 normoalbuminuric patients of similar age and duration of diabetes and 8 normoalbuminuric patients with diabetes of short duration. The CMR was similar among normoalbuminuric groups (1.47• and 1.39• mean• and microalbuminuric patients (1.47• 2P=NS), but was significantly lower in patients with clinical nephropathy (i.18• 2P<0.001). Glycaemie control was similar in patients with microalbuminuria and in those with overt nephropathy (HbAIc: 9.8• vs 10.3+-3.5%, 2P=NS) and could not explain the difference in CMR. Among all patients with elevated urinary albumin excretion, glomerular filtration rate (51Cr-EDTA GFR) was directly related with the CMR (n=13; r=0.797, 2P<0.005). We suggest that abnormally low CMR is a late feature in diabetic nephropathy, probably reflecting intrarenal haemodynamic changes secondary to reduced GFR and/or interstitial fibrosis.

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URINE ALBUMIN/CREATININE RATIO ESTIMATES ALBUMIN EXCRETION AND PREDICTS NEPHROPATHY IN TYPE 2 (NON-INSULIN-DEPENDENT) DIABETES. P.H. Bennett, R.G. Nelson, W.C. Knowler, S.A. Hardy, G.W. Williams, D.J. Pettitt, M.F. Saad, W.E. Mitch, and B.D. Myers for the Diabetic Renal Disease Study and the National Institute of Diabetes and Digestive and Kidney Diseases, Phoenix, AZ, Cleveland, OH, Atlanta, GA, and Palo Alto, CA, U.S.A.

E V A L U A T I O N OF A NEW S E M I - Q U A N T I T A T I V E D I P - S T I C K FOR M I C R O A L B U M I N U R I A (MICRAL-TEST). H.-J. Bangstad, K. Try, K. D a h l - J ~ r g e n s e n and K.F. Hanssen. Aker U n i v e r s i t y Hospital, Oslo, Norway. The m e a s u r e m e n t of u r i n a r y albumin e x c r e t i o n rate (UAR) is b e c o m i n g an important p a r a m e t e r of clinical care of b o t h insulin and n o n - i n s u l i n d e p e n d e n t diabetics. We compared a semiq u a n t i t a t i v e d i p - s t i c k (Micral-Test, BoehringerMannheim) w i t h test areas 0, i0, 20, 50 a n d i00 mg/l w i t h an i m m u n o t u r b i d i m e t r i c m e t h o d (CV 5%). Timed overnight urines (albumin < 200 mg/l) from 181 patients w i t h diabetes m e l l i t u s were examined. The c o r r e l a t i o n c o e f f i c i e n t between d i p - s t i c k and i m m u n o t u r b i d i m e t r i c concentration was 0,82 and d i p - s t i c k and UAR 0,46. We defined the UAR as normal b e l o w 15 ug/min. 116 of 181 of the urine samples were b e l o w this level. A d i p - s t i c k response of I0 mg/l or less identified 93 of the 116 n o r m o a l b u m i n u r i c patients, h o w e v e r 16 of the 65 w i t h elevated UAR were also included. The sensitivity, spesi•177 positive and negative p r e d i c t i o n value of the d i p - s t i c k m a r k e d 20 mg/l or m o r e were 75,4%, 87,7%, 72,0% and 85,3% respectively. D e f i n i n g e l e v a t e d a l b u m i n e x c r e t i o n as > 20 mg/l changed this figures to 94,1%, 81,5%, 66,7% and 97,2%. We conclude that this s e m i - q u a n t i t a tive d i p - s t i c k test has an a c c e p t a b l e correlation to a t u r b i d • m e t h o d in a clinical setting. It is sensitive in d e t e c t i n g e l e v a t e d albumin e x c r e t i o n (< 20 mg/l), but not s u f f i c i e n t l y sensitive to detect slightly e l e v a t e d albumin e x c r e t i o n rate d e f i n e d as > 15 ug/min, in o v e r n i g h t urine.

Urine albumin to creatinine ratios (ACR) and urine albumin excretion rates (AER) measured over 2h were compared to determine if the ACR ac~rately estimates AER. Urine volume and albumin and creatinine concentrations were measured in timed urine specimens from 120 Pima Indians; 25 with normal glucose tolerance, 23 with impaired glucose tolerance, and 72 with Type 2 (non-insulin-dependent) diabetes. A highly significant linear correlation was found between log ACR and log AER (r = 0.97). To determine the clinical significance of ACR, the incidence of overt nephropathy (ACR >_300 mg/g) was determined by proportional hazards in 320 diabetic Pima Indians aged >25 years who had normal albumin excretion (ACR < 30 mg/g) or microalbuminuria (30-299 mg/g) and were followed an average of 3.8 years. Compared to subjects with an ACR <30 mg/g, those with an ACR of 30-99 mg/g bad 9.9 times (95% confidence interval 3.9-25.2) the incidence of overt nephropathy; and those with an ACR of 100-299 mg/g had 18.8 times (95% confidence interval, 7.7-45.4) the incidence. Thus, in Pima Indians ACR reliably estimates the AER, and a single ACR predicts overt nephropathy.

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HYPERINSULINAEMIA IN NORMOGLYCAEMIC MT-HGH TRANSGENIC MICE DEVELOPING GLOMERULOSCLEROSIS

RENAL INSULIN-LIKE G R O W T H FACTOR 1 BINDING IN EXPERIMENTAL DIABETES S.M. Marshall, L. Korsgaard, A. Flyvbjerg, J. Frystyck and H. Orskov. Department of Experimental Medicine, Aarhus University, Denmark.

Th. Buchm~ller*, R. Wanke**, E. Wolf***, V. Maier*, W. Hermanns** and G. Brem ~**

F. Bischof*,

*

Medizinische Klinik und Poliklinik der Universit~t Ulm ** Institut f~r Tierpathologie, Ludwig-Maximi!iansUniversit~t Ulm *** Molekulare Tierzucht, Ludwig-Maximilians-Un• M~nchen Transgenic mice (NMRI) harbouring methallothionein-human growth hormone (MT-hGH) fusion genes were produc%d using the microinjection technique. Transgene-expression resulted in high serum-lewels of hGH ranging from 8 to 600 ~g/ml. Clinico-chemical and pathological investigations were carried out in 20 transgenic mice (mean age 113 days) and an equal number of controls. Insulin levels were measured by RIA using rat insulin as calibration standard. Fasting insulin levels of the transgenic mice were significantly increased as compared to controls, in several animals up to 3-fold. Serum concentrations of glucose in the transgenic animals did not differ from controls. Creatinine, urea, potassiom, cholesterine, and total protein concentrations were significantly elevated in the transgenic mice. Trigly~ ceride levels remained unchanged. Renal failure was found to be due to pathological lesions of the kidneys, which were a constant finding and were predominantly characterized by diffuse segmental or global glomerulosclerosis associated with signs of proteinuria. The glomeru~ar lesions in MT-hGH transgenic mice mimicked diabetic glomerulopathy in man. However, clinico-chemical data did not indicate diabetic metabolic Changes.

Renal growth after the induction o f experimental diabetes is preceeded by a rise in the renal content o f insulin-like growth factor I(IGF-1). To clarify whether this increase is due to local synthesis or uptake from the circulation, we have measured IGF-1 binding to plasma membranes from control, diabetic and insulint r e a t e d d i a b e t i c m a l e W i s t a r r a t s , b o d y w e i g h t 190+3 g(mean+SEM). W e t kidney weight after 7 days of diabetes was 988+20 mg in the diabetic group, 828_+20.4 in the insulin-treated group and 849-+17.9 in the controls (p<0.01). The kidney content of IGF-1 rose from 505+40 ng/g wet kidney weight to 674_+30 on day 2 (p<0.05) in the diabetic group but remained constant in the control and insulin-treated animals. Maximum IGF-1 binding was 8.2+1.4 x l 0 u mol/25ug membrane protein and affinity constant 7.0+0.15 x l 0 s on d a y 0 a n d b o t h r e m a i n e d u n c h a n g e d throughout the study in the Control and diabetic groups. In the insulin-treated group, m a x i m u m binding rose to 11.0+1. l x l 0 -n mol/25ug on day 7(p<0.05) and affinity constant decreased to 6.1+0.1 l x l 0 s. We conclude that IGF-1 binding to renal cell membranes in untreated diabetic rats is unchanged for 7 days after the induction of diabetes, despite an acute rise in the renal content of IGF- l.

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HIGH GLUCOSE STIMULATES PRODUCTION OF TYPE N COLLAGEN AND LAMININ P1 FROM HUMANENDOTHELIAL CELLS. K. N e g i s h i , S. M o r i t a n i , T. Watanabe, S. Shunto, N. Sevizawa, M. Suzuki, M. Takahashi, S. Katayama, 3. I s h i i and S. Kawazu, Saitama and Gunma, Japan

LOCALIZATION OF T A M M - H O R S F A L L G L Y C O P R O T E I N IN S T R E P T O Z O T O C I N DIABETIC RAT KIDNEYS.

I n c r e a s e d p r o d u c t i o n o f basement membrane components such a s type f f c o l l a g e n and l a m i n i n from t h e e n d o t h e l i a l c e l l s may be linked to t h e development o f d i a b e t i c c o m p l i c a t i o n s . In t h i s s t u d y we i n v e s t i g a t e d t h e e f f e c t t h e e f f e c t of high g l u c o s e on t h e p r o d u c t i o n of type c o l l a g e n and l a m i n i n P1 from c u l t u r e d human u m b i l i c a l v e i n e n d o t h e l i a l c e l l s ( HUVEC ). HUVEC were c u l t u r e d d i f f e r e n t c o n d i t i o n s a t c o n f l u e n c e , and t h e c o n c e n t r a t i o n s of t y p e N c o l l a g e n and l a m i n i n P1 in t h e medium were measured by type N c o l l a g e n s p e c i f i c EIA newly developed and lamini P1RIA k i t ( Hoechst Co. ). We compared c o n t r o l ( 5mM D - g l u c o s e ) with 30mM D - g l u c o s e ( HG ), 30mM L - g l u c o s e ( LHG ) a t 48hr or 96hr i n c u b a t i o n period. HG i n c r e a s e d the p r o d u c t i o n of type N c o t l a g e n n e a r l y 3 f o l d ( p<.O1 ) to t h e c o n t r o l l e v e l s both a t 48hr and 96hr i n c u b a t i o n . T h i s e f f e c t was i n c u b a t i o n t i m e dependent and r e p r o d u c i b l e . LHG did n o t a f f e c t type c o l l a g e n p r o d u c t i o n from HUVEC. In c o n t r a s t , t h e l e v e l s of l a m i n i n P1 in HG and LHG were not s i g n i f i c a n t l y d i f f e r e n t from t h o s e in c o n t r o l . Morphorogical s t u d i e s showed t h a t t h e s e e f f e c t s o f HG and LHG medium did not appear to a c c o u n t f o r HUVEC d e s t r u c t i o n and growth. These r e s u l t s s u g g e s t t h a t i n c r e a s e d p r o d u c t i o n o f type N c o l l a g e n from HUVEC i s induced by HG but n o t LHG.

N.O. Jacobsen and R. Rasch. Institute of Pathology, A a r h u s Kommunehospital, Aarhus, D e n m a r k and D e p a r t m e n t of Cell Biology, Institute of Anatomy, University of Aarhus, A a r h u s Denmark. In streptozotocin (SD) diabetic rats glycogen accumulations are present in the thick ascending limb of Henies loop (TAL); the Armanni-Ebstein lesion (AE). Since A E is present in the n e p h r o n segment which contains the Tamm-Horsfall glycoprotein (TH) we studied the histochemical distribution of this protein in SD rats with 10 days diabetes duration. The kidneys were perfusionfixed with 4% formaldehyde, e m b e d d e d in paraffin and 4 u thick sections were exposed to an immunoperoxydase reaction with an antibody against TH. The volume density of stained cells was m e a s u r e d by a morphometric technique and expressed in % of the m e a s u r e d kidney zones. T h e result of the studies showed, that the volume density of stained cells was significantly lower in cortex in the diabetic animals than in the controls; 1.5 and 4.5 %, respectively, and the staining was irregular. In medulla the central part of the inner stripe was unstained in diabetes. The T H is of importance for establishment of the low water permeability of the T A L and hence for the formation of the concentration gradients for the countercurrent multiplier system in the kidney. A decrease in T H in diabetes could be of significance for the abnormal kidney function in diabetes.

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Impaired mesangial basement membrane heparan s u l f a t e proteoglycan synthesis caused by elevated glucose. E.D.Schleicher,B.Olgem611er,S.Schwaabe,E.M.Wagner,K.D.Gerb i t z , Munich, FRG Thickening o f the glomerular basement membrane and expansion o f the mesangial matrix associated with compositional changes i s a c h a r a c t e r i s t i c f e a t u r e o f d i a b e t i c nephropathy. We have studied synthesis o f f i b r o n e c t i n and basement membrane heparan s u l f a t e proteoglycan in c u l t u r e d porcine mesangial c e l l s . Basement membrane associated heparan s u l f a t e proteoglycan (HSPG) was determined by enzyme immunoassay using an antiserum r e c e n t l y shown to s p e c i f i c a l l y recognize the core p r o t e i n o f HSPG. Bath f i b r o n e c t i n and HSPG were observed to be synthesized during the l o g a r i t h mic phase of growth. High e x t r a c e l l u l a r glucose l e v e l s decreased HSPG content o f c e l l s in a concentration dependent manner. 40 mmol/1 glucose reduced HSPG-synthesis by about 50 Z. F i b r o n e c t i n synthesis was only s l i g h t l y r e duced. P r o l i f e r a t i o n and metabolism o f c e l l s was not a f fected as i n d i c a t e d by constant DNA and unchanged l a c t a t e production, r e s p e c t i v e l y . No s i g n i f i c a n t e f f e c t o f i n s u l i n on basement membrane components was found n e i t h e r f o r high

THE EFFECT OF INSULIN ON MACULA DENSA IN DIABETIC RATS.

nor for low glucose. Similar results were obtained with porcine aortic smooth muscle cells, another cell type of mesenchymal origin. Our results indicate that elevated glucose levels may be the underlying cause of dysregulations of basement membrane associated components leading to the development of diabetic nephropathy.

R. Rasch. Department of Cell Biology. Institute of Anatomy. University of Aarhus, Aarhus, Denmark. Kidney autoregulation is located to the juxtaglomerular apparatus (JGA). The macula derma senses the composition of tubular fluid and consequently the glomerular arterioles are activated. Kidney function is abnormal in diabetes and structural abnormalities are present in the JGA. The effect of insulin on the abnormal macula derma (MD) lateral intercellular spaces (LIS) has been studied in SD rats after 50 days duration of diabetes. SD animals were given one dose of 6 i.u.(DIxl) of insulin (Heat treated Ultralente for rats, N o v a ) and studied 4 hours later when blood glucose concentration (BGC) was normalized. The insulin effect was also studied after 5(DI5) and 15(DI15) days normalization of BGC. The kidneys were perfusion fixed with 1% glutaraldehyde and embedded in Epon. On electron micrographs volume density of the LIS was measured as fraction (%) of the MD region. Results. Volume density C D Dlxl DI5 DI15 (%) (n=6) l(h0.7 1.2_+0.2 8.3.+5 9.5_+2 10.2.+0.3 The MD LIS are small in diabetic animals and insulin treatment normalizes LIS fast and continuously. The dependency of LIS on BGC indicate that glucosuria probably is of significance for the autoregulatory function in diabetes.

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ACARBOSE INHIBITS EXCESS NONENZYMATIC GLYCATION (NEG) AND ADVANCED GLYCATION END PRODUCT (AGEP) FORMATION IN DIABETES. M.P.Cohen, University City Science Center, Philadelphia, PA. Excess NEG and increased AGEP formation of matrix proteins are believed to play important roles in the pathogenesis of diabetic complications. Direct evidence that modest hyperglycemia increases NEG and AGEP of matrix proteins in vivo, and that reduction of integrated blood glucose levels influences these processes, is fundamental in evaluating the therapeutic potential of new agents for treatment of diabetes. We examined the ability of Acarbose, an alpha-glucosidase inhibitor that restricts post-prandial glycemic excursions, to influence the level of NEG in glomerular basement membrane (GBM) and collagen AGEP formation. Three groups of experimental animals, maintained for 8 weeks after induction of diabetes, were used: age-matched control (C), streptozotocin diabetic (SD), and Acarbose-treated SD ~-SD) rats. Blood glucose and HbAlc levels were increased in all diabetic rats, but HbAlc levels were significantly less in A-SD than in SD rats. Kidney-to-body weight ratios were significantly increased in SD rats but were completely normal in A-SD rats. NEG of GBM was significantly greater in SD than in C animals (.362+--41vs.200+23 ug glycated lysine/mg GBM), but did not differ significantly from control values in the A-SD rats (290~54 ug/mg). AGEP, determined as fluorescence normalized to protein or OH-proline content, was significantly increased in skin (1100+170 vs.670+240) and tail tendon (1430+250 vs.890+135)_ col~agen of SD--rats, but did not differ from control in skin (865+210) and tail (530+75) of A-SD rats. The results indicate ~hat increased NEG ~nd AGEP formation occur with modest hyperglycemia, and that Acarbose improves integrated glycemic control, prevents the renal hypertrophy, and reduces the increased NEG andAGEPformation associated with experimental diabetes. The results further suggest that the anti-hyperglycemic effects of Acarbosehave a salutary influence on the pathogenesis of diabetic nephropathy.

PREVENTIVE EFFECT OF EXERCISE TRAINING ON DIABETIC RATS WITH NEPHROPATHY K.Nomura, K.Utsunomiya, N.Yoshizawa, H.Kurata and Y.Ikeda, Tokyo, Japsn The effect of chronic exercise training on diabetic nephropathy was studied in streptozotocin (STZ) - diabetic rats. Male Wistar rats (200g) were randomly assigned to diabetic exercise group (DM-E, n=lS), diabetic sedentary group (DM-S, n=15), non-diabetic exercise group (NC-E, n= 5) and non-diabetic sedentary group (NC-S, n=5). Diabetes was induced with STZ 40mg/kg. After the induction of diabetes, exercise groups were subjected to swimming in a water bath 60 minutes a day For 3 months. Blood sampling and 24 hours urine collection were repeated every each month. At 3 months, urinary albumin excretion was increased in DM-S compared to NC-S (4.0 h i.i vs 1.3 ! 0.5 rag/day p<0.01). Urinary albumin was significantly reduced in DME (2.4 + I.i rag/day, p
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ALDOSE REDUCTASE AND CELLULAR OSMOREGULATION T.C. Hohman and D. Carper*, Wyeth-Ayerst Research, CN 8000, Princeton, NJ 08543 and *The National Eye Institute, Building 6 Room 232, National Institutes of Health, Bethesda, MD 20892

Direct s t i m u l a ~ r y action of insulin on the plasma membrane Ca ~- pump of kidney basolateral membrane: the effect of high fat feeding

Compelling evidence from animal studies has implicated aldose reductase (AR) in the development of the long-term complications of diabetes. Recent studies have suggested that AR catalyzed sorbitol production may also be involved in cellular osmoregulation. In these studies hypertonically stressed renal medullary cells adjusted their tonicity by accumulating sorbitol. Our aim was to determine whether a similar response occurs in other cells. AR activity, sorbitol accumulation and cellular viability and proliferation were measured in osmotically stressed glomemlar endothelial cells. Transfer of cells from normal (310 mosm/kg) to hypertonic medium (610 mosm/kg), resulted in sorbitol accumulation (after 8d, 311 _+33 nmol/mg protein). Simultaneously, AR activity increased 32-fold. Cellular myo-inositol (MI), another potential cellular osmolyte also rapidly increased from 39 4- 4 nmol/mg protein to a plateau of 214 + 22 within 48h. Similar results were obtained with kidney mesangial and lens epithelial cells. Hypertonic stress decreased cell viability and proliferation by 50-70%; however, AR inhibition with tolrestat, or MI removal from the media caused no further decrease. We conclude that neither sorbitol nor MI accumulation is essential for the survival of hypertonically stressed cells. The results also suggest that these cells may have additional mechanisms for responding to osmotic stress.

K. Nagy, L. Romics 3rd Department of Medicine,

Semmelweis Univ.Med.Sch.

High fat feeding causes insulin resistance in ~ boratory animals and man. Abnormal cellular C a ~ homeostasis was proposed as a common m e c h a n i s m leading to insulin resistance in n o n - i n s u l i n dependent diabetes and obesity. Therefore our aims were: I ~ to prove the direct a@~ion of insulin on the plasma membrane (PM) Ca =T pump in kidney basolateral membrane (BLM) 2. to d i s t i ~ $ u i s h the insulin effect on the PM a s s o c i a t e d Ca~---ATPase activity 3. to detect the effect of high fat feeding on these two activities. S p r a g u e - D a w l e y rats were given high fat diet (31,4% fat~ 32,1% carbohydrate, 25,6% protein) for 5 weeks after weaning. New h o m o g e n i s a t i o n method was worked out %o incorporate insulin into i n s i d e - o u t s i d e vesicles. S u b s e q u e n t l y calcium transport and Ca~+ ATPase activities were measured. Results: I. In control membranes insulin stimulated calcig~ transport which was ATP, C a l m o ~ l i n and Mg ~- dependent lowering the Km for Ca~-while leaving the Vmax identical. 2. The insulin s t i m u l a t o r y effect on the PM associated Ca~--ATPase was nucleosid aspecific, calmodulin ig{ependent and required only submicromolar Mg- . 3. High fat feg{ing increased the Km of the pump enzyme for Ca ~- and decreased its stimulation by insulin by 30%. ,In summary w ~ + e m p h a s i s e the direct stimulation of the PM Ca pump of kidney BLM by insulin and the necessity to d i s t i n g u i s h this action from the PM associated aspecific CaS+-ATPase activity. High fat feeding impairs insulin action also by interfering with the calcium pump en~me which might contribute to %he insulin resistant cond~ion.

PS 16 Pathogenesis of Nephropathy P549

P548 SODIUM-LITHIUM COUNTERTRANSPORT ALBUMINURIC, N O ~ M O T E N S I V E D I A B E T I C

IN NORMOPATIENTS.

R Friedman, JB Lopes de Faria, SR Ferreira, MacDonald, C Hill and GC Viberti. Unit for M e t a b o l i c Medicine, Guy's Hospital, London,

FA UK.

An o v e r a c t i v i t y of s o d i u m - l i t h i u m countert r a n s p o r t (Na+/Li+ CT) is a s s o c i a t e d with proteinur• in i n s u l i n - d e p e n d e n t d i a b e t i c s (IDDs). However, its p r e v a l e n c e in normoa l b u m i n u r i c IDDs is unknown. We m e a s u r e d Na+/Li + CT in 70 (41M:29F) n o r m o a l b u m i n u r i c IDDs (geometric m e a n a l b u m i n e x c r e t i o n rate 5.6, 5.3-5.9 ~g/min, 95% CI), w i t h d i a s t o l i c blood pressure <95 mmHg, as part of a diabetic clinic screening. Nine patients (12.9%) had Na+/Li + CT above the upper limit of normal in our l ~ b o r a t o r y (>0.41 m m o l / L RBC/h). Systolic (SBP) and d i a s t o l i c blood p r e s s u r e (DBP) were higher in the group with high Na+/Li + CT (SBP 126• vs 120 • DBP 79• vs 75• mmHg) as was BMI (26• • vs 24• kg/m~), though these d i f f e r e n c e s just failed to achieve s t a t i s t i c ~ l significance. There was no d i f f e r e n c e in age (34• vs 34• y), diabetes d u r a t i o n (13• vs 14• y), insulin dose (0.69• vs 0.65• U/kg/day), f r u c t o s a m i n e (546• vs 559• ~mol/l) and a l b u m i n e x c r e t i o n rate (geometric mean 5.3 vs 5.0 ~g/min) b e t w e e n the two groups. D i v i d i n g by d u r a t i o n of diabetes e l e v a t e d Na+/Li + CT was p r e s e n t in 12% of patients w i t h d u r a t i o n <10 years, 17% of patients with d u r a t i o n b e t w e e n 11-20 and 11% of patients w i t h d u r a t i o n >20 years. Our findings suggest that in n o r m o a l b u m i n u r i c patients Nat/Li + CT a c t i v i t y is a s s o c i a t e d w i t h m a r g i n a l e l e v a t i o n s in blood p r e s s u r e and BMI i n d e p e n d e n t ly of diabetes control and duration.

SODIUM-LITHIUM COUNTERTRANSPORTACTIVrrY OF RED CELLS IN PATIENTS WITH TYPE 1 AND TYPE 2 DIABETES MELLITUS

D. M~ller-Wieland, G. JUrgens, B. Emmenbach, C. HebellSiewers, S. Wilhelmi, D. Look, J. K~hnau, W. Krone, H. Greten, W. D~rr. MedizinischeKernklinik und Poliklinik, UKE, Hamburg; Klinik Hellbachtal der BfA, M611n. Recently i t has beenpostulated that increased sodium-lithium countertransport activity of red cells might be a genetic marker not only for essential hypertension, but also for diabetic nephropathy in type 1 patients. We have examined the Na+/Li+ countertransport activity of red cells and microalbuminuria in 21 normal individuals, 66 patients with type 1, and 54 patients with type 2 diabetes. Normal individuals had a meancountertransport rate of 0.24 mmol/l cells/h. Type 1 patients with incipient diabetic nephropathy had a significantly increased Na+/Li+ countertransport activity of 0.39 mmol/l cells/h. Meantransport rate in type I diabetics without renal disease was only slightly elevated by 20~. There was no correlation betweenthe transport rate and the level of HbA1 glycosylation or the duration of the disease. Interestingly, increased countertransport rates were also found in a group of individuals with newly discovered type 1 diabetes and normal blood pressure. Patients with diabetes mellitus type 2 had elevated tranport activity rates (0.38 mmol/l cells/h) and there was no relation to the degree of proteinuria. These results demonstrate, that increased rates of Na+/Li+ countertransport may relate to renal disease only in patients with type 1 diabetes and indicate, that the disease diabetes mellitus per se might be a factor affecting countertransport activity.

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Insulin resistance and cardiac and renal hypertrophy are associated with high erythracyte sodium-lithiun countertransport (Na+/Li+CT) in essen-

E F F E C T S OF INSULIN, G L U C O S E AND G L U C A G O N ON L I T H I U M - S O D I U M C O U N T E R T R A N S P O R T IN VITP.O. W.]. F O Y L E AND P.L. D R U R Y . D E P A R T M E N T O F DIABETES, K I N G ' S C O L L E G E HOSPITAL, D E N M A R K HILL, L O N D O N , SE5 9RS.

tial hypertension (EH).Nosadini R., Fioretto P., Deria A., Avegaro A., Trevisan R.,Donadon V.,Velussi M.,Viberti G.C.Padova, Italy, london, UK. EH has been reported to be a state of insulin resistance end of higher risk of cardiovascular disease. However only a subgroqo of KH patients with elevated Na+/LJ~CT appe.ar~ to be at greater risk of cardiovascular and reno] complicatians. To test whether insulin resistance is associated with high Na+/Li+CT in I~ we measured insulL-I sensitivity (using euglycemic-insulin i00 pU/ml clsmp), and forearm arterial-venous differences in 9 EH patients with Na+/Li+ CT below (HI) end in 9 EH patients with Nat/Li*CT above the upper limit of normal subjects (0.41 mmol/l RBC/hr) (~). Eight normotensive subjects (C) served as ccntrols. Diastolic arterial pressure was similar in HI and in ~ patients (98k2 vs 98~_S mmHg). Hype~insulinemia inhibited glucose production similarly in HI, H2 and C. Whole body glucose utilization was lower in [{2 (3._8iO.4 mg/Kg/min) t/qsn in HI (5.7_+0.5 p$O.Ol) and in G (6.8m0.6 p
M a x i m a l r a t e ( V m a x ) of l i t h i u m - s o d i u m c o u n t e r t r a n s p o r t (Li + - N a + CT) is r a i s e d in s o m e d i a b e t i c s u b j e c t s . We e x a m i n e d t h e i n f l u e n c e of insulin, g l u c o s e a n d g l u c a g o n on Li + - N a + CT in vitro. On t h r e e o c c a s i o n s b l o o d w a s t a k e n f r o m e i g h t n o n - d i a b e t i c n o r m o t e n s i v e m e n w i t h no f a m i l y h i s t o r y of h y p e r t e n s i o n . V m a x f o r s o d i u m - s t i m u l a t e d lithium e f f l u x w a s d e t e r m i n e d as d e s c r i b e d b y C a n e s s a / M a n g i l ~ , e a c h s a m p l e a t f o u r insulin, g l u c o s e or g l u c a g o n c o n c e n t r a tions. S t a t i s t i c a l a n a l y s i s w a s b y o n e - w a y a n a l y s i s of v a r i a n c e . V m a x is e x p r e s s e d as m m o l / l e r y t h r o c v t e s / h . 9 + -1Insuhn r e d u c e d V m a x f r o m 0 . 2 5 6 - 0.013 ( m e a n - SEM) a t zero to 0.169 + 0.013 a t 10 mU/1, 0.113 + 0.015 a t 25 mU/1 a n d 0.091 + 0.015 at 50 m U / l . (p < 0.001). G l u c o s e h a d no e f f e c t on c o u n t e r t r a n s p o r t , V m a x being 0 . 2 3 2 + 0.01% 0.262 + 0.01% 0 . 2 4 4 + 0.015 a n d 0.258 3 0.015 a t z e r o , 5 retool/l, 10 m m o l / l a n d 20 mmol/1 r e s p e c t i v e l y (p > 0.1). G l u c a g o n , a t 20 p m o l / l , 50 pmol/1 a n d 100 pmol/1, also h a d no e f f e c t , V m a x r a n g i n g f r o m 0.227 + 0.018 to 0 . 2 4 0 + 0.019, a n d b e i n g 0 . 2 3 0 + 0.016 in its a b s e n c e , (p > 0.1). Insulin a p p e a r s t o r e g u l a t e V m a x . T h i s is p r o b a b l y n o t m e d i a t e d b y g l u c o s e c o n c e n t r a t i o n or t r a n s p o r t . T h e e n e r g y r e q u i r e m e n t s of t h e t r a n s p o r t s y s t e m m a y be s m a l l , s i n c e t h e a b s e n c e of g l u c o s e h a s no effect.

high Na+ /Li+ CT, high triglyceridas levels and cardiac and renal hypertrophy in EH. FurTher studies are needed to assess whether this abnormality in cell membrane cation transport plays a direct pathogenetie role or is simply a genetic marker of cardiac, renal and metabolic complications.

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Hyperfiltratian and i~paired response to &qgiotensin LI (AnglI) in hypertensive patients with insulin-dependent diabetes and elevated e r ~ soditln-lithium cotmtertransport activity (Na~Li§ Role of prostaglandins (PCs) and AngII. Giorato C., Fioretto P., Sambataro M., Cipollina M., De Don~ C., Trevisan R., Nosadini R. Padova, Italy.

INCREASED N-ACETYL GLUCOSAMINIDASE AND TRANSFERRIN E X C R E T I O N IN H Y P E R F I L T E R I N G TYPE I DIABETIC PATIENTS.

The pathogenesis of hypertension in patients with insulin-dependent diabetes before Overt nephropathy is poorly tmdenstood. The majority of diabetics have high POs and low AngII activity. These hormonal abnormalities could play a role in the pathogenesis of hypertension in diabetes. The presence of high Na+/Li+ CT in diabetics wil/q hypertension may indicate a susceptibility to nephropathy. Our aim was to investigate the response to AngII in 7 nonngJ~ (C), in 9 normotensive diabetics (6]3), in 9 hypertensive diabetics with Na+/Li+CT below (HID) and in I0 hypertensive diabetics above (H2D) 0.41 nlml/L RBC/nour (0.41 is the mean value +2$D of Na+/LI+CT in normal subjects). Baseline glomerular filtration rate (GFR) and ~?enal plasma flow (RPF) was 105+-3 and 50~_18 ml/min/l.TS m2 irl C, 117--+6and 549k22 in CD (p<0.05), i19~7 and 555k24 in HID (p
J.N. Harvey and P. Martin, D e p a r t m e n t of Medicine, General Infirmary, Leeds LSI 3EX, UK. The e x c r e t i o n of N - a c e t y l - B - D - g l u c o s a m i n i d a s e (NAG) and t r a n s f e r r i n are i n c r e a s e d early in d i a b e t e s b e f o r e c l i n i c a l l y evident nephropathy. This study was u n d e r t a k e n to d e t e r m i n e if their i n c r e a s e d e x c r e t i o n c o u l d be r e l a t e d to increased GFR. N i n e normal c o n t r o l s and 18 type I d i a b e t i c p a t i e n t s w i t h o u t m i c r o a l b u m i n u r i a u n d e r w e n t GFR m e a s u r e m e n t by 51 Cr-EDTA clearance. Insulin was g i v e n i n t r a v e n o u s l y to m i n i m i s e hyperglycaemia. NAG was m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y and t r a n s f e r r i n by R I A on u r i n e c o l l e c t e d d u r i n g GFR measurement. GFR in the normal c o n t r o l s was 108 • 12 (M • SD) m l / m i n / 1 . 7 3 m 2. H y p e r f i l t r a t i o n was d e f i n e d as GFR >132 (M + 2SD). Seven d i a b e t i c subjects e x h i b i t e d h y p e r f i l t r a t i o n (146 ~ 14) and 11 had normal GFR (111 10). N A G e x c r e t i o n [geometric m e a n (I SD range)] was i n c r e a s e d in h y p e r f i l t e r i n g p a t i e n t s versus normal GFR p a t i e n t s (p < 0.01) and normal controls (p < 0.01): 451 (247-822) v 160 (85-304) v 124 (49-319) m U / m i n / 1 . 7 3 m 2. T r a n s f e r r i n e x c r e t i o n was increased in the h y p e r f i l t e r e r s v normal G F R p a t i e n t s and c o n t r o l s (p < 0.05): 302 (791156) v 114 (53-245) v 86 (37-197) n g / m i n / 1 . 7 3 m 2 . G l u c o s e e x c r e t i o n and HbAIc were not d i f f e r e n t b e t w e e n the two d i a b e t i c groups. NAG c o r r e l a t e d with GFR (r = 0.44, p < 0.025) and glucose e x c r e t i o n (r = 0.51, p < 0.01). T r a n s f e r r i n c o r r e l a t e d w i t h GFR (r = 0.48, p < 0.02) and g l u c o s e e x c r e t i o n (r = 0.65, p < 0.001). NAG and t r a n s f e r r i n e x c r e t i o n are increased in h y p e r f i l t e r i n g d i a b e t i c patients, c o n s i s t e n t with a p a t h o l o g i c a l effect of g l o m e r u l a r h y p e r f i l t r a t i o n on the kidney.

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RENIN-ANGIOTENSIN-ALDOSTERONE AXIS IN HYPERFILTERING AND NOR}{OFILTERING INSULIN-DEPENDENT DIABETIC PATIENTS J.L.Gross, M.J.Azevedo and O.L.Ramos. Endocrine Unit of Hospital de Clinicas de Porto Alegre, Porto Alegre, Brazil

HYPERFILTRATION IS ASSOCIATED WITH ENHANCED GROWTH HORMONE SECRETION AND INCREASED RENAL PLASMA FLOW.

To investigate whether abnormalities of the renin-angiotensin-aldosterone axis (RAA) are associated to hyperfiltration, 21 insulin-dependent normotensive, normoalbuminuric diabetic patients were studied. Plasma renin activity (PRA) and aldosterone (radioimmunoassay-Travenol), blood pressure were measured after an overnight fast, I0 min rest, sitting position, at 0 and 2 hours after 25m~ captopril po. Seven normal subjects formed the control group and was matched by age, sex and body mass index with the patients. Glomerular filtration rate (GFR) was measured by 51CrEDTA and the patients were divided into hyperfiltering, G F R > 134.0 ml/min/l.73m2 (GFE = 158.7 • 18.7ml/min/l.73m2; 5F, 6~i; age 35.0 + 4.6 years; duration of diabetes: 7.3 • 2.4 years) and normofiltering (GFR = 115.9 ~ 13,6; 4F, 6M; age 33.6 • 6.5 years); duration of diabetes 12.4 • g.l years). No difference was found in 24h urinary sodium excretion and metabolic control (HbAlc and frutosamine) between the 2 groups. Basal PRA was higher in diabetic patients than in control group (2.4 • 1.7 x 1.2 0.8 ng/ml/h; p < 0 . 0 5 ) . PRA post-captopril (9.9 i 8.3 ng/ ml/h-patients; 5.9 f 5.6 ng/ml/h-control), basal plasma aldosterone (Ii.0 • 5.8 ng/dl-patients; 10.5 • 2.9 ng/dl-control) and pos-captopril plasma aldosterone (6.6 • 3.3 ng/dl-patients; 8.5 • 5.2 ng/dl-control) were similar (p> 0.05) in both groups. A decreased blood pressure was observed in all individuals ( p < 0 . 0 5 ) , but diabetic patients tend to have a greater decrease than the control group (11.8 i 6.5 mmHg x 6.7 • 5.2 mmHg; 0.i0< p < 0.05). No difference was observed between hyperfiltering and normofiltering patients. It is concluded that hyperfiltration is not associated with PAA abnormalities, although diabetic patients as a group present higher

PJ Blankesiijn, FHM Derkx, SWJ Lamberts, MADH Schalekamp, L Verschoor and RFA Weber. Departments of Internal Medicine I and III , Erasmus University Rotterdam, The Netherlands To elucidate the role of growth hormone (GH) in glomerular hyperfiltration in early type 1 diabetes mellitus (DM), we measured GH response after GH-releasing factor (1 p.g/kg) as area under the curve (GHAUC) in patients (n=38) with hyperfiltration and normofiltration. Hyperfillration, GFR>130 ml/min/1.73m2 (1251.thalamate clearance), was present in 22 (aged 34+8 year, mean+SE) and normofiitration in 16 patients (aged 42_+7year), GFR respeotively 148_+2 and 113_+4 and renal plasma flow (RPF, 1311.hippurate clearance) respectively 578_+17 and 451_+16 ml/min/1.73m 2 (p<0.001). Duration of DM, HbAlc, renin and catecholamines and blood glucose during GFR measurements were not different in patients with hyper- and normofiltration. In all patients albuminuda was less than 300 mg/24h, but 8 patients with hyperfiltration and 8 with normofiltration had microalbuminuria (30-300 mg/24h). GHAUC was 1726_+218 Bg.h.L-1 in patients with hyperfiltration and 1044_+289 in those with normofiltration (p<0.02). In the absence of microalbuminuria (<30 mg/24h) GHAUC was 2130+383 in patients with hyperfiltration (n=15) and 959+480 in patients with normofiltration (n=7) (p<0.001) and GHAUC was correlated with GFR (r=0.60, p<0.01) and RPF (r=0.63, p<0.001). In the presence of microalbuminuria (n=16) GHAUCwas not different between patients with hyperfiltration and normofiltration and it was not correlated with GFR and RPF. Thus enhanced GH secretion is associated with increases in both RPF and GFR in patients without microalbuminuria. GIomenJlar hyperfiltration may be secondary to increased RPF.

basal PRA levels.

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NATRIURETIC HORMONES (ANF, 0LF) AND RENAL HAEMODYNAMIC IN TYPE 2 (NON-INSULIN-DEPENDENT) DIABETES J.D. LALAU, F. TENENBAUM, P.F. WESTEEL, G. MAUREL, J. NUSSBERGER*, H. FAVRE**, A. FOURNIER, J. QUICHAUD. HSpital Sud, Amiens, France, *Service d'hypertension, Lausanne, Suisse, **Service de M@decine, Gen~ve, Suisse

TAMM-HORSFALL PROTEIN IN TYPE 1 (INSULIN-DEPENDENT) DIABETES MELLITUS. O. T o r f f v i t 1,2, C.-D. A g a r d h 2, B. K j e l l s s o n s a n d J. W i e s l a n d e r 1. Departments of INephrology, 2Internal medicine, University o f L u n d , a n d a C l i n i c a l I m m u n o l o g y , U n i v e r s i t y of G S t e b o r g , Sweden. Urinary excretion rate of Tamm-Horsfall protein was m e a s u r e d to s t u d y t u b u l a r f u n c t i o n in 75 T y p e 1 d i a b e t i c p a t i e n t s w i t h m e d i a n d u r a t i o n o f d i a b e t e s o f 15 y e a r s a s well a s in 76 h e a l t h y c o n t r o l s u b j e c t s . T h e i n t r a - a n d interassay and intra-individual variation of the TammH o r s f a l l p r o t e i n ELISA w a s 8.9%, 13.6% a n d 41%, r e s p e c t i v e l y . T h e s e n s i t i v i t y w a s 4 ~g/1. T h e c o r r e s p o n d i n g v a l u e s f o r t h e a l b u m i n a s s a y w e r e 6%, 9% a n d 40%, r e s p e c t i v e l y . T h e s e n s i t i v i t y w a s 15 pg/1. 2 4 h u r i n e c o l l e c t i o n s w e r e a n a l y z e d . T h e a l b u m i n e x c r e t i o n r a t e w a s h i g h e r in d i a b e t i c patients (40.3x/:7.4 vs. 4.7x/:2.3 og/min; p<0.01; geometric m e a n x/: t o l e r a n c e f a c t o r ) w h e r e a s t h e T a m m - H o r s f a l l p r o t e i n e x c r e t i o n r a t e w a s n o r m a l c o m p a r e d to c o n t r o l subjects (34.0x/:1.9 og/min). Tamm-Horsfall protein excret i o n r a t e w a s n e g a t i v e l y c o r r e l a t e d to d u r a t i o n of d i a b e t e s (r=-0.25,p<0.05), systolic blood pressure (r=-0.38;p<0.001), serum creatinine (r=-0.37,p<0.01) and HbAlc levels (r=0.33,p<0.01). However, Tamm-Horsfall protein excretion rate c o r r e l a t e d t o H b A l c o n l y in p a t i e n t s w i t h a d u r a t i o n of <15 y e a r s ( r = - 0 . 5 2 , p < 0 . 0 1 ) o r w i t h a l b u m i n e x c r e t i o n r a t e of < 2 0 p g / m i n ( r = - 0 . 4 9 , p < 0 . 0 1 ) . In c o n c l u s i o n t u b u l a r d y s f u n c tion as measured by the excretion of Tamm-Horsfall protein coexisted with degree of inadequate metabolic control.

Evidence that increase in ANF mediates, at least in part, glomerular hyperfiltration in diabetic rats prompted us to study the relationship between natriuretic factors and renal haemodynamic in hyperglycaemic and normoglycaemic states. Eleven normotensive non-macroproteinuric patients with type 2 diabetes and secondary drug failure were studied without dehydration (natriuresis was > i00 mmol/24H). Before and after glycaemic normalization by i.v. continous insulin infusion (from day 0 to day 7), the following parameters were determined : glomerular filtration rate (GRF) and renal plasma flow (RPF) by 99mTc-DTPA and 131I-hippuran clearances ; the DTPA diffusion volume, reflecting the extra-cellular volume ; plasma ANF and urinary ouabaine-like factor (0LF) by RIA and receptor-binding assay respectively. Renin activity, aldosterone, antidiuretic hormone, and cateeholamines concentrations were also measured. With normoglycaemia GFR declined from a high to a normal mean value (138 + 27 and 115 + 6 ml/min, p < 0.001), RPF declined also but not significantly, and the DTPA diffusion volume did not vary. Concentrations of all hormones, initially in the normal range, did not vary except for 0LF that declined (p < 0.05). There was no corelation between i) GFR and hormonal concentrations on day 0 and on day 7 ; 2) variations of GFR and of natriuretic hormone concentrations. In conclusion the natriuretic hormones are apparently not involved in the pathogenesis of glomerular hyperfiltration nor in the GFR variation with glycaemic changes.

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URINARY ACTIVITY OF N-ACETYL-8-D-GLUCOSIDASE (NAG) IN NON-INSULIN-DEPENDENT DIABETICS F. Schnoell, R. Weitgasser, A. Straberger and I. Pretsch II. Medizinische Abteilung LKA Salzburg / Austria

KIDNEY PROTEIN PERMSELECTIVITY DIABETES MELLITUS

Urinary activity of NAG is increased in patients with essential hypertension and can be reduced by successful antihypertensive therapy; severity of atherosclerotic disease is associated with high levels. We wanted to determine whether these correlations can also be found in type-2-diabetics. 204 patients were randomly selected, and 79 excluded because of urinary infections or renal diseases. We measured blood pressure, urinary NAG-activity, excretion of urinary creatinin, albumin, a-l-microglobulin (a-I-MG) and b-2microglobulin (b-2-MG) in the remaining 125 subjects and assessed atherosclerotic disease. Because of the skewed distribution results are given as mean/median values, the Mann-Whitney-test and Spearman's rank-correlation-coefficient were used for statistical testing. The NAG-Index (ratio NAG/urinary creatinin) and b-2-MG did not differ significantly in hypertensive versus normotensive subjects, but albumin and a-I-MG did. The NAG-Index for patients with severe vascular disease (1.46/1.02; n = 17) was higher (p < 0.03) than for those without overt atherosclerosis (1.01/0.82; n=27). In hypertensives the NAG-Index was correlated to albuminuria (r=0.33/p < 0.02) and to blood glucose (r=0.31/p < 0.03). Whether NAG-activity, excretion of a-I-MG or b-2-MG are of any predictive value for the developement of renal disease and/or atherosclerosis in type-2-diabetics remains to be shown in an ongoing longitudinal survey.

IN

TYPE

2

S. M o r a n o , P. P i e t r a v a l l e , G. Mariani, M.G. De Rossi, G. Cristina, P.L. M a e s t r i p i e r i * , A. Clementi**, U. Di M a r i e , Endocrinology, University " La S a p i e n z a "; * C l i n i c a S. Anna; **Diabetology, Ospedale Forlanini Roma,Italy In o r d e r to i n v e s t i g a t e the selectivity p r o p e r t i e s of the kidney f i l t r a t i o n barrier in t y p e 2 d i a b e t e s , c l e a r a n c e s of p r o t e i n s d i f f e r i n g in size and/or e l e c t r o s t a t i c c h a r g e w e r e s t u d i e d . A g r o u p of 55 p a t i e n t s was selected as r e p r e s e n t a t i v e of t h e t y p e 2 d i a b e t i c s a t t e n d i n g our c l i n i c s (age -median, IR- 57,51-61 years; duration of d i a b e t e s 9 . 5 , 4 - 1 7 years), i01 a g e - c o m p a r a b l e n o r m a l s r e p r e s e n t e d the control group. C l e a r a n c e s of p r o t e i n s d i f f e r i n g in size (albumin,IgG) and charge (anionic proteins: albumin,IgG4; neutral/ cationic: total IgG) w e r e evaluated. Clearances of a l b u m i n , IgG4 a n d t o t a l I g G were (median,IR) 0.14, 0 . 6 - 0 . 4 1 ( p < 0 . 0 1 vs normals) ; 0.03, 0.01-0.65(p<0.005 vs normals); 0.12, 0.8-0.19(p< 0.01 vs normals) ml/min x 10 -3 respectively. The anionic/cationic immunoglobulin ratio was (median,IR) 0.25, 0.12-0.43 and e x c e e d e d 0.25 (the u p p e r limit of normal values) in 37% of the n o r m o a l b u m i n u r i c and in all of the m i c r o and m a c r o a l b u m i n u r i c patients. IgG4 c l e a r a n c e was p o s i t i v e l y c o r r e l a t e d w i t h a l b u m i n c l e a r a n c e (r = 0.69). P r o t e i n c l e a r a n c e s d i d not c o r r e l a t e w i t h the duration of diabetes. The anionic/cationic immunoglobulin ratio highlights some patients w i t h s e l e c t i v e p r o t e i n u ria a b n o r m a l i t i e s but still n o r m o a l b u m i n u r i c .

P560

P561

PLASMA ATRIAL NATRIURETIC PEPTIDE AND CYCLIC GMP IN EARLY STAGES OF DIABETIC NEPHROPATHY.

URINARY KALLIKREIN EXCRETION IN TYPE I DIABETES A. Manto, P. Cotroneo, P. Magnani, P. Til li, G. Porcelli*, G. Ghirlan da and A.V.Greeo. Ist.Clin.Med.Univ.Catt. ,~]NR, Roma. Renal kallikrein-kinin system is thought to be involved in renal h a e m o c ~ c . Renal kallikrein has not been studied in patients with diabetes mellitus, a disease in which abnormalities of r e n a l h a e m o d ~ c occur. The aim o f our stydy was to determime whether abnormality in urinary kallikrein-kJ_nin system is present in type I diabetes. We mea sured the 24h urinary excretion of renal-kallikrein in i0 normal subjects (Group A) and in 26 diabetic patients (Gro up B); 5 patients of Group B were mieroalbuv/nurie (urinary-alb~nin-excretion:97.8+l~g/min). All subjects were mat ehed for age and duration of disease. Mean of ~oA IC was 4. 4~LO_.8% in normal and 6.3_+0.6% in diabetic subjects. Urinary-kallikrein excretion was evaluated by measuring its esterolytic-aetivity on the substrate L-prolyl-l-phenylalanyl -L-arginine-7-anddo-4-methylcou~qrin salt. Besides the fol lowing parameters were studied: creatinine and blood-ureanitrogen (BUN). Results of our study indicate no significant difference between group A and B in kallikrein-urina ry excretion (140_+37 UE/24h vs 145+77 UE/24h, p=ns). However 5 patients with mieroalbLrninuria significantly showed lower values (37+6 UE/24h, p
K.Matoba, T.Moriya, Y.Fujita and Y.Yajima Dept of Intern Med, Kitasato Univ Sch of Med, Sagamihara, Japan. To investigate the role of plasma atrial natriuretic peptide (ANP) and the inhibition of angiotensin converting enzyme in the pathogenesis of early renal changes in Type 1 (insulin-dependent) diabetic patients, we examined plasma ANP, cyclic GMP (cGMP), GFR and urinary albumin creatinine ratio (AI) in 33 normotensive Type I diabetic patients; 9 without nephropathy, 15 with glomenflar hyped'titration (HF) and 9 with incipient nephropathy. ANP in three diabetic groups was significantly higher than that in 28 normals. In HF, ANP (70.1+30.3 (mear~+SD) pg/ml) was significantly higher than the other two diabetic groups. In diabetic groups, ANP correlated with GFR (r=0.402, p<0.05), but did not with blood pressure (BP). Plasma cGMP in diabetic groups was significantly higher than that in normals, cGMP correlated with AI (r=0.774, p<0.001), but did not with ANP. After enalapril administration, AI .decreased (p<0.05), but ANP, BP and GFR did not change significantly. These results suggest that elevated Plasma ANP and cGMP levels mediate, in part, glomerular hyperfflwation and urinary albumin excretion, and the reduction of AI after enalapfil administration is brought about by the mechanisms other than hyper-ANP-emia and glomerular hyperfillration.

A154

P562

P563

INSULIN-LIKE GROWTH FACTOR-I AND BINDING PROTEIN-I IN DIABETIC NEPHROPATHY

ROLE OF LIPID COMPOSITION IN THE PROGRESSION OF URINARY ALBUMIN EXCRETION IN 90 DIABETICS.

J D Q u i n , J G Fox, A M T a y l o r , M A P r e e c e , J M P H o l l y , J A H W a s s , Y Y Ng, G H B e a s t a l l and A C MacCuish Royal Infirmary, Glasgow, Institute of Child Health, London and St Bartholomews Hospital, L o n d o n , U K.

S. Suraniti, A. Girault, Ph. Fressinaud, and M. Marre - CHRU ANGERS - 49033 - FRANCE.

A role has been proposed for insulin-like g r o w t h f a c t o r - i (IGF-I) in t h e p a t h o g e n e s i s o f diabetic nephropathy. To investigate this, we s t u d i e d 25 a d u l t s w i t h l o n g s t a n d i n g (>I0 years) Type 1 (insulin-dependent) diabetes w i t h a l b u m i n e x c r e t i o n r a t e (AER) o f 6 - 3 6 7 0 mg/24 hours and creatinine clearance of 38-135 m l / m i n / l . 7 3 m 2. Serum IGF-I was determined by ELISA, insulin-like growth factor binding p r o t e i n - i (IBP-I) b y R I A a n d I G F - I i n h i b i t o r y bioactivity by porcine costal cartilage bioassay. We found no significant r e l a t i o n s h i p s b e t w e e n IGF-I, I B P - I concentration or IGF-I inhibitory bioactivity and AER. IBP-I was significantly correlated w i t h c r e a t i n i n e c l e a r a n c e (r=-.57, p < O . 0 1 ) b u t IGF-I and IGF-I inhibitory bioactivity were not. In conclusion, serum IGF-I and IGF-I i n h i b i t o r y b i o a c t i v i t y a r e n o t a l t e r e d in t h e e a r l y s t a g e s of n e p h r o p a t h y d u e t o T y p e - i diabetes. I m p a i r e d r e n a l f u n c t i o n is associated with elevation of serum IBP-I levels probably because of reduced renal c l e a r a n c e o f t h i s 28 k d p r o t e i n .

Variables accounting for progression of Urinary A l b u m i n Excretion (UAE) in diabetics must be delineated. Fifty six type 2 (non insulin-dependent) and 34 type 1 (insulin-dependent) diabetics were studied at one year interval ; 62 diabetics had normal UAE, 22 microalbuminuria and 6 macroalbuminuria. During one year interval, drug treatments were unchanged. Changes expressed in percent of initial value (mean-range) were for the following variables : HbA1C : - 8 % (- 56 to + 41%), weight : + 3 % (- 9 to + 24 %), systolic blood pressure (SBP) : + 2 % (- 29 to + 33 %), diastolic blood pressure (DBP) : 0 % (- 28 to + 50 %), Apoprotein B/Apoprotein A1 ratio (Apo B/A1) : - 6 % (- 48 to + 71%) and U A E : + l % ( - 8 9 t o + 2 3 1 % ) . In die whole group, changes in UAE were related positively to changes in Apo B/A1 (p = 0.01 ; r = 0.25) but not to modifications of HbA1C (p = 0.31 ; r = 0.09), weight (p = 0.42 ; r = 0.08), SBP (p = 0.19 ; r = 0.14) o r D B P (p = 0.16 ; r = 0.15). Variations of Apo B/A1 were related to variations of HbA1C (p = 0.001 ; r = 0.33). In the microalbuminuric group, multivariate analysis showed progression of UAE associated to increase in Apo B/A1 (F = 10.6) and increase in SBP (F = 9.4).In the normoalbuminuric group, changes in Apo B/A1 were related to changes in HbA1C (F = 24). In conclusion, at one year interval, progression of UAE in microalbuminuric diabetics is related to lipid composition, which depends of changes in metabolic control.

P564

P565

URINARY EGF-EXCRETION IS CORRELATED TO RENAL FUNCTION LOSS PER SE AND NOT TO THE DEGREE OF DIABETIC RENAL DISEASE. G.J.van Kamp, H.J.G.Bilo, R.0.E.Oans and A.J.M.Donker. Amsterdam and Nijmegen, The Netherlands. Urinary epidermal growth factor (EGF-)excretion is seen as a tubular function, and some studies conclude that EGF-excretion can be reduced already early in the development of diabetic renal disease. We investigated various groups of subjects, controls (C, n=5), patients with chronic renal insufficiency (CRI, n=8), and normo-albuminuric (N, n=9), micro-albuminuric (M, n=8) and nephropathic (DN, n=6) type I (insulin-dependent) diabetic patients. GFR (ml/min, 125I-iothalamate) and ERPF (ml/min 131I-hippuran) were measured under (near)normoglycaemic conditions. During the testing proeedure~ EGF-exeretion (in ng) was measured as well. Although absolute EGFexcretion/min was lower in patients with impaired renal function (C=9.98+3.54, CRI=6.79+6.98, N=8.90+5.45, M=8.46+2.93 and DN=4.37~3.97 ng/--min, with GF~'s of C=136!~, CRI=62+_42, N=139~15, M=122+25 and DN=71+35 ml/min, CRI an DN vs others p<0.05)T fractional EGFexcretion (EGF/GFR) was comparable in all groups (C=0.075+0.030, CRI=0.083+_0.045, N=0.064+_0.035, M=0.069+~.020 and DN=0.052+0.035 ng/ml, respectively). Furthermore, in our M- and DN-patients we found no correlation between EGF-excretion and urinary albumin excretion. We ocnclude that impaired EOF~excretion is correlated to renal function loss per se and not specifically to the degree of diabetic renal disease as expressed by both renal function and urinary albumin excretion.

Relationship between urinary laminin PI levels and blood pressure in normotensive Type 2 (non-insulindependent) diabetic patients without clinical proteinuria Y.Yano,Y.Sumida,C.Seguchi,K.Shirayama,T.Shima,M.Misaki and S.Suzuki. The Third Department of Internal Medicine, Mie University School of Medicine, Tsu, Japan Laminin is one of the major non-collagenous components of basement membrane (BM) and it is found to be increased in the capillary BM with diabetic changes. We reported that urinary laminin P1 (U-LPI) and serum laminin P1 (S-LPI) levels were significantly increased in normotensive type 2 diabetic patients without clinical proteinuria compared with normal subjects. We investigated S-LPI, U-LPI, blood pressure, glomerular filtration rate (GFR), urinary excretion of albumin (AER), N-acetylB-D-glucosaminidase (NAG), ~2-microglobulin (B2-MG) and ~l-microglobulin (~I-MG) in 26 normotensive Type 2 diabetic patients (M/F:13/13, age:55• years) without clinical proteinuria (AER<50 ~g/min). 24 hour-urine collections were carried out three times. The urine samples, previously adjusted to pH 7.4, were concentrated 20 times. Then U-LPI was measured by RIA (Hoechst). There was a significant correlation between U-LPI levels and systolic (r=0.40 p<0.01), diastolic (r=0.40 p<0.01) blood pressures. There was also a significant correlation between U-LPI and S-LPI levels (r=0.42 p<0.01). There was no correlation between U-LPI and GFR, AER, NAG, ~2-MG, ~I-MG levels. No correlation was found between HbAzc and U-LPI levels. These data suggest that metabolic alterations of BM occurs in the early stage of diabetic nephropathy and they become progressive with blood pressure elevations in normotensive diabetic patients.

A155

P566

P567

P L A S M A A T R I A L N A T R I U R E T I C P E P T I D E IN T Y P E 1 DIABETIC PATIENTS B E F O R E AND A F T E R I S O T O N I C V O L U M E E X -

URINARY TRANSFERRIN EXCRETION IS ELEVATED DURING EXERCISE IN TYPE I (INSULIN-DEPENDENT) DIABETES. M.J. O'Donnell 1, P. Martin 2, D.A. Cavan1, A. Parkes 3, C. Chapman2 and A.H. Barnett 1. Depts. of Medicine, University of Birmingham and East Birmingham Hospital I, University Depts. of Medicine and Chemical Pathology, Leeds General Infirmary 2 and Respiratory Physiology Laboratory, East Birmingham Hospital 3.

PANSION.

H.-H. Lervang, E. Toft, J.H. K r i s t i a n s e n , G. Espersen, J. Br~chner-Mortensen, and J. Ditzel. S e c t i o n of E n d o c r i n o l o g y , D e p a r t m e n t of M e d i c i n e and D e p a r t m e n t s of C l i n i c a l C h e m i s t r y and Clinical Physiology, A a l b o r g R e g i o n a l Hospital, Denmark. S t u d i e s in a n i m a l s and findings in Type 1 (insulin-dependent) d i a b e t i c subjects m i g h t i n d i c a t e a l i n k i n g of atrial n a t r i u r e t i c peptide(ANP) to g l o m e r u l a r h y p e r f i l t r a t i o n and the d e v e l o p m e n t of d i a b e t i c nephropathy. We t h e r e f o r e i n v e s t i g a t e d p l a s m a ANP levels (RIA-assay) and g l o m e r u l a r filt r a t i o n rate (51Cr-EDTA clearance) in 32 well c o n t r o l l e d Type 1 d i a b e t i c p a t i e n t s as c o m p a r e d to 15 h e a l t h y subjects b e f o r e and after i s o t o n i c saline infusion. M e d i a n age was 29 (range 17-41) years in the d i a b e t i c s u b j e c t s and 31 (24-39) years in the c o n t r o l group. Eight of the 32 diabetic s u b j e c t s had c l i n i c a l proteinuria. At baseline m e d i a n p l a s m a ANP levels w e r e s i m i l a r in the d i a b e t i c p a t i e n t s w i t h or w i t h o u t c l i n i c a l p r o t e inuria as c o m p a r e d to the h e a l t h y c o n t r o l g r o u p (6.4 vs 5.0 vs 4.4 p m o l / l ; K r u s k a l l - W a l l i s test p=0.10). A f t e r isotonic v o l u m e e x p a n s i o n p l a s m a ANP i n c r e a s e d in the c o n t r o l group (4.4 to 7.7 p m o l / l ; W i l c o x o n test p<0.01) and in the d i a b e t i c s u b j e c t s w i t h o u t c l i n i c a l p r o t e i n u r i a (4.7 to 6.1 p m o l / l ; p < 0 . 0 0 0 1 ) but r e m a i n e d u n c h a n g e d in p a t i ents w i t h overt d i a b e t i c n e p h r o p a t h y (6.4 to 6.9 p m o l / l ; p > 0 . 0 5 ) . In c o n c l u s i o n overt d i a b e t i c nep h r o p a t h y was a s s o c i a t e d w i t h a b l u n t e d r e s p o n s e of ANP to i s o t o n i c v o l u m e expansion. We did not d e m o n s t r a t e a r e l a t i o n s h i p of g l o m e r u l a r f i l t r a t i o n rate to ANP levels.

Elevated urinary albumin excretion rate (AER) a f t e r exercise has been reported in diabetes m e l l i t u s . Urinary t r a n s f e r r i n excretion rates (TER) may p r e d i c t nephropathy and microvascular complications. We studied (AER), (TER) and urinary excretion of N-acetyl-B-D-glucosaminidase (NAG) and A-1-Microglobulin (AIM) in 8 normoproteinuric diabetics without complications (age 20-35, median 28.5 years and duration 7-19, median 15 years) (DM) and 8 age, sex matched controls (CM) before, immediately and 1 hour a f t e r exercise t e s t i n g . Workloads were s i m i l a r (DM 210w, CM 260w, p=NS). TER was higher in DM before and a f t e r exercise (0.19 vs 0.12, p
PS 17 Eye Disease P568 PROTECTIVE EFFECT OF LIVER CIRRHOSIS AGAINST DIABETIC RETINOPATHY AND INSULIN-LIKE GROWTH FACTOR I. Y.Sakamoto, O.Mokuda, E.Ubukata, R.Kawagoe and N.Shimizu. 3rd. Dept. of Int. Med. Teikyo Univ. Ichihara, Japan. To i n v e s t i g a t e the r e l a t i o n s h i p between the low incidence of p r o l i f e r a t i v e r e t i n o p a t h y in diabetic patients w i t h liver cirrhosis and i n s u l i n - l i k e growth factor I (IGF-I), plasma IGF-I levels were m e a s u r e d in 9 diabetic patients w i t h liver c i r r h o s i s ( D u r a t i o n of diabetes: over 8 years), 18 diabetic patients w i t h p r o l i f e r a t i v e retinopaty, 8 diabetic patients w i t h o u t r e t i n o p a t h y ( D u r a t i o n : over i0 years), 8 liver cirrhotic p a t i e n t s w i t h o u t diabetes and 12 normal controls. Diabetics w i t h liver cirrhosis and cirrhotic patients had s i g n i f i c a n t l y lower mean IGF-I levels than the normal controls (Diabetics w i t h cirrhosis: 264.0 mU/ml, Cirrhosis: 211.8 mU/ml, Controls: 392.2 mU/ml). In contrast, mean IGF-I level in d i a b e t i c s w i t h p r o l i f e r a t i v e r e t i n o p a t h y was s i g n i f i c a n t l y h i g h e r than the controls (603.1 mU/ml). The mean IGF-I level in d i a b e t i c s without r e t i n o p a t h y and controls did not differ s i g n i f i c a n t l y (382.3 mU/ml). Only one case had p r o l i f e r a t i v e r e t i n o p a t h y in the group of d i a b e t i c s w i t h liver cirrhosis, but the IGF-I level was 466 mU/ml. This study d e m o n s t r a t e d the low incidence of p r o l i f e r a t i v e r e t i n o p a t h y in d i a b e t i c s w i t h liver cirrhosis and high c o n c e n t r a t i o n of IGF-I may be involved in a v a r i e t y causes of the p r o g r e s s i o n of diabetic retinopathy.

P569 LOSS OF VISUAL FUNCTION ASSOCIATED WITH MICROALBUMINURIA IN DIABETES MELLITUS. Usha Dhanesha, Jim G i l c h r i s t , David Miles, Nell Bradford and John Weatherill, Department of Optometry, U n i v e r s i t y of Bradford and Airedale General Hospital, West Yorkshire. This study investigates whether the presence of microalbuminuria in d i a b e t i c patients is associated with loss of visual function. Thirty-seven non-insulin dependent diabetics showing microalbuminuria were compared with twenty-six age-matched diabetics with no evidence of microalbuminuria. The inclusion c r i t e r i a were visual acuity >6/9 and minimal background retinopathy. Retinopathy was assessed by ophthalmoscopy and fundus photography and c l a s s i f i e d according to the ETDRS scheme. Microalbuminuria was defined as an albumin/creatinine r a t i o >1.5 measured on two successive occasions at an o u t - p a t i e n t c l i n i c by immunoturbidimetry. Contrast s e n s i t i v i t y , using a Pelli-Robson chart, and f l i c k e r fusion thresholds (CFF) were measured. Results show that CFF and Pelli-Robson values c l e a r l y separated the microalbuminuria group from the control group (Mann-Whitney, p<.O001). Using kappa values, the best c r i t e r i a for d i f f e r e n t i a t i n g between the two groups have been suggested f o r each t e s t , (CFF:41Hz; Pelli-Robson:1.43) and a f a i r association was demonstrated, (CFF:k=O.58; Pelli-Robson:k=0.66). Combining the results from the two tests separated the two d i a b e t i c groups more e f f e c t i v e l y at the same minimal levels of retinopathy.

A156 P570 DIABETIC RETINOPATHY AND URINARY ALBUMIN EXCRETION RATE IN TYPE 2 (NON-INSULIN-DEPENDENT) DIABETIC PATIENTS I.N. Migdalis, N. Dimakopoulos, A. Tel• A. Likoudi, K. Koutoulidis and M. Samartzis. Depts. of Internal Medicine IT and Diabetes, NIMTS Hospital, Athens, Greece.

P571 CHANGES IN P U P I ~ Y

LIGHT REFLEX BY ACUTE HYPERGLYCAEMIA

Associations between overnight urinary albumin excretion rate (AER) and d i a b e t i c refinopathy and i t s major r i s k factors were examined in a cross-sectional study of 105 Type 2 (non-insulin-dependent) d i a b e t i c p a t i e n t s . Forty-seven of these patients had no retinopathy, 32 had background retinopathy(BR) and 26 had p r o l i f e r a t i v e retinopathy (PR). Patients with background and p r o l i f e r a t i v e retinepathy were older than those without retinopathy (p
A. de Vos, T.W. Van Haeften, O. de Weerdt, G.J. Blok and E.A. van der Veen, Department of Endocrinology, Free University Hospital, Amsterdam, The Netherlands. Abnormalities of pupillary function are well known in diabetes mellitus, presumably due to autonomic neuropathy. The effect of acute hyperglycaemia on pupillary reflexes in man is unknown. We performed hyperglycaemic glucose clamps in 8 healthy volunteers (mean age (• s.d.): 29.5 (• 3) years, mean (+ s.d.) body mass index: 23.2 (• 3.2) kg/m2). Blood glucose was increased in three steps from the fasting level to 8, 12 and 16 ~mol/l at 15 minute intervals and kept at the level of 16 mmol/l for 150 minutes. Pupillary reflexes were recorded with a standardized infra-red reflection technique, at 10 minutes after each acute rise in blood glucose level and at 30 minute intervals during the remainder of the clamp. Latency of the constriction, defined as the time between the moment of light stimulation of the pupil and the moment of its constriction, was shortened as compared with baseline at all three blood glucose levels (mean • sd: 230 • 13, 229 • 17, 229 • 20 versus 245 • 13 ms, all p < 0.01). Constriction latency was not significantly different from baseline during the remainder of the clamps. No changes in dilatation latency were observed (p > 0.2). We conclude that acute hyperglycaemia induces a shortlasting reduction in the latency of pupillary constriction in man, presumably due to increased parasympathetic activity.

P572

P573

DETERMINATION OF CORNEAL AUTOFLUORESCENCE IN DIABETIC PATIENTS BY FLUOROPHOTOMETRY

L A M I N I N P1 AS S E R U M M A R K E R O F D I A B E T I C M I C R O A N GIOPATHY. R.Sin6, RM.Segura, L.Garcia-Pascual, J.Mesa, C.Pascual and JM.Martinez-Vazquez. Division of Endocrinology and Service of Biochemistry. Hospital General Vall d'Hebron,Barcelona, Spain.

Th. R. Stolwijk~ J.A. van Best, J.P. Boot and J.A. 0$sterhuis. Department of Ophthalmology, University Hospital, Leiden, The Netherlands. At fiyst the fluorophotometer signal representing the cornea was verified to originate mainly from corneal fluorescence and not from scattered excitation light at the corneal surface. The minimal percentage fluorescence in the signal as determined using a fluorescence blocking filter amounted to about 9OZ. The corneal autofluoreseence was determined in 23 "Type I (insulin-dependent)" diabetes mellitus and 18 "Type 2 (non insulin-dependent)" diabetes mellitus patients by fluorophotometry and compared to that of 22 healthy controls to detect possible alterations in autofluorescence as a result of diabetes. The mean corneal autofluorescence values in the Type I and Type 2 patients were significantly higher than the value of the healthy controls (mean values in ng. equivalent fluorescein/ml ~ SD: 20.6 ~ 5.1, 18.0 + 4.2 and 13.7 ~ 3.7, respectively; P < 0.01). The mean values in both patient groups did not differ significantly (P > 0.1). The autofluorescence values were independent of age in all groups (r < 0.47) and were linearly correlated with the diabetes duration in the joint patient groups (r = 0.6, P = 0.02; increase: 0.36 ng.eq, fluorescein/ml per year of diabetes). These results show that corneal autofluorescence is a useful parameter in ophthalmology which might be of assistance for in vivo evaluation of corneal metabolism.

Laminin is a m a j o r n o n - c o l l a g e n o u s glycoprotein of b a s e m e n t m e m b r a n e s . It is w e l l Known that thickness of b a s e m e n t m e m b r a n e s is h i g h l y characteristic of d i a b e t i c m i c r o a n g i o p a t h y . In o r d e r to investigate whether serum laminin concentration is a s s o c i a t e d w i t h the s e v e r i t y of d i a b e t i c m i c r o a n g i o p a t h y , we d e t e r m i n e d s e r u m l a m i n i n PI ( l a r g e r fragment,pepsin-resistant p r o d u c t of l a m i n i n ) by radioimmunoassay (Lam Pl, B e h r i n g w e r k e AG) in 89 diabetic patients (age:47.8• T y p e i (insulin-dependent) and 58 Type 2 ( n o n - i n s u l i n - d e p e n dent). P a t i e n t s w i t h r e n a l failure , h e p a t o p a t h y or a l c o h o l i s m w e r e e x c l u d e d . A c c o r d i n g to retinal status,diabetic p a t i e n t s w e r e divided/nto 3 ~-oups: l=no r e t i n o p a t h y , 2 : n o n - p r o l i f e r a t i v e retinopathy, 3:proliferative r e t i n o p a t h y . S t a t i s t i c a l methods: a n a l y s i s of v a r i a n c e and l i n e a r r e g r e s s i o n anal• sis. S e r u m l a m i n i n Pl was h i g h e r in group 3 (1.87• 0.39 U/ml) than in g r o u p 2 ( 1 . 7 4 • U/ml) a n d in g r o u p i ( 1 . 4 3 • U/ml), ( p < 0 . 0 0 1 ) . T h e s e res u l t s w e r e i n d e p e n d e n t of a g e , b o d y m a s s index and l e v e l s of f a s t i n g b l o o d g l u c o s e or H b A l c detemmined at same time. In c o n c l u s i o n , o u r f i n d i n g s sug g e s t that p l a s m a l a m i n i n Pl r e f l e c t s the severity of d i a b e t i c m i c r o a n g i o p a t h y and m a y he u s e f u l as s e r u m m a r k e r of m i c r o v a s c u l e r disease progression.

A157

PS 18 Endothelium and Rheology P574

P575

IFR~UNE NEUTRALIZATION OF PLATELET ANIONIC SITES IN DIABETES. ROLE OF CROSS-REACTIVE ANTI-ssDNA. G. Triolo, E. Giardina, G. Scarantino, G. Seddio~ D. Casiglia and G.D. Bompiani Istituto di Clinica Medica, University of Palermo, Italy 21 of 50 sera from children with type i (insulin-dependent) diabetes (duration of diabetes 4.4 • 2.9 yrs) showed increased platelet IgG binding. This anti-platelet reactivity was significantly (p< 0~025) associated with the presence of heparan-sulfate cross-reactive anti-ssDNA antibod-ies (10/14, anti-DNA+; 11/36, anti-DNA-). The binding was ~ot prevented by immune complex precipitation with PEG-6000 and as well as for DNA-anti-DNA interaction, increas iug salt concentration of the dilution buffer there was a decrease in the binding of positive sera. Other studies showed that neutralization of platelets by treatment with poly-l-lysine or with heparitinase caused a further increase in the igG binding that was not prevented by preincubation with ssDNA and/or heparan-sulfate. The addition of purified (ammonium sulphate precipitation, Protein ASepharose 4B chromatography) IgG (300 ug/ml) from anti-DNA + but not from anti-DNA - sera to washed platelets caused an increased collagen-induced aggregation (from ii • 5 to 42 • 7 %) similar to that obtained with the addition of poly-l-lysine (from 12 • 5 to 32 • ii %). ionic interaction between anti-negative charged molecules antibodies (i.e.-~ anti--ssDNA) and platelet surface negative charges may be a pathophysiological mechanism contributing to the altered platelet function observed in diabetes. The reduction of piatelet anionic sites may lead also to the binding of "anionic" non antigen-specific immunoglobuiins that furtherly may cause platelet alteration.

ENDOTHLIUM-DEPENDENT INHIBITION OF VASCULAR Na+/K+/ATPase ACTIVITY BY HIGH GLUCOSE. S.GUPTA, R.A.COHEN, N.B.RUDERMAN and I.SUSSMAN. BOSTON UNIVERSITY MEDICAL CENTER, BOSTON, MA 02118. Hyperglycemia has been reported to alter both vascular eicosanoid production and the modulation of vascular reactivity by endothelium-dependent relaxants. It has also been shown to decrease Na§ § ATPase activity (NaKA) in a number of tissues including the aorta. To determine whether endothelium influences decrease in NaKA, rabbit aortic rings with or without endothelium were incubated in 5.5 mM or 44 mM glucose. NaKA was measured as ouabain(0.2mM)sensitive (O.S.) ~Rb(2uCi/ml)-uptake. Data is represented as K § nmol/min/mg (dry tissue weight). Removal of the endothelium did not significantly affect total or O.S. ~Rb-uptake in 5.5 mM glucose (With endothelium 0.31+_0.04; Without endothelium 0.25+0.04). After 3 hrs of incubation in 44 mM glucose, O.S. uptake was significantly decreased in rings with intact endothelium (0.17+0.03). In contrast, in the absence of endothelium O.S.uptake by the aortic rings was similar to that at 5.5 mM glucose(0.34+0.06). Incubation of intact rings for 6 hrs led to lower NaKA at both glucose concentrations (0.11 +_0.01 and 0.15+0.06 for 5.5 and 44mM glucose respectively). Also, differences between the high and low glucose groups were not observed. The results suggest that endothelium may alter vascular NaKA and may also play a role in the decrease in vascular NaKA produced by hyperglycemia.

P576

P577

EFFECT OF METABOLISM Department School of T.Arisaka, K.Sawada,

INSULIN AND CORTISOL ON GLYCOS~qINOGLYCAN(GAGs) IN CULTURED ENDOTHELIAL CELLS of Internal Medicine, Juntendo University Medicine T.Tohjima, K.Fujii, K.Mochizuki, M. Sakamoto, H.Funayama, T.Kurosawa, S.Hirose.

Acute vascular complications such as ischemic heart disease, cerebral stroke etc., under stressed condition are frequently found in diabetics. But, the relationship between vascular lesion and stress is still unknown. On the other hand, the diabetic state, so called insulin insufficiency, has been implicated in the pathogenesis of atherosclerosis. We studied the effect of insulin and cortisol on synthesis of glycosaminoglycan(GAGs) by cultured porcine endothelial cells, and also studied the effect of somatostatin. [Method] Endothelial cells were obtained from the intima-media of the porcine aorta. Subconfluent culturs were incubated with ~H-glucosamino-hydrochloride at each concentration of insulin(10-6,10-7,10-s,10-9,0M) hydrocortisone(10-3,10-5,10-7,0M) and somatostatin (10-s,lO -~, I0-7,10-8,10-9,0M) for 72 hrs. Metabolically labelled GAGs in cultured medium and cell layer were analysed. [Results] I. Cortisol had a dose-dependent suppression on synthesis of Hyaluronic acid at each insulin concentration, but did'nt have a dose-dependent suppression on synthesis of sulfated GAGs below 10-SM. 2. Insulin dosedependently stimulated synthesis of sulfated GAGs significantly (p<0.05), but had no effect on synthesis of Hyaluronic acid. 3. Somatostatin had no effect on synthesis of HA~ sulfated GAGs. [Discussion] Our observation at cellular level suggests that reduced production of heparan sulfate in endothelial cell may take part in the pathogenesis of vascular diseases.

DECREASED SIALYLATION OF GLYCOPHORIN IN DIABETIC RED CELLS M. Rogers,D. Williams, R. Niththyananthan, M. Rampling*, K. Heslop and D.G. Johnston. Departments of ClinicalEndocrinology and Physiology and Biophysics*,St Mary's Hospital Medical School,Norfolk Place, London, W2, England. Loss of negative charge on the red cell membrane may contribute to the abnormal blood rheology observed in diabetes. This negative charge on the erythrocyte membrane has been attributed partially to sialic acid. Glycophorin is the major sialic a c i d - c o n t a i n i n g p r o t e i n in e r y t h r o c y t e membranes, and this study has investigated therefore its glycosylation. Red cells ghosts were prepared from diabetic and non-diabetic patients. Sialoglycoproteins were extracted from the erythrocyte ghosts by treatment with butanol and glycophorin A was purified by chromatography (Sepharose 6B). On the final purified samples, total protein was estimated and 50-150 ~tg of purified glycophorin were obtained. Content and purity were assessed by gel electrophoresis with staining for carbohydrate using a periodic acid method. Quantitative carbohydrate analysis was performed by gas chromatography:mass spectrometry. Sialic acid content was decreased in the diabetic red cells (mean decrease 76% p= 0.015, comparing results from 9 diabetics and seven controls). N-Acetylgalactosamine content was also reduced (by 42%), but no difference was observed in the content of mannose. These data provide a precise chemical basis for the reduction in negative charge on diabetic erythrocyte membranes. The decrease in sialic acid and other carbohydrate analysis suggest a decrease in 0-1inked oligosaccharides.

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P578

P579

INCREASED RED CELL AGGREGATION 1N TYPE 2 (NONINSULIN-DEPENDENT) DIABETICS WITH HYPERTENSION

IS HIGH GLUCOSE RESPONSIBLE FOR ALTERED FIBRINOLYSIS? CLINICAL AND EY~ERIMENTAL OBSERVATIONS. D. Boeri, M. Maiel]o, E. Cag]iero ~, M. Lorenzi *, G. Falzetti, F. Podesta', A. Tigani, hl. Vichi and L. Adezati - Cliniea 5~diea RE I.S.M.I. University of Genova (ITALY) - * Eye Research Institute Harvard Boston (USA) Reduced fibrino]ysis is reported in diabetics; endothelium modulates it producing tissue plasminogen activator (t-PA) and its inhibitor (PAI-I). We documented that exposure of human endothelial cells (HEC) to high glucose enhances their proeoagulant activities. We have now explored whether high glucose affects t-PA and PAI-I production in HEC in vitro and if there is a correspondence between clinics/ finding and in vitro effects. In HEC grown in 5 or SO mM glucose, harvested at conf]uency, medium eoneentrations of t-PA and PAI-I toghether with the corresponding mRNAs were determined. In diabetics and controls plasma concentrations of t-PA, PAI-I and B-beta 15-42 peptide were also evaluated. HEC grown in SO mM glucose showed significantly higher t-PA (235+/-162% of controls), PAI-I (256+/-156%) mRNAs and protein concentrations in the medium (t-PA:S2+/-7 vs 45+/-9 ng/m] p
SM MacRury, P McColl, R Balendra and GDO Lowe. Diabetic Unit and University Department of Medicine, Royal Infirmary, Castle St., Glasgow, UK. Red cell aggregation (RCA) is a major determinant of whole blood viscosity which is increased in diabetes and hypertension, and correlates with left ventricular mass, a risk factor for cardiovascular disease. Diabetic patients have a greater incidence of hypertension and earlier onset of vascular complications and we therefore measured red cell aggregation photometrically as an index using the Myrenne red cell aggregometer in 60 type 2, non-insulin-dependent diabetic patients compared with 50 healthy control subjects to investigate the association with hypertension and macrovascular disease. RCA index was significantly higher in diabetics (5.51 +__1.53) compared with controls (3.15 + 1.30, p<0.001, unpaired Wilcoxon test). Hypertensive diabetics had significantly higher RCA index (n=20, 6.3 + 1.8) compared with type 2 normotensive diabetics (n=40, 5.2 + 1.3, p<0.02) and RCA index correlated wkh diastolic blood pressure (r=0.25, p<0.05, Spearman rank test). Hypertensive diabetics with clinical evidence of macrovascular disease had a higher RCA index (7.8 _+ 1.2) compared with hypertensive diabetics without macrovascular disease (5.6 + 1.6, p<0.02), while in normotensive patients RCA index was similar in those with vascular disease (5.1 + 1.3) and those without (5.2 + 1.3). Red cell aggregation is increased in hypertensive diabetics and further increased with concomitant vascular complications. The red cell aggregation index may have a prognostic value in non-insulindependent diabetics with hypertension.

P580

P581

CONTRASTING EFFECTS OF TYPE I (INSULIN-DEPENDENT) AND TYPE 2 (NON-INSULIN DEPENDENT) DIABETES ON FIBRINOLYSIS. D Walmsley, KK Hampton, MH Stickland and PJ Grant. University Department of Medicine, The General Infirmary, Leeds, LSI 3EX, UK. The fibrinolytic enzyme system is important for maintenance of vascular pateney. To study fibrinolysis in relation to microvascular diabetic complications, 20 controls were compared to age matched type I diabetic patients with a: no complications (n=20), b: lasertreated netinopathy (n=17) and e: neuropathy (n=13). None were smokers, hypertensive or had macrovascular disease. Pre- and post-venous occlusion (V0) blood samples for tests of fibrinolysis were taken. Median basal tissue plasminogen activator (tPA) activity was lower in controls (100 mIU/ml) than diabetic patients (a: 145 mIU~ml, p=0.015; b: 180 mIU/ml, p=0.037; c:210 mIU.ml r p=0.004 respectively). Basal tPA inhibition was higher in controls (5.9 IU/ml) than diabetic patients (a:4.0 IU/ml, p=0.001; b:4.5 IU/ml, p=0.058; c:4.0 IU/ml, p=0.015 respectively). Post-V0 tPA antigen was higher in controls (10.2 ng/ml) than neuropathic patients (5.5 ng/ml, p=0.004). Other tests showed a consistent, but non-significant, trend towards increased basal fibrinolysis in the type I patients. The results indicate that type I diabetic patients have enhanced basal fibrinolysis. The diminished response to V0 isconsistent with an endothelial cell defect. These findings are in marked contrast to those in 20 uncomplicated type 2 patients who had diminished basal and post-V0 fibrinolytic activity with high t-PA inhibition. The differing fibrinolytic responses may contribute to the heterogenous nature of vascular disease in diabetes.

HIGHER MEAN PLATELET VOLUME IN DIABETICS G. Aliberti, E. Vecci, C.M. Oddo and I. Pulignano II Clinica Mediea, University of Rome "La Sapienza" Abnormalities of platelet function are documented in patients with diabetes mellitus (DM). The aim of the present study has been to evaluate in DM some platelet indices, that seem not fully investigated. Platelet count (PLT), platelet haematocrit (Pct), mean platelet volume (MPV) and platelet distribution width (PDW), measured with Coulter Counter Mod. S Plus II, were studied in $75 DM, 87.4 + 18 mean aged, 74 with type i, 41 men and 33 women, 301 with type 2, ISO men and 171 women, and in 90 sex and age matched control subjects (C). PLT (x I09/L) 258.5S3 + 82.150, Pet (%) 0.224 + 0.069, MPV (fl) 8.74 + 1.04, PDW (%) 16.75 + 0.59 in C and respectively 278.218 + 93.110, 0.252 + 0.087*, 9.S0 + 1.23", 17.01 + 0.81 in DM (*: p < 0.001) were found. Results show higher MPV and Pct in DM. There are studies indicating megathrombooyte count as an indirect evaluation of accelerated rate of platelet production in response to shortened systemic platelet survival. The large platelet have been shown to be more active functionally and to have a greater adhesiviness and a more rapid aggregability and to progress with age to smaller size. The increased MPV observed in DM may be belived an index of increased peripheral consumption, due to the enhanced intravascular platelet adhesion and aggregation, expression of the associated excess of vascular disease. The significantly higher Pct may be in part explained with the higher MPV.

A159

P582

P583

I n c r e a s e d p l a t e l e t a c t i v i t y d u r i n g e x e r c i s e in i n s u l i n - d e p e n d e n t d i a b e t i c p a t i e n t s w i t h albuminuria. T Jensen, B F e l d t - R a s m u s s e n , A B H a a b e r and T Deckert. Steno M e m o r i a l Hospital, Gentofte, Denmark.

ALTERED "INTEGRIN" EXPRESSION AND DNA-PLOIDITY PATTERN IN MEGAKARYOCYTES OF DIABETIC BB-RATS D.Tschoepe, B.Schwippert, B.Schettler, U.Kiesel, U.Schawach, P.Roesen and F.A.Gries. Diabetes Research Institute at the Heinrich Heine University, Duesseldorf, FRG.

I n s u l i n - d e p e n d e n t d i a b e t i c p a t i e n t s w i t h clinica] n e p h r o p a t h y have an e ~ t r e m e l y high m o r t a l i t y ofc a r d i o v a s c u l a r diseases. I n c r e a s e d p l a t e l e t activity, p o s s i b l y leading to i n t r a v a s c u l a r thrombosis and r e l e a s e of a t h e r o g e n i c g r o w t h factors has been s u g g e s t e d to be of i m p o r t a n c e for the d e v e l o p m e n t of a t h e r o s c l e r o s i s . P l a t e l e t activity, at rest and d u r i n g exercise, was m e a s u r e d in 28 i n s u l i n - d e p e n d e n t d i a b e t i c males m a t c h e d as to age and d i a b e t e s d u r a t i o n in two groups: group I (urinary a l b u m i n e x c r e t i o n (UalbV) <30 mg/24h; n=15) and group II (UalbV >30 mg/24h, i n c i p i e n t and c l i n i c a l d i a b e t i c nephropathy; n=13). Ten a g e - m a t c h e d non d i a b e t i c males served as controls. In vivo p l a t e l e t activity, m e a s u r e d as the plasma c o n c e n t r a t i o n of the p l a t e l e t sec r e t e d p r o t e i n b e t a - t h r o m b o g l o b u l i n , was similar at rest in the three groups. F u r t h e r m o r e , no s i g n i f i c a n t d i f f e r e n c e in p l a s m a c o n c e n t r a t i o n of b e t a - t h r o m b o g l o b u l i n d u r i n g e x e r c i s e was found b e t w e e n the c o n t r o l group and group I (47 ng/ml vs. 60 ng/ml), but it was s i g n i f i c a n t l y h i g h e r in group II (89 ng/ml) (p<0.05). In vitro platelet a c t i v a t i o n was ruled out by s i m u l t a n e o u s m e a s u r e m e n t of p l a t e l e t factor 4. These findings suggest that p l a t e l e t a c t i v a t i o n is a c c e l e r a t e d d u r i n g exercise in i n s u l i n - d e p e n d e n t d i a b e t i c p a t i e n t s w i t h albuminuria. We b e l i e v e that gener a l i z e d e n d o t h e l i a l injury, r a t h e r than p r i m a r y p l a t e l e t h y p e r a g g r e g a b i l i t y , is r e s p o n s i b l e for this abnormality.

Glycoprotein (GP) liB/IliA expression is enhanced on hyperactive diabetic platelets indicating altered thrombopoiesis. Therefore, we directly investigated the expression of the "integrin" GPIIB/IIIA and the ploidity pattern of bone marrow megakaryocytes [mkc] in 8 acute diabetic [AD] (plasma glucose [pg] = 444_+90 mg/dl) and 20 insulin treated diabetic [ITD] BB-rats (pg=361.5+62.7 mg/dl) vs 22 non-diabetic [ND] BB-rats (pg= 97.5 +23.7 mg/dl). Bone marrow was aspirated from both femura and mkc's were enriched by double density gradient centrifugation. The enriched cell layer was resuspended and fixed in separate tubes. Mkc- membrane bound GPIIB/IIIA was directly stained with the monoclonal antibody conjugate PLT1-FITC (Coulter Hialeah, USA). Simultaneously the complementary cell DNA-content was labelled with propidiumjodid. The cells were then analysed with two colour fluorescence single cell analysis (FACS). PLT-FITC binding was increased in both diabetic groups: [ND] 294_+77 logarithmic scale units vs 335+82 [AD] and 434+96 [ITD, p<0.0001]. The maximal frequency peak of the modal ploidity pattern was distributed to 16n _+4.3 [ND]) vs 2.5n -+0.9 [AD, p<0.0001] and 14.9n + 12.9 [ITD]. Thus, acute development of diabetes induced an altered megakaryocytic ploidity pattern which was reversed by insulin. Megakaryocytic expression of GPII/IIIA was however consistently increasing providing direct evidence that peripheral platelet glycoprotein enhancement is primarily conditionated during thrombopoiesis.

PS 19 Autonomic Neuropathy P584

P585

SWEAT GLAND ACTIVATION AND SYMPATHETIC SKIN RESPONSE AS INDEPENDENT MEASURES OF SMALL-FIBRE DIABETIC NEUROPATHY. RR ABRAHAM, DM LEVY, D ROWLEY, T JULSRUD BERG, G REID and

SIMULTANEOUS RECORDINGOF SKIN SYMPATHETIC AND CARDIAC PARASYMPATHETIC FUNCTIONS IN DIABETICS

C. Ionescu-Tirgoviste, S. Prena, and I. Mincu, Clinic of Diabetes, Bucharest, Romania

S McHARDY-YOUNG. Diabetic Neuropathy Research Group, Central Middlesex Hospital, London NWl0 7NS, UK. Sudomotor function of the foot was assessed in 71 diabetic (45yr (SD 15), 34 Type 1) and 44 age-matched normal subjects using both pilocarpinestimulated sweatspot counting (computerised image-analysis) and the sympathetic skin response (SSR) (mean of 12 responses to a deep breath) and related to tests of small and large fibre function. Mean normal sweat spot density was 78/cm2 (SD 27) (CV 20%); all 15 (21%) diabetic patients with low densities (<24/cm2) and 5/6 with high densities (>132.cm2), had neurophysielogical evidence of peripheral neuropathy, even though the latter were younger (28yr vs. 50yr); only one was symptomatic. Mean SSR onset latency was longer and peak amplitude (first wave) lower in diabetic patients (1836ms. vs 1564ms (p<0.001); 254uV vs 350uV (p=0.006)); 13(18%) had prolonged responses. Patients with symptomatic autonomic neuropathy had prolonged latencies (p=0.02) and lower amplitudes (p=0.07). Sweatspot density was associated with SSR amplitudes (r=0.32, p=0.02) but not with onset latency which was associated with different nerve tests (warm threshold (Somedie) @=023, p=0.07), sural sensory amplitude (r=0.37, p=0.05), and respiratory RR variation (r=.0.26, p=0.05). Both methods reliably detect neuropathy though they reflect different aspects of peripheral nerve function and sweatspot counts particularly are valuable in identifying patients with denervation hypersensitivity.

Skin sympathetic a c t i v i t y was recorded as electrical skin impedance transient response (ESITR) using a dual-channel self-balancing autonomic reactometer. Peak of the electrical signal (g/s change) elicited by a standard stimulus was obtained from palm and sole of the foot with two pairs of surface electrodes. Parasympathetic cardiac a c t i v i t y was assessed by a cardiorhytmometer which automatically records the difference in heart rate (AHR bpm) between two successive measurements of 8 photoplethismographic signal finger pulse, one in prolonged inspiration and the other in prolonged expiration. Three sets of successive measurements were obtained from each subject in 91 randomly selected diabetic patients (57M/37F; mean age ~ SD, 47 12 yrs; duration of disease 8 . 2 • 7.1 yrs; 56 insulin-treated and 35 non-insulin treated). The data were compared with those recorded in 89 age and sex matched non-diabetic controls. There were significant differences (p
A160

P586

P587

Assessment of 24-hour sympatho-vaga] activity in ambulant diabetics by power spectral analysis of heart rate variability

Persistence of respiratory sinus arrhythmia in diabetics with low heart rate variability: vagal or intrinsic mechanism?

L.Ricordi, P.L.Solda', A.Calciatl, P.Lazzari, M.R.FerrarJ', I.Vandea*, A.Giardullo, P.Fratino and L.Bernardl. Dept.Internal Medicine, University of Pavia and IRCCS S.Matteo, Pavia, *Carlo Poma Hospital, Mantua, Italy

L.BernardJ, L.Ricordi, S.Perlini, P.L.Solda', S.LeuzzJ, A.CaIciati and P.Fratino. Dept.Internal Medicine, University of Pavia and IRCC$ S.Matteo, Pavia, Italy

In order to study the circadian modulation of sympatho-vagal activity in diabetes we performed continuous power-spectral analysis (PSA) of 24-hour electrocardiographic RR-tntervaI variability in 15 diabetic subjects with mild to severe autonomic neuropathy (AN), in 9 age-matched diabetic subjects without AN and In 29 age-matched controls. PSA is a new technique allowing to quantify both the RR-interval oscillations between 0.15-0.4Hz, synchronous with respiration and under vaga] control (HF), and the oscillations around 0.1Hz (LF),independent from respirationand under both vaga] and sympathetic control.Compared to diabeticswithout AN and to controls, diabetics with AN showed a significant reduction in LF (9.4+-2.1msec= vs 13.0+_1.6 vs 14.3+1.2, p<0.05) and an even greater reduction in HF (7.3+t.tmsec 2 vs 73• vs 14.7+_!.6, p<0.0I) during the night and the first morning hours, whereas no differences were observed during afternoon (LF: 8.9+2.gmsec= vs 11.1• vs 13.3+1.3, p=ns; HF: 5.3+_1.7msec= vs 5.2• vs Z8.4• p=ns). Thus, diabetic subjects with AN have an abnormal relative decrease of vagal activity (and hence a relatively higher sympathetic activity) at the same day-time during which a higher frequency of sudden death has been reported. These findings may explain the increased risk for cardiac accidents related to diabetic AN.

The proportion of 0.1Hz non-respiratory (LF) and respiratory-related (HF) components (LF/HFratio) of heart rate variability(HRV), obtained by autoregressJve power-spectral analysis, is a marker of sympatho-vagal control of the heart under various physiological conditions. To evaluate the sympatho-vagal balance in autonomic dysfunction we measured LF and HF in supine position and after tilt-induced sympathetic activation in ? diabetics (D-) (age 38+_3years, mean+SEN) with low (20beats/rain) (D+) and in 10 age-matched controls (C). After t i l t the LFIHFratio increased in C (I.68+_0.95 to 31.71+15.32, p<0.005) and D+ (2.28+0.49to 8.15+2.07,p<0.001),due to a reduction in HF, but remained unchanged in D- (0.12+0.09to 0.13+0.10,p:n.s).The low LF/HFratio in D- was due to a large predominance in HF both before and after t i l t . Furthermore, atropine injection (0.05mg/kg,i.v.) in one D- subject failed to abolish HF. Thus, despite the parasympathetic damage reported in diabetics with low HRV, respiratory-related oscillations remained the largest part of HRV regardless of changes in autonomic tone. This suggests that in D- the residual HF can be due to intrinsic sinus node activity, independent of parasympathetic tone and revealed by loss of autonomic modulation.

P588

P589

THE ASSESSMENT OF THE BARO REFLEX SENSITIVITY IN DIABETIC PATIENTS. TH.J.C. Faes (i), P.Lanting (2), F.W. Bertelsmann (2), B. ten Voorde (i) and 0. Rompelmann (I). (i) Department of Medical Physics, Free University and (2) Dept. of Neurology, Free University Hospital, Amsterdam, The Netherlands. The Baro Reflex Sensitivity (BRS) is a measure of the strength of the baro reflex arc, conventionally obtained by administering a vasoconstrictor. BRS is defined as the change of heart rate relative to the change in blood pressure and expressed in ms/mmHg. The aim of the study was to investigate the value of BRS as a test for cardiac autonomic neuropathy. Two age matched groups of 63 non-diabetic and 33 diabetic adults were studied. During forced breathing manoeuvres (6 breath/min) beat to beat changes in heart rate (HR) and blood pressure (BP) were measured by conventional ECG-techniques and a non-invasive blood pressure monitor (Pinapres). BRS was obtained by spectral analysis of ER- and BP-variability. Compared with the control group, the BRS was significantly reduced in the group of diabetic patients (mean • SEM): 19 • 1 ms/mmHg, versus 8 • I ms/mmHg, p
SPECTRAL ANALYSIS OF SPONTANEOUS HEART RATE VARIABILITY IN DIABETIC PATIENTS WITH AND WITHOUT CARDIOVASCULAR AUTONOMIC DYSFUNCTION. P. Lanting, Th.J.C. Faes, F.W. Bertelsmann and J.J. Heimans. Free University Hospital, Dept. of Neurology, Amsterdam, The Netherlands. Spectral analysis of spontaneous heart rate variability was performed in 63 control subjects and 61 diabetic patients. The power in het mid-frequency band (MF;0.04-O.12 Hz), related to baroreceptor activity and high-frequency band (MY;0.12-0.5 Hz), which is respiratory linked, was measured with the patient in supine and standing position. The diabetic patients were divided into three groups according to the degree of abnormality of cardiovascular reflex tests (heart rate variation with deep breathing and heart rate response to standing). It was possible to define normal age related values for the power in the MF and HF band, supine and standing. Compared with the control subjects, the patients with normal cardiovascular reflex tests had reduced power in the MF and HF band, supine and standing: MF supine (mean • SEM): 1340• Hz versus ~ 0.619 • 0.128 HZ~ p<0.05; MF standing (mean • SEM): 3.068 • 0.523 HzZversus 1.358 • 0.299 Hz~ p
A161

P590

P591

aUTONOMIC NEUROPATHY OF THE URINARY BLADDER IN

ERECTILE IMPOTENCE IN DIABETIC PATIENTS

CORRELATION TO GLYCEMIC CONTROL

W.D. Hetzel, C. Neyerhoff, Chr. Schmid and E.F. Pfeiffer, abteilung Inhere I, Medizinische Zlinik und Poliklinik der Universit~t Ulm We studied the function of the urinary bladder under the influence of glycemic control. In 65 unselected diabetics we estimated sonographically the volume of first sensation to void (VFS} and residual urine (RES}. Diabetic complications were documented. VFS was dependent upon Hbal: Group I (n=6, Hbal 5-8%, mean 6.9% • VFS 366 • ml, Group II (n=13, HbAI>8-10%, m. 9.3_+0.4) VFS 432 • ml, Group III (n=12, Hbal>lO-12%, m. 11.O• VFS 466 • (p12%, m. 13.7~1.6) VFS 529 • ml (p5OO ml, 35% of the patients) was significantly often associated with autonomic polyneuropathy. Peripheral polyneuropathy (52%), impotence (50%), retinopathy (49%) and nephropathy (29%) were dependent upon age and duration of diabetes. 29% had autonomic polyneuropathy. Diminished sensation of a filled bladder apparently is a sensitive parameter of asymptomatie autonomic neuropathy of the urinary bladder. It is dependent upon glycemic control and associated with cardial autonomic polyneuropathy.

C. Neyerhoff, N.D. Hetzel, F. Bischof and E.F. Pfeiffer, abteilung Innere I, Medizinisehe Klinik und Poliklinik der Universit~t Ulm Erectile impotence is due to neuropathy or angiopatby. We studied 50 men. Group I consisted of 30 subjects with erectile failure, Group II consisted of 20 male diabetics, who had no erectile problems. Glycemic c o n t r o l was poor i n both groups (Hbaz 10.7%!2.3% and 11.5%• Diabetic complications were monitored by physical examination, fundusscopy, heart rate variability and microalbuminuria. In 16 of the impotent patients (age 29-56, median 47) we estimated penile blood pressure (PBP) by Doppler sonography and bulbocavernosus reflex latency (BCR). Statistical analysis was done using Mann-Whitney UTest and Chi=-Test. Plasma testosterone levels of all patients were normal. 74% (35%*; *p
P592

P593

IS DIABETIC NED~ROPATHIC CACHEXIA DUE TO MALABSORF210N?

T h e s e c r e c t i o n of p a n c r e a t l c p o l y p e p t i d e ( P P ) after the loadlng of a t e s t m e a l in type IIdiabetic patients with autonomic neuropathy (AN).K. B r o Z y ~ s k i , J. Loba, P. W o Z n i a k , W . T o r z e c k a . Academy of M e d i e i n e , ZOdZ, POLAND. T h e a i m of t h i s s t u d y w a s the e v a l u a t i o n of t h e s t i m u l a ted s e c r e t i o n of P P in p a t i e n t s w i t h t y p e II diabetes mellitus complicated by latent or overt AN, A N w a s d i a g n o s e d o n the b a s i s of widely used diagnostic tests and clinical criteria. 20 patients were classified fop hormonal examinations. Group I(control)-8 patients w i t h o u t a n y s i g n s of AN, g r o u p II-6 patients with latent AN and III-$ patients with overt AN. T h e glycemla, IR-PP and i n s u l i n ( I R I ) w e r e e x a m i n e d i n all the p a t i e n t s after loading of the m i x e d t e s t meal(about 1870 kJ). The blood was taken fasting and 15",50",60',90" a n d 120". The statysticai analysis(Student % - t e s t ) w a s p e r f o r m e d . It h a s been s t a t e d t h a t a f t e r the m i x e d m e a l in the g r o u p If a n d III t h e r e w a s n o p e a k secretion of IR-PP in the f i r s t ~O" (no Ith phase of s e e r e c t i o n ) a n d t h e r e w a s a d e f i c i e n c y of the secretion b e t w e e n 3 0 " , a n d 120' (in 2 n d phase of the s e c r e t i o n ) . T h e l o w e s t i n c r e a s e s of IRPP secretion were observed in the group III(240 n g / l v s 937 ng/l, p < O, OOi). In the c a s e of IRI secretion the s i m i l a r c h a r a c t e rlStic of curves was observed in all 3 groups. N o s i g n i f i c a n t d i f f e r e n c e s as f a r as the b e h a v i o u r of glycemia w e r e observed. T h e results of the p e r f o r m e d s t u d i e s show that I R - P P s e c r e t i o n a f t e r m i x e d m e a l is d e f l c i e n % in t y p e II d i a b e t e s c o m p l i c a t e d b y l a t e n t a n d overt AN. The observed changes in IR-PP s e c r e t i o n do n o t d e p e n d o n h y p e r g l y c e m i a .

D F D'Costa, D E Price and A C Burden. Diabetes Research, leicester General Hospital, leicester, LE5 ~PW, UI<. Diabetic neuropathic cachexia is characterised by a weight loss of greater than 20% and severe neuropathic pain - the cause is unknown. We describe ~ cases with co-existent malabsorption. The clinic features of the 3 males and 1 female: mean age 53 (range 45-67)years, mean duration of known diabetes 3.3 (0.4-6)years, mean weight loss 18 (10-21) kg, mean haemoglobin AI 8% (?.6-8.2%)i( normal range 4-8.5%) all had neuropathic pain, anorexia and depression. The malabsorption tests showed: a mean seldom butterfat difference of 4 units (0-9) (normal greater than 60 units), low serum xylose and a delayed urinary xylose excretion, low serum vitamin BI2 and abnormal schilling tests. Bacterial overgrowth and coeliac disease were excluded. Ultra sound scans of liver, gall bladder and pancreas and the endoscopic retrograde cholangiopsncreatogram were normal. They were treated with pancreatic supplements and an increased calorie intake. 2 have completely recovered and the other 2 are improving. The malabsorption tests were no~_~l in diabetic controls. The abnormalities are due to reduced pancreatic exocrine function and gastric st~sis. Tests of malabsorption and subsequent specific treatment are recommended in diabetic neuro pathic cachexia.

A162 P594 GALL BLADDER FUNCTION IN INSULIN DEPENDENT DIABETICS

F. Fuchs, C. Meyerhoff, W.D. Hetze/ and E.F. Pfeiffer, Abteilung Innere I, Nedizinische Klinik und Poliklinik der Universit~t Ulm Aim of this study was to investigate the contractility of the g a l l b l a d d e r i n i n s u l i n e dependent d i a b e t i c s . Three d i f f e r e n t s t i m u l i of the g a l l bladder were used on t h r e e d i f f e r e n t days: c a r b a t h o l e , c e r u l e t i d e ( c h o l e c y s t o k i n i n e - a n a l o g o n ) and s t a n d a r d i z e d o r a l meal, c o n s i s t i n g o f 1 7 . 9 g s o r b i t and 20 g d r i e d e g g y o l k i n 200 ml w a t e r . The volume of the g a l l b l a d d e r was f o l l o w e d up f o r one hour s o n o q r a p h i c a l l y a f t e r t h e s t i m u l u s . C o n t r a c t i o n speed was e x p r e s s e d a s p e r c e n t u a ] d i m i n i s h i n g of v o l u me/time ( V o l % / m i n . ) . D i a b e t i c c o m p l i c a t i o n s were s c r e e n e d f o r by p h y s i c a l e x a m i n a t i o n , d e t e r m i n a t i o n of m i c r o a l b u m i n u r i a , h e a r t r a t e v a r i a b i l i t y and f u n d u s s c o p y . C e r u l e t i d e and c a r b a c h o l e r e s u l t e d i n c o m p l e t e c o n t r a c t i o n of t h e g a l l bladder i n d i a b e t i c and c o n t r o l p e r s o n s . C o n t r a c t i o n speed was d i m i n i s h e d i n d i a b e t i c s a f t e r t h e s t i m u l a t i n g meal ( 2 . 1 Vol%/min • 0 . 3 v s . 3 . 9 Vol%/min • 0 . 4 , p < O . 0 5 ) . There was no d i f f e r e n c e i n t h e d e g r e e o f g a l l b l a d d e r emptying, but i n t h e time of emptying ( ~ < 0 . 0 5 ) . Impaired c o n t r a c t i l i t y did not c o r r e l a t e t o HbA,, h e a r t r a t e v a r i a b i l i t y or o t h e r d i a b e t i c c o m p l i c a t i o n s . T h e r e f o r e d i m i n i s h e d c o n t r a c t i l i t y of t h e g a l l bladder in diabetics apparently is a result of d i s t u r b e d c o o r d i n a t i o n between q a s t r o i n t e s t i n a l t r a n s p o r t and c o n t r a c t i o n a l answer of t h e g a l l b l a d d e r . This d y s f u n c t i o n i s not due to d i m i n i s h e d r e s p o n s i v e n e s s to v a g a l or hormonal s t i m u l i .

P595 A ~ L I N E ~ . ~ A S E IN EXPERIMENTAL AUTONOMIC NEUROPATHY L. Uceioli*, P. Magnani, P. Til li, P. Cotroneo, A. Manto, L. Mane•177 A.V.Greco and G.Ghirlanda. *Malattie del Ricambio,Univ.Tor Vergata,Roma. Ist, Clin.Med; ,Univ.Cattolica S.Cuore,Roma. The aim of our study is to evaluate autonomic neuropathy by determining somatostatin(SS)- and potassitm(K)-evoked Ach-release from postsynaptie parasynpathetic fibers in the atria of control and streptozotocin diabetic rats S(3 m) and 6(6m) months, with and without tetrodotoxin (TTX). Slices (Imm) of atrial tissue wd~re incubated for 60'in Krebs-Ringer buffer containing H-choline and physostigmine. After addition of Hemichilinium-3, sequential ~IcdDations were performed over 90'. The effect of SS(IO ) and K(IO0 hiM) were examined at SO'. 8m and 6ni Aeh-release is not significantly different in diabetic (SS=14.5+5% and 25.6_+10%; K=12.8_+II% and 21_+13%) and in control rats (SS=17.2_+~% and 21.8+8.6%; K=II.8+13% and 26s TTX modifies K-evoked Aeh-release neither in diabetic (3m:12+5%; 6m:16+15%), nor in control rats (~n:i0.7+4%; 6m:21+7%). In the presence of TTX SS-induced Aeh-release is decreased in control (~n:4.4 +3%, p
P596

P597

P A N C R E A T I C P O L Y P E P T I D E R E S P O N S E T O A M E A L IS INDEPENDENT OF GLYCAEMIC CONTROL. M.H.Rasmussen, S.List, T.W.Schwartz and J.Hilsted Department of Endocrinology, Hvidovre hospital; Lab. o f M o l e c u l a r E n d o c r i n o l o g y , R i g s h o s p i t a l e t ; Copenhagen, Denmark.

THE VAGAL CONTROL OF GALLBLADDER MOTILITY IS IMPAIRED IN DIABETICS WITH AUTONOMIC [~UROPATHY. L. Scionti, *S. Fiorucci, *R. Bosso, F. Poreellati, C. Mar• no, G.P. Reboldi and F. Santeusanio. Istituto di Patologia Speciale Medica e Metodologia Clinica e *Cattedra di Gastr~ enterologia, University of Perugia, Italy. The aim of this study was to evaluate the separate effects of neural and hormonal stimulation on gallbladder emptying in lO normal subjects (C), 12 diabetic patients without (D) and 9 with autonomic neuropathy (DAN). We measured gallblad der volume (GV) after modified sham-feeding (MSF) and after cerulein ( C R ) infusion. MSF acts on gallbladder motility by vagal pathways, whereas CR (a CCK-analogue) by hormonal stimulation. CR infusion was performed in three steps(0.25, 1 and 4 ~g/kg/min), each lasting 20 min and separated by 60 min wash-out periods. GV was evaluated by sonography using a 3.5 MHz convex transducer. GV showed a significant (p ~ 0.001 vs basal) reduction only in C and D (-7.0!i.0 ml and -6.2+1.0 ml respectively) after MSF, without differences between the groups. On the contrary, GV increased in DAN after MSF (+3.4~1.7 ml; p~ 0.05 vs basal). GV did not show any difference among the groups after CR infusion at different rates (0.25 ~g/kg/min: C -45%, D -53%, DAN -46%; l~g/kg/min: C -69%, D -73%, DAN -71%; 4 yug/kg/min: C -87%, D -89%, DAN -88%). Conclusions: i) DAN have an impaired ne~ ral control of gallbladder emptying, whereas the hormonal control is preserved; 2) D have both normal neural and hormonal control.

T h e m e a l - i n d u c e d i n c r e a s e in P a n c r e a t i c P o l y p e p t i d e (PP) c o n c e n t r a t i o n s is u s e d as a m e a s u r e of v a g a l i n t e g r e t y in d i a b e t i c p a t i e n t s . S h o r t - t e r m c h a n g e s in m e t a b o l i c c o n t r o l m i g h t , h o w e v e r , ind u c e an a l t e r e d PP r e l e a s e , i n d e p e n d e n t of v a g a l nervous activity, since hyperglycaemia inhibits PP r e l e a s e f r o m canine pancreas. Seven Type 1 ( i n s u l i n d e p e n d e n t ) d i a b e t i c p a t i e n t s (age 2 6 f 3 yr (mean• d u r a t i o n of d i a b e t e s 4• yr) were studied on two occasions. They had no signs of autonomic neuropathy (beat-to-beat variation in h e a r t r a t e a n d p l a s m a c a t e c h o l a m i n e r e s p o n s e s to s t a n d i n g w e r e n o r m a l ) . T h e m e a l - i n d u c e d PP response was measur~after an overnight glucose c l a m p ( o v e r a l l m e a n b l o o d g l u c o s e 5.3 m m o l / 1 ) as w e l l as a f t e r an o v e r n i g h t h y p e r g l y c a e m i c g l u c o s e clamp (overall mean blood glucose 15.2 mmol/l). T h e P P c o n c e n t r a t i o n s 1 h a f t e r t h e b e g i n n i n g of the meal were indentical on the two occasions; p e a k c e n c e n t r a t i o n s w e r e f o u n d a f t e r 15 m i n (99• 5 p m o l / l ( h y p e r g l y c a e m i a ) vs. 1 0 2 • (normoglycaemia, n.s.) a n d s i g n i f i c a n t l y l o w e r t h a n t h a t o f h e a l t h y , a g e - m a t c h e d s u b j e c t s (165• P<0.05). C o n c l u s i o n : T h e P P r e s p o n s e to a m i x e d m e a l is i n d e p e n d e n t o f a c u t e c h a n g e s in m e t a b o l i c c o n t r o l in T y p e 1 ( i n s u l i n d e p e n d e n t ) p a t i e n t s . T h e m e t h o d is s u i t a b l e for r o u t i n e d e t e r m i n a t i o n of v a g a l i n t e g r i t y in d i a b e t i c p a t i e n s .

A163

P598

P599

THE LEVEL OF GASTRIN IN INSULIN-DEPENDENT DIABETES MELLITUS

OT e I N T E R V A L

N. Car, A. Car~ M. Orani6 and Z. Skrabalo, Vuk Vrhovac Institute for Diabetes, 4a Dugi dol, Zagreb, Yugoslavia The aim of this study was to link increased gastrin level in diabetic patients with diabetic gastroenteropathy. Fasting and postprandial gastrin levels were investigated in 20 insulin-dependent diabetic patients with signs os impaired parasympathetic innervation (group g) and 10 patients without such signs (group k). Fasting gastrin level was ~g=123.95560.89 in group g and Xg:47.74~4.62 in group k (p<0.05). Postprandial gastrin level was Xg=270.20+ 211.80 in group g and Xk=64.22523.25 in group k (p
AUTONOMIC

IN O I A B E T I C

NEUROPATHY

PATIENTS

WITH

- A FOLLOW-UP

CAROIAC

STOOu

G . J e r m e n d y , P . V O r ~ s , L . T 6 t h , M . Z . K o l t a i and G. P o g ~ t s a . I s t v @ n H o s p i t a l and N a t i o n a l I n s t i t u t e of C a r d i o l o g y , B u d a p e s t , H u n g a r y QI c i n t e r v a l

prolongation

betic

patients

this

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red

in 57 Type

2 (non (age: for

to s u d d e n resting

43.732.0

for c a r d i a c

years;

In this

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50:15

ratio:

p
ms;

13 p a t i e n t s

non-cardiac

subjects

5 years.

siBn

mechanism

death

in

in

diabetic

change

54.4•

p
while

ms

years) n=8,

died

QTo:

QT c : 4 5 9 / 1 5

45639

ms;

in 40 a g e - m a t h e d

ms vs 3 9 3 3 3

of CAN the

decreased

p
ms vs 4 5 1 3 4

cause:

n=5,

QT c p r o l o n g a t i o n

additional for

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cause:

(39233

of t e s t s

(CAN)

vs 1 . 2 5 3 0 . 0 5

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followed

vs ? . 2 3 1 . 2

(42433

16 Type

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dia-

To test

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diabetics.

mean/SEW)

1.1930.05

ratio:

OT c p e r i o d

death.

QT c i n t e r v a l

group

autonomic

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predispose

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insulin-dependent)

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patients.

P600

P601

THE LONG Q-T INTERVAL IN DIABETES: IS IT ARRYTHMOGENIC? M.F. Saad, M.A. Pfeiffer, H.A. Richter, Q. Liu, P.H. Bennett, and W.C. Knowler, Phoenix Epidemiology and Clinical Research Branch, NIDDK, NIH, and VA Medical Center, Phoenix, Arizona, and Diabetes Research and Analysis Association, Lexington, Kentucky, U.S.A.

~IONUCLIDE V E N T R I C O L O G ~ P H Y I N TYPE I YOUNG DIABETIC PATIENTS TO INVESTIGATE CARDIAC FUNCTION. T u r c o S., F e r r a r o S., D e S i m o n e G., Mossetti G., Santomauro M., Maddalena G., M a t e r a M . G . , M a r m o E., G r a v i n a E., F a z i o S. Medical School University of Naples, Italy

A long Q-T interval in diabetic patients is attributed to autonomic neuropathy and is thought to predispose to arrhythmias and sudden death. We studied the relationships between long Q-T interval in the resting electrocardiogram, corrected for heart rate (Q-To) , autonomic neuropathy, and the occurrence of arrhythmias in 47 Pima Indians with non-insulin-dependent diabetes. Twenty-six patients had long Q-T o (>440 msee) (mean 459) and 21 normal Q-T c (mean 421). The two groups were comparable in age (45.2 vs. 42.1 yrs), diabetes duration (12.2 vs. 11.5 yrs), and glyoemic control (HbA1 10.7 vs. 11.3%). None had ischemic heart disease. Patients underwent five cardiovascular autonomic function tests and had 48-h ambulatory electrocardiographic monitoring. Of the subjects with long Q-To, 18 (69.2%) had one or more abnormal autonomic tests compared with 14 (66.7%) of those with normal Q-T c . Ambulatory electrocardiographic monitoring revealed no significant ventricular arrhythmias in either group. In conclusion, a long Q-T c interval was often found with diabetic autonomic neuropathy, but was also observed in diabetic subjects with normal autonomic tests who may have diabetic eardiomyopathy or autonomic dysfunction undetectable by the tests used. These data do not demonstrate a potential arrhythmogenic effect of, or suggest a possible role in predisposition to sudden death for long Q-T interval in diabetic Pimas.

This study was aimed at evaluating cardiac function and s i m p a t h e t i c a c t i v i t y in type I young diabetics without evidence of micro and macroangiopathy. 16 t y p e I d i a b e t i c s (D) (8M and 8F mean age 25.2+5.2 years) with mean duration of diabetes 9.3+5.4 years, in good metabolic c o n t r o l (mean H b A l c 7.4+1.5%, mean fasting glycaemia 118.8+18.9 mg/dl absence of g l i c o s u r i a in t h e l a s t t h r e e m o n t h l y controls) and I0 n o r m a l s u b j e c t s (N) o f c o m p a r a b l e age, body mass index and blood pressure were studied. The diabetic patients were in insulin t h e r a p y at t h e m e a n d o s e o f 4 2 + 1 4 . 8 U . I . / d i e in 2 or 3 daily administrations. Ischaemic heart disease was excluded by a treadmill maximal effort Ecg. C a r d i a c a u t o n o m i c neurepathy was excluded by clinical tests according to Ewing. Radionuclide ventriculography (RNV) was perf o r m e d at r e s t a n d d u r i n g e f f o r t . P l a s m a l e v e l s of catecholamines were also measured. At rest Stroke Volume, Cardiac Output and Ejection Fraction (EF) were comparable in the two groups, while Peak Ejection Rate and Peak Filling Rate were higher in D then in N (p<0.O01). EF showed a comparable increase in the two groups during effort. Epinephrine and norepinephrine w e r e s i g n i f i c a n t l y h i g h e r in D t h e n in N w h i l e d o p a m i n e w a s n o t significantly different. Our data indicate increased flow velocities, both sistolic and diastolic, in well controlled type I diabetes which may be accounted for by sympathetic overactivity.

A164

P603

P602 MICROCIRCULATORY APPLICATION IN

BLOODFLOW RESPONSES TO PRESSURE DIABETIC AUTONOMIC NEUROPATHY.

R.J. van Marum I, A. Kruit 2, J.II. Meyer 3, Th.J.C. Faes 2, F.W. Bertelsmann 4, N. Schneider 2 and M.W. Ribbe I. iDepartment of Nursing Home Medicine, 2Department of Medical Physics, 3Institute for research in extramural medicine and 4Department of Neurology, Free University, Amsterdam, Netherlend. Diabetic autonomic neuropathy plays an important role in the development of pressure-ulcers iil diabetes mellitus, i.a. by disturbing cutaneous micrseirculatory bloodflow regulation. To investigate the effect of autonomic neuropathy on skin microcirculatory responses after local pressure application, we examined 18 patients with proven diabetic autonomic neuropathy (decreased E-I ratio) and I0 non-diabetic controls matched for age and sex. On the medial site of the heel a local pressure (I00 kPa) was applied for i0 minutes. Skin temperature (ST) was taken as a measure for dynamic bloodflow and registered, during and after pressure application, by means of a thermistor placed under the weight. Temperature curves in the diabetic patients showed characteristic differences from those of the controls. The slope of the STdecrease during pressure application in the diabetic group was significant larger than in the control group: (mean• 0.Z2• controls: (meaniSEM) 0.07• (p<0.05). The diabetic group showed also a significant larger period of latency between pressure relief and starting of the ST increase: (mean• SEiJ) 133• sec., controls (meaniSEM) 30• sec. (p< 0.05). This test shows that diabetic autonomic neuropathy leads to an impaired nicrocirculstory bloodflow response during and after pressure load.

P604 HAS THE RELATIONSHIP BETWEEN AUTONOMIC NEUROPATHY AND NEPHROPATHY A PATHOGENETIC SIGNIFICANCE IN DIABETES ? V.Spallone, S.Gambardella, S.Frontoni, M.R. Maiello,M.G. Felici, R.Gatta and G.Menzinger, Dpt. of Internal Medicine, Metabolic Diseases, II University of Rome,Italy To evaluate whether autonomic neuropathy (AN) is related to albumin excretion rate (AER), 44 insulin-dependent diabetic patients were divided in two groups (matched for age, diabetes duration and HbAI) according to presence of AN, assessed using deep breathing, Valsalva manoeuvre, lying to standing and postural hypotension. On the basis of the 4 tests a score was then calculated. Group A included 26 pts with AN(age:45.7~9.9;diabetes duration:20+8.4 yr) and group B 18 without AN(41.4! I0.6;16.8!7.7 yr). Albumin concentration was measured on timed 24-h urine collections to determine 24-h AER, daytime AER and overnight AEE;percentage change from day to night in AEB was calculated(~day-night). Group A showed higher 24-h and daytime AER than B(24-h AER:85.6 +121.5 vs 22.7+31.5 ug/min,p < O.03;day AER:80.6+IO0.8 vs 19.3+18 ug/min,p < O.Ol).Deep breathing was inversely r e l a t e d to day AEN(r=O.31,p < O.04),AN score was related to 2A-h AER(r=O.50,p
DISSOCIATION BETWEEN SYMPATHETIC NERVE ACTIVITY AND AUTONOMIC REFLEX DYSFUNCTION IN DIABETIC NEUROPATHY. C. Tsigos, A. White and R.J. Young, Dept. of Medicine, University of Manchester, U.K. Plasma nor-adrenaline (NA) is a measure of sympathetic nerve activity while adrenaline reflects adrenomedullary tone. In diabetic polyneuropathy, cardiovascular reflexes are widely used to assess autonomic nerve damage. We investigated whether autonomic reflex dysfunction is proportionate to abnormal sympathetic activity. Supine (S) and erect (E) circulating adrenaline and NA were measured by HPLC in II normal controls, 14 diabetic patients without detectable neuropathy and 20 diabetic patients symptomatic of autonomic neuropathy who all had postnral hypotension and abnormal heart rate responses to deep breathing, Valsalva manoeuver and standing up. 7 of these patients also suffered from painful neuropathy and Ii had painless neuropathy (neurotrophic foot ulceration).Groups were matched for age, glycaemia and renal function. NA levels were indistinguishable between diabetic patients with (1.67 nmol/L (S), 2.90 nmol/L (E)) and without autonomic neuropathy (1.52 nmol/L (S), 2.71 nmol/L (E)), but reduced compared to normal controls 2.34 nmol/L(S), 3.96 nmol/L (E)) (p<0.Gl). However, NA was higher in painful (2.36 nmol/L (S), 4.02 nmol/L (E)) than in painless neuropathy (1.27 nmol/L (S), 2.35 nmol/L (E)) (p
P605 INTRACAVERNOUS INJECTION OF PROSTAGLANDIN E I IN DIAGNOSIS AND TREATMENT OF IMPOTENCE IN DIABETIC PATIENTS M Bavnik-Oblak,

C Oblak, DB Vodu~ek, V Kristl, S Ziherl,

University Medical Centre Ljubljana, Yugoslavia The prevalence of erectile dysfunction in diabetic patients is high (approximately 50%). The aim of our study was to find the efficiency of intracavernous injection of prostaglandin E I (PGEI). 41 diabetic patients with organic impotence (aged 32-70 yrs, mean 51,7 yrs; duration of diabetes 1-31 yrs, mean 11,5 yrs; duration of erectile dysfunction 0,5-11 yrs, mean 3,8 yrs) received 20 ~g of PGE I (Prostin VR, Upjohn) into one corpus cavernosum. In 29 patients (70,8%)

the result was positive (full erection las-

ted at least 30 minutes). In the remaining patients only tumescence without rigidity was achieved and they underwent further investigations.

24,4% of patients reported

slight pain during injection or erection. One patient had prolonged erection. There were no systemic side effects. Patients with positive test were selected for autoinjection. They injected 10-40 pg of PGE I. The erection lasted 0,5-3 hours (mean 1,1 hours). 21,9% of patients reported ecchymosis and/or haematoma which resolved without consequences. After 1-12 months of therapy no scarring of the cavernous tissue was apparent in any one of the patients. In conclusion, PGE I is an effective and sa~e diagnostic and therapeutic agent in erectile dysfunction of diabetic men.

A165 P606 MUSCLE SYMPATHETIC NERVE RESPONSE AFTER ORAL G L U C O S E IS A T T E N U A T E D IN T Y P E 1 D I A B E T E S . H.Frandsen, J.Fagius and C.Berne. Dept. of Clinical Physiology, Herlev University Hospital, 2730 Herlev, Denmark and Departments of Neurology and Internal Medicine, University H o s p i t a l of U p p s a l a , S w e d e n . Baroreceptor governed muscle sympathetic nerve activity (MSA) i n c r e a s e s a f t e r i n t a k e of oral g l u c o s e . T h e c a u s e of t h i s r e s p o n s e is u n k n o w n , although insulin during euglycaemic infusions increases MSA. T o e v a l u a t e t h e r o l e of i n s u l i n and other potential contributing factors to the MSA response, patients with Type 1 diabetes were studied after oral glucose. 6 normal controls and 6 well regulated patients, without signs of a u t o n o m i c n e u r o p a t h y a n d w i t h normal body weight were given 100 g of glucose orally. The patients did not receive insulin substitution during the glucose load. MSA (bursts/min) was determined with microneurography from a microelectrode in the right peroneal nerve. Basal MSA levels were 17 • 2 (mean• i n t h e c o n t r o l a n d 20 • 1 b u r s t s / m i n in t h e p a t i e n t g r o u p . 30 m i n a f t e r glucose MSA had increased significantly in both groups (p<0.05), i n t h e c o n t r o l s b y 82% to 31 • 3 bursts/min and considerably less in the p a t i e n t s b y 25% t o 25 • 2 bursts/min. Blood pressure was stable in both groups. The MSA response after oral g l u c o s e is a t t e n u a t e d i n IDDM patients. Insulin is p r o b a b l y e s s e n t i a l for a normal MSA response. Other factors may be of significance. T h e r o l e of i n s u l i n u p o n the autonomic nervous system is being further investigated using both human and porcine insulin.

PS 20 Peripheral Neuropathy P607

P608

PREVALENCE OF SUBCLINICAL NEUROPATHYIN TYPE I (INSULIN-DEPENDENT) DIABETIC CHILDREN

E F F E C T S OF D I F F E R E N T P E R I O D S OF O R A L E S S E N T I A L F A T T Y A C I D S ON N E R V E C O N D U C T I O N IN D I A B E T I C R A T S P.O.O. Julu, D e p a m t m e n t of P h y s i o l o g y , U n i v e m s i t y of Z i m b a b w e , H a r a r e . ( P r e s e n t l y L e e t u m e r , U n i v e m s i t y of G l a s g o w , S c o t l a n d ) .

t.Barkai and L.Mad~csy (II Dept.of Pediatrics,Postgrad. Med.Univ.,Miskolc,Hungary) To investigate the prevalence of autonomic nervous system dysfunction,two simple bedside tests,beat-to-beat variation during forced respiration and blood pressure response to standing (for parasympathetic and sympathetic nervous function) were applied to llO children with Type 1 (insulin-dependent) diabetes,mean age 13 years,(range 6-18 years).The duration of diabetes varied from 1 month to 16 years (mean 6 years).130 healthy age-matched children were used as control group.Confidence limits of 95% (2SO) from means of control subject were considered as limits of normality.The prevalence of autonomic nervous system dysfunction (parasympathetic and/or sympathetic) in the whole population of diabetic children was 29%.Isolated parasympathetic and sympathetic disturbances were detected in 16% and 7% of diabetic children,respectively.6% of diabetics had coinciding parasympathetic and sympathetic dysfunctions.A clear correlation between duration of diabetes and tests for autonomic neuropathy was shown (beat-to-beat variation,r=-O.4%p
E s s e n t i a l f a t t y a c i d s (EFAs) can p r e v e n t or c o r r e c t s l o w e d n e m v e e o n d u e t l o n v e l o c i t y (NCV) in s t r e p t o z o t o c i n (STZ) d i a b e t e s . To e s t i m a t e the t u r n o v e r p e r i o d of n e r v e c o n s t i t u e n t ( s ) b e i n g r e p l e n i s h e d , a t i m e - c o u r s e s t u d y is vital. 5 w e e k s a f t e r b u f f e r or STZ i n j e c t i o n s (65 mg/'Kg I.P.), r a t s w e r e e i t h e r g i v e n 0.3 ml,/day of E p o g a m (75% l i n o l e l s and 9Z ~ - l i n o l e n i e acids) by g a s t r i c g a v a g e for 3 , 5 . 1 0 and 35 d a y s Or left w i t h o u t . T e r m i n a l s e n s o r y NCV w a s m e a s u r e d inv i v o u n d e r uretl]ane a n a e s t h e s i a (l. S g / K ~ I.P) by s t i m u l a t i n g and r e e o r d i n ~ f r o m the s a p h e n o u s n e r v e trunk. NCVs w e r e ( m e a n s ! S E M , m/s) aT.a ! 0 . 8 ( n = 1 2 ) in 3 and 5, a 6 . 7 ~ 3 . 1 ( n = 5 ) in I0, and a T . 6 ~ l . 8 ( n = 9 ) in 35 days of t r e a t m e n t a m o n g c o n t r o l s . T h e y w e r e 52.9-!1.7(n=7), a5!l.8(n=5), aa.3• and a S ~ l . 6 ( n = 8 ) f o l l o w i n ~ 3 , 5 , 1 0 and 35 d a y s of EFAs t r e a t m e n t . W i t h o u t treament, N C V s in STZ d i a b e t e s w e r e 3 9 . 5 + 1 . 3 ( n = 5 ) at 3 and 5, a l . 3 t a . 5 ( n = 5 ) at io, and 3 6 . 2 • (n=5) at 35 days. EFAs c a u s e d an o v e r s h o t in N C V by lit (p
A166

P609

P610

ESSENTIAL FATTY ACID SUPPLEMENTATION CAN IMPROVE NERVE FUNCTION AND VASCULAR SUPPLY IN DIABETIC RATS. N.E. Cameron, M.A. Cotter and S. Robertson, School of Biomedical Sciences, University of Aberdeen, Scotland, U.K. The aim was to compare effects of gamma-linolenic acid (evening primrose oil, Efamol), alone and mixed with eicosapentaenoic acid (Efamol Marine, Scotia Pharmaceuticals), on nerve function and vascularization in experimental diabetes. Three streptozotocin-diabetic and one non-diabetic group of male rats were used. Diabetic groups were treated with 10% dietary supplements of either Efamol, Efamol Marine, or corn oil for 2 months. Sciatic motor and sensory conduction velocity were measured in vivo. Hypoxic resistance was measured on sciatic trunk in vitro while the preparation was gassed with nitrogen. Sciatic vessel density was estimated using alkaline phosphatase staining. Motor and sensory conduction deficits (1a-25%), apparent with corn oil, were absent with Efamol treatment (p
LONG TERM GLYCAEMIC CONTROL AND DIABETIC NEUROPATHY. D.R. McCance, D.R. Haddem, A.B. Atkinson and L. Kennedy. The Sir George E Clark Metabolic Unit, Royal Victoria Hospital, Belfast BTI2 6BA.

N. Ireland.

220 patients with type I (insulin-dependent) diabetes (age at onset ~25y, duration <18y) were examined for the presence/absence of ]ower limb reflexes and determination of vibration perception thresholds (VPT) at each medial mal]eolus and great toe (mean of 5 readings) using biothesiometry. These parameters were related to the most recent HbAI and the mean of serial measurements "mean HbA~' over the previous 6y. Ankle and knee reflexes were absent in 59,28 (right) and 41,27 (left) patients respectively. Mean (R + t) ankle and toe VPT's were 8.7• and 6.5• (mean• (arbitrary units) respectively. Toe thresholds were more sensitive than ankle thresholds. Both mean HbA] and the latest HbAI d i f f e r e d significantly in patients with absent (A) or present/present with reinforcement (P) ankle reflexes: ll.6• ]O.5• p=O.O0O]; 12.2• 10.5• p=0.0OO0 respectively. Similarly, the R ankle reflex (A/P) was related to mean HbA] arbitrarily divided into groups ~10~, lO-12~, >12~ (p=0.OOO9). In contrast, mean ankle (8.0• 8.8• l, ]0.3+6.2) and toe (5.5• 6.]+2.9, 8.5• VPT's did not increase significantly with poor glycaemic control (p>0.05). Age, retinopathy, 24h urinary albumin excretion and erect systolic blood pressure were the only independent variables predicting toe VPT using linear regression aoaiysJs. These findings may support only a permissive role for glycaemic control in diabetic neuropathy and question the usefulness of biothesiometry in its evaluation.

P611

P612

RELATIONSHIP BETWEEN CARDIAC AND PERIPHERAL AUTONOMIC NEUROPATHY ASD DURATION OF DIABETES MELLIT~]S.

VARIABILITY ACCORDING

O. de Weerdt, G.A. IJff, Th.J.C. Faes, E.A. van der Veen and F.W. Bertelsmann, Free University Hospital, Amsterdam, The Netherlands.

1, ,rue C I . V e l l e f a u x 7 5 0 1 0 PARIS (France] Aim: To evaluate the e f f e c t s of blood glucose levels v a r i a t i o n on the r e l i a b l l l t y of the tests for n e u r o p a t h y . P a t i e n t s : Group I : 10 T y p e I d l a b e t l c s , mean age 51 + 7 y e a r s , diabetes d u r a t l o n 8.5 + 8 y e a r s . G r o u p I i : IO diabetics (q t y p e I , 6 t y p e l l ) , - m e a n age 50 + 6 y e a r s , diabetes d u r a t l o n (6.3 4- 7.7 y e a r s ) . Methods: V l b r a m e t r y b y - b i o s t h e s i o m e t e r , v a r l a b l l l t y of cardiac f r e q u e n c y a f t e r l a y i n g - s t a n d i n g (L.S.) deep brea t h i n g ( D . B . ) and Valsalva manoeuvre ( V . M ) were s t u d i e d . Tests were done for each p a t i e n t s at day I and a f t e r 3 d a y s . G r o u p I p a t l e n t s were tested h a v i n g same blood glucose levels (8.3 + 2.2, v e r s u s 8.q + 1.q mmolll) G r o u p II p a t i e n t s - w e r e tested e i t h e r w l t h n~-rmoglycaemia o r w l t h h y p e r g l y c a e m i a in randomlzed r a n g e ( 5.8 4- 0.ammolll v e r s u s 17 4- 2 m m o l l l ) . C o e f f i c i e n t oi~ v a r i a b l l l t y ( C . V ) was calculated for each test. Results were as followlng V B G r o u p 1 9.5 + 3.1 v e r s u s w + 3.1 (r= 0,83) (CV=11%]. G r o u p I I - 1 6 . q + 6.9 v e r s u s 17.1 + 7.3 (r= 0.96) (CV = 9%}. L . S . G r o u p I 1.18 + 0 . 1 8 - - v e r s u s 1.21 + 0 . i 5 (r=0,95) (CV= ,%). GroupII 1.20 40.I0" versus 1.26 + 0.21 {r=0.62)(CV=q%] b B - G r o u p I 18 4" 7.9 v e r s u s 1"7 + 8.4 {r= 0.7q) {CV=16%)Group II 16 u 8.3 v e r s u s 16 u 7.1 (r=0.89} (CV=9%} VM G r o u p I O,85 + 0,36 v e r s u s 0~85 40.29 (r=0.97) (CV=6%) Group II 0.9w + 0.45 v e r s u s 0.99 + 0.q8 {r=0.92) (CV=9%). C,onclusion : In thls s t u d y blood glucose levels w l t h e x c e p t i o n of hypoglycaemla do not a f f e c t r e s u l t s o f used t e s t s for n e u r o p a t h y .

OF TESTS FOR D I A B E T I C NEUROPATHY TO BLOOD GLUCOSE LEVEL

G. C A T H E L I N E A U ,

To study the relationship between cardiac and peripheral autonomic neuropathy and the duration of diabetes mellitus, ~ evaluated heart rate variability and sympathetic vasomotor control in the extremities of 25 patients with diabetes mellitus type i, age 48.4 + 16 years, duration of diabetes 17.2 + 10.6 years, HbAlc 8.7 + 1.9% (mean + SD). Cardiac autonomic function was evaluated by heart rate response to deep breathing. Peripheral sympathetic function was assessed by rate of skin temperature decrease (RST) in the leg during cooling. All patients had abnormal (< 5th centile of age-related normal value) values for E/I ratio compared with controls. The rate of skin temperature decrease was significantly reduced in the diabetic patients as compared to controls, 0.07 + 0.02 vs 0.23 + 0.02 ~ (mean + SEM, p<0.001). 18 of 25 patients had abnormal values for both E/I ratio and RST. E/I ratio was not correlated with vasomotor control. Duration of diabetes showed negative correlation with RST (r~0.41, 'p=0.04). We conclude that cardiac autonomic function tests do not reflect peripheral autonomic neuropathy. Measurement of skin vasomotor control is required to study distal autonomic nerve function, which decreases with duration of diabetes mellitus.

116pital S a i n t

Louis

P. V E X l A U . -

A167

P613

P614

The effects of the aldose reductase inhibitor 'Statil' on diabetic peripheral neuropathy. E. Dietrich, H. Stracke and K. Federlin. Department of Internal Medicine, University of GieBen, FRG In a randomised double-blind study we evaluated the effect of the aldose reductase inhibitor Statil (ICI 128436; 600mg/d) on peripheral nerve function in 60 Type I (insulin-dependent) and Type 2 (non-insulin-dependent) diabetic patients (38 M, 22 F; mean age 47,4• years; mean duration of diabetes 14,9• years) with neurographic signs of peripheral neuropathy. After a placebo period of 4 weeks respectively after 30 and 56 weeks nerve function was measured using nerve electrophysiology, vibration perception thresholds and heat perception thresholds in the upper and lower extremities. Symptoms (pain, dysesthesia, paresthesia) were assessed with visual analogue rating scales. 50 patients completed the study. No significant treatment effect was shown in the Statil group compared to placebo treatment for following parameters: motor conduction velocity of the median (p=0,14) and peroneal nerve (p=0,22), vibration perception at the dorsum of the hand (p=0,35) and the foot (p=0,81), temperature perception at the index finger (p=0,67), the thenar eminence (p=0,61) and the dorsum of the foot (p=0,74). Symptoms were slightly improved in both groups but there was no significant difference between the patient groups in favour of Statil treatment. Side effects were not attributable to Statil treatment. This long term study suggests that Statil treatment may not beneficially influence peripheral nerve function in diabetics with established neuropathy.

PARALLEL STUDYOF VIBRATION PERCEPTIONTHRESHOLDAND THERMAL PERCEPTION THRESHOLDS Iosif Cristiana, Ionescu-Tirgoviste C., Pruna S., Clinic of Diabetes, Bucharest, Romania

Unmyelinated (C)-small myelinated (B) and large myelinated (A~) fibre functions were assessed by parallel determination of two neurophysiological parameters: thermal perception thresholds (TPT) both for cooling (-A~ and warming (+A~ and vibration perception threshold (VPT). For this we used two original devices: Thermosensitometer and Neurovibrometer. The quantitative informations obtained were compared between two groups of subjects: 20 non-dlabetic controls (lIM/9P; mean age ~SD: 38.8~I0.2 yrs.) in which the following values were recorde4: cooling sensitivity 1.4~o.sOc; warming sensitivity 1.2~0.3~ VPT (arbitrary units) in hand 3.5~1.3 U and VPT in foot 6.5~2.6 U. The corresponding values obtained in 29 type J diabetics (2OM/9F, mean age 41.0~15.0 yrs.; duration of diabetes 4.8~5.2 yrs.) were: 2.0~I.3~163 1.9~l.O~ 4.2~1.7 U and 7.3~3.] U. In 37 type 2 diabetics (31M/6F, meanage 58.9~9.4 yrs.) duration of diabetes 15.0~8.1 yrs.), the values were: 3,7~I.90C; 3.351.8~ 9.3~3.2 U and 14.152.6 U. In diabetic patients at least one abnormal parameter was recorded in 86.6% of cases. The relationships between TPT and VPT in diabetics were: both functions normal in 13.4%; both functions abnormal 48.8%; only VPT abnormal 22.7%; only TPT abnormal 15.]%. In conclusion the simultaneous determination of TPT and VPT increase the accuracyof nerve dysfunction assessement in diabetics.

P615

P616

DOES VIBRATORY PERCEPTION THRESHOLD OR TEMPERATURE PERCEPTION THRESHOLD CORRELATE WITH SENSORY- AND MOTOR NERVE CONDUCTION VELOCITY? B.Bravenbeer, I.Wegner, P.H.Hendrikse, P.L.Oey, A.C.van Huffelen, D.W.Erkelens. Departement of Internal Medicine a n d Neurophysiology, University State Hospital, Utrecht, Holland. Aim of the study: to evaluate the m e a n i n g of a n abnormal vibratory perception threshold(VT) a n d temperature perception threshold for w a r m t h a n d cold (TTw a n d TTc) as compared with m e a s u r e m e n t of nerve conduction velocity nerves. Methods: 51 type 1 diabetic patients were studied a n d selected with either a n a b n o r m a l VT or TTw for f u r t h e r electrophysiological studies. VT was measured at the second metacarpal of the r i g h t h a n d using a Vibrameter (&)medic AB,Sweden), TTw a n d TTc were meastmed at the r i g h t wrist using a Temperature Threshold Tester (Medelec Ltd.,England). Motor nerve conduction velocity was measured in the u l n a r nerve (NUM) a n d tibial nerve (NTM),sensory nerve conduction velocity in the u l n a r nerve (NUS) a n d sural nerve (NSS), a n d HM-interval (HM) using the tibial nerve. Statistical analysis: correlation coefficients were calculated between all parameters. Regression analysis was performed using the method of the least squares (lO<0.01 shown by *). Results: correlation coefficients were VT/TTw 0.332,VT/TTc 0.387*,VT/NSS 0.430,VT/NUS 0.230,VT/NUM 0.263,VT/NTM 0.314,VT/HM 0.500*,TTw/TTc 0.659*,TTw/NSS 0.266,TTw/NUS 0.309,TTw/NUM 0.397*,TTw/NTM 0.415*,TTw/B:M 0.495*,TTc/NSS 0.243,TTc/NUS 0.633*,TTc/NUM 0.431*,TTc/NTM 0.316 a n d TTc/HM 0.454*.Conclusions: 1.VT,TTw a n d TTc correlate best with HM-interval. 2.TTc correlates better t h a n VT a n d TTw with nerve conduction velocity resxflts.

C E N T R A L N E R V O U S SYSTEM A B N O R M A L I T I E S A P P E A R EARLY BUT A R E L A T E R A S S O C I A T E D TO S E L E C T I V E PROTEINURIA IN TYPE i DIABETIC PATIENTS P.Pietravalle,S.Morano,G.Cristina,U.Di Mario, A.M. M a r r a n g h i n o * , E . V a l l e * , G . P o z z e s s e r e * and P.A.Rizzo*, Endocrinology and N e u r o l o g y * , Universita' "La Sapienza"; Roma, Italy Recent works have shown central nervous system electrophysiological (NP) a b n o r m a l i t i e s in recent onset diabetic patients. To explore the possible relationship between NP abnormalities and signs of incipient diabetic nephropathy or of retinopathy two groups of type 1 diabetics were studied: group 1 (n=lS), with no p r e v i ously known clinical signs of complications, < 5 years from diagnosis; group 2 (n=15), w i t h and without clinical signs of diabetic complications, > I0 y e a r s of the disease. A g e a n d sex matched normals were included in t h e study. Multimodal evoked potentials (visual, brainstem auditory, median and tibial neurosomatosensory) and selective protein c l e a r a n c e (albumin, anionic immunoglobulins, neutral/cationic immunoglobulin) were evaluated. In Group i, 27% of patients showed significant NP abnormalities vs controls, one showed proteinuria and one background retinopathy. No correspondence has been found between the complications studied. In G r o u p 2, 60% of patients showed NP abnormalities, 67% showed abnormal charge or size selective proteinuria and 33% background or proliferative retinopathy. A significant association has been found b e t w e e n NP a n d p r o t e i n u r i a abnormalities (p
A168

P617 BONE MASS IN THE F O O T AND AXIAL SKELETON IN DIABETIC

PATIENTS WITH PERIPHERAL NEHEOPATHY A.W. PATRICK, A. COLLIER, J.J. NICOLL, D.W. PYE, R.J. YOUNG and B.F. CLARKE. Diabetic Department and Department of Medical Physics, Royal Infirmary, Edinburgh

EH3 9Ylq Bony d i s o r g a n i s a t i o n ( d i a b e t i c o s t e o p a t h y ) os t h e f e e t i s a recognised complications of diabetic nearopathy. It has been suggested that osteopenia is more severe in neuropathic patients and this contributes to the development of osteopathy. The aim of this study was to investigate the possible neurogenic a e t i o l o ~ of osteopenia in diabetes. Diabetic patients with painful neuropathy (n=20; mean (SD); age 56(8)yrs; 9 Type i, ii Type 2), painless neuropathy (n=20; age 55(10); 7 Type i, 13 Type 2), no clinical evidence of neuropathy (n=20; age 56(10); 8 Type i, 12 Type 2) and a comparable control group (n=20; age 54(11)) were studied. Foot and heel bone mineral was measured using single photon absorptiometry, the bone density of the calcaneum was estimated using Broadband Ultrasonic Attenuation while spine bone mineral was assessed using dual photon absorptiometry. There was no difference in the parameters measured between the four groups. This suggests that neuropathy does not contribute towards the development of osteopenia, and that diabetic osteopathy results from repeated trivial trauma in an insensitive neuropathic foot.

PS 21 Diabetic Foot P618

P619

Abnormal Vascular P.esponses in tlre Diabetic Foot are a Result of Neuropathy and not Microangiopathy.

SCREENING FOR HIGH FOOT PRESSURES IN PATIENTS AT POTENTIAL RISK OF FOOT ULCERATION. AJM Boulton, DJS Fernando, IB Borowiecka, EA Masson and A Veves. University Department of Medicine, Manchester Royal Infirmary, UK.

Stevens, Id.J., Edmonds, M.E. and Watldns, P.J. Diabetic Dept., King's College Hospital, London, SE5 9P.S. Paradoxical vasoconstriction rather than the expected vasodilatation occurs in response to skin heating in diabetic neuropathy: we have now demonstrated this same response in the limbs of non diabetics with t r a u m a t i c neuropathy, which contrasts with the physiological vasodilatation in the normal limb. Skin blood flow in 4 non diabetic p a t i e n t s with a unilateral t r a u m a t i c neuropatby were compared to 13 neuropathic diabetics (12 type 1, insulin dependent), 10 diabetic controls (7 type 1, insulin dependent), and 10 normal subjects. A laser Doppler measured the microcirculat ory response to heating the toe pulp to 44~ for 7 minutes. Blood flow decreased during heating in the t r a u m a t i c neuropathy limbs and diabetic neuropathy patients, from 11.6(3)S.D. volts to 3.3(4) volts and from 13.3(9) volts to 11.9(10) volts, p < 0.05, respectively. This contrasted with the vasodilatation in the c o n t r a l a t e r a l normal limb, diabetic controls and normal subjects, flow increasing from 19.4(4) volts to 37(6) volts, from 14.8(13) volts to 38.8(20) volts, and from 19.2(12) volts to 43.3(27) volts, p < 0.05, respectively. Since the non diabetics with t r a u m a t i c neuropatby do not have microangiopathy, the paradoxical responses occurring in both these subjects and diabetic neuropathy patient s must result from vascular denervation and not microangiopathy. The role of these very abnormal responses in the development of foot disease now needs to be determined.

We have previously demonstrated an association between high foot pressures and diabetic foot ulceration. Using the optical pedobarograph, dynamic (walking) foot pressures were measured in 143 diabetic patients and 60 healthy controls. Diabetic subjects were clinically subdivided to those with neuropathy (DN, 98 patients) and without neuropathy (D, 45 patients). Mean peak foot-pressure of the diabetic patients was significantly higher than the healthy controls (10.3 kg cm ~ 4.9 SD versus 7.9 • 3.4: p < 0.001). Mean peak pressure of neuropathic group was 11.1 ~ 5.2, higher than both nonneuropathie (8.3 • 3.5) and the control groups (p < 0.001), while no difference existed between the last Lwo groups. A higher proportion of DN (44~) had high peak pressures, (> 11.5 kg.em ) comparing to D (20~, p < 0.001) and to controls (12.5%, p < 0.001). All but one of the non-neuropathic diabetic patients with high foot pressures were found to have limited joint mobility on clinical examination. Regression analysis showed correlation between foot pressures and age (r = 0.3, p < 0.005) but not with weight and duration of diabetes. We conclude that high foot pressures are common in diabetic patients, especially in those with neuropathy, are age related and that neuropathy and limited joint mobility are candidate aetiologic factors.

A169

P620

P621

fs onycho~nycosis a risk factor for major lesions of diabetic foot?

AORTIC AND LOWER LIMB ARTERY CALCIFICATION IN PATIENTS WITH TYPE 2 (NON-INSULIN-DEPENDENT) DIABETESAND CONTROL SUBJECTS L.K. Niskanen, M. Suhonen, O. Siitonen and M.I. Uusitupa, Departments of Medicine, Clinical Nutrition and Radiology, K u o p i o University Central Hospital, Kuopio, Finland The aim was to assess among a respresentative group of 133 middle-aged newly diagnosed Type 2 diabetic patients and 144 control subjects the baseline prevalence and 5-year incidence of radiological abdominal aortic (ABC-C), lower limb intimal (INT-C) and medial (MED-C) c a l c i f i c a t i o n s . Their relationship to the development of intermittent claudication (IC+) and r i s k factors f o r ABD-C in diabetic patients werestudied. At baseline the age-adjusted prevalence of ABD-C and INT-C was higher in diabetic than in control subjects (ABD-C: 29 vs. 17 % f o r men, p=.O.05; 26 vs. 19 % f o r women; p=.O.06, INT-C: 24 vs. 12 % f o r men, p=O.02; 34 vs. 23 % f o r women; p=NS), the respective figures f o r MED-C were: 15 vs. 21% f o r men, 9 vs. lO % f o r women. The incidence of lower limb c a l c i f i c a t i o n s was higher on]y in diabetic women than in control women (INT-C: 33 vs. II %,

L.Mancini, L. Uecioli*, A.Manto, G.Careddu, P.Magnani, P.Cotroneo, G.Ghirlanda and A. V.Greco. Ist. of Internal Medicine, Catholic Uni~ versity of Rame.*Cattedra dim. del metabolismo, Univ. Tor Verga~ ta di Roma. Onychomycosis is a frequent finding in diabetic population but its prevalence is unknown. The aim of our work was to evaluate the prevalence of onychomycosis in three groups: A- a sample of general population; B - 450 diabetics randomly selected in the outpatients clinic; C - 130 diabetics treated in the foot clinic because of major problems (ulcers, infections, etc.), (age 61.5+15.Syrs vs 60.4+13.2yrs vs 63.3+_ll.5yrs; diabetes duration ll.24+9.22yrs

vs

16.5+_7.5yrs). Onychomycosis was evaluated

by clinical and coltural investigation

in all suspected nails.

Group C showed the highest prevalence of aaychomycosis (34%) than group B (20%) (p<0.001) and group A (10%)(p<0.05). In diabetic groups onychomycosis was not correlated with age and duration of disease. Microscopy examination was positive in 50% of cases while coltures were positive in more than 70% (Tricophiturn Rubrum, Rhodoturula Rubra, Candida Torulopsis and Parapsilosis). Conclusions: diabetics have higher prevalence

p=O.O09; MED C: 29 vs. 14 %, p=O.05). At baseline the prevalence of-MED-C (23 vs. 7 % p=O.03) was higher in patients with IC+ than in IC-. High systolic blood pressure (p< 0.05) and abnormalities in VLDL-metabolism were associated with the development of ABD-C. To conclude, at diagnosis the ABD-C and INT-C, but not MED-C were more common in diabetic patients than in control subjects. MED-C predicted the development of IC in diabetic patients.

of onychomycosis than general population; direct relationship (as cause and~or consequence) with major foot problems is hypothesizable

and onychomycosis may be considered a risk

factor for diabetic foot.

P622

P623

Diabetic foot: the importance of a specialized foot clinic after

COST AND HEALING EFFICACY OF TREATING D~ABETIC FOOT ULCERS IN A WOUNDMANAGEMENTPROGRAMWITH GROWTHFACTOR THERAPY. C.P. FYLLING and P.C. MCKEOWN. Curative Technologies, Inc. Minneapolis, Minnesota. The aim of the study was to evaluate the healing and cost efficacy of two d i f f e r e n t approaches of treating diabetic foot ulcers; conventional treatment(C) versus a comprehensive wound care center program(WCC) using growth factor therapy. Charge and outcome data over 489 years were tabulated, n-C=28, WCC=79 patients. Average OP charges per patient were C=$867, WCC:$1,058(p=O.19). Average AS charges per patient were C=$286, WCC=$528. One(4%) of the C patients and 17(21%) of the WCC patients had vascular reconstructive surgery(VR)(p=O.02166), thus the inpatient(IP) charges were tabulated including VR and excluding VR for uniform comparison of treatment modalit i e s . Average IP data per patient including VR were: hospitalizations-C=3, WCC=I.5(pO.5); TMA, C=12%, WCC=1%(p=O.0459); Ray, C=4%, WCC=0%; BKA, C=20%, WCC=2%(p
30 months ractivity. G.Ghirlanda, L.Mancini, L.Uccioli*, A.Manto, G.Careddu, P.Magnani, P.Cotroneo, and A.V.Greco. Ist. of Internal Medicine, Catholic University of Rome.*Cattedra dirn. del metabolismo, Univ. Tor V~rga~a di Roma. After 30 months' activity we assessed the utility of a foot care clinic. Follow up included i i 0 diabetics, 25% of which were already known for presenting foot problems, 41.3% came from the first aid with acute foot lesions, 33% came from diabetic outpatients

clinic

after

routine

examination.

The

starting

problems were: ulcers (75), soft tissue infections (37), necrosis (21), osteornyelitis (12), hyperkeratosis (57) and mycosis (45). Patients were

divided

in 3

groups: neuropathic

(IV) (47),

vasculopathic (V) (42) and mixed (M) (21). During follow up N underwent

local treatment, orthopedic device and 2 plaster

Castings; healing

rate

was

98%

with

8

relapses,

3 new

osteomyelitis and no amputation. In V in addition to locai treatment 8 subjects were submitted to vascular bypass, 2 endoluminal angioplastic with healing in 94% of cases, 3 relapses, 22 new ulcers, 1 foot amputation,

2 minor amputations,

2

osteomyelitis. All ulcers of M healed after local care, including 4 relapses and 5 new ulcers. We observed a decrease of number (42 vs 9) and length (12+--4 vs 6+3 weeks) of admissions (pcO.05). The high healing rate, the foot lesions severity reduction, the decrease of admissions prove the medical and ~'ocioeconomical importance of foot clinic.

A170

P624 LIMITED JOINT MOBILITY: RELATIONSHIP FOOT PRESSURES AND FOOT ULCERATION. DIS Pernando, EA Masson, A Veves Manchester Royal Infirmary U.K.

TO

ABNORMAL

and AIM Boulton

Diabetic foot ulceration and abnormal plantar foot pressures (PFP> have been attributed to neuropathy and possibly limited joint mobility (LJM). We examined the role of foot LIM in causing foot ulceration by studying 65 patients in 5 matched groups : (l) non-neuropathic diabetic patients with LJM : (2) him and neuropathy : (3) neuropathy : (4> diabetic controls and : (5) nondiabetic controls. Joint mobility was assessed in the foot at subtalar and metatarsophalangeal joints, PFP by optical pedebarography and neuropathy by biothesiometry. Joint mobility was reduced at both sites in groups 1 and 2 compared with groups 3, r and 5 (p. PFP was significantly higher in groups i and 2 compared to 3, 4 and 5 (p <0.01). No differences in FFP were observed between groups 1 and 2. There were strong correlations between PFP and LIM (r= -0.7, p <0.001). Plantar ulceration occurred in 65% of patients in group 2: 5% of group 3: none in group i. These data suggest that: (I) LJM is a major factor causing abnormally high PFP: (2) abnormal PFP without neuropathy does not lead to foot ulceration and :<3) LIM contributes to foot ulceration in susceptible neuropathic feet.

PS 22 Diabetic Complications P625

P626

MICROCIRCUIATION IN DIFFFJ~ENT STAGES OF TYPE I (INSULINE-DEPBNDF~NT) DIABETES MELLITUS. A.C. N i e u w e n h u i j z e n Kruseman, A.J.H.M. Houben, D.W. S l a a f * , N.C. Schaper. Dept. o f I n t e r n a l M e d i c i n e , University Hospital Maastrieht, Maastrlcht; *Dept. o f B i o p h y s i c s , U n i v e r s i t y o f Limburg, M a a s t r i c h t , The Netherlands. To s t u d y s k i n m i e r o c i r c u l a t i o n i n type I p a t i e n t s i n r e l a t i o n to d u r a t i o n o f d i s e a s e and p r e s e n c e o f c o m p l i c a t i o n s , b a s a l blood f l u x and r e a c t i v e hyperemia a f t e r 4 minutes a r t e r i a l o c c l u s i o n were s t u d i e d u s i n g l a s e r - D o p p l e r f l u x m e t r y ( P e r i f l u x PF3, Perimed). Patients: d u r a t i o n of d i s e a s e < 2 yrs., mean a g e • SD: 29 • 8 y r s . (N=I2, S); d u r a t i o n of d i s e a s e > I0 y r s . , age: 38 • 14 y r s . , e i t h e r w i t h r e t l n o p a t h y (N=12, R+), m i c r o - a l b u m i n u r i a (N=12, M+) or no s i g n s o f r e t i n o p a t h y / m i c r o - a l b u m i n u r l a (N=12, R-M-). Data were compared w i t h t h o s e o f age matched h e a l t h y c o n t r o l s (C). R e s u l t s : M e t a b o l i c c o n t r o l between p a t i e n t groups was comparable. In S p a t i e n t s s k i n temperature ( 3 2 . 6 ~ was higher than that in C (30.3 ~ P < 0.05). In S and R+ patients rest-flux was increased compared with C (34 vs 16 and 37 vs 19 perfusion units, P < 0.05). Post-reactive hyperemic response after 4 min. arterial occlusion was markedly decreased in S patients compared with C (238% vs 519%, P < 0.01). Conclusion: EarSy after onset of type I diabetes, changes, probably functional, in skin mierocirculation can be observed. In older patients most of these differences seem to disappear, possible due to age related alterations.

ANTi-ssDNA ANTIBODIES IN TYPE 2 (NON INSULIN-DEPENDENT) DIABETES. ASSOCIATION WITH VASCULAR COMPLICATIONS. E. Giardina, G. Scarantino, F. Novara, S. Verga and G. Triolo istituto di Clinica Medica, University of Palermo, Italy Anti-saDNA antibodies cross-reactive with heparan-sulfate (HS) were determined by ELISA in the serum of 202 patients with type 2 (non insulin-dependent) diabetes (mean age 59.4 ql 10.2 yrs; duration of diabetes 12.4 • 9.5 yrs) and of 135 healthy subjects. The prevalence of anti-ssDNA was higher in patients with angiopathy (43/128) rather than in patients without (5/74; p< 0.0001) or controls (9/135; p< 0.0001). There was no relationship between anti-ssDNA positivity and age, disease duration, sex, treatment. AntisaDNA were more frequent in patients with stable proteinuria (17/43) than in those with retinopathy or macroangiopathy or both (26/94~ p< 0.05). The presence of complications was significantly associated with the anti-ssDNA positivity in patients with short duration diabetes (J 2 yrs: 7/7 vs 4/16, p< 0.001~ ~ 4 yrs: 10/11 vs 9/29, p< 0.005; < 8 yrs: 11/14 vs 20/58, p< 0.01) but not in patients with duration of diabetes of 8-16 yrs (15/17 vs 31/48, NS) or > 16 yrs (12/12 vs 33/43, NS). Recently, we demonstrated the occurrence of HS cross-reactive antissDNA antibodies in type 1 diabetes. Here, we show that anti-HS/DNA antibodies are present also in type 2 diabetes associated with angiopathy. Because of their potential activity with HS-associated basement membranes and cell surfaces, these antibodies may be considered a factor in the pathogenesis and/or in the accelerated progression of diabetic angiopathy.

A171

P627

P628

EFFECT OF HYPERINSULINAEMIA ON RISK FACTORS FOR CARDIOVASCULAR DISEASE IN PATIENTS WITH NON-INSULIN-DEPENDENT DIABETES MELLITUS T. Lindstri~m, R.G. Olsson, H. yon Schenck, L. Wallentin and H.J. Arnqvist, Departments of Internal Medicine and Clinical Chemistry, Faculty of Health Sciences, Linki~ping University, Linki~ping, Sweden

PATHOGENETIC FACTORS OF MACROVASCULAR COMPLICATIONS IN T Y P E II D I A B E T E S M E L L I T U S . D.Travia, E.Bonora, V.Cacciatori, M.Zenere, F.Saggiani, M.G.Artioli and M . M u g g e o . D e p a r t m e n t of Metabolic Diseases, University of Verona, Italy. In the present study we compared several c l i n i c a l a n d l a b o r a t o r y d a t a in t y p e II d i a b e t i c subjects with or without macrovascular complications. 160 diabetic patients with no evidence of m a c r o v a s c u l a r complications and 160 diabetic subjects with coronary heart disease, cerebrovascular disease and/or peripheral vascular disease, diagnosed with clinical and/or instrumental criteria, were studied. The two g r o u p s w e r e m a t c h e d f o r sex, age, and d u r a t i o n of d i a b e t e s . Diabetic subjects with and without manrovascular ~omplioations d i d not d i f f e r in body mass index~ skinfold-estimated body composition, an~ fat topography, although physical activity at w o r k or at leisure was significantly lower in subjects with complications. The latter showed higher concentrations o f H b A l c ( 7 . B 2 0 . 0 1 vs. 6 . 8 2 0 . 0 1 Z , p . P l a s m a l i p i d s and p r e v a l e n c e of dislipidaemia, family history of macrovascular disease~ cigarette/day consumption, a n d y e a r s of s m o k i n g w e r e similar in the two groups, w h i l e the prevalence of hypertension was higher in subjects with macrovascular d i s e a s e (60 vs. 40Z~ p
Hyperinsulinaemia is considered to be a risk factor for atherosclerosis and is related to o t h e r risk factors for cardiovascular disease. The aim of this study was to investigate how the hyperinsulinaemia caused by insulin t r e a t m e n t of patients with non-insulin-dependent diabetes mellitus influences risk factors for cardiovascular disease. Ten patients with secondary failure to oral hypoglycaemic agents and body mass index 26.0 + 1.3 k y / m L (mean _+SE) were studied while still taking oral agents and a f t e r 8 weeks of insulin t r e a t m e n t . Fasting and postprandial plasma insulin concentrations increased while C-peptide concentrations were lowered. HbA1c was reduced from 8.9 + 0.3 to 6.3+- 0.2 %. There was a weight gain of 2.8 -+ 0.7 ky. Plasma concentrations of total cholesterol, VLOL cholesterol, total triglycerides, VLDL-, LDL- and HDL triglycerides were all reduced. Also the plasma concentration of apolipoprotein B was lowered. Supine diastolic blood pressure and both standing systolic and diastolic blood pressures were lowered, t-PA antigen a f t e r venous occlusion showed u significant reduction while PAl activity was 26.0 .+. 9.8 on oral t r e a t m e n t and 18.2 _+ 4.7 I U / m l on insulin t r e a t m e n t (NS). Microalbumiuria was reduced. In conclusion, several risk factors for cardiovascular disease are improved when hyperinsulinaemia is produced by insulin t r e a t m e n t of patients with non-insulin-dependent diabetes mellitus and secondary failure ~o oral hypoglycaemic agents.

P629 PREVALENCE OF SILENT MYOCARDIAL ISCHEHIA IN MIDDLE-AGED DIABETIC PATIENTS. PRELIMINARY RESULTS. C. P a i l l e l e , F. Paycha*, P.G. Steg, J.H. J u l i a r d , R. Gourgon and Ph. Lo Passa**. Department o f Cardiology and * Department o f Nuclear Medicine - H 6 p i t a l B i c h a t 46, rue Henri Huchard - 75018, P a r i s , France. ** Department o• Endocrinology - H ~ p i t a l S a i n t - L o u i s - 1, avenue Claude V e l l e f a u x - 75010, P a r i s , France. The aims o f the study were t o determine the prevalence o f s i l e n t myocardial ischemia in the d i a b e t i c p o p u l a t i o n and t o assess p r o s p e c t i v e l y in t h i s r e s p e c t the usefulness o f 3 noninvasive t e s t s . Were e l i g i b l e type 1 ( i n s u l i n dependent) and type 2 (non i n s u l i n - d e p e n d e n t ) d i a b e t i c p a t i e n t s , age 40 t o 60 years, d u r a t i o n o f diabetes > 10 years, w i t h B 2 a s s o c i a t e d c a r d i o - v a s c u l a r r i s k f a c t o r % l a c k o f c a r d i a c symptoms and normal r e s t E.C.G. T h i r t y seven p a t i e n t s : 28 men and g women, mean age 50.4 + 6 years, mean d u r a t i o n o f diabetes 22.3 + $0.7 years under-went 24 hour ambulatory E.C.G., maximal e x e r c i s e e l e c t r o cardiography and d i p y r i d a m o l e t h a l l i u m myocardial s c i n - t i g r a p h y . I f e i t h e r o f these t e s t s evidenced signs o f myocardial ischemia, a coronary angiography was performed. H o l t e r m o n i t o r i n g was p o s i t i v e t w i c e , e x e r c i s e E.C.G. 7 times and t h a l l i u m imaging 5 t i m e s , r e s u l t i n g o v e r a l l in performing 8 coronary angiographies (22%). Five p a t i e n t s had severe ( > 75%) and m u l t i p l e coronary s t e n o -sea ; 3 p a t i e n t s had normal angiograms. I n t h i s ongoing study, prevalence o f s i l e n t myocardial ischemis was 13.5%. The p o s i t i v e p r e d i c t i v e value f o r maximal e x e r c i s e t e s t i n g and d i p y r i d a m o l e t h a I l i u m imaging was r e s p e c t i v e l y 71% and 100%. I n middle-aged asymptomatic d i a b e t i c p a t i e n t s , c l i n i c a l examination and r e s t E.C.G. may miss severe coronary a r t e r y disease : t o d e t e c t s i l e n t myocardial ischemia t h e choice o f the more accurate noninvasive test is still controversial.

P630 SILENT MYOCARDIAL !SCHAEMIA AND AUTONOMIC NEUROPATHY IN ASYMPTOMATIC DIABETIC MALES. DAS Marshall, AA-M Hamdy, BM Fisher, JD Quin, PW Macfarlane, AC MaoCuish and SM Cobbe. Departments of Medical Cardiology and Diabetes~ Royal Infirmary, Glasgow, Scotland, UK. Silent myocardial ischaemia is commonly found in patients with coronary disease. We have investigated male diabetic patients without angina pectoris but with a high risk of coronary disease to ascertain the prevalence of occult ischaemia. I00 patients (aged 53• yrs) underwent exercise testing (ETT) and 24 hour Holter monitoring for ST segment changes. The duration of diabetes was 4.9• yrs in 69 non-insulin dependent subjects and 14.9• yrs in 31 receiving insulin. Resting ECG showed ischaemia(9), LVM(5) and Q waves(5). 25/100 ETT were electrically positive, 21/100 with significant (>0.1mV)planar ST depression and 5/100 with ventricular arrhythmias. In an age and sex-matched non-diabetic control group, I/i00 positive ETT occurred. Ambulatory monitoring revealed significant ST depression in 16/100 diabetics of whom ii had positive ETT. 31/89 (35%) patients had evidence of a u t o n o m i c dysfunction by standard criteria, with 13/31(34%) having positive ETT. In conclusion, silent myocardial ischaemia is common in asymptomatic diabetics and lack of symptoms may in part be due to cardiac autonomic neuropathy.

A172

P631 DO DIABETIC PATIENTS HAVE MORE FREQUENTLY VENTRICULAR ARRHYTHMIAS THAN NON-DIABETIC D. S t r 6 d t e r , A. M 6 h r i n g , H . - P . H a m m e s , K. F e d e r l i n III. Med. D e p . , U n i v e r s i t y , G i e S e n , F R G

P632 ?

Diabetic patients (D) h a v e a h i g h e r risk of myocardial infarction and, independently from infarction, a higher incidence of heart failure. However, the influence of premature ventricular b e a t s (VPB) o n t h e m o r t a l i t y of D without infarction is currently unknown. Therefore, 2 4 h l o n g - t e r m E C G o f 185 D (76 m, 109 f, m e a n a g e 5 5 . 8 y e a r s , 2 9 . 7 % t y p e I) w e r e analyzed and compared with long-term ECGs of 129 n o n - d i a b e t i c c o n t r o l p a t i e n t s (ND; 67 m, 62 f, m e a n a g e 53 y e a r s ) . E x c l u s i o n criteria for b o t h g r o u p s : i n f a r c t i o n s in h i s t o r y o r ECG. RESULTS: 7 3 . 5 % o f D, 77 % o f N D h a d VPB. 3 6 . 8 % o f D a n d 34.1 % o f N D h a d L O W N g r a d e s III a n d IV. Additionaly, there was no difference in different age~groups. - A c o r r e l a t i o n o f V P B to d i a b e t e s d u r a t i o n w a s n o t found. - Metabolic control, measured by HbAI, was not c o r r e l a t e d t o L o w n g r a d e s o f VPB. 100 % o f D w i t h c h r o n i c o c c l u s i v e arterial d i s e a s e (COAD) h a d V P B in c o m p a r i s o n t o 6 9 . 4 % of D without COAD. Hypertensive D had significantly more often V P B L O W N I V t h a n n o r m o t e n s i v e D (32 % v s 10 %). CONCLUSION: D have no increased ventricular electrical ins t a b i l i t y c o m p a r e d t o ND. O n l y D w i t h C O A D as indicator for progressive atherosclerosis and D with hypertension have a higher incidence of ventricular arrhythmias.

L E F T V E N T R I C U L A R F U N C T I O N IN T Y P E - I D I A B E T E S MELL.: H E M O D Y N A M I C A N D D O P P L E R E C H O C A R D I O G R A P H I C DATA. H. H a u n e r ~, G. GroBmann, A. Schmidt, M. Stauch*, E.F. Pfeiffer+, V. Hombach. Diabetes Research Institut, D ~ s s e l d o r f ~ a n d D e p t I n t e r n a l M e d i c i n e I+, IV a n d V I*. U n i v e r s i t y of U l m , F R G

L o n g - t e r m d i a b e t e s is a s s o c i a t e d f r e q u e n t l y w i t h c o n g e s t i v e h e a r t f a i l u r e e v e n a f t e r e x c l u s i o n of c o r o n a r y a r t e r y d i s e a s e or h y p e r t e n s i o n . To a s s e s s e a r l y c h a n g e s of left v e n t r i c u l a r function, we s t u d i e d 28 d i a b e t i c s (D, age 28• diabetes d u r a t i o n 17• a n d 39 a g e - m a t c h e d c o n t r o l s u b j e c t s (CS) b y m e a n s o f m i t r a l f l o w v e l o c i t y e x a m i n a t i o n . In 6 s u b j e c t s of e a c h g r o u p p u l m o n a r y a r t e r y pressures (PAP) were measured simultaneously. E c h o c a r d i o g r a p h i c p a r a m e t e r s : P e a k f l o w v e l o c i t y in t h e e a r l y d i a s t o l i c p h a s e (Ve) a n d d u r i n g a t r i a l f i l l i n g (Va). Results: H e a r t r a t e (HR) was s l i g h t l y h i g h e r in D vs CS (D:77• vs CS: 72• n.s.). A c l o s e c o r r e l a t i o n b e t w e e n V e a n d PAP (r = 0,91) c o u l d b e demonstrated, p<0,05* Ve 53• 52•

CS D

Va 36• 42•

Ve/Va 1,6• 1,3•

PAP(mmHg) 6• 9•

The e a r l y i n f l o w v e l o c i t y V e m i g h t be n o r m a l i z e d b y the i n c r e a s e d PAP in D. L V f u n c t i o n seems t o b e altered, s i n c e a t r i a l fill i n g was m o r e p r o n o u n c e d , w h e r e a s p r e l o a d c o n d i t i ons w e r e d i f f e r e n t b e t w e e n D a n d CS. This m i g h t b e an e a r l y sign of a h e a r t d i s e a s e in d i a b e t i c s .

P633

P634

RESTING DIASTOLIC AND EY~RCISE SYSTOLIC FUNCTI{~ IN INSULIN DEPENDENTS WITH NOR~AL RESTING SYSTOLIC FUNCTION. P.Ubaldi, * F. Bruzzone,F. Catalani*, S.Borziani, C. Ferraro, G.Gnecco, G.P.Bezante,A.Cambiano,G.L.Viviant,* L. Adezati* 9 Dpt. of Internal Medicine RR; Dpt. of Cardiovascular Diseases - University of Genoa. Aim of our study was to assess non-invasively the resting diastolic and the exercise systolic function in insulindependent patients with resting normal systolic fbnction. Seventeen insulin-dependent diabetics without complications, mean age 32+-9 ys (Group A), 17 insulindependents with mieroangiopathie complications, mean age $9+-9 ys (Group B), both in good metabolic control and 20 sex-age matched controls (Group C) were studied. They underwent simultaneous Echodopp]er (ED) and First Pass Radionuclide Angiography (RA) at rest and First Pass RA during maximal exercise test. Were considered : the early peak flow velocity (EPFV),the atrial peak flow velocity (APFV) and the RA global ejection fraction. Results: GROUP A GROUP B GROUP C basal E~{ 42+-7 ns 44+-5 ns 45+-5 effort EFt 51+-8 ns 53+-6 ns 54+-7 EPFV (cm/see) 45+-6 ns 48+-7 p 0.01 54+-9 I...............p 0,01 ................ I APFV (em/sec) 65+-8 ns 64+-7 p 0.01 58+-11 I...............p 0,O1 ................ I EF showed a normal, exercise increase in all groups.Significant statistical differences about EPFV and APFV of Groups A and B vs controls were noted; not differences between Groups A and B. In conclusion diabetics with resting normal systolic function have a normal increase of EF% during exercise and show resting diastolic function abnormalities, not related to microangiopathie complications.

ISOMETRIC

EXERCISE

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RELAXATIOH

1 DIABETES

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A173

P635

P636

MITRAL FLOW VELOCITY AND PULMONARY HEMODYNAMICS AFTER VOLUME LOADING IN TYPE-I DIABETES MELLITUS A. Schmidt, G. GroBmann, H. Hauner I, M. Stauch*, E.F. Pfeiffer+, V. Hombach. Dept Internal Medicine I+, IV and VI*, University of U l m and Diabetes Research Institut ~, D~sseldorf, FRG

BONE MINERAL DENSITY (BMD) MEASURED BY DUAL PHOTON AB5ORBTIOMETRY (DP~) SN WOMENWITH DIABETES TYPE S (fDDN).

F r o m several investigations it had been suggested that diastolic properties are altered in long-term diabetes mellitus due to non coronary factors. 16 diabetics (D; mean age 30 • 8y, diabetes duration 18 • 7y) and 13 age-matched control subjects (CS) were investigated to determine hemodynamic changes measured by pulmonary artery pressures (PAP, 6 persons in each group) and transmittal flow velocity at rest (R) and after volume loading (V, 15 mi/kg.). Parameters were the maximal flow velocity in the early diastole (Ve) and at time of atrial filling (Va) as well as the PAP. Results: A close correlation between Ve and PAP (rest:r =0,91; volume: r =0,9) could be demonstrated.p<0.05*, p<0,01** Rest Ve Va Ve/Va PAP(nTnHg) D 32• 25• 1.3• 9• CS 31• 19• 1.7• 6• Volume D CS

Ve 38• 36•

Va 28• 24~5

Ve/Va 1,4• 1,5•

PAP 19• 16•

Mrevlje F, Salobir B. OeDartment o f Endocrinology and Metabolism, U n i v e r s i t y Med ica l Centre, Ljubljana, Yugoslavia BMD l s measured by OPA in 9d women with IDOM (A) and in 41 healthy women (B) in age group o f 30 to 60 yrs. There i s no s i g n i f i c a n t d i f f e r e n c e i n percentage off postmenopausal (postN) women (30% in A, 27% in B, l a s t l n g f o r d.5 and 5.4 y r s . r e s p . } as well as in the body welght. Comparing BMD between ~ and B withln the same decade (dec) we f i n d s i g n i l . lower BMD in IDDM in 4th dee. (A v. B: 1.01 +/-0.02 v. 1.08 + / - 0 . 0 2 g/cm2) but not in 5th and dth dee. Comparing BND between A and B separately f o r preM and posen IDDM we f l n d that BMD i s s i g n l f i e a n t S y Sower in preM (A v.B: 1.01 + / - 0.02 v. 1.07 + / - g/cm2) as well as in postM d ia b e ti c s (V v.B: 0.83 + / - 0.01 v. 0.92 + / - 0.05 g/cm2, p< O.05).The observed differences cannot be explained by age or by menopausal perlod. A e i g n i f , negative c o r r e l a t i o n e x i s t s between BMD and age in both groups as well as with durat io n o f diabetes, serum alkalSne phosohatase, urea and osteoealcln, and wlth urlne OH-prolln excretion. We f i n d no c o r r e l a t i o n between BMO and parameters o f metabolic control o f diabetes, body weight, or calclum, phosphorus and e r e a t l n l n in serum as well as in urine calcium excret io n in IDDM. Conclusion: we found that younger women wlth diabetes type I can have lower BMD but la t e : r t h i s d if f e re n ce can diminish.

Conclusion: Although the increase of Ve and Va after volume loading was similar in diabetics and control subjects, pulmonary artery pressure and Va at rest were significantly higher in diabetics. These changes might be early abnormalities of relaxation and compliance in diabetics.

P637 Calcium phosphorus regulation . A study Non-Insulin-Dependent

P638 metabolism and its hormonal in Insulin-Dependent (type I) and (type II) Diabetes Mellitus

L T A U V E R O N , S. MOINADE, E. ALBUISSON, Ph. T H I E B L O T , G. G AIL L AR D , C. G E N T O U . D6partment of Diabetes and Endocrine Diseases, Biostatistics, In Vitro Nuclear Medicine and Biochemistry - CHRU - CLERMONT-PERRAND 63000 - France. T h e pathophysiology of diabetic bone metabolism remains unclear, either microvascular or metabolic. Thus, this study was made in three groups of patients : group 1 type I n = 42 age 44.5 + 2.2' years - duration of diabetes 14.5 + 1.5 years - BMI 24.2 + 3.4 kg/m2 group 2 type II n = 42 age 55.7 +_. 1.3 years - duration of diabetes 10 + 1.1 years - BMI 27.6 + 0.7 kg/m2 and group 3 controls (C) n = 44 abe 41.2 +. 1.8 years - BMI 23.5 + 0.9 kg/m2 . All subjects were free of severe kidney or liver disorder . While the levels of total and ionized blood calcium and phosphorus did not differ, urinary calcium excretion increased in group 1, as did urinary phosphorus Bone turnover was lower in type II patients as indicated by rates of osteocalcin ( g r l : 7.85 + 0.416 ng/ml - gr2 : 6.94 + 0.402 - gr3 : 7.82 + 0.442), especially in poorly controlled subjects (negative correlation with glycosylated haemoglobin r = - 0.3 p < 0.05) . Serum intact parathormone levels were similar in all groups ( g r l : 33.05 + 2.196 pg/ml - gr2 : 28.71 + 2.08 - gr3 : 29.66 + 2.265), as were thyrocalcitonin and 25 OH vitamin D3 ( g r l : 12.33 + 1.046 ng/ml - gr2 : 11.72 +.+_ 1.574 - gr3 : 11.77 + 0.942) . 1.25(OH)2 vitamin D3 was slightly lower in type II subjects ( g r l : 47.93 + 2.646 pg/ml- gr2 : 44.72 _% 3.915 - gr3 : 47.046 + 2.955), but more interesting was the negative correlation in type I diabetes between 1.25(OH)2 D3 rates a nd glycaemic balance (glycosylated haemoglobin r = - 0.317 p < 0.05 and fructosamin r = - 0.397 p < 0.02) . Conclusion : even in rather well controlled diabetic patients, disturbance of calcium-phosphorus m e t a b o l i s m r e g u l a t i o n occurs, in our study mainly reduced osteocalcin in type II diabetes and a negative linear correlation between 1.25(OH)2 vitamin D3 rates and glycaemic control in type L These alterations might be involved in the genesis of diabetic osteopathy .

NORMAL BONE MINERAL DENSITY IN TYPE 1 (INSULINDEPENDENT) DIABETIC PATIENTS WITH POSTPUBERTAL ONSET. M. Salvatore, C.V. Albanese, P. Astazi, M.A.S. Di Leo, A.V. Greco and G. Ghirlanda. Institute of Internal Medicine, Catholic University, Rome, Italy. Osteopenia seems to be one of the complications of diabetes and the bone mass appears to decrease over the first years of disease. To study the relationship between the diabetes onset and bone mineral density (BMD), we evaluated trabecular bone using an X-ray dual-photon bone densitometry of the lumbar spine and femoral neck of 19 Type 1 (insulin-dependent) diabetic patients with disease duration < 7 years. All patients were diagnosed after the puberty (18.6 ~ 3.5 years). None had other diseases, BMD of the L2-L4 region, the femoral neck and Ward's triangle (lowest density) were evaluated. No differences for sex, height, weight, and age between the 2 groups were observed. We did not find any differences between patients (unpaired t test; mean • SD, lumbar: 1.037 • 0.12 g/c~; neck: 0.841 • 0.14 g/cm2; Ward's: 0.730 • 0.12 g/ore2) and control subjects (lumbar: 1.034 • 0.14 g/cm2; neck: 0.824 • 0.12 g/cm2; Ward's: 0.699 • 0.12 g/cm2), also when analyzed according to sex. A positive correlation between age at diabetes onset and BMD (regression analysis; lumbar: r=0.49, p=0.03; neck: r=63, p=0 004; Ward's: r=53, p=0.02) was observed. No correlation was found with disease duration, insulin requirement, HbAlc. Our results suggest that osteopenia is an uncommon feature in patients with short-standing disease and postpubertal diagnosis, although a lower BMD may be related with a younger age of Type I diabetes onset.

A174

P639

P640

JUVENILE O S T E O P E N I A I N TYPE 1 DIABETICS: LOSS OF B O T H O R G A N I C A N D I N O R G A N I C M A T R I C E S T.Yflmaz, G.Yfimaz, E.Ano~lu, i.Satman, U.Korugan, S . D e v r i m and F.Biyal, Department of Diabetes, Istanbul and Cerrahpa~a Medical Schools, Istanbul University. In Type I diabetes juvenile osteopenia characterized w i t h loss of both organic a n d inorganic matrices can be detected. In this study, 35 Type I diabetics (mean age + SD=19.45:9.1) and a matched control group (mean age :t:SD= 21.11-9.6) w e r e i n v e s t i g a t e d for different p a r a m e t e r s of this loss. Cases a n d controls were s t u d i e d in three different a g e groups; group 1:5-14 years, group 2:15-25 years and group 3:>25 years. The parameters u s e d i n c l u d e d bone-mineral-content (BMC) of left r a d i u s as d e t e r m i n e d b y I ~Ztphoton absorbance; urine calcium, m a g n e s i u m a n d p h o s p h a t e excretion ( all excretory values are corrected w i t h 1 / u r i n e creatinine (uCr) r n g / d l to eliminate polyuria factor in diabetics) a n d h y d r o x y p r o l i n u r i a in 24 hours. The latter is a n e a r l y i n d e x of i m p a i r m e n t in organic matrix; whereas all the former ones indicate imbalance in m i n e r a l incorporation. Metacarpal indices (MCI) of the two g r o u p s w e r e also compared. BMC as m e a s u r e d in g r / c m 2 w a s 13.2% decreased in diabetics (0.4785:0.103 versus 0.551i-0.101; p<0.01). Urine caldum, m a g n e s i u m and phosphate excretion were all increased b y 291, 196 a n d 142 % respectively (0.218_+0.115 versus 0.1015=0.075 urine C a / u C r mg%, 0.315:h0.121 v e r s u s 0.170+0.098 u r i n e M g / u C r m g % 1.067i-0.541 versus 0.752._+0.277urine P04/uCr rag%, p<0.01 in all three conditions). As to h y d r o x y p r o l l n e excretion, it w a s also significantly h i g h e r in cases as o p p o s e d to controls (by 179%; 6.147:t5.810 versus 3.432-%1.852 u r i n e h y d r o x y p r o l i n e / u C r m g %; p<0.02). T h e s e differences were most p r o m i n e n t in G r o u p 2 a n d were not statistically significant in G r o u p 3 (p>0.05). Also excretory calcium, m a g n e s i u m , p h o s p h a t e a n d h y d r o x y p r o l i n e levels w e r e n e g a t i v d y correlated to BMC (r < -0.550 in all conditions). MCI (a m u c h more practical index) w a s significantly l o w e r (18.2%, p<0.01) in the diabetic g r o u p as well and had a strong positive correlation with BMC (r= 0.712). In conclusion, juvenile osteopenla gains importance d u r i n g the period of active bone g r o w t h a n d formation. Because of h i g h correlation to other parameters a n d its convenience MCI is proposed as a routine-method for detecting early osteopenia.

1.25(OH)2 vitamin D3 status is related to glycaemic control in insulin-dependent (type I) diabetes mellitus patients . S. MOINADE, L TAUVERON, E. ALBUISSON, Ph. THIEBLOT, G. GAILLARD ,C. GENTOU .Department of Diabetes and Endocrine Diseases, Biostatistics, In Vitro Nuclear Medicine and Biochemistly - CHRU - 63000 CLERMONT-FERRAND - France. We performed a study in 42 type I diabetic (DM I) patients and 44 controls (C). All patients presented without severe kidney (normal creatinine clearance microalbuminuria allowed) or liver disease. Mean age was 44.5 + 2.2 years (DM I) (41.2 + 1.8 C) and durailon of diabetes 14.5 + 1.5 years. Total (2.38 +_ 0.1 mmol/1 DM I / 2.34 + 0.13 C) and ionized (1.17 + 0.005 mmol/l DM I / 1.17 + 0.006 C) blood calcium, linked together (r = 0.551 p < 0.01), phosphorus (1.44 + 0.331 mmol/l DM I / 1.25 +. 0.026 C, p < 0.01) as well as urine calcium and phosphorus excretion and phosphorus reabsorption rate (86.8 + 0.678% DM I/88.36 + 0.803 C) were measured. We also determined markers of bone turn over, serum alkaline phosphatase, osteocalcin and hydroxyprolinuria, which were similar in both groups. Hormonal results included intact parathormone (33.05 + 2.196 pg/ml DM I/ 29.66 + 2.263 C), thyrocalcitonin (5.88 + 0.487 pg/ml DM I/6.16 + 0A28 C), 25 OH vitamin D3 (12.33 + 1.046 ng/ml DM I/11.77 + 0.942 C) and 1.25(OI5)2 vitamin D3 (47.93 + 2.646 pg/ml DM I/47.046 +_.2.955 C). This parameter, already observed at lower levels in very poorly controlled patients, was negatively correlated with both HbAlc (mean 8.07 + 0.248% - usual values 4 - 6%) (r = 0.317 p < 0.05) and fructosmnin (mean 3.32 + 0.073 - usual values 2.52 - 2.98) (r = - 0.397 p < 0.02). These data are also confn'med by ANOVA test. We conclude that even in regulary treated with quite satisfactorily controlled type I diabetic patients, without major disorders of calcium phosphorus metabolism nor kidney or liver disease, 1.25(OH)2 vitamin D3 levels are inversely correlated with glycaemic balance, estimated by frutosamin or glycosylated haemoglobin.

P641

P642

LIMITED JOINT MOBILITY AND M I C R O A N G I O P A T H Y IN TYPE I (INSULIN-DEPENDENT) DIABETES MELLITUS. J.Soler, E.MontaNa, A.Rozadilla, JM.Nolla, C.Ligorria, N.Virgili and C.Vinzia. Endocrine Unit. Hospital Bellvitge, Barcelona, Spain.

STUDY OF SIALITIS IN NOD MICE AND INTO SYNGENEIC HEALTHY NEONATES.

The aim of the study was to analyse the r e l a t i o n s h i p between limited joint mobility (LJM), metabolic control and m i c r o a n g i o p a t h y in type I (insulin-dependent) diabetes mellitus. We studied 61 type I (insulindependent) patients, age (X• 30.9• years (range ii-57), diabetes duration: ii.7• years (5-30); 52.5% were males. We analysed chronic metabolic control (mean HbA 1 during the last two years) and the presence of retinopathy (direct oftalmoscopy) and microalbuminuria (24 hour samples, RIA). Joint mobility was assessed by the prayer sign and by the range of joint movement (in degrees) recorded bilaterally with a goniometer in 3 and 5 metacarpophalangeal (MCP) joints, wrist and elbow. The prayer sign was positive in 47.5% of patients and was associated with retinopathy (p< 0.0001) and m i c r o a l b u m i n u r i a (I 20 Dg/min) (p=0.025), but not w i t h m e t a b o l i c control. Patients with r e t i n o p a t h y (52.4%) had a limited extension and flexion of 3 MCP (p= 0.020 and p= 0.016 respectively) and limited extension of 5 MCP (p= 0.048) and wrist (p= 0.001). Patients with m i c r o a l b u m i n u r i a (31.5%) had a limited flexion of 3 and 5 MGP (p= 0.042 and p= 0.046 respectively) and limited wrist extension (p= 0.001). In conclusion LMJ is associated w i t h r e t i n o p a t h y and microalbuminuria in type I (insulin-dependent) diabetes, but has no relation w i t h chronic metabolic control

E. GOILLOT, M. MUTIN and J. L. TOURAINE, URA 1177 and UCBL Lyon France.

ITS TRANSFER

INSERM U 80,

NOD mice exhibit insulitis, and also a sialitis, resembling that of Sj6gren's syndrome. We studied sialitis spontaneous incidence in NOD males and females aged 4, 8, 12, and 24 weeks. In both sexes, sialitis appeared later than insulitis. At any age, though lower, frequency of sialitis increased with that of insulitis. At 24 weeks, incidence of sialitis (about 75%), as well as that of insulitis (85%) was not different between sexes. Sialitis appeared only in mice with pancreas showing insulitis. Frequency of sialitis increased with severity of insulitis. Most infiltrating cells, surrounding ducts and blood vessels, proved to be T lymphocytes (Thy 1.2.+), with a majority of L3T4 +. Transfer of sialitis was attempted into syngeneic neonates by injecting either total splenocytes or purified T cells. About 40% of males and females injected with total splenocytes showed sialitis at 4 weeks, whereas controls reached this percentage between 8 and 12 weeks. Similar infiltrations were obtained with purified T cells. These results enable to postulate that auto-reactive T cells infiltrating pancreas islets in NOD mice may either contain or recrute effector T cells invading secondarily salivary glands.

A175

P643

P644

ALTERATIONS OF ALVEOLO-CAPILLARY P E R M E A B I L I T Y IN D I A B E T E S . Ch. Daumerie and M. Zona-Vielvoye. Univ. Louvain Med. Sch., Brussels, Belgium.

IMPAIRED PHAGOCYTOSIS OF CIRCULATING POLYMORPHONUCLEAR NEUTROPHILS IN PATIENTS WITH DIABETES MELLITUS A. Kuzuyama, H. Senmaru, H. Mori and K. Kasbima

The 3rd Department

of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, Long term diabetes mellitus is characterized by widespread thickening of the capillary basement membrane in s e v e r a l o r g a n s s u c h as k i d n e y , retina and muscle. In o r d e r t o a s s e s s the status of the alveolo-capillary barrier, an aerosol of [Tc-99m]-DTPA was administered and the half-life o f its c l e a r a n c e f r o m t h e l u n g s was u s e d as a n i n d e x of a l v e o l o - c a p i l l a r y p e r m e a b i l i t y . Measurements w e r e c a r r i e d o u t in f o u r g r o u p s of s u b j e c t s , namely diabetics, smokers (n = 20; a g e 5 7 . 3 • years; mean• and non-smokers (n = 28; age: 61.6• and controls, smokers (n = 8; age: 4 9 . 8 • and non-smokers (n = 16; age: 5 4 . 1 • . The rate of DTPA clearance was drastically i n c r e a s e d in smoker (23.0• min) v e r s u s n o n - s m o k e r control subjects (76.5• p<0.001) . In addition, the clearance was s i g n i f i c a n t l y s l o w e r in s m o k e r d i a b e t i c s (35.1• than in smoker control subjects (23.0• p=0.003), and s l i g h t l y but not s i g n i f i c a n t l y s l o w e r in non-smoker diabetics (84.9• relative to non-smoker controls (76.5• No correlations were found b e t w e e n c l e a r a n c e r a t e s a n d p a t i e n t age, d u r a t i o n or c o m p l i c a t i o n s of d i a b e t e s , and HbAIC levels. Our findings suggest that lung alveolo-capillary permeability m a y be a b n o r m a l in d i a b e t e s .

Japan

P645

P646

D-m_jLo-INOSITDL-I,2,6-TRIPHOSPHATE PREVENTS GLUCOSEINDUCED VASCULAR DYSFUNCTION IN NORMAL RATS. R.G. Tilton, K. Chang, C. Kilo, and J.R. Williamson. Department of Pathology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri, 63110 USA. This study was undertaken to investigate the effects of D-myo-inositol-l,2,6-triphosphate (PP-56) (M.3. Siren, 1984, US patent number 4735936, developed by Perstorp Pharma, Sweden) on vascular dysfunction induced by topical application of 30 vs 5 mM D-glucose to granulation tissue forming in skin chambers. After removal of 2 cm circles of skin, b i l a t e r a l plastic chambers were mounted on either side of the back of normal male Sprague-Dawley rats. Beginning l week l a t e r , l ml of 5 or 30 mM D-glucose • 0.02, O.l, or l.O mM PP-56 in PBS was added to the chambers twice daily for lO days. After lO days, vascular clearance of plasma 131I-BSA and blood flow (assessed by use of 15 ~m radiolabeled microspheres) were determined, n=4 f o r each group. Albumin clearance and blood flow were both s i g n i f i c a n t l y increased in tissues exposed to 30 mM vs 5 mM glucose (albumin clearance = 296 • 42 (SD) vs 58 + 15 nl/g wet weight/min, r e s p e c t i v e l y , P < O.OOl; b l o o d flow = 0.70 • 0.08 ml/g wet wt/min vs 0.45 • 0.03, P < 0.005). 0.I and 1.0 mM (but not 0.02 mM) PP-56 markedly reduced albumin clearance (141 • 39 and 134 • 16, r e s p e c t i v e l y , P < 0.005 f o r each vs 30 mM glucose) and normalized blood flow (0.43 • 0.07 and 0.47 • 0.06, r e s p e c t i v e l y ) . PP-56 had no e f f e c t on elevated s o r b i t o l l e v e l s in 30 mM glucose t i s s u e s . These observations i n d i c a t e t h a t PP-56, l i k e m w o - i n o s i t o l , pyruvate, and aldose reductase inhibitors, prevents vascular dysfunction induced by elevated glucose l e v e l s .

PREVENTION OF GLUCOSE-INDUCED VASCULAR FUNCTIONAL CHANGES BY ACETYL-L-CARNITINE J.R. Williamson, K. Chang, C. K i l o , W.R. Sherman*, M. Montenero+, and E. A r r i g o n i - M a r t e l l i w Departments of Pathology and * P s y c h i a t r y , Washington U n i v e r s i t y School of Medicine, St. Louis, M i s s o u r i , USA; +Eums Pharma, New York, New York, USA; w S.p.A., Rome, I t a l y .

Phagocytic function of polymorphonuclear neutrophils (PMN) was studied by the rapid method using Micrococcus Lysodeiktics (M. lys) in 89 diabetics (insulin: 32, oral hypoglycemic agents: 39, diet: 18) and 60 controls. Heparinized blood was incubated with M. lys in saline containing 6% hydroxyetbyl starch up to 10 minutes. Samples were taken at 1 min intervals for the quantitative determination of phagocytosis. In all controls 100% of phagocytosis was observed within 7 min after incubation and therefore percentage phagocytosis reaching to less than 100% at 7 rain was assessed as impaired phagoeytosis. Impaired phagocytosis was found in 42% (37/89) of diabetics. Glycosylated hemoglobin and /3-hydroxybutyrate concentrations significantly increased in diabetics with impaired phagocytosis compared to those with normal phagocytosis (10.8 + 2.2 vs 9.8 -+ 1.9%, 79.9 + 86.4 vs 45.3 • 53.1 #mol/1, mean + SD). When washed PMN from diabetics with impaired phagocytosis were suspended in plasma from controls, impaired phagocytosis completely improved. In several subjects sorbitot content of PMN was also measured. No significant difference in sorbitol content was found between diabetics with impaired phagocytosis and controls. Chronic hyperglycemia and increased /3 -hydroxybutyrate concentration may affect the phagocytosis of PMN in diabetics and impairment of phagocytosis might be due to humoral factors, and not to PMN itself.

This study was undertaken to examine effects of acetylL-carnitine (ALC) (provided by Sigma-Tau SpA) on glucose-induced vascular dysfunction in skin chamber granulation tissue. After removal of 2 cm circles of skin, b i l a t e r a l plastic chambers were mounted on either side of the back of normal male Sprague-Dawley rats. After l week, l ml of 5 or 30 mM D-glucose • 0.2 or 2.0 mM ALC in PBS was added to the chambers twice daily f o r lO days. IO days l a t e r 131I-BSA clearance and blood flow (assessed by use of 15 ~m 85Sr microspheres) were determined. Albumin clearance and blood flow in 5 mM glucose tissues (n=6 for a l l groups) were 109 • 41 (SD) nl/g wet wt/min and 0.44 • 0.07 ml/g wet wt/min, respectively. They were increased by 30 mM glucose to 429 • I07 and 0.90 • 0.05, respectively, P < O.OOl. Albumin clearance was reduced by 0.2 mM and 2.0 mM ALC to 131 • 59 and I03 • 18 (P ~ O.OOl), and 2 mM (but not 0.2 mM) ALC v i r t u a l l y normalized blood flow (0.52 • 0.14, P < O.OOl vs 30 mM glucose). 2 mM ALC had no effect on blood flow or albumin clearance in 5 mM glucose tissue. ALC had no effect on elevated sorbitol levels in 30 mM glucose tissues. In view of what is known about effects of ALC on intermediary metabolism, these observations suggest that glucose-induced vascular dysfunction may be linked to impaired energy metabolism.

A176

PS 23 Glycation P647 ENZYME IMMUNOASSAY: A NEW APPROACH TO THE MEASUREMENT OF HbA~c D L Bates ~, J Beacham 2, I Gibb 3 and J-O Jeppsson 4. 1. Novo Nordisk Diagnostics, Cambridge UK. 2. Hammersmith Hospital, London UK. 3. Royal Victoria Infirmary, Newcastle UK. 4. Malmo General Hospital, Sweden. Measurement of HbAtc is widely used to monitor diabetic control. To date, assays for this analyte have relied on classical biochemical fractionation methods; we report on a novel enzyme immunoassay (NovoClone TM) that measures HbAlc directly. The assay uses a monoclonal antibody to detect the structural epitope of the glycated residue at the N-terminaI of the haemoglobin [~-chain. The EIA does not recognise non-glycated haemoglobin (HbA0), other glycation sites or any haemoglobin variant lacking the epitope. It is not subject to interference by 'pre-HbAlc', other modified haemoglobins, hyperlipidaemic samples or other common intereferences. The assay is extremely precise; the within assay CV's are 3.3%, 2.1% and 2.0% at HbAlc values of 3.0%, 6.4% and 10.5% respectively. The EIA co~relates well with a routine HPLC method (HbAlc) [EIA = 0.09 + 0.81 HPLC, r=0.95, n=241], agar electroendosmosis (HbA1) [EIA = 0.94 + 0.81 Electro, r=0.90, n=339] and affinity chromatography (GHb) [EIA = 0.76 + 0.70 Affin, r= 0.95, n=178]. In a sample of 178 non-diabetic patients, the normal range of HbAlc was shown to be 2.6-4.9%. This method will provide a useful tool for the direct and specific measurement of HbAlc in diabetic patients.

P648 A PROPOSED MATHEMATICAL MODEL FOR IN VITRO INTRAERYTHROCYTE H A E M O G L O B I N GLYCOSYLATION E.Arlo~Iu, T.Yfimaz, i.Satman, M.Haclbekiro~lu and S.Devrim, Center for Experimental Medical Research and Applications (DETAM), Istanbul University. The aim of this study was to observe the variations in the level of in-vitro haemoglobin-glycosylation with increasing glucose concentration and incubation-period. For this, blood samples were drawn from 10 healthy volunteers (mean age + SD = 25.7+4.1 years; m e a n glycemia + SD= 91.6+9.2 m g d l % The erythrocytes were w a s h e d three-times with heparinized-Krebs-Ringer-Buffer (KRB) and incubated in 5 different KRB solutions containing 0, 2.8, 5.6, 11.2 and 16.8 mMol glucose. G l y c o s y l a t e d - h a e m o g l o b i n (HbA~C) levels of the erythrocytes determined from their haemolysates after 15, 30, 60, 90, 120 and 180 minutes of incubation using the manual-calorimetric kitt for HbA~C (Biotroll, France). The results (300 measurements) show a positivelineer-relationship between HbA~C 0-I, in nmol fr.mgHb 4) and in vitro glucose-concentration (G, in mMols) which can be formulated as H=e+m (G) with r ranging between 0.92-0.98 for different incubation periods, c is a constant representin~ the average-basal HbA~C level and in this experimental set c= 6.11i-0.32.m is the rate of glycosylation and logarythmically dependent on t in minutes. This can be expressed as m= (-0.043)+ 0.056 In t (r= 0.99). In conclusion H= c+(-0.043+ 0.056 In t) (G) and can be calculated for any incubation period greater than 1minute in any glucose-concentration. This relationship also points out that in vitro glycosylation starts within the first minutes of incubation and detectable after a few minutes. Glycosylation does not saturate, but the speed of the process slows down to a great extent after the first few hours. These might imply that in diabetics the effect of short-term hyperglycemia-periods on HbA~Cqevels can be greater than that expected. If this is true, samples for HbA~C measurements should be collected before OGTT or IVGTT if they are s i m u l t a n e o u s l y scheduled.

P649

P650

~s

I H VITRO GLYCATION OF HUIfAN/kNDP~T HAI~OGLOBIN BY ~-GLUCONOLACTONE, ~ DICARBONYL ANALOGUEOF GLUCOSE R.M. Lindsay, V. Ayre and J.D. Baird, H e t a b o l i c Unit, University of Edinburgh Department of Medicine, Nestern 6eneral Hospital, Edinburgh, U.K.

C~I'ROL~,~&YiN~LD~CE ~ INCREASEDSO'PEROXIDE~ON PRODUCTION IN T~E DIABETIC SERL~ A. Quatroro, A. Ceriello, D. Giugliono, P. Lefebvre

Catt. Diabetologia,i Fac. Med. ,Napoli-CentroDiabe%ol. ~Casadi Cura S.Rita Tarente,Div. of Diabetes,Univ. of Li@ge,Li@ge;Belgit~ A general theory considers the involvement of oxy-free radicals in the development of aging tissue as well as in the diabetic camplicatians. S~0eroxide anion (0~) is an oxy-frse radical.The evidence that in vitro coth glucose and glycated proteins are a source of O~ has been recently reported (Diabetologia31,27A,IS88).inthis study N~e serum O~ production in the sara of i0 type I diabetics,camparedto i0 matched controls,has been evaluated.Glycatedproteins (9.3!0.48vs 4.9_+O.17%,p(O.OCl;Y_wSE), fastingglyc~emia and 02 ganeratien (0.62+0.02 _ vs 0 .2_+0.02 ~ tmol/dl/15', p
It has been proposed that auto-oxidation of monosaccharides produces dicarbonyl compounds which covalently bind to proteins ("auto-oxidatlve Iycosylation"). Ne have studied the effect of

~-g1~conolactone,

a dicarbony1 analogue of glucose on vitro glycation of haemoglobin in blood samples from diabetic and non-diabetic human subjects and spont~neously-diabeUc, Insulin-dependent BB/E rats. Blood samples were incubated with either glucose or ~-gluconolactone (final concentration = 25 mmol/l and 50 mmol/l for studies with human and rat blood respectlveIy) at 37oC for 5h. Glycosylated haemoglobin was determined in these samples by ion-exchange HPLC (HbAlc) and electroendosmosis (Hb^l) respectively. Mean ( S ~ ) values of KbA1c in samples from nondiabetic human subjects (n=18) were 5.1% (0.14), 5.34 (O.14), and 6.54 (0.24) for control, glucose-treated and 6-gluconolactone-treated samples respectively. Equivalent values for samples from diabetic outpatients (n=20) were 8.0~ (0.4~), 8.24 ~0.44) and 8.64 (0.44) respectlveIy. In each case, 6-gluconolactone significantly increased g I y c o s y l a U o n of haemoglobin (p<0.OOl, paired t-test). ~-Gluconolactone also significantly increased the HbAI values of samples from non-dlabetlc (6.1% • 0.2% vs control 5.0~ • 0.2~; n=6; p<0.UOl) and diabetic (7.5% • vs'control 6.6~ • 0~44 ;n=11; p
the hypothesis of oxidative 91ycosylaUon.

A177

P651

P652

The

MEMBRANE KINETIC AND GLYCAT• OF M O N O N U C L E A R M E M B R A N E P R O T E I N S :iN V i V O A N D IN V I T R O S T U D I E S M.Vanhaeverbeek, D.Broh6e, A.Lef~vre, P.Piro, B . K e n n e s a n d P.Neve, Lab. Exp. Med., CHU VESALE, Monnigny-le-Tilleul, Belgium. By • V l V O studiest we llave si~own enllanced glycatlon of circulatlng mononuclear cells m e m b r a n e p r o t e i n s ( M C M P ) in d i a b e t i c s c o m p a r e d uo n o r m a l s ; this g l y c a r z o n is c o r r e l a t e d win~l H ~ 1 p r o v i d e d t h a t t h e i m m u n e s y s t e m is in the r e s t i n g stare. The a i m of the p r e s e n t w o r k is to c o m p a r e the r e l a t i v e r o l e s of g l u c o s e and m e m b r a n e turn-over in the g l y c a t z o n of M C M P in vitro. M o n o n u c l e a r cells of i0 n o r m a l volunteers are cultured four days, without stimulation, with i00, 300 and 500 mg/dl glucose; M C M P are i s o l a t e d w l t h a d e t e r g e n t (Triton X-100) a n d c e n t r i f u g a t i o n at 96 000 g; g l y c a t i o n is m e a s u r e d at day O and 4 by spectrofluorimetry of a fluorophore formed by condensation of OH-m6thyl-furfural with resorcinol, after adjustment of proneln concentration (lowry method); CV innraassay:10%; interassay:15%. Comparisons of g l y c a t i o n b e t w e e n d a y s 0 a n d 4, at any glucose concentration show a negative correlation (r=-.91,-.83 and-.83,p=O.OO3 respectlvely), without statistical difference b e t w e e n the t h r e e g r o u p s at day 4. Friedmann analysis s h o w s a s i g n l f i c a n u e n h a n c e m e n t of glycauion for low-glycated patients (n=4,p
mechanism and biological importance of v i t a m i n C in l o w e r i n g of n o n e n z y m a t i c g l y c a t i o n

P.Stolba, J.Stra~kov6, O.Vohradnikov~, A.Krndkov~ and M.Adam, R e s e a r c h I n s t i t u t e of E n d o c r i n o l o g y , Prague, R h e u m a t i s m R e s e a r c h Czechoslovakia

Institute,

Prague,

P r e v i o u s e x p e r i m e n t s have d o c u m e n t e d that vitamin C i n h i b i t s n o n e n z y m a t i c g l y c a t i o n of serum proteins, c o l l a g e n s and renal b a s e m e n t m e m b r a n e s in vivo and in vitro. The aim of this study was to analyse • m e c h a n i s m of this effect and its i m p o r t a n c e in vivo and in vitro. In vitro effect on human albumin, serum p r o t e i n s and ~ l l a g e n s type I , I I , I V we i n v e s t i g a t e d using o(gC)-ghcose, fructosamine, thiobarbituric acid and s p e c t r o f l u o r i m e t r i c assays after 3-28 days of incubation. B i o l o g i c a l i m p o r t a n c e we tested on serum p r o t e i n s of 36 Type 1 ( i n s u l i n - d e p e n d e n t ) d i a b e t i c s before and after treatment with 3x5OOmg vitamin O per day per os for 3 weeks (ll% decrease, p=O,OlB) and on renal b a s e m e n t m e m b r a n e s of STZ rats t r e a t e d with vitamin C for 120 days (14% decrease, p=O,O18, n=20). We found that the place of primary effect of v i t a m i n C in physiological c o n c e n t r a t i o n s was the i n h i b i t i o n of aldimine f o r m a t i o n in all tested proteins. Oecreased levels of k e t o a m i n e and advanced g l y e a t i o n e n d p r o d u c t s resulted as a consequence. We suppose redox m e c h a n i s m b e c a u s e d e h y d r o a s c o r b i c acid exh i b i t e d a d v e r s e effects and i n h i b i t i o n was pH dependent. We c o n c l u d e that vitamin C could be i n v o l v e d in adjuvant therapy of d i a b e t i c complications.

P653

P654

PREFERENTIAL LOSS OF NON-GLYCATED ALBUMIN THROUGH THE PERITONEAL MEMBRANE IN CAPD-TREATED DIABETICS. R.O.B.Gans, H.J.G.Bilo, G.J.van Kamp, N.Lameire and A.J.M.Donker. Nijmegen and Amsterdam, The Netherlands, and Ghent, Belgium. The transcapillary leakage of glycated proteins is thought to be preferential over the leakage of nonglycated proteins. To prove this assumption has been difficult. Tracer studies even showed the preferential transcapillary loss of non-glycated over glycated albumin. Since patients treated with continuous ambulatory peritoneal dialysis (CAPD) lose albumin in their CAPD-exchange fluid we measured the amounts of nonglyeated and glycated albumin in this fluid and compared the results with serum measurements in the same patients. Nine diabetic patients ( 6M, 3F, 54+12 years) were studied. Only glycerol-containing exchange fluid users were selected. Total albumin, non-glycated and glycated albumin were measured in serum and in at least 8 bags of exchange fluid per patient. Albumin was fraetionated on m-aminophenyl-boronic acid columns (Pierce). The fractions were quantitated either nephelometically or enzyme-immuno-assay. Total serum albumin was 30.7+2.5 g/l, 2.2+_0.6% of which was glycated. CAPDexchange fluid albumin was 0.43+0.13 g/l, with only 0.4+_0.2% glycated. We conclude That non-glycated albumin is lost preferentially over the peritoneal membrane. The results are in accordance with the-abovementioned tracer studies, showing a preferential loss from the circulation of non-glycated albumin after injection of labeled albumin.

EFFECT OF VENOUS STASIS AND DIURETICS ON FRUCTOSAMINE CONCENTRATION WITH AND WITHOUT CORRECTION BY TOTAL PROTEIN OR ALBUMIN B. Willms, G. Ahrens and E. Schumann, Fachk]inik fur Diabetes und Stoffwechse]krankheiten, Bad Lauterberg im Harz, Germany Introduction: Fructosamine is considered as measure of glycated albumin and as such a control parameter of diabetes f o r a period of 2-3 weeks. To evaluate the diagnostic relevance of the fructosamine assay we investigated i f v a r i a t i o n s in t o t a l serumprotein concentration i n f l u ence the fructosamine concentration. Methods: In 14 d i a b e t i c s we studied the e f f e c t of a venous ~ o r I0 minutes on e l e c t r o l y t e s , hematokrit, blood glucose, HbAIc, fructosamine, t o t a l protein and albumin. In a second study with II diabetics we studied the e f f e c t of 40 mg furosemide in comparison to placebo on body weight, urin production, e l e c t r o l y t e s , hematokrit, blood glucose, HbAlc, fructosamine and t o t a l p r o t e i n . Results: A f t e r venous stasis the blood glucose concentrat1-T6-6--decreased s i g n i f i c a n t l y . A s i g n i f i c a n t increase was observed of serum calcium, hematokrit, fructosamine (+ 22%), t o t a l p r o t e i n (+ 28%) and albumin (+ 27%). After correction of fructosamine to t o t a l protein or albumin resp. no s i g i f i c a n t difference was observed a f t e r venous stasis. After 40 mg fruosemide the urin production t r i p l e d , t o t a l protein and fructosamine increased s i g n i f i c a n t l y . A f t e r correction of fructosamine to t o t a l protein and albumin no s i g n i f i c a n t change was observed. Conclusion: Blood f o r fructosamine should be taken under standardised conditions. To increase the diagnostic relevance the fructosamine concentration should be corrected by the actual serum protein concentration.

A178 P655 INFLUENCE OF ORTHOSTASIS ON THE DIURNAL VARIATIONS OF FRUCTOSAMINE IN DIABETIC PATIENTS E. Schumann, G. Ahrens, J. Wehmeyer, and B. Willms, Fachk l i n i k f u r Diabetes und S t o f f w e c h s e l k r a n k h e i t e n , Bad Laut e r b e r g im Harz, Germany I n t r o d u c t i o n : Fructosamine is considered as measure of glycated albumin and as such a c o n t r o l parameter of diabetes f o r a period of 2-3 weeks. To evaluate the d i a g n o s t i c relevance of the fructosamine assay we i n v e s t i g a t e d i f v a r i a t i o n s in t o t a l serum p r o t e i n concent r a t i o n i n f l u e n c e the fructosamine value. Methods: We took d a i l y p r o f i l e s o f blood glucose, HbAlc, ~ o t e i n and fructosamine and measured the q u o t i e n t fructosamine x 7/g t o t a l p r o t e i n in I0 d i a b e t i c p a t i e n t s . At 4 a.m., when the p a t i e n t s were asleep, during the day and at I I . 0 0 p.m. when the p a t i e n t s were sleeping again. In a second study with 16 d i a b e t i c s in a d d i t i o n albumin was measured. Results: HbAIc remained constant throughout the whole studyed. Total p r o t e i n , albumin, hematokrit and f r u c t o samine increased s i g n i f i c a n t l y {max. + 15.3%) a f t e r o r t h o s t a s i s . In r e l a t i o n to t o t a l p r o t e i n or albumin the i n crease during the day was less (max. + 5.5%) and not at a l l time points s i g n i f i c a n t l y . Conclusion: The study shows t h a t the c o r r e c t i o n o f the measured fructosamine value in r e l a t i o n to t o t a l p r o t e i n or albumin decreases the d i u r n a l v a r i a t i o n s of f r u c t o s a mine, but does not completely e l i m i n a t e s i g n i f i c a n t v a r i ations of fructosamine during the day. Blood f o r f r u c t o s a mine should be taken under standardised c o n d i t i o n s (30 minutes l y i n g or s i t t i n g ) . To increase the d i a g n o s t i c r e l e vance the fructsamine c o n c e n t r a t i o n should be corrected bw the actual serum p r o t e i n c o n c e n t r a t i o n .

PS 24

ExperimentalTransplantation P656

P657

METABOLIC CONTROL AFTER CANINE ISLET TRANSPLANTATION M.P.M. van der Burg, O.R Guicherit, J.P. Scherft, H.H.P.J. Lemkes, M. Fr61ich and H.G.Gooszen. Departments of Surgery, Cell Biology and Endocrinology, University Hospital Leiden, Leiden, The Netherlands. Technically, islet transplantation as a therapeutical approach to human diabetes has become more realistic. The quality of metabolic control is however largely unknown. We studied isolated islet function after autotransplantation in dogs. In 5 dogs pancreatic islets were isolated by collagenase digestion and density gradient purification, and autotransplanted by intrasplenic venous infusion. Graft function was assessed up to 3 mo by determining the glucose and insulin response to an intravenous glucose injection (IVGTT), i.v. arginine injection during 35 mmol/L glucose clamp (AT), and a meal. The islet dose at transplantation ranged from 3500-13000 islets/kg b.w. One animal became overtly hyperglycemic within 7 days after receiving 3500 islets/kg b.w. The other grafts (>6000 islets/kg b.w.) were successful (fasting glucose < 7 mmol/L) but demonstrated, compared to preoperative values, a 50% reduced glucose clearance and insulin response at IVGTT, and a 90% reduced insulin secreting capacity at AT (P<.01). Postprandially hyperglycemia (~10 mmol/L) and in contrast to i.v. glucose and arginine, a normal insulin response was observed. In dogs "one-to-one" transplantation was successful in 4/5 recipients of isolated islets. The difference in the effect of islet transplantation on the insulin response to intravenous glucose or - arginine and a meal, may be related to the postprandial activation of the entero-insular axis.

LONG-TERM FUNCTION OF TRANSPLANTED ISLETS IN STREPTOZOT0CIN-DIABETIC RATS A.Ar'Eajab, B.Ahr~n, J.Alumets and S.Bengmark, Depts. of Surgery, Pharmacology and Pathology, Lund University, Lund, Sweden Islet transplantation improves glucose homeostasis in diabetes. To study the long-term function of transplanted islets, streptozotocin-diabetie rats were transplanted with 400 or i000 freshly isolated islets into the kidney subeapsule and studied after 3 months. Eats transplanted with i000 islets had then normoglycemia, normoinsulinemia and a normal insulin response to i.v. glucose. Furthermore, at electron microscopy, the transplanted islets looked normal. In contrast, rats transplanted with 600 islets were hyperglycemic and had an impaired insulin response to glucose, and in the transplanted islets only a few B-cells were seen. The functional innervation of the transplanted islets was studied by infusion of the ~2-adrenoceptor antagonist yohimbine. Whereas yohimbine induced hyperinsulinemia and potentiated glucose-stimulated insulin secretion in control rats, no such effect was observed in rats transplanted with i000 islets. Furthermore, the islet adaptation to insulin resistance was studied using nicotinic acid or dexamethasone. Thereby, rats transplanted with i000 islets responded with hyperinsulinemia to retain normoglycemia as did control rats. We conclude that i)i000 islets, but not 400, are necessary for restoring insulin secretion when transplanted in streptozotocin-diabetic rats, 2)transplanted islets seem functionally denervated after 3 months, but 3)they retain the normal capacity to adapt to insulin resistance.

A179

P658

P659

INTRATHECAL ALLOT~NSPLANTATION OF PANCREATIC ISLETS WITHOUT IMHUNO-

PORTAL OR P E R I P H E R A L SITE FOR TRANSPLANTATION OF D I F F E R E N T A M O U N T S OF ISLETS. L. Falqui, L. Kowalski, A. M. Donnelly, M.L. Chivetta, G. Pozza and P.E. Lacy. Scientific Institute S.Raffaele, Milano, Italy and W a s h i n g t o n University, st. Louis, Usa.

SUPPRESSIVE AGENTS Y.Hara, H.Taniguchi, K.Ishihara, A.Tsutou and K.Narutaki, Nyngo Reha bilitatioo Center, i070, Akebono-cho, Nishi-ku, Kobe, Japan. [Aim] The i s l e t transplantation without immunosuppressive agents is s t i l l unsuccessful because of rejection. We searched for the immunotolerant region for the i s l e t transplantation and examined the intrathecal space as one of such candidates. [Methods] Streptozetocin-induced diabetic Wistar rats showing fasting blood glucose over 400 mg/dl were selected for recipients. I s l e t s iso lated from Fisher rats were collected by eollagenase method, and transplanted into the intrathecal space by our lumbar puncture method. In these recipients, 0.2 g glucose/kg body weight was loaded intravenously after overnight fasting. The recipient rats were sacrificed to morphological studies as well as determination of insulin content of the spinal cord. Intact Wistar rats were subjected to the similar studies as control. [Results] Blood glucose level measured at 4 p.m. f e l l to normal range about 7 days after transplantation and this level was maintained for more than 350 days. The blood glucose and serum insulin levels after the glucose load were similar between the transplantrd and the intact rats. Insulin in i s l e t s stained by avidin biotin a f f i n i t y cytochemistry were found in the cauda equina, while extremely higher content of insulin was extracted from the eanda equina of the recipients than the control rats.

TO c o m p a r e portal versus p e r i p h e r a l site of islet t r a n s p l a n t a t i o n , two d i f f e r e n t amounts of fresh rat islets, 1000 or 2000, were transplanted either into the liver via the portal vein (PV) or u n d e r the k i d n e y capsule (KC) of s y n g e n e i c Lewis rats, made diabetic (BG >350 mg/dl) w i t h STZ. Normal rats served as controls (C), 5/group. D u r i n g a standard m i x e d meal test (6 K c a l / k g bw) all the animals showed a normal glycemic tolerance (BG
P660

P661

PROLONGATION OF ALLOGENE~C PANCREATIC ISLETS GRAFTED INTO DIABETIC BB-RATS H.J.Hahn~ I. K16ting, A. Dunger and T. Diamantsteint Central Institute of Diabetes Karlsburg (GDR) and I n s t i t u t e of Immunology, Free U n i v e r s i t y B e r l i n (FRG) Pancreatic islets or f u l l skin o b t a i n e d from LEW. 1A rats ( g e n e t i c background (gb) LEW'; MHC: RT1a ) were d e s t r o y e d i n 4 3 . 6 • 2 . 0 d, and 12.6 • 3.3, r e s p e c t i v e l y , when g r a f t e d into BB-rats (gb: WISTAR-BB~ MHC: RTI~-BB). To clarify the r o l e o f gb a n d / o r MHC we s e l e c t e d LEW. 1BB (gb: LEW ! MHC: RTlu-BB) and BB.1A (gb: WISTAR-B~, MHC: RT1~) as donors and g r a f t e d p a n c r e a t i c i s l e t s and s k i n i n t o B B - r a t s . LEW. 1BB s k i n g r a f t s were r e j e c t e d i n > 44.5 • 4.8 d (n = 10), when g r a f t e d into BB r a t s . In c o n t r a s t BB r a t s g r a f t e d w i t h LEW. 1BB i s l e t s r e l a p s e d i n t o h y p e r g l y c a e m i a i n 24.3 • 6 . 4 d (n = 6) indicating the participation of autoaggression in d e s t r u c t i o n . Full skin grafts o b t a i n e d f r o m B ~ . I A r a t s were r e j e c t e d i n 14.3 • 2.3 d (n = 10), islet grafts were d e s t r o y e d within 13.4 • 2 . 8 d (n = 5) by t h e BB r a t s . Treatment o f BB r a t s , which had been gra~ted w i t h LEW. 1BB i s l e t s , w i t h an a n t i - I L - 2 R mab i n c o m b i n a t i o n w i t h low doses o f C y c l o s p o r i n A f o r 10 d resulted in a marked islet graft prolongation (> 60 d; n = 7), whereas an identical treatment resulted in invariable graft s u r v i v a l i n BB r a t s , which had been t r a n s p l a n t e d w i t h BB.1A i s l e t s . The r e s u l t s u n d e r l i n e f i r s t l y that the genetic background o f BB r a t s is i m p o r t a n t f o r autoimmune B - c e l l d e s t r u c t i o n and s e c o n d l y t h a t t h e s e l e c t e d f o r m o f immunotherapy is able to prevent the islets destruction induced or facilitated by the genetic background.

15-DEOXYSPERGUALIN PROLONGS ISLET ALLO- AND XENOGRAFT SURVIVAL IN MICE J-O. Sandberg, O. Korsgren, C-G. Groth and A. Andersson. Department of Cell Biology, Uppsala University, Uppsala and Department of Surgery, Karolinska Institute, Stockholm, Sweden. The immunosuppressant 15-deoxyspergualin (DSG) was evaluated with regard to its efficacy in allogeneic and xenogeneic islet transplantation. AIIoxan-diabetic C57BL/Ks mice were transplantated under the renal capsule with 500 collagenase-isolated C57BL/6 mouse islets. DSG (5mg/kg) was injected once daily for 14 days and then every second day until day 28. Six of eight animals became normoglycemic one week post-transplantation and four mice were still normoglycemic on day 70. All eight controls became hyperglycemic within four weeks. Light microscopy showed graft rejection in hyperglycemic mice but only scarce infiltration of lymphocytes in the grafts of normoglycemic animals. A total of 34 C57BL/Ks mice were transplantated with porcine islet-like cell clusters (ICC). The mice were killed after 4,7,14 and 32 days. Light microscopical evaluation by morphological scoring showed that DSG treatment (2.5 mg/kg; daily injections) prolongated the ICC graft survival with the greatest difference between DSG treated animals and controls appearing after 14 days (p<0,01; rank sum test). After 32 days there was no difference between DSG treated mice and controls. Thus, DSG is a potent immunosuppressive agent demonstrated in this study by the prolongation of survival of allogeneic islets grafts and delayed rejection of xenogeneic porcine ICC grafts.

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P662 FACTORS INFLUENCING THE EFFICACY OF A N T I - I L - 2 RECEPTOR THERAPY IN PREVENTING AUTOIMMUNITY IN DR-RATS B. Kuttler, L. V o g t , I. K l O t i n g , S. L u c k e , A. D u n g e r , H.D. V o l k , T. D i a m a n t s t e i n a n d H.J. H a h n C e n t r a l I n s t i t u t e of D i a b e t e s " G e r h a r d t K a t s c h " , Karisburg, GDR A temporary anti-IL-2 receptor targeted therapy prevents autoimmune destruction of syngeneic islets when grafted into diabetic BB-rats. In further investigations 2000 syngeneic islets were grafted subrenal into diabetic BB/OK recipients (n = 40) t r e a t e d f o r I0 d with 5 different anti-IL-2R mAb-batches (1.0 mg ART 18/kg b.w.) a n d C y c l o s p o r i n A (1.5 m g / k g b . w . ) . Plasma glucose was monitored to evaluate the metabolic effect. Altogether we observed 9 therapy f a i l u r e s (22.5 %) and asked which § may i n f l u e n c e t h e m e t a b o l i c success (normoglycaemia f o r 120 d ) . Among t h e ART 18b a t c h e s we found 3 which were v e r y e f f e c t i v e i n 5/5, 6/6 and 6 / 7 t r e a t e d r a t s whereas 2 were less effective ( m e t a b o l i c success o n l y i n 4/8 and 7/10 t r e a t e d r a t s ) . A retrospective a n a l y s i s of t h e t r e a t e d R B - r a t s revealed no correlation between sex, age at diabetes onset, diabetes duration and metabolic outcome. The 40 B B / O K r e c i p i e n t s came from 7 generations and 9 families. Surprisingly, 75 % of the therapy failures belonged to the same generation (FIG). M o r e o v e r , 5 0 % of t h e t h e r a p y failures belonged to one DR~OK § Just this family was characterized by a low diabetes incidence (< 3 0 %, p < 0.05). The availability of effective mAb preparations and the careful analysis of the recipients case history including family diabetes incidence may help to predict the metabolic outcome to individualize the immunotherapy in D R - r a t s .

P664 INCREASED CLEARANCE IN ISLET TRANSPLANTED DOGS CONTRIBUTES TO SUB-NORMAL INSULINEMIA D.W. O'Brien, G.D. Molnar, D.C. O'Brien and R.V. Rajotte. University of Alberta, Edmonton, Canada. Is hepatic insulin (IRI) clearance changed after islet autotransplantation and does it affect peripheral insulinemia? Normal (N) and transplanted (T) dogs had flow probes on the portal vein (PV) and hepatic artery, sampling catheters in the PV, carotid artery, hepatic and external jugular veins. Fasting normoglycemic conscious dogs had IRI and glucose (G) fluxes measured at two Biostator-coutrolled glucose damps (5.5 and 11 mM) and arginine (A) 5 g bolus plus infusion (0.33 g/min). Islet responsiveness (IRI pM/G mM/kg) and hepatic IRI clearance (ml/min) were compared by two-way ANOVA (N versus T and G5.5 versus Gll). Regardless of the level of the clamp, or the presence of arginine in N compared to T, IRI hepatic clearance was increased (175 _+ 7.8 to 245 _+ 11.9, p < 0.001) and islet secretory responsiveness reduced (0.106 _+ 0.029 to 0.026 + 0.004, p<0.05). Peripheral IRI was lower (p < 0.05) in T than N at each clamp level with or without A. Despite fasting normoglycemia IRI responses in T dogs to glucose and arginine are impaired. This impairment is worsened in the periphery due to increased hepatic IRI clearance.

P663 IS

SLUCOSE T O X I C FOR

IMPLANTED

PANCREATIC

ISLETS?

A. D u n g e r , C. K a u e r t and H.d. Hahn, Central Institute of Diabetes "Gerhardt Katsch", K a r l s b u r g , G.D.R. Hyperglycemic s t r e s s is reported to elecit an a d v e r s e e f f e c t on t h e e n d o c r i n e pancreas. Since e x p e r i m e n t a l s u p p o r t f o r t h i s concept i s mainly based on i n v i t r o f i n d i n g s t h e aim o f t h i s s t u d y was t o i n v e s t i g a t e t h e phenomenon o f "glucose toxicity" in vivo. Therefore, Streptozotocin diabetic LEW.1W r a t s (plasma g l u c o s e > 25 mmol/l) were g r a f t e d w i t h 250 syngeneic i s l e t s . Based on t h e i n s u l i n c o n t e n t the number o f islets was i n s u f f i c i e n t t o r e v e r t d i a b e t e s . The r e c i p i e n t s were m o n i t o r e d f o r body w e i g h t and plasma g l u c o s e up t o 120 days. No i n s u l i n t h e r a p y was p r o v i d e d . D e s p i t e t h e d i a b e t i c s t a t e all a n i m a l s were c h a r a c t e r i z e d by a s i g n i f i c a n t increase in body weight. U n e x p e c t e d l y , two third o f t h e r e c i p i e n t s became normoglycemic i n 46 • 9 days a f t e r islet implantation. Their g l u c o s e t o l e r a n c e was w i t h i n t h e normal range. The removal of the i s l e t g r a f t resulted in a recurrence of the hyperglycemia. Independent o f t h e r e c i p i e n t s blood g l u c o s e l e v e l t h e r e was a significant increase of the graft insulin c o n t e n t compared t o t h a t a t t r a n s p l a n t a t i o n . The results c l e a r l y d e m o n s t r a t e t h a t a long l a s t i n g and s e v e r e h y p e r g l y c e m i a has no injurious effects on i m p l a n t e d i s l e t s . On t h e contrary, they r a t h e r s u p p o r t t h e v i e w o f an i s l e t growth and/or function promoting e f f e c t of high glucose.

P665 CHANGES IN CD4+/CD8 + RATIO OF PERIPHERAL LYMPHOCYTES: A POSSIBLE MARKER OF DESTRUCTION OF TRANSPLANTED ISLETS P.Djordjevi6,S.Brki6~N.Lalid,M.Zamaklar~M.Draga~evi6 and Lj.Igrutinovi6, Imstitute for Endocrinology~ and City Clinical Center Zvezdara, Belgrade~ Yugoslavia In five patients with Type 1 (insulin-dependent) diabetes, after transplantation of human fetal islets~ we analysed the ratio between CD4 and CD8-positive lymphooytes in peripheral blood, in order to clarify initial mechanims of destruction of transplanted islets. Human fetal islets were isolated using eollagenase digestion~ cultured 2-4 weeks (37 C,5xo COp) and • under fasela of rectus abdominis musele.-Funotional capacity of transplanted islets was evaluated by C-peptide plasma level determinations before and after glucagon stimulation (Img iv,0 vs 6 minutes). Phenotypes of peripheral blood lymphooytes were+ determined by immunofluoreseence. We found that CD4+/ CD8 was increased on day 40 after transplantatmon (I.70 + § . . - 0.12) compared to day+-1(1.32-0.14). The ratlo rema• constant on day 90(1.74-0.19) and rapidly declined on day + . 120(I.J?-0.09). Simultaneously we detected and increase in functional capacity of transplanted islets (day - I: <0.09/ <0.09 nmol/l:day 30: 0.24~0.07/0.31~0.09nmoi/i) and then declined by the end of the fourth month (day 120: 0.16~0.05/0.19~0.06nmol/l,p <0.05)9 which corresponded to the observed decrease in Cf4~f~ ra%mo.0ur results slgnmfy that slight increases in CD4 /CD8 ratio might reflect an early activation of the islet destruction mechanisms and the decrease of the ratio might be a marker of the ongoing destruction of transplanted islets presumably due to the prevalence of CD8 + effector cells. . .

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A181

P666 ACTIVATION OF COMPLEMENT BY ALGINATEPOLYLYSINE MICROCAPSULES S. Darquy, M.E. Pueyo, F. Capron and G. Reach. Service de Diabftologie, H6tel-Dieu Hospital, 1, Place du Parvis Notre Dame, 75004 Paris, France. Although microencapsulated islets of Langerhans are able to correct experimental diabetes, the biocompatibility of the material used for microencapsulation (alginate-polylysine) is questionable since the occnrence of a fibrotic reaction surrounding microcapsules implanted in the peritoneal cavity of rats and mice has been observed. Complement activation, and generation of anaohylatoxins, such as C3a, is supposed to be involved in the biocompatibility of foreign surfaces. This work was undertaken to assess complement activation in human serum incubated with empty sterile alginate-polylysine microcapsules, in parallel in veronal buffer solution, or in EDTA buffer, in which complement activation is not possible. Complement activation was determined by the measurement of CH50 (i.e. the volume of serum required to induce half of the maximal lysis of sheep erythrocytes sensitized with anti-erythrocytes antibodies), and by C3a determination (RIA). When the serum was incubated with increasing amounts of microcapsules, a decrease in CH50 was observed, which was more pronounced in VBS than in EDTA buffer (for 100 microcapsules, 52 + 26 vs 85 + 16 % of CH50 determined in the absence of microcapsules, n=3) suggesting that complement activation occured in addition to adsorption. Moreover, serum C3a concentration was higher after incubation in VBS than in EDTA, for all microcapsule amounts tested. This study represents a first attempt to investigate the mechanisms of fibrosis generation around the microcapsules.

P668 IN VITRO EVALUATION OF ACTIVATION OF PERITONEAL CELLS BY ALGINATE-POLYLYSINE-ALGINATE CAPSULES D. Chioheportiehe, M. Waterfall, G. Reach and J.D. B a i r d , M e t a b o l i c Unit, U n i v e r s i t y of Edinburgh Department of Medicine, Western General Hospital, Edinburgh, U.K. The alginate-polylysine membrane used for microencapsulation of islet grafts has been shown to i n d u c e an i n f l a m m a t o r y r e s p o n s e w h e n the peritoneal cavity is used as the site for implantation. This could be more important in animals with spontaneous auto-immune, insulindependent diabetes. The aim was to develop an in vitro system for assessing activation of peritoneal c e i l s by c a p s u l e m e m b r a n e s . Peritoneal cells were recovered from non-BB W i s t a r , d i a b e t e s r e s i s t a n t (DR) BB/E, and diabetes prone (DP) BB/E rats which had been implanted with empty alginabe-polylysine microcapsules 14 days previously and incubated with microencapsulated rat islets. The effect of the peritoneal cells was assayed by determining release of insulin by the microencapsulated islets. After 5 days incubation the insulin release decreased by 66%, 39%, 83% respectively, whereas islets cultured without cells showed a 35% increase in insulin secretion. Decreased s e c r e t i o n of i n s u l i n was also f o u n d w h e n peritoneal cells removed from non-BB Wistar, DR BB/E, and DP B B / E a n i m a l s not p r e v i o u s l y implanted with empty capsules were incubated with miercencapsulated islets (48%, 36% and 32% respectively). These results cast doubt on the feasibility of using intraperitoneal alginatepclylysine microencapsulated islet grafts as a t r e a t m e n t for i n s u l i n - d e p e n d e n t (Type I) diabetes.

P667 IN VIVO EVALUATION OF INFLAMMATORY RESPONSES TO INTRA-PERITONEAL IMPLANTATION OF POLY-L-LYSINEALGINATE MICROCAPSULES M. Waterfall, D. Cole, D . Chicheportiche, M. M c I a t y r e and J.D. Baird, M e t a b o l i c Unit, University of Edinburgh Department of Medicine, Western General Hospital, Edinburgh, U.K. It has been suggested that the transplantation of pancreatic islets within a biocompatible membrane could circumvent the problems of graft rejection and autoimmune destruction in insulin-dependent (Type I) diabetes. In this study we have icoked at the inflammatory response to poly-L-lysinealginate capsules in vivo. Empty capsules, believed to be biocompatible, were implanted into the peritoneal cavity of non-diabetic, non-BB Wistar (18), diabetes resistant (DR) BB/E (11), diabetes prone (DP) BB/E (25 diabetic, 5 nond i a b e t i c ) and s t r e p t o z o t o c i n - i n d u c e d (STZ) diabetic Wistar (9) rats. Capsules recovered at 14 days post-implantation were sectioned and stained with Haematoxylin and Eosin. In all groups an inflammatory response was associated with damaged capsules. By contrast, only intact capsules recovered from BB/E rats exhibited signs of a major inflammation response DR-BB/E (5/11) and DP-BB/E (19/25 diabetic, 5/5 non-diabetic) whilst those from STZ (I/8) and non-diabetic Wistar (0/18) rats were associated with minimal response. This result taken together with reports that eytokines, produced during inflammatory responses, have been demonstrated in vitro tc be toxic for islet cell function, suggest that the use of such capsules for the protection of intraperitoneal islet grafts may not be successful as a treatment for spontaneous, autoimmune, insulindependent diabetes mellitus.

A182

PS 25 Immunology in Man P669

P670

DOES CONTINUOUS SUBCUTANEOUS INSULIN INFUSION (CSII) INCREASE INSULIN ANTIBODY PRODUCTION?

Proinsulin autoantibodies(PAA) are closer associated with Islet cell antibodies (ICA) and predlabetes than Insulin autoantibodies (IAA)

G. Kerum, Z. Metelko, M. Granid and Z. Skrabalo, Vuk Vrhovac Institute for Diabetes, Zagreb, Yugoslavia

BOhmer, Kirsten 1, Keilacker Hubert 2, Hiibinger, Achim 1, Gries, Arnold I, Michaelis, Dietrich ~, Kolb, Hubert 1, Kuglin, Bernd 1 1Diabetes Research Institute, Diisseldorf,2Zentralinstitut far Diabetes, Karlsburg

Some previous studies indicated increased insulin antibody production during the CSII treatment using highly purified porcine insulins in contrast to conventional therapy. The aim of the study was to evaluate the immunogenic effect of human insulin during CSII treatment. 20 Type i (insulin-dependent) diabetic subjects (IS females and 7 males, mean age: 36.1• years, 103• of standard body weight) were switched from various insulin preparations to human insulins (Actrapid HM, Monotard HM, 40U/ml, NOVO). After a S-month run-in period on conventional insulin therapy, patients were treated with CSII (DISETRON!C -HOECHST MRS i INFUSORS) using human insulin (Actrapid HM, lOOU/ml, NOVO) during the following 6 months, insulin antibody concentration was assessed by the determination of total, antibody-bound and free insulin according to Gennaro and Van Norman. Metabolic control improved significantly during CSII (HbAI fell from 7.5• to 6.0• p
PAA have initially been described by us using a competitive ELISA for detection. We have now used a fluid phase assay (RIA) to study the high affinity subset of PAA and to compare its prevalence and disease association with that of RIA determined IAA. For better comparison, aequimolar concentrations of radiolabelled proinsulln and insulin have been used and competition was performed with both peptides. Sera of 18 prediabetic subjects (PRE), mean of 2.5 yr prior to diabetes manifestation, and 18 patients with newly diagnosed type I diabetes (PAT) have been evaluated. IAA were present in 5/18=28% PRE and 1/18=6% PAT (mean+ 4 SD of 99 normal controls), respectively in 6/18=33% PRE and 4/18=22% PAT with 3 SD cutoff. In comparison 6/18=33% (11/18=61%) PRE and 3/18=17% (10/18=55%) PAT bound proinsulin above 4 SD (3 SD) of normals. At 3 SD the prevalence of PAA was significantly (p<0.05) higher than that of IAA. PAA were significantly (p<0.002) associated with ICA: ICA were present in 20/21 (95%) PAA p~ but only in 6/15(40%) PAAneg(3 SD), and 9/9(100%) PAAP ~ vs. 17/27(63%) PAA neg (4SD) probands. About 80% (29/36) of total sera reacted concordant for PAA and ICA (vs 53% for IAA and ICA, p< 0.025). These results suggest that high affinity PAA detect more prediabetics than IAA. The use of the precursor molecule proinsulin as target for autoantibodies against beta cell products may increase sensitivity in the prediction of Type 1 diabetes.

P671

P672

A U T O A N T I B O D I E S TO I N S U L I N (IAA) A R E E Q U A L L Y POTENT MARKERS FOR TYPE I (INSULIN-DEPENDENT) D I A B E T E S AS T H O S E TO P R Q I N S U L I N (PAA) H. K e i l a c k e r , B. K u g l i n ~, I. R j a s a n o w s k i , K.P. W o l t a n s k i , K.D. Kohnert, M. Z i e g l e r e n t r a l I n s t i t u t e of D i a b e t e s K a r l s b u r g , GDR, Diabetes Research Institute D~sseldorf, FRG

ISLET CELL SURFACE ANTIBODY D E T E C T E D ON HUMAN ISLET CELLS BY FLOW C Y T O M E T R Y M. Peakman, M.J. Hussain, R.D.G. Leslie and D. Vergani, Dept. Immunology, King's College School of Medicine, London. Islet cell surface antibody (ICSA) is a p o t e n t i a l l y p a t h o g e n i c a u t o a n t i b o d y in Type 1 diabetes but its role remains p o o r l y defined for both technical reasons and the use of nonhuman substrate in its detection. We have d e v e l o p e d a novel technique using single cell suspensions prepared from human islets to detect ICSA by indirect i m m u n o f l u o r e s c e n c e and flow cytometry. Islets prepared by c o l l a g e n a s e d i g e s t i o n are separated with trypsin and EDTA into a cell suspension and cultured overnight. Islet cells (viability 70-95%) are then incubated with serum (diluted 1/10), w a s h e d and ICSA revealed using FITC rabbit a n t i - h u m a n IgG. Using a FACSCAN, islet cells are identified by autofluorescence (FLI) and light scatter and dead cells excluded using p r o p i d i u m iodide detected on the FL3 channel. The level of green f l u o r e s c e n c e m e a s u r e d is p r o p o r t i o n a l to the degree of autoantibody binding. Median fluorescence levels in 34 r e c e n t l y - d i a g n o s e d patients with type I diabetes were h i g h e r than in controls (p<0.05). This technique offers a new, q u a n t i t a t i v e m e t h o d for the d e t e c t i o n of ICSA, which may have a role in beta cell damage.

~

A u t o a n t i b o d i e s a g a i n s t i n s u l i n have b e e n f o u n d in i n s u l i n - n a i v e Type I ( i n s u l i n - d e p e n d e n t ) d i a b e t i c p a t i e n t s (IDDM) as w e l l as in f i r s t d e g r e e r e l a t i v e s of d i a b e t i c p a t i e n t s (FDR). ~o test the h y p o t h e s i s that p r o i n s u l i n r a t h e r than i n s u l i n w o u l d be the a u t o a n t i g e n f o r these a n tibodies, we m e a s u r e d the f r a c t i o n of b o u n d ~ I - l a b e l e d insulin, C - p s p t i d e a n d p r o i n s u l i n w h i c h can be d i s p l a c e d by the a d d i t i o n of excess c o l d i n s u l i n a n d p r o i n s u l i n , r e s p e c t i v e ly. Sixty five n e w - o n s e t IDDM, 7 8 F D R a n d 89 healthy controls were investigated. Binding v a l u e s e x c e e d i n g m e a n + 3 SD of c o n t r o l group values were considered antibody-positive. IAA w e r e found in 2 5 % (16/64) of I D D M a n d in 1.3% (I/78) of FDR, C - p e p t i d e s u t o a n t i b o d i e s (CAA) in 1.6% (I/62) of I D D M a n d in 1.9% (I/53) of FDR; P A A in 2 2 % (14/65) of I D D M a n d in 1.5% (I/68) of FDR. N o n e of the c o n t r o l s w e r e p o s i t i v e f o r e i t h e r type of a u t o a n t i b o d y . There w a s a s t r o n g c o r r e l a t i o n (r=0.89, s l o p e = 1 . 0 1 ) b e t w e e n i n s u l i n a n d p r o i n s u l i n b i n d i n g data in d i a b e t i c p a t i e n t s but not in the two o t h e r g r o u p s (r<0.1). In conclusion, d e s p i t e the o c c a s i o n a l o c c u r r e n c e of CAA, IAA a n d P A A are s t r i k t l y r e l a t e d to e a c h other. Thus, P A A do not r e p r e s e n t a m o r e s e n s i t i v e m a r k e r f o r Type I ( i n s u l i n - d e p e n d e n t ) d i a b e t e s than IAA.

A183

P673

P674

INSULIN AUTOANTIBODIES: AN ADDITIONAL IMMUNOPATHOLOGIC EXPRESSION IN AUTOIMMUNEDISEASES OF THE THYROID M. Modan, Z. Ish-Shalom, D. Modan, D. Barzilai and P.Vardi Chaim Sheba Medical Center, Tel Hashomer, Tel Aviv; Rambam Medical Center, Haifa, ISRAEL. In this study we evaluated the presence of i n s u l i n autoantibodies (INSAAB) in immune and non immune diseases of the t h y r o i d , u t i l i z i n g a q u a n t i t a t i v e f l u i d phase competitive radiobinding assay. We studied 37 patients with Graves' disease, 44 patients with Hashimoto, 11 patients with non immune thyroid (NIT) diseases and 30 normal controls. In 5/37 (13.5%), 7/44 (15.9%) patients with Graves' and Hashimoto disease INSAAB levels exceeded our upper l i m i t of normal range (50nU/ml) respectively. Positive INSAAI levels ranged between 50-150 nU/ml. In contrast to INSAAB levels in type I diabetics, f l u c t u a t i o n of INSAAB levels was found. None of the NIT and control groups were INSAAB p o s i t i v e . No association was found between p r o p y l t h i o u r a c i l treatment and levels of INSAAB. Early phase i n s u l i n secretion of the intravenous glucose tolerance test was under the f i r s t percentile in 2/10 (20%) INSAAB p o s i t i v e patients. In conclusion: this study indicates t h a t in 15% of patients w}th immune thyroid diseases, INSAAB are detected but t h e i r levels seem to f l u c t u a t e . Whether 'such phenomenon r e f l e c t s associated immune disregulation in autoimmune thyroid diseases, causing d i f f e r e n t degree of beta cell damage, has to be elucidated.

DO ISLET CELL AND INSULIN A U T O A N T I B O D I E S R E F L E C T D I F F E R E N T PATTERNS OF ISLET A U T O I M M U N I T Y ? E. Bosi, S. Genovese, M. Caruso, A. Sergi, M.R. Pastore, F. Meschi, N. Dozio, G. Chiumello and G. Pozza. San R a f f a e l e Institute, Milan, Italy Islet cell {ICA) and insulin (IAA) autoantibodies are both p r e d i c t i v e markers of type-i diabetes. Aim of this study was to evaluate cross-sectionally the p r e v a l e n c e and q u a n t i t a t e ICA and IAA in first degree relatives of type-i diabetic patients p a r t i c i p a t i n g to a prospective family study. 106 families for a total of 342 non-diabetic individuals were screened. ICA were detected by standard immunofluorescence on f r o z e n unfixed human pancreas and then q u a n t i t a t e d in JDF-units; IAA were m e a s u r e d by a competitive ELISA corrected for n o n - s p e c i f i c b i n d i n g by insulin excess. The d e t e c t i o n limits for the assays w e r e 5 JDF-units for ICA and 0.371 d i f f e r e n t i a l O.D. for IAA (95% C.I. of 100 normal controls). Overall, ICA were detected in 13 (3.8%) n o n - d i a b e t i c relatives (27.2• 5-73 JDF-units): I0 were males (4 fathers, 5 brothers, 1 son) and 3 females (all mothers) (p=0.053); ICA e x c e e d e d 10 JDF-units in 7 relatives (2.0%). IAA were d e t e c t e d in 13 (3.8%) non-diabetic relatives (2 fathers, 7 mothers, 2 brothers, 2 sisters), w i t h a slight female preponderance. None had ICA and IAA associated. These findings indicate that ICA and IAA occur at comparable frequencies in u n a f f e c t e d m e m b e r s of diabetic families, but are differently d i s t r i b u t e d and seldom associated, p o s s i b l y r e f l e c t i n g d i f f e r e n t patterns of islet cell autoimmunization.

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AGE-RELATED CHANGES OF ACUTE INSULIN RESPONSE TO IV GLUCOSE (AIRG) IN SUBJECTS AT RISK FOR TYPE 1 DIABETES AND PROGRESSION TO DIABETES IN ICA+ IAA+ SUBJEClX3. M.G. Joseph, S. Bardet, H. Stetieh, M. Marre, H. Allannic, A. Antonelli, V. Rohmer, B. Charbonnel and P. Sai'. N a n t e s ,

F A S T I N G C - P E P T I D E AT O N S E T OF TYPE 1 D I A B E T E S AS A P R E D I C T O R OF R E M I S S I O N A N D C - P E P T I D E L E V E L S A F T E R 5 YEARS. O. Snorgaard, L.H. L a s s e n and C. Binder. Steno M e m o r i a l Hospital, DK-2820 Gentofte, Denmark.

Angers, R e n n e s - F R A N C E .

Three IVGTTs (0.5 g/kg ; 2 min) were performed in 310 subjects at risk for type 1 diabetes classified in "pubertal" subgroups : ~ p r e - p u b e r t a l , ( ~ ) T a n n e r ' s stages 2-3, 9 stages 4-5, D~ adults. ICA (international standards) and IAA (RIA) were tested at least twice. A group I of 272 ICA- IAA- first degree relatives (FDR) (3-48 years) ( ~ : n=50, ~ ) : n=20, ~ : n = 8 5 , ( ~ : n=l16) was compared to a group II (5-43 y e a r s ) ~ ) : n=6,~: n=2,~: n=7,~: n=23) including 27 ICA + IAA-, 5 ICA + IAA +, and 5 ICAIAA + subjects. With 30 FDR and 7 subjects with previous transient hyperglycaemia. In group I : IRI 1+3 rain was higher (p1.5 years) (particularly in transient hyperglycaemic subjects), or very late. III) Some subjects display persistently low AIRGs without progression to diabetes within 2 years.

P l a s m a C - p e p t i d e was f o l l o w e d p r o s p e c t i v e l y from o n s e t of Type 1 d i a b e t e s and d u r i n g the subsequent 7 . 4 ( 6 . 0 - 9 . 0 ) y e a r s (median and quartiles) in 204 subjects. The initial d i a g n o s i s was b a s e d on clinical criteria and r e - e v a l u a t e d at 5 years. F a s t i n g C - p e p t i d e was m e a s u r e d at onset, i, 3, 6, 9, 12 and then every 6 m o n t h s until 106(104-135) weeks. Thereafter postprandial C-peptide was measured. F a s t i n g C - p e p t i d e was 0 . 1 7 ( 0 . 1 1 - 0 . 2 5 ) nmol.l -I at onset f o l l o w e d by an annual i n c r e a s e rate of 0 . 1 6 ( 0 . 0 6 - 0 . 4 8 ) n m o i . i "I to a p e a k of 0 . 2 8 ( 0 . 2 3 - 0 . 3 4 ) n m o i . 1 "I a f t e r 2 5 ( 1 2 - 3 9 ) w e e k s . The s u b s e q u e n t annual d e c l i n e rate of fasting Cpeptide was 0.08(0.05-0.12) nmol.l "I and of postprandial C-peptide 0 . 0 3 ( 0 . 0 2 - 0 . 0 6 ) n m o i . I "I. N o n e of t h e s e p a r a m e t e r s s h o w e d bimodality. At a critical level of fasting C - p e p t i d e b e t w e e n 0.14 (0.09-0.18) and 0.20 (0.16-0.26)nmoi.i "I a m o r e than 50% i n c r e a s e of i n s u l i n d o s e was seen. 74% had lost d e t e c t a b l e C-peptide (<0.1nmol.l "I) at 5 years and 88% failed to a c h i e v e i n s u l i n free remission. However, if f a s t i n g C - p e p t i d e was b e l o w 0.18 nmol.l "I at onset, 86% lost C - p e p t i d e and 95% failed to a c h i e v e r e m i s s i o n a g a i n s t 62% and 81% if C - p e p t i d e ~ 0 . 1 8 n m o l . l 1 ; p < O . 0 0 1 and p<0.01. Conclusion: These r e s u l t s speak against a m a j o r h e t e r o g e n e i t y in the d e c l i n e of B-cell function in Type 1 d i a b e t e s and s u g g e s t some v a l u e of f a s t i n g C - p e p t i d e at onset as a p r e d i c t o r of r e m i s s i o n and future C - p e p t i d e levels.

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P678

FIRST PHASE INSULIN RESPONSE IN RELATION TO HLA AND ICA IN 216 RELATIVES OF DIABETIC PATIENTS. J.J. Robert, C. Boitard, B. Rakotoambinina, E. Gardais and I.Deschamps. Service Endocrinologie et Diab~te de I'Enfant, INSERM U30 AND INSERM U25, H6pital Necker, Paris. In order to test the reliability of the first phase insulin response for predicting development of Type 1 diabetes, we determined peak plasma insulin (PPI) values (1+3 min. after 0.5 g/kg glucose i.v.), islet-cell antibodies (ICA) and HLA-A,B,DR in 216 children or young adults (190 siblings, 26 offspring of diabetic patients) aged 3 to 29 years. PPIs correlated significantly with age in children: m (log transformed) = 53.7+/-1.95, 87.1+/-1.82 and 114.8+/-2.04 mU/L for age groups 0-5, 6-10 and 11-15 yrs, respectively (p<0.01). Values <30mU/L were not uncommon, namely in young children, without any associated anomaly. PPIs were not related to HLA, but significantly related to ICA status: 69.8+/-14.8 and 101.3+/-19.2 mU/L in ICA+ subjects <10 or >10 yrs old, respectively, versus 94.9+/-6.8 and 138.0+/-8.4 mU/L in ICA- subjects (p<0.05 and <0.01, respectively). Reproducibility of subsequent tests in 75 subjects (r=0.70, p<0.01) proved inadequate for interpretation of individual variations. PPIs were usually higher on the 2nd test, except in ICA+ subjects in whom they remained unchanged or declined. During follow-up, 4 of 9 ICA+ children developed diabetes. PPIs dropped acutely in 2 and remained stable borderline for several years in the 2 others, but none showed a progressive decline allowing the prediction of onset of diabetes.

SEX, AGE AND ICA INFLUENCE ON RESIDUAL BETACELL FUNCTION IN TYPE I (INSULIN-DEFENDENT) DIABETES MELLITUS. E.Monta~a, M . F . C a s t a ~ e r , P.Rosel~ J . F . A l v a r e z J.Gdmez, C . V i l l a b o n a and J.Soler. Endocrine Unit, Hospital Bellvitge, Barcelona, Spain. The aim of the study was to a n a l y s e the influence of sex, age and ICA on beta-cell function and metabolic control. We studied p r o s p e c t i v e l y 40 consecutive type I (insulind e p e n d e n t ) diabetic patients during the first year after diagnosis. The age of the patients was 16.3• years (X• (range 7-28); 62.3% were males. Basal and glucagon s t i m u l a t e d C - p e p t i d e were d e t e r m i n e d at 0, 3, 6 and 12 months. ICA values (indirect immunofluorescence) were expressed in JDF units. A m u l t i v a r i a t e analysis of variance for repeated measures over time was u s e d (MANOVA test) and sex, age and ICA were considered as the independent variables. W o m e n had a higher C-peptide secretion than men over time (p= 0.027), and adult p a t i e n t s (~ 18 years) h a d a better m e t a b o l i c control (HbA I ) than younger patients (p= 0.006). Differences between ICA + (75~) and ICA" patients were not significant, but ICA + patients w i t h a moderate or high ICA value (>I0 JDF units) (60~) had a lower C-peptide secretion (p= 0.067) and a poorer m e t a b o l i c control (p= 0.056) than ICAand ICA + patients with a weak ICA value (~I0 JDF units). We conclude that sex, age and ICA values influence the evolution of residual beta-cell function a n d m e t a b o l i c control over the first year after type I (insulind e p e n d e n t ) diabetes mellitus diagnosis.

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C-PEPTIDE A F T E R DIAGNOSIS IN R E L A T I O N T O ISLET C E L L ANTIBODIES A T DIAGNOSIS OF DIABETES A.Gottsater, M. Landin-Olsson, K-O Nilssun, A.Lernmark, G.

THE USE OF GLIPIZIDE FOR REMISSION INDUCTION IN N E W ONSET TYPE I DIABETES. M. A. Charles, J.L. Selam, E. Chan, L. Woertz, M. Robinson, and Irvine, California, USA. Immunosuppression for the induction of remissions in new onset diabetes is efficacious but concern relates to side effects. Safer methods for remission induction could be useful by permitting lower dose immunosuppression. In a randomized controlled o p e n trial, we have evaluated 27 type I patients (<90 days). T y p e I diabetes was c o n f i r m e d by I C A positivity, ketoacidosis or non-glucagon r e s p o n s i v e C-peptide. Patients w e r e t r e a t e d with i m o n t h of intensive subcutaneous insulin and then insulin was discontinued until fasting blood glucose levels exceeded 140 mg/dl. The experimental group was treated with glipizide after day 30 of intensive insulin; the control group received no treatment. Remissions (no insulin r e q u i r e m e n t to maintain normal fasting blood glucose for >i month) o c c u r r e d in 3/14 (24%) controls a n d 7/13 (54%) experimental patients (p<.05). Remission duration was similar in both groups (8-10 months respectively). At six months, remission rates w e r e identical at 20%, Patients entering remissions had lower mean fasting blood glucose levels (95_+2 mg/dl) than non-remitters (105+2 mg/dl p<.05). Basal and stimulated p e p t i d e levels were similar in both groups. The~e data indicate that the a) level of blood g l u c o s e control m a y be important for remission induction, b) efficacy of glipizide appears unrelated to circulating C-peptide levels, suggesting an effect by immunomodulation or improved insulin sensitivity, a n d c) glipizide can safely and easily be used to induce remissions in new onset t y p e I diabetes.

Sundkvist.Departments of Medicine and Pediatrics, Department of Medicine, University of Lund, Malmo General Hospital, Malmo and Department of Medicine, University of Washington, Seattle, USA Fasting C-peptide (fCP) after diagnosis of diabetes was related to islet cell antibodies (ICA) and insulin treatment at diagnosis in 216 consecutive patients (age 3-88 years, mean 56). fCP was unchanged during the first two years after diagnosis though higher (p<0.001) in initially non-insulin (n=175) than insulin-treated (n=41) patients (at diagnosis: 0.36_+0.05 nmol/l and 0.85• nmol/l; two years after: 0.31_+0.07 nmol/l and 0.88_+0.07 nmol/l). Among initially non-insulin treated patients, 17/175 (10 %) were ICA positive with lower (p<0.001) fCP than ICA negative (n=158) patients (at diagnosis: 0.30_+0.04 nmol/1 and 0.90:L0.04 nmol/; two years after: 0.32i0.15 nmol/1 and 0.93_+0.07 nmol/l).Though fCP was unchanged 15 of 17 ICA positive initially non-insulin treated patients later needed insulin. Among the 41 initially insulin-treated patients, ICA lacked in 17 showing higher fCP than ICA positive subjects (at diagnosis: 0.56+0.10 nmol/l and 0.22_+0.02 nmol]l,p<0.01 ; two years after: 0.75_+ 0.09 nmol/l and 0.18+0.04 nmol/l, p<0.005); 6 ICA negative patients left insulin later.There were no correlations between the levels of ICA and fCP. In ICA positive patients, non-insulin treated had higher (p< 0.02) ICA levels than insulin-treated. In conclusion, fCP though unchanged during the first two years after diagnosis was clearly related to the presence of ICA at diagnosis. ICA and fCP simplify classification of diabetes.

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SECRETION OF I N T E R L E U K I N - 2 AFTER SMALL DOSES OF MITOGEN IN TYPE I ( I N S U L I N - D E P E N D E N T ) DIABETES

~-3-POLYUNSATURATED F A T T Y A C I D S (PUFA) D E C R E A S E MONONUCLEAR CELL (MNC) I L - I B C O N T E N T BUT NOT SECRETION. J M~ivig, T M a n d r u p - P o u l s e n , F Pociot, H Worsaae, LD Wogensen, L Bmk, P Christensen, K Andersen, P Madsen, J Dyerberg, and J Nerup, S t e n o M e m o r i al H o s p i t a l and N O V O - N o r d i s k ; R i g s h o s p i t a l e t and I n s t i t u t e of E x p e r i m e n t a l Medicine, U n i v e r s i t y of C o p e n h a g e n ; H e r l e v and A l b o r g Hospitals, Denmark. M o n o k i n e s m a y be e f f e c t o r m o l e c u l e s in the p a t h o g e n e s i s of t y p e 1 (insulin-dependent) diabetes. P U F A r e d u c e the c o n t e n t of IL-I and TNF~ in M N C lysates. The aim was to study IL-IB and TNFa secretion, IL-IB c o n t e n t and PHA s t i m u l a t e d p r o l i f e r a t i o n (P) of s t i m u l a t e d MNC or a d h e r e n t mon o c y t e s (Mo) i s o l a t e d from h e a l t h y individuals, who w e r e r a n d o m i z e d to t r e a t m e n t for I0 w e e k s w i t h 2 (n=8) or 4 g/d (n=9) of P U F A or p l a c e b o (n=8), and from 8 r e c e n t - o n s e t IDDM p a t i e n t s g i v e n 4 g/d of PUFA. P U F A i n c r e a s e d M N C and t r o m b o c y t e m e m b r a n e e i c o s a p e n t a e n o i c acid two- to t h r e e - f o l d a s c e r t a i n i n g compliance, s i g n i f i c a n t l y i n h i b i t e d M N C - P and r e d u c e d the MNC and Mo content of IL-IB, with no d i f f e r e n c e s between h e a l t h y and IDDM subjects. However, P U F A did not a f f e c t IL-IB, TNF~, PGE 2 or LTB 4 s e c r e t i o n in h e a l t h y s u b j e c t s or IDDM patients. Thus, a l t h o u g h d i e t a r y s u p p l e m e n t a t i o n w i t h 2-4 g/d of P U F A inhibits M N C - P and IL-IB c o n t e n t it d o e s not alter m o n o k i n e s e c r e t i o n in h e a l t h y or I D D M subjects. The p r e v e n t i v e e f f e c t of P U F A on l o w - d o s e s t r e p t o z o t o c i n d i a b e t e s in m i c e m a y not be due to i n h i b i t i o n of i m m u n e - e f f e c t o r p a t h w a y s d e p e n d e n t on m o n o k i n e s .

Milidevid Z.~Knegevid N.~Paveli6 K.~Grani6 M.~ $krabaio Z. and M a r u ~ i 6 - G a l e ~ i d S.; Institute for d i a b e t e s ~ e n d o c r i n o l o g y and m e t a b o l i c diseases "Vuk V r h o v a c " ~ 4 a , D u g i d o l ~ Z a g r e b ~ Y u ~ o s l a v i a o C h a r a c t e r i s t i c s of i n t e r l e u k i n - 2 (IL-2) secretion is important in type 1(insulin-dependent) diabetics because they are infection-proneo Along with the eapacity for seeretion of this lymphokine~ it is also n e c e s s a r y to determine the dependency of IL-2 secretion by helper T cells on s t i m u l a t i o n intensity i.e. s e n s i t i v i t y . A f t e r s t i m u l a t i o n of p e r i p h e r a l blood lymphocytes with the increased e o n e a n a v a l i n e A doses~ a supernatant with IL-2 was added to the culture of citotoxie T lymphocytes line (CTLL). For measuring the IL-2 level, the intensity of 3/H/thymidine i n c o r p o r a t i o n into target CTLL cells was used~ The results were presented as count per minute (epm+SEM). 9 healthy persons~18 with type I and 8 with type 2 ( n o n - i n s u l i n - d e p e n d e n t ) diabetes were examined~ The IL-2 seeretion in three groups of subjects, after the s t i m u l a t i o n with optimal c o n c a n a v a l i n e A doses (5 and 10 ug/ml) did not differ. After the a p p l i c a t i o n of 5ug/ml the i n c o r p o r a t i o n of 3 / H / t h y m i d i n e was 2 2 4 6 5 + 9 0 5 4 c p m in eontrols and did not differ in type ] (22605+3413) and type 2 diabetics (22130 +3659). The o b s e r v a t i o n s were identical when the iowest (1o25Ug/ml) or i n t e r m e d i a r y (2.5ug/ml) c o n e a n a v a l i n e A concentrations were being used. In eonclusion~ the s e n s i t i v i t y and capacity for IL-2 secretion remains unchanged in type I diabetic patients~

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T-CELL LINES FROM TYPE I DIABETIC AND PREDIABETIC SUBJECTS: FLOW CYTOMETRY AND LYMPHOKINE PRODUCTION ANALYSIS. C. Giordano~ R. De Maria, F. Panto', A. Mattina, T. De Franchis~ G. D'Acquisto, A. Pugliese, G. ,Stassi and A. Galluzzo . Laboratory of Immunology & Endocrinology, Institute of Clinica Mediaa, University of Palermo, Italy

T CELL A C T I V A T I O N AND LOW CD4+ L Y M P H O C Y T E S IN F A M I L I E S OF PATIENTS W I T H TYPE I DIABETES. D. Vergani. T. Warnock, M.J. Hussain and M. Peakman, Dept. Immunology, King's College School of Medicine, London. To examine the possibility that genetic s u s c e p t i b i l i t y to Type I diabetes is d e t e r m i n e d t h r o u g h the immune response, we s t u d i e d Tlymphocyte sub-populations in m e m b e r s of 29 families with an index case of the disease. To e n h a n c e the a c c u r a c y and c l a r i t y of the data, cells were a n a l y s e d by flow c y t o m e t r y u s i n g a lysed whole blood t e c h n i q u e w h i c h does not r e q u i r e a s e p a r a t i o n step. L y m p h o c y t e s g a t e d on their characteristic light scatter were a n a l y s e d for c o - e x p r e s s i o n of CD3, CD4, CD8 and HLA-DH. The absolute n u m b e r of CD4+ l y m p h o c y t e s was r e d u c e d in diabetics, s i b l i n g s (n=35) and parents (n=52) as c o m p a r e d with a g e - m a t c h e d children (n=30) and adult (n=20) controls (p<0.05). In addition, high levels of a c t i v a t e d CD4+ l y m p h o c y t e s were found in the diabetics, siblings and p a r e n t s (p<0.01). These r e s u l t s indicate a familial tendency to immune abnormalities characterised by subtle i m m u n o d e f i c i ncy and h y p e r - r e a c t i v i t y and HLA t y p i n g will reveal w h e t h e r these are l i n k e d to the MHC.

To determine the role of cell-mediated immunity in the outcome of Type I diabetes, we studied the phenetypic characteristics of PBL from 12 normoglycemic prediabetic subjects (PDS) with IAA and ICA positivities, i0 newly diagnosed Type I diabetic patients ~nd 7 controls, after 120 hr culture in vitro of 2.5 x I0 cells with (a) only tissue culture medium (TC); (b) 25 meg of PHA; (c) 20 U/ml of rlL2; (d) 200 U/ml of IFN~. The cells from (a), (b), (c) and (d) were analyzed by flow cytometry (FACS 440) for CD3, CD4, CD8, CD2, CD25, CD45R, CDIIb, CD71, CD69 and DR phenotypes and TCR /6. The respective supernatents were analyzed by ELISA method for production of ILI~, IL2, soluble IL-2R, soluble CD4 and CD8, IL6, TNF and IFN~. PDS did not show any single cell phenctype deficit or increase when examined freshly or after condition (a). T-cell lines after stimulation with+ PHA or + rIL2+ were+ characterized by an increase in TAC , DR , + + + + CD8 IL2R , CD4 CD45R and CD8 CDIIb cell phenotypes. As regards lymphokine production, an increase in ILl, IL6 and TNF was found in PDS and Type I DM with a simultaneous decrease in IL2 and IFN, with a significant difference vs controls (p range 0.01-0.001). Our findings show that the numerical alterations in phenetypic expression of T-cells in prediabetes are associated with the hyperpreduction of the lymphokines deputed to the growth of autoreactive T cell clones.

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D e p l e t i o n of a c t i v a t e d CDR-p0sitive T - c e l l s in t h e p r a - d i a b e t i c p e r i o d oF Type I D i a b e t e s m e l l i -

SPECIFIC T CELL RESPONSES IN RECENT ONSET TYPE I DIABETES PATIENTS AGAINST INSULIN SECRETORY GRANULE DETERMINANTS

tus. F. B e c k e r , A. H. H o f f m a n n , R. G. B r e t z e l and K, Federlin, Medizinische Klinik III und P o l i k l i n i k der Justus-Liebig-Universit~t G i e 6 e n , R o d t h o h l 6, 6300 G i e ~ e n , FRG A number o f c h a n g e s i n t h e T - c e l l subpopulation o f Type I d i a b e t i c s have been d e s c r i b e d a t t h e o n s e t o f t h e d i s e a s e . Most o f t e n , a reduction of CD4-positive T-cells and i n c r e a s e d m a r k e r s o f activation were f o u n d . I n t h e s t u d y p r e s e n t e d h e r e we h a v e p r o s p e c t i v e l y looked at a group of 15 p r e - d i a b e t i c patients by means o f d o u b l e f l o w cytometry analysis of T-lymphocytes. The p r e diabetic s t a t e was d e f i n e d by p r e s e n c e o f i s l e t cell antibodies (IC A) and a r e d u c e d f i r s t phase insulin secretion to intravenous g l u c o s e . By means o f t h e d o u b l e f l o w - c y t o m e t r y the state of activation of the single T-cell compartments c o u l d be e v a l u a t e d . M e a s u r e m e n t s were c a r r i e d out with a Becton-Dickinson FACSCAN u s i n g t h e simultest with standard monoclonal antibodies against T-cell s u b s e t s and e n t i - I L p - R e c e p t o r and a n t i - D R as a c t i v a t i o n m a r k e r s . R e s U l t s : Compared t o an age and sex m a t c h e d c o n t r o l group the prediabetic patients showed s i g n i f i c a n t l y reduced numbers o f a c t i v a t e d CDR-positive T-cells (3.7 vs. 7.6, p<.05 for IL2-Recepter; 7 . 7 vs 1 4 . 2 , p<.01). Simultaneously, a number o f t h e p r e - d i a betics displayed elevated figures of activated CD~-positive cells, but this feature could not be-demonstrated in all patients. These f i n d i n g s are evidence of a disturbed immunological distrib u t i o n o f a c t i v e and i n a c t i v e T-cell subpopulat i o n s i n t h e p r o d r o m a l phase o f Type I d i a b e t e s mellitus, possibly demonstrating a s u p p r e s s o r Tcell defect.

B.O.Roep, A.A. Kallan, W.L.W.Hazenbos, G.J.Bruining*, J.C.Hutton** and R.R.P.de Vries; Dept. Immunohaematology & Blood Bank, U n i v e r s i t y Hospital Leiden, *Dept. P e d i a t r i c s , Erasmus U n i v e r s i t y Rotterdam, the Netherlands; **Dept, C l i n i c a l Biochemistry, U n i v e r s i t y o f Cambridge, U,K.

P687 LOSS OF HLA DR - POSITIVE, BUT NOT T6 - POSITIVE LANGERHANS CELLS AT THE ONSET OF TYPE 1 (INSULINDEPENDENT) DIABETES.

T. Konrad, A.-G. Ziegler, E. Standl. Diabetes, Hunich, FRG.

Forschergruppe

Langerhans cells (LC) represent the antigen presenting cells of the skin. In normal epidermis they are the only cells which express HLA DR- and CDIa (T6)antigens. We have reported a marked decrease of DR+ LC/mm2 at the onset of Type 1 ( i n s u l i n dependent) diabetes. Now we have investigated LC in epidermal sheet preparations of new onset diat~etics (n=21), longterm diabetics (n:12), i s l e t c e l l a n t i b ~ y (AB)-po~itive (n=5) and AB-negative (n=16) r e l a t i v e s , and controls (n=31) using monoclonal anti-DR and anti-T6 antibc~ies and i n d i r e c t immunofluorescence. A s i g n i f i c a n t decrease o f DR* LC/mm2 was found in new onset diabetics compared with long-term diabetics and controls (448_+20 vs 759-+26 vs 621+22, P
There is strong evidence

t h a t Type I

(insulin-dependent)

diabetes mellitus is an autoimmune disease with a significant T cell component. Unclear is the nature of the target antigen(s) and the events that i n i t i a t e the autoimmune process. In the present study the T cell response of T cell lines generated f r o m the peripheral blood against,a subcellular fraction of a beta cell line was analyzed. We report that the T cell s p e c i f i c i t y described is not unique, but rather generally found in the type I diabetes population at time of onset of the disease (14 out of 19 newly diagnosed patients; 74%). In contrast, very low or no r e a c t i v i t y towards t h i s determinant was detected in an HLA- and age-matched healthy control group (2 out of 16 individuals; 12,5%; p
A187

PS 26 HLA and Diabetes P688

P689

TWO HLA-DQB ALLELES ARE ASSOCIATED WITH PROTECTION AGAINST TYPE I (INSULIN-DEPENDENT) DIABETES. C. M i j o v i c , K. Jacobs~ D. Jenkins, M. Penny, J. Fletcher and A.H. Barnett. Dept. of Medicine, U n i v e r s i t y of Birmingham and East Birmingham Hospital, U.K.

REGULATORY REGION POLYMORPHISMIN HLA CLASS I I GENES. L.C. Andersen, J.S. Beaty, C.E. Seyfried, L. Shewey, B.S. Nepomand G.T. Nepom. Virginia Mason Research Center, 1000 Seneca Street, Seattle WA 98101, USA.

Genetic s u s c e p t i b i l i t y to Type I (insulin-dependent) diabetes has been associated with genes in the HLA-D region. HLA-DR2 has been negatively associated with the disease but t h i s association is not consistent in a l l races. HLA-DQ a l l e l e s are in linkage d i s e q u i l i b r i u m with HLA-DRB1 a l l e l e s and so could be a l t e r n a t i v e candidates for p r o t e c t i v e a l l e l e s . HLA-DQB polymorphism in diabetics and healthy racially-matched controls from three ethnic groups was investigated using a l l e l e s p e c i f i c o l i g o n u c l e o t i d e h y b r i d i s a t i o n . Two DQB a l l e l e s were e i t h e r or both reduced in a l l three racial groups. DQw6 was negatively associated with Type I diabetes in Caucasians (0/163 diabetics v 11/88 controls, pc2xlO -4, RR 0.02) and Afro-Caribbeans ( i / 3 8 diabetics v 21/87 controls, Pc 8xi0-4, RR 0.07) but was rare in Asian Indians. DQw1.18 was s i g n i f i c a n t l y reduced in Caucasian diabetics (0/163 diabetics v 8/88 controls, Pc 6x10-4, RR 0.03) and in Asian Indian patients (1/43 diabetics v 31/92 controls, Pc 4x10-I, RR 0.12) but was rare in Afro-Caribbean subjects. DQw6 and DQw1.18 d i f f e r by a single nucleotide. This s i m i l a r i t y of the expressed DQ~ chains suggests a possible common functional role in protection against Type I diabetes.

P690 HLA

Studies have correlated the DQB3.2, but not the closely related DQB3.1 a l l e l e , with susceptibility to type I diabetes. The promoter regions of these two DQ alleles, including the closely related DX8 gene, were cloned and sequenced from several B-LCLs. All three promoters contained the conserved sequence motifs, W, X and Y boxes, occurring in all class II genes. However, several sequencedifferences exist between the two DQB alleles and between these and the DX8 geneo The DQ and DX promoters were subcloned into the expression vector pTCAT upstream of the chloramphenicol acetyl transferase gene. Transfection into human B-LCLs showed high, albeit very different CAT a c t i v i t y levels for the DQ83.2 and DQ83.1 constructs, and no measurable levels for the DXB construct. Mobility shift assays were performed, where the binding of nuclear proteins to labeled promoter fragments were competedwith unlabeled promoter fragments or oligonucleotides from either homologousor heterologous promoters, correlating reporter gene function with the capacity of DNA binding proteins to d i f f e r e n t i a l l y recognize DQ and DX sequences. DNAsel footprinting further explored differences in nuclear protein binding patterns. These findings identify an important, previously unrecognized, source of MHC class II diversity, regulatory region polymorphism, which may contribute to susceptibility to autoimmunedisease.

P691 TYPING

IN

314

ITALIAN

TYPE

1

DIABETIC

P A T I E N T S : R E L A T I O N S H I P S W I T H AGE AND S E A S O N AT ONSET.

M.Caruso,F.Meschi,E.Bognetti,C.Malavasi, ~ MT.Illeni,~ C.Maffeis, ~ L.Pinelli,G.Chiumello.Scientific Institute San Raffaele, Dept of Pediatrics, Endocrine Unit, University of Milano; ~ Laboratorio di Immunoematologia, Istituto dei Tumorij Milano; ~ Dept of Pediatrics, University of Verona. HLA typing (A,B,C,DR,DQ,) was performed in 314 patients (184 males,130 females).Mean age at onset was i0 ~ 5.7 yrs.All the patients were unrelated and of caucasian origin. The frequency of DR3 and DR4 antigens resulted significantly increased in diabetic population compared to controls (DR3: 50% vs 17%,X2=52.15; DE4: 43% vs 13%;X 2 =47.94). DR2 frequency resulted significantly decreased in diabetics compared to the controls (9% vs 30%;X 2 =34.B2)as well as the frequency of DR7 (16% vs 30%;X 2 =13.77) and of DRwll (6% vs 25%;X z =34.47) while the frequency of DR5 did not show a significant decrease. We have found a significant increase in frequency of B8 (20% vs II%;X2=17.45); BIB (27% vs 13%;X2=34.65);DQw2 (41% vs 22%;X 2 =I7.3);DQw3 (47% vs 39%;X 2 =15.17) while the frequency of BI5 did not show an increase in our diabetic population. Diabetic patients under i0 years of age at diagnosis (mean age 5.8 -+ 2.7 yrs) showed an increased DR3 antigen frequency compared to older diabetics (mean age 17 -+ 7.7 yrs): 57% vs 41%;Xa= 5.85.No correlation has been observed between HLA antigens frequencies and season at onset.Breast-fed diabetics developed d i a b e t e ~ later than bottle-fed ones

(mean age~ sem = 12.6 -+ 0.9 yrs vs

9.7 ~ 0.5 yrs; P~O.Ol),indipendently from HLA status.

ANALYSIS OF DR7 HAPLOTYPES SUPPORTSDQw2 AS A DETERMINANT OF SUSCEPTIBILITY TO TYPE I (INSULIN-DEPENDENT) DIABETES. D. Jenkins, M. Penny, K. Jacobs, C. M i j o v i c , J.A. Fletcher and A.H. Barnett. Dept. of Medicine, University of Birmingham and East Birmingham Hospital, U.K. DR3 is strongly associated with Type I diabetes. The Caucasian DR3 haplotype encodes a DQ# chain producing the DQw2 s p e c i f i c i t y which may predispose to diabetes d i r e c t l y . DR7 haplotypes encode one of 2 DQ~ chains producing the s p e c i f i c i t i e s DQw2 and DQw9. A diseasepredisposing e f f e c t of DQw2 should r e s u l t in d i f f e r i n g disease associations of the 2 DR7 haplotypes. This hypothesis was tested by examining 165 Caucasian Type I diabetics and 90 racially-matched controls. DR and DQ types were determined by r e s t r i c t i o n fragment length polymorphism (RFLP) analysis. Nineteen d i a b e t i c subjects were typed as DR7 compared with 23 controls, RR=0.38, Pc
A188

P692

P693

THE CD3 EPSILON LOCUS IS ASSOCIATED WITH TYPE I DIABETES IN FEMALES. F.S.Wong, S. Moore, S.Orisio, B.A.Millward and A.G. Demaine.Departments of Medicine and Diabetes, King's College School of Medicine and Dentistry, London, U.K. Type 1 diabetes is associated with MHC and T-cell receptor p chain genes. In the NOD(Non Obese Diabetic) mouse model of diabetes, there are 3 recessive genes, one of which is located on chromosome 9 near to THu This chromosome has a high degree of homology with chromosome iiq23 in man. Probes to region Iiq23 were used to investigate the possibility of a susceptibility gene in this region in man. Informative RFLPs of 8 and 9 kb were detected using Taql restriction endonuclease and a probe to CD3~, found between THY-I and neural adhesion molecule NCAM. No differences were found in the frequency of the CD35 alleles or genotypes between patients or controls. However, a highly significant difference was found in the frequency of CD3E genotypes and alleles between male and female diabetics and between female diabetics and controls. There was an excess of 8 kb allele in diabetic females (frequency 0.524 v 0.26 in diabetic males and 0.264 in normal females, p<0.0005 and 0.001 respectively). These results suggest that different factors may determine susceptibility to diabetes in males and females and point to genetic heterogeneity of the disease.

HLA GENE PROBING IN CHINESE SUBJECTS DEMONSTRATES A POSITIVE ASSOCIATION BETWEEN TYPE I DIABETES AND DR3. M. Penny, D. Jenkins, K.H. Jacobs, C. M i j o v i c , V. Yeung, C. Cockram, J. Fletcher and A.H. Barnett. Dept. of Medicine, U n i v e r s i t y of Birmingham, UK and Dept. of Medicine, The Chinese U n i v e r s i t y of Hong Kong. Mapping of genetic s u s c e p t i b i l i t y to Type I diabetes is aided by t r a n s - r a c i a l analysis. This allows i d e n t i f i c a t i o n of disease associations which are secondary to linkage d i s e q u i l i b r i u m between marker genes and primary disease s u s c e p t i b i l i t y a l l e l e s . DRB and DQB r e s t r i c t i o n fragment length polymorphism (RFLP) analyses were used in subjects of Chinese o r i g i n to study HLA associations with Type I diabetes. Eight of 17 Type I d i a b e t i c subjects and 8 of 51 control subjects possessed DRB/DQB RFLPs c h a r a c t e r i s t i c of the DR3 haplotype, RR=4.6, p< 0.01. Five of 17 d i a b e t i c subjects possessed RFLPs c h a r a c t e r i s t i c of DR4, compared with 13 of 51 control subjects, RR=I.3, NS. No DQB RFLP was s i g n i f i c a n t l y associated with Type I diabetes. These data confirm a p o s i t i v e association between Type I diabetes and DR3 in this race. The absence of s i g n i f i c a n t association between DR4 and the disease contrasts with other races. This suggests that DR4-associated s u s c e p t i b i l i t y to Type I diabetes is secondary to linkage d i s e q u i l i b r i u m between the DR4 a l l e l e of the DRBI gene and an a l l e l e at a d i s t i n c t locus.

P694

P695

HLA-DQI3 ALLELES ARE LINKED TO EARLY-ONSET AND LATE-ONSET FORMS OF TYPE I DIABETES

RESTRICTION FRAGMENT LENGTH POLYMORPHISM STUDY OF HLA HAPLOTYPES IN MULTIPLEX FAMILIES WITH TYPE I DIABETES. J. Jos, M.-F. De Tand, E. Tozzo, M. Thuriaux, D. Farkas, J. Cartron, I. Deschamps, H6pital des Enfants Malades, Paris, France. Restriction fragment length polymorphism (RFLP) of HLA-class II genes was analysed in 14 multiplex families with type I diabetes mellitus. DNA was digested with three restriction enzymes (Taq I, EcoRI and BamHI) and

H Heimberg*, E Quartier*, G Somers ~ I De Leeuw$ and F Schuit* Dept of Biochemistry* and Diabetology~ Vrije Universiteit Brussel, Brussels, Belgium; dept of EndocrinologyS, Universitaire Instelling Antwerpen, Antwerp, Belgium. The association between HLA-DQB alleles encoding Asp or nonAsp amino acids in codon 57 and, respectively, resistance or susceptibility to develop type I diabetes has been well documented for Caucasian patients younger than 20 years at diagnosis but not for patients with later appearance of this disease. We therefore compared DQg-gene polymorphism of the following three groups: 58 type I diabetics diagnosed between age 20-40 (D20-40), 91 type I diabetics diagnosed under age 20 (D<20) and 81 healthy controls (CTR). The allele DQwl.2 was more often encountered in CTR (22%) than in D<20 (2%) or D20-40 (6%) while DQw3.2 was less frequent in CTR (6%) than in D<20 (33%) or D20-40 (27%). The DQw2/3.2 genotype was observed in 34% of D<20 and 33% of D20-40 but only in 4% of CTR (P<0.0001; relative risk=13.4). The genotype DQwl.1/3.2 was also common in D<20 (11%) and D20-40 (9%) but not in CTR (1%). Asp/Asp phenotypes were commonly found in CTR (31%) but rarely in D<20 (1%) and D2040 (5%). Non-Asp/non-Asp phenotypes were observed in 70% of D<20, 68% of D20-40 but only in 18% of CTR (P<0.0001; relative risk=9.5-10.4). These data support the importance of the HLADQB gene as a marker for type I diabetes. Secondly, patients with early or late clinical manifestation of type I diabetes appear genetically indistinguishable at the HLA-DQfi locus.

hybridization was performed with HLA DRB1 and DQB1 p32 labelled probes. Applied procedures and reported RFLP data were in conformity with the 1@ H i s t o c o m p a t i b i l i t y Workshop standards. 82 haplotypes were analysed, 52 from diabetic sibs and 36 from non affected sibs. The results confirmed the high frequency of DR4-DQw8 and DR3-DRw17-DQw2 haplotypes in diabetic patients. All affected sibs shared DR3-DRw17-DQw2 and/or DR4-DQw8 haplotypes compared to 83% in non affected sibs. There was a much higher frequency of DR3-DRw17-DQw2/DR4-DQw8 heterozygotes (30.8%) in affected children than in non affected ones (5.6%, p < 0.05). The protective effect of DR7-DQw2-Dw17 haplotype is also supported by our study (none of diabetic children shared this subtype contrary to 11.1% of non affected sibs, p < 0.03). Thus, RFLP studies in multiplex families demonstrated the segregation of disease susceptibility on some high risk haplotypes. The identification of such HLA subtypes may provide additional means for the detection of susceptible sibs in affected families.

A189

PS 27 Spontaneous Diabetes, Antigens and Viruses P696

P697

R E D U C T I O N O F D I A B E T E S I N C I D E N C E IN BB R A T S BY NEONATAL INJECTIONS STIMULATING DRUGS

OF

BETA-CELL

K. Buschard, M. Jcrgensen, T. Kjmr, T. Bock, K. A a e n and K. Josefsen. Bartholin Institute, Kommunehospitalet, 1399

THE SERUM IMMUNOGLOBULIN CLASS AND SUBCLASS V A R I A T I O N IN RELATION TO DIETARY EXPERIMENTS WITH BB-RATS.

Department of Animal Physiology and Biochemistry, Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Denmark.

A. Lassen, J. Hoorfar, F. Scott and C.-H. Brogren..

C o p e n h a g e n K, Denmark. T h e aim was to study the possible ability of different betacell stimulating drugs to influence the incidence of diabetes in BB rats. Newborn BB rats were injected intraperitoneally or subcutaneously with different drugs twice a day for six days, whereas control BB rats received saline or were untreated. 39.6% of 265 experimental BB rats developed diabetes during a 200-day observation period as opposed to 63.8% of 163 control BB rats (p<10-5). A m o n g the different subgroups of experimental BB rats the diabetes incidence was as follows: Glucagon: 37% (n=93, p<0.0001); Tolbutamide + glucose: 36% (n=58, p<0.0005); Theophylline + glucose: 39% (n=41, p<0.005). The optimal dosage/time of t r e a t m e n t and the definite mechanisms have to be established. However, the possibility exists that the injected drugs, by accelerating betacell maturation, induce tolerance. W e conclude that neonatally injections of beta-cell stimulating drugs can lower diabetes incidence in BB rats.

Correlation between dietary exposure to various protein diets and the incidence of diabetes have been described earlier. This indicates a possible stimulation o f the immune system through the dietary immunogens. In these studies the total level of serum lgM, IgA, tgG1, IgG2a, lgG2b and IgG2e were determined by rocket immnnoelectrophoresis analysis from three groups of BB-rats given a diet with respectively skim milk powder (Affi17 rats), casein (B=15 rats) and hydrolysed lactalbumin (C=12 rats) as the only source of amino acids from weaning until 120 days of age. The incidence of diabetes (A=35%, B=20% and C=8%) and the levels of individual immunoglobulins were compared. The best correlation between high incidence and higher immunoglobulin levels was seen in the IgG1 (A=1.04, B=0.64, C=0.38 mg/ml; p<0.01 A/C) and IgG2b (A=1.48, B=1.95, C=1.28 mg/ml; p<0.05 B/C). The two other lgG-snbclasses showed lower level of immnnoglobulins with decreasing incidence. The IgM had the same tendency (A--L28, B=0.99, C=0.83 mg/ml), but IgA showed no correlation. In conclusion, these data indicate that higher diabetes incidence might correlate with higher level of serum immunoglobulin titre in general, indicating a possible polyclonal activation mecanism behind the dietary influenee on the ineidenee of insulin-dependent diabetes in the BB-rat model.

P698

P699

THE SPECIFIC IMMUNE RESPONSE AGAINST FOOD PROTEINS IN RELATION TO DIETARY EXPERIMENTS WITH BB-RATS.

PROLIFERATIVE ACTIVITY OF PANCREATIC ISLET PARENCHYYP~L AND INFILTRATING CELLS IN NON OBESE DIABETIC (NOD) MICE J.Krug, A.Williams, P.Beales~ E.Gale and P.Pozzilli Dept.Diabetes & Immunogenetics, St.Bartholomew's Hospital London ECIA7BE, UK The aim of this study was to assess the proliferative activity of islet parenchymal cells and infiltrating lymphocytes in the NOD mouse. Female non-diabetic NOD mice (age 10-14 weeks) received 300 ug Bromodeoxyuridine (BrdU), which labels proliferating cells, by i.p. injections over a period of 48 hours. Cryocuts from snap-frozen pancreata were stained with monoclonal antibodies to BrdU and to T-lymphocyte subsets (L3T4~ Lyt2 and IL2R). Insulitis was found in 83% of the pancr~ata. In infiltrated islets between 12 and 30% (24.3:7.4) of lymphocytes were positive for BrdU. BrdU+ve nuclei were found both in L3T4+ve and Lyt2+ve lymphocytes. However more Lyt2+ve and BrdU+ve cells were observed in the population of infiltrating lymphocytes bordering the uninfiltrated islet tissue and as single infiltrating lymphocytes. Lymphocytes in the proximity of lymph- and blood vessels (probably representing trafficking l%mphocytes) and lymphnodes were found to be 10-20% (13.7L5.4) positve for BrdU, even in pancreata without insulitis. Less than 2% of islet cells in infiltrated islets were found to be positive for BrdU, which is comparable to the percentage in noninfiltrated islets (1.8• vs. 1.6Ti.5%, n.s.). In conclusion, there was no evidence for a significant islet cell proliferation in female NOD mice; in contrast infiltrating lymphocytes show high mitotic activity.

Worm. Department of Animal and Biochemistry, Royal Veterinary and University, DK-1871 Frederiksberg C, Denmark.

C.-H. Brogren, J. Hoorfar and K.

Physiology Agricultural

Previous nutritional experiments have indicated that the protein sources given in diets to BB-rats strongly influence the incidence of diabetes. In order to investigate this further, we have performed dietary experiments with three different sources of soya protein different in immunogeneeity; soyaflour (A), alcohol-treated hypoallergic Danpro-S (B) and alcalase-hydrolysed Danpro-S (C). Three groups of 20 BB-rats were exposed to a common weaning on casein diet and subsequently exposed exclusively to the three soya diets from day 45-48 to day 150 of age. The incidence of diabetes correlated with the immunogenecity of the diets (A=60%) B=30% and C=35%, p<0.05 A/B+C). The measurement of specific dietary antibodies were done by ELISA using soya trypsininhibitor (TI) and glycinln (G) as antigen. The antibody titres (in abitrary O.D.-units) showed a correlation between the immuuogeuecity of the diets and the dietary immune responses (798i275(A), 712~151(B), 661.~190(C) for G-antibodies 311+208(A), 283i145(B), 254.-t:154(C) for IT-antibodies). The titres from diabetic animals are often higher than those from non-diabetics (904+263(A) versus 653~234(A), 723* 116(B) versus 707.+.168(B), and 754~225(C) versus 607~209(C) for Gantibodies, and 373:1:306(A) versus 252,~82(A), 350d:64(B) versus 255+161(B), 239~:102(C) versus 262i181(C) for Tl-antibodies). These data elucidate an immuno-stimulatory influence from food proteins on the aul:oimmune behaviour of BB-rat diabetes.

A190 P700

P701

E C H O 4 V I R U S I N F E C T I O N I N M I C E IS A S S O C I A T E D W I T H I M P A I R E D ISLET FUNCTION. T.M.Szopa, A . U r i a r t e * and K.W.Taylor. D e p a r t m e n t of B i o c h e m i s t r y , L o n d o n H o s p i t a l M e d i c a l College, London, UK. D e p a r t m e n t of I m m u n o l o g y , N a t i o n a l Institute of E n d o c r i n o l o g y , C u b a * .

VIRUS AND DIABETES: CYTOMEGALOVIRUS SEQUENCES IN THE PANCREAS OF PATIENTS WITH TYPE 2 (NON-INSULIN-DEPENDENT) DIABETES MELLITUS.

F o l l o w i n g an earlier report of the i n d u c t i o n of g l u c o s e i n t o l e r a n c e and islet cell a n t i b o d i e s in children infected by echo 4 virus, the effects of this virus on islet f u n c t i o n in m i c e was i n v e s t i g a t e d . D i a b e t e s - s u s c e p t i b l e m i c e were i n o c u l a t e d w i t h virus i s o l a t e d from i n f e c t e d children~ Rates of i n s u l i n release, (pro)insulin and total p r o t e i n s y n t h e s i s w e r e m e a s u r e d in c o l l a g e n a s e - i s o l a t e d islets from i n f e c t e d mice and n o n - i n f e c t e d controls. The most m a r k e d effect found was the increase in the basal rate of insulin r e l e a s e at 2 m m o l / l glucose concentration (3 months p o s t - i n f e c t i o n : c o n t r o l s 8.6~0.6 p g / i s l e t / m i n , i n f e c t e d 17.1+0.9 pg/islet/min, p<0.025; 4 months p o s t - i n f e ~ t i o n : c o n t r o l s 7.1~ 1.6 p g / i s l e t / m i n , i n f e c t e d 14.1~i.i pg/islet/mi~ p<0.005). Rates of ( p r o ) i n s u l i n b i o s y n t h e s i s w e r e u n c h a n g e d at either 2 and 20 m m o l / l g l u c o s e in islets from infected mice. Total ~ r o t e i n synthesis, however, was d e c r e a s e d at both low and high g l u c o s e c o n c e n t r a t i o n at 4 m o n t h s posti n f e c t i o n (2 m m o l / l : c o n t r o l s 663~58 c p m / i s l e t / h , infected 422+41 cpm/islet/h, p<0.01; 20 m m o l / l g l u c o s e ; c o n t r o l s 5095+336 cpm/islet/h, i n f e c t e d 3259~249 c p m / i s l e t / h , - p < 0 . 0 0 1 ) . These changes r e s e m b l e those o c c u r i n g in islets from C o x s a c k i e B 4 - i n f e c t e d mice. The C o x s a c k i e B v i r u s e s have been i m p l i c a t e d as c a u s a t i v e agents of diabetes, and since echo 4 virus is a c l o s e l y r e l a t e d p i c o r n a v i r u s and a common human pathogen, it may be yet a n o t h e r virus involved in the d e v e l o p m e n t of diabetes.

M. LShr, R. Maekawa, M.B.A. Oldstone and G. Kl~ppel; Dept. of Medicine I, Univ. of Erlangen, FRG; Scripps Clinic and Research Foundation, La Jolla, CA and Dept. of Pathology, Free Univ. of Brussel, Belgium.

For long, viruses have been suspected to be involved in diabetes, both type 1 (insulin-dependent) and type 2 (non-insulin dependent). To test this hypothesis, we studied RNA extracted from the pancreas of patients with type 2 diabetes by RNA-slot-blot hybridization with eDNA probes of viruses known to infect human beta-cells, namely cytomcgalovirus (CMV), mumps, rubella and coxsackie. The functional morphology of the islets was studied by immunocytochemistry for insulin and glucagon. Forteen of 32 pancreata from diabetic patients (mean age: 69 _+ 7 yrs) hybridized to two different CMVprobes, but only 1 of 50 (none of 30 age- a n d sex matched, mean age: 63 + 14 yrs) controls was positive. Mumps, rubella and coxsackie were all negative. For confirmation and to localize the CMV signal to the islets, in situ hybridization was performed, which revealed positive but spare signal over endocrine cells, presumably beta cells. DNA amplification with the PCR and subsequent Southern blottting and direct sequencing was performed to further proof the presence of CMV. However, no antigens could be detected by immunocytochemistry. Hybridization of the pancreatic RNA with probes to insulin and somatostatin did not reveal any significant differences in RNA levels by comparative laser-densitometry. No morphologic abnormalities could be detected in the CMV-positive pancreata, i.e. insulitis, perivasculitis or cytopathic changes. Patients positive for CMV tend to have a shorter duration of diabetes. These results indicate that, first, CMV can persist in the endocrine pancreas of patients with type 2 diabetes, second, that this is neither associated with expression of viral proteins nor signs of cellular injury. In conclusion, we would suggest that some cases of type 2 diabetes may be associated with CMV infection, however, the role the virus plays in the pathogenesis of diabetes remains to be elucidated.

P702

P703

MUMPS VIRUS INFECTS AND IMPAIRS FUNCTION OF HUMAN FETAL BETA CELLS IN VITRO

Detection of human retrovirus like sequences in DNAs from patients with polyendocrinopathies (Grave's disease and Type 1 diabetes).

T. Vuorinen, G. Nikolakaros, T. Hyypi~, R. Vainionp&&, A. H~nninen and O. Simell. Departments of Virology, Anatomy and Pediatrics, University of Turku, Finland

P.~S~ut, S.~A~ ~, v . ~ , J.~Y-T~mJ, A...~om'~L J.co~, J~t F . S ~ 2, c . ~ I m B~B~1, G.CATH~.T~AU I, R . ~ O I L - R A V I E R 2, J.PERIES2

We investigated if mumps virus can infect human fetal islet cells in culture. The influence of the virus on beta cell function was also evaluated. Human fetal pancreas cultured as isletlike cell clusters was infected with mumps virus. Double immunofluorescence of dispersed cells revealed that virus infected both beta cells and non-beta cells. Release of infectious virus continued for 14 days. Infected cells could still be detected at the end of the 22-days observation period indicating the possibility of a latent form of infection. The amount of immunoreactive insulin released in culture medium was initially 3-5 mU/I per day. In mumps virus infected cultures it declined steadily and became undetectable 7 days post infection, whereas insulin release in control cells remained detectable at least 14 days. Our results display that mumps virus infects human fetal beta cells in vitro and decreases their insulin release. Mumps virus may persist in beta cells and play a role in the etiopathogenesis of type 1 diabetes mellitus.

1-Service de Diab~tologie, 2-UPR A0043 CNRS, 3-INSERM U.248, 4-INSERM U.93, CHU et HSpital Saint Louis, 75010 Paris, France.

Relationships with retroviruses have recently been found in different human pathologies as autoimmune diseases, which would be associated with the presence and eventually the expression of retroviral sequences. Samples of 18 patients with polyendocrinopathies (Grave's disease and type 1 diabetes) were studied by serological and genomic tests, to detect antibodies and HTLV-I, Foamy virus and HIV-I retroviral sequences. No antibodies to HTLV-I, Foamy virus and HIV-I could be detected by western blot and Elisa tests. HTLV-I-related sequences were revealed by southern blot (SB) in 5 out of 18 patients DNAs. Analysis of all DRAs were performed by polymerase chain reaction (PC]{). Seven DHAs, including the 5 previously positive in SB, contained HTLV-I-Gag related sequences, but neither Pol nor Px. Foamy virus related sequences were found in 10 patients, 4 were positive for Foamy-Gag related sequences (2 out of these were also positive for HTLV-I-Gag), and all ten patients were positive for Foamy-Bel related sequences. Concerning HIV-I, all 18 DNAs examined were negative by both methods. Ten clinical healthy donors' DNAs were found to be negative with the same tests. In s,mmary, 12 out of 18 patients were positive for one or more retroViral tested related sequences. These preliminary results indicate the presence of HTLV-I and/or Foamy virus related sequences in some patients with autoimmune diseases.

A191 P704

P705

C O X S A C K I E B4 V I R U S I N D U C E S A R E D U C T I O N I N P R E PROINSULIN mRNA IN CULTURED MOUSE ISLETS.

GLYCOSYL-PHOSPHATIDYL-INOSITOL ANCHORED, SIALILATED PROTEINS ARE INVOLVED IN THE INCREASED B-CELL ADHESION OF T-SPLENOCYTES FROM NOD MICE. P. Ritz, B. F~ve, B. Charbonnel and P. Sai'. Laboratoire Immunologic du Diab~te, Facult6 de M6decine, 1 rue G. Veil, 44035 Nantes C6dex, FRANCE. 300 NOD mice displayed a higher number (p<0.001) of B(RINm5F)-cells binding splenocytes ("Diabetic rosettes"="DR") than 100 controls. These "DR" involve L 3 T 4 and Lyt2 T-cells and 50
N.D. P o r t w o o d , T. W a r d a n d K.W. T a y l o r , D e p a r t m e n t of B i o c h e m i s t r y , L o n d o n H o s p i t a l M e d i c a l C o l l e g e , T u r n e r S t r e e t , L o n d o n E1 2 A D . U K S t r a i n s of C o x s a c k i e v i r u s h a v e b e e n s h o w n to p r o d u c e c h a n g e s in i n s u l i n r e l e a s e a n d b i o s y n t h e s i s in c u l t u r e d m o u s e i s l e t s u p to 48 h o u r s post-infection. To s t u d y f u r t h e r t h e e f f e c t s of t h i s v i r u s in v i t r o u p o n ( p r o ) i n s u l i n b i o s y n thesis, islets were infected with virus and c u l t u r e d , w i t h a p p r o p r i a t e c o n t r o l s , f o r 48 h o u r s in R P M I 1640 m e d i u m , a f t e r w h i c h R N A species were phenol/chloroform extracted. R e l a t i v e l e v e l s of p r e p r o i n s u l i n m R N A w e r e measured by dot blot hybridisation using rat p r e p r o i n s u l i n I c D N A as a p r o b e . Preproinsulin m R N A w a s f o u n d t o b e s i g n i f i c a n t l y r e d u c e d in i n f e c t e d i s l e t s [ c o n t r o l s , 1 . 0 0 + 0.06 (n=8); i n f e c t e d , 0.38 + 0.06 (n=9), p < 0 ? 0 0 1 , a r b i t r a r y units]. S i m i l a r e f f e c t s h a v e b e e n o b s e r v e d in islets infected with encephalomyocarditis virus, another picornavirus. The above results i n d i c a t e t h a t C o x s a c k i e B4 v i r u s c a u s e s an i n h i b i t i o n of p r e p r o i n s u l i n m R N A s y n t h e s i s , or an i n c r e a s e in its r a t e of d e g r a d a t i o n . Such a l t e r a t i o n s in p r e p r o i n s u l i n m R N A t u r n o v e r m a y e x p l a i n C o x s a c k i e B4 v i r u s - i n d u c e d p e r t u r b a t i o n s in p r o i n s u l i n b i o s y n t h e s i s . These results may b e d i r e c t l y r e l a t e d to t h e d i a b e t o g e n i c e f f e c t s of t h i s v i r u s .

P706

P707

I S L E T C E L L A N T I B O D I E S AND A N T I G E N S : A N T I G E N H E A T S T A B I L I T Y AND F U R T H E R C H A R A C T E R I S A T I O N R . R a j u , S . S r i k a n t a and N . K o c h u p i l l a i . D e p t of E n d o c r i n o l o g y M e t a b o l i s m , All I n d i a Inst M e d i c a l S c i e n c e s , N e w D e l h i 110029, I n d i a

INCREASED EXPRESSION OF ICA ANTIGEN IN ISLETS OF NOD VS. C57 MICE IS PARALLELED BY A CHANGED GANGLIOSIDE PATTERN F.Dotta, L. Peterson,M. Previti, J. Metzger ,C.Tiberti ,L.Wfcker and U.Di Mario. University of Rome, Merck Res. ,Rahway,USA The target antigen of cytoplasmic islet cell antibodies (ICA) on pancreatic frozen sections has properties of a monosialo-ganglioside migrating between GM2 and standards. Following on the observation of increased ICA antigen expression on pancreatic frozen sections of Non Obese Diabetic (NOD) mouse vs. Balb/c or C57/BI0 mice, we characterized the ganglioside pattern of isolated islets from 4wk old NOD (n=40) or from age matched C57/BI0 mice (n=40). Islets have been isolated using an intraductal collagenase distention procedure. Gangliosides have been extracted in chloroform:methanol:water, purified on SepPak C18 cartridge, and analyzed by TiC and HPLC. Isolated NOD islets expressed GM3 and a monosialo-ganglioside (GM2l) migrating between 6~42 and GMI standards; C57/BI0 islets expressed GD3 and the G~2-1 ganglioside. Quantitative analysis showed a 3-fold increase in the amount of GM21 expression in NOD vs. C57/BI0 islets (p(0.001). In conclusion, increased ICA antigen expression in NOD islets is paralleled by the increased expression of the GM21 monosialo-ganglioside, but not by the presence of GM3 or GD3. Interestingly, genes encoding for ganglioside synthesis are associated to the Class II region that has been shown to contain at least one diabetogenic gene in the NOD mouse.

To f u r t h e r c h a r a c t e r i s e the m o l e c u l a r n a t u r e of p a t h o g e n e t i c a l l y relevant islet autoantigen(s) we h a v e r e c e n t l y g e n e r a t e d a s e r i e s of m u r i n e m o n o c l o n a l i s l e t cell a n t i b o d i e s [ n = l S ; immunogen= human insulinoma homogenates; screening=pancreas immunohistochemistry]. Differentiation antigens [ICAgs] r e c o g n i s e d by t h e s e N A b s d i s p l a y e d v a r i e d i n t r a c e l l u l a r and ' s u r f a c e - l i k e ' c y t o l o g i c a l d i s tribution; M A b s 1-39 and -45 r e a c t e d w i t h all i s l e t e n d o c r i n e c e l l s , 1-51 and -52 r e a c t e d w i t h p e r i p h e r a l i s l e t a l p h a c e l l s only. B i n d i n g of MAbs 1-39,-45,-51,-52 and H I S L - 5 [ E n d o c r i n o l o g y : 1 2 2 , 1 2 6 3 , 1 9 8 8 ; 100kd p r o t e i n ] t o p a n c r e a t i c i s l e t s w e r e not i n h i b i t e d by p o l y c l o n a l I C A b ( a u t o a n t i body) p o s i t i v e p a t i e n t sera. E x p o s u r e to 100~ for lhr [dry h e a t i n g of p a n c r e a t i c s e c t i o n s ] did not s i g n i f i c a n t l y a l t e r i m m u n o r e a c t i v i t y of i s l e t a n t i g e n s r e c o g n i s e d by h u m a n ICAb and N A b s 1-39 and -52; t h o s e r e c o g n i s e d by 1-45 and H I S L - 5 w e r e less h e a t s t a b l e . The e x t r a o r d i n a r y h e a t s t a b i lity of the i s l e t a u t o a n t i g e n has p o t e n t i a l for e x p l o i t a t i o n in its i s o l a t i o n , and m a y be c o n s o n a n t w i t h its p r o p o s e d c a r b o h y d r a t e ( g l y c o l i p i d / glyeoprotein) nature. P r e e x p o s u r e to pH 2 [ 0 . 0 5 M p h o s p h a t e , lOmin] a b o l i s h e d the i m m u n o r e a c t i v i t y of the a u t o a n t i g e n ; I C A g s r e c o g n i s e d by M A b s 1-45 and -52 w e r e a l s o s e n s i t i v e to l o w pH.

A192

P708

P709

"Antibodies to trypsin solubilised fragments of 64kD antigen in identical twins discordant for Type i (insulin dependent) diabetes" Y.M.Tun, M.R.Christie and R.D.G.Leslie, Westminster Hospital, London and Dept of Biochemistry, University of Oxford.

PANCREATIC GANGLIOSIDES AND AUTOIMMUNITY IN BB RATS: DIFFERENCES BETWEEN DIABETES PRONE (DP) DIABETES RESISTANT (DR) STRAINS AND WISTAR RATS. M. P r e v i t i , F. D o t t a , C. T i b e r t i , R. G i a n a n i , E. A n a s t a s i , G.S. E i s e n b a r t h , L. L e n t i a n d U. Di Marie, University of Rome, Italy and J o s l i n C l i n i c , B o s t o n , USA. Glycolipids may play a role in pancreatic autoimmune phenomena: the monosialoganglios i d e G M 2 - 1 , s i m i l a r l y e x p r e s s e d in h u m a n a n d rat islets, inhibits the ICA binding on human and rat pancreata. The pancreatic ganglioside p a t t e r n in B B - D P , B B - D R a n d a g e matched Wistar rats has been characterized. Acidic glycolipid extracts were analyzed by TLC and HPLC. BB-DP pancreas contained the following gangliosides: GM3 (64.6%), GM2-1 ( 1 0 . 3 % ) , G T I b (6.6%) a n d a g a n g l i o s i d e migrating between GD3 and GDIa standards (22.1%). BB-DR pancreas expressed the above components: GM3 (64.6%), G M 2 - 1 (26.%), G T I b (4.9%), GD3-1a (4.5%). In Wistar rat panc r e a s t h e g a n g l i o s i d e p a t t e r n was: G M 3 (15%), G M 2 - 1 (5%), G M I (10%), GD3 (17%), G D I a (28%), a n a s s u m e d GT3 c o m i g r a t i n g f r a c t i o n (7%) a n d G T I b (15%). Interestingly (i) G M 2 - 1 e x p r e s sion was similar in the rats studied; (2) GD3-1a was high in B B - D P , l o w in B B - D R a n d a b s e n t in W i s t a r r a t s ; (3) GM3 a n d G T I b w e r e similar i n D P a n d DR. G M 2 - 1 p r e s e n c e in DP and DR explains ICA+ sera binding both in DP and DR pancreata. The GD3-1a presence in B B - D P m a y b e a p o t e n t i a l m a r k e r of s u s c e p t i bility to autoimmune diabetes , since genetic studies have shown an association between genes encoding for ganglioside synthesis and c l a s s II M H C g e n e s in t h e B B rat.

Type i diabetes is associated with autoantibodies to a detergent solubilised 64kD islet cell antigen. Partial tryptic proteolysis of 64kD antigen yields fragments of 50kD, 40kD and 37kD. 93% of recent onset diabetics contain antibodies to these tryptic fragments. To define their predictive value we determined the prevalance of these antibodies in 17 identical twin pairs discordant for diabetes at the time of sampling, within 3 years of diabetes of their diabetic twin. Autoantibodies to tryptic fragments were found in I0 diabetic twins (9 to 60kD, 6 to 37kD) and i0 non-diabetic (7 to 50kD, 7 to 37kD) co-twins; of the 8 non-diabetic twins who later developed IDDM (i to 156 months after sampling), all had autoantibodies to at least one islet fragment as compared with 2 of 9 twins who have not developed diabetes (p,O.Ol) and none of 14 normal controls (p
P710 PREVENTION OF CYCLOPHOSPHAMIDE-INDUCED D I A B E T E S IN N O D M I C E B Y A N T I - A S I A L O G M 1ANTIBODY T. M a r u y a m a , K. W a t a n a b e , T. Y a n a g a w a , T. K a s a t a n i , I. T a k e i , Y. S u z u k i , K. K a t a o k a a n d T. S a r u t a , S a i t a m a a n d Tokyo, J a p a n To e l u c i d a t e the r o l e o f n a t u r a l k i l l e r (NK) c e l l s in the p a t h o g e n e s i s of d i a b e t e s in n o n - o b e s e d i a b e t i c (NOD) m i c e , we e x a m i n e d whether treatment with anti-Asialo GM-I a n t i b o d y (AGI) p r e v e n t s c y c l o p h o s p n a m i d e induced diabetes. 0 . 1 m l of A G I ( d i s s o l v e d 3ml of d i s t i l e d w a t e r p e r v i a l ) w a s i n j e c t e d intraperitoneally 2-8 t i m e s a w e e k . Three d a y s a f t e r the f i r s t A G l - i n j e e t i o n , cyelophosphamide (200mg/kg body weight) was injected intraperitoneally. In c o n t r o l s , rabbit immunoglobulin was injected instead of AGI. N o n e o f the 16 A G l - t r e a t e d N O D m i c e d e v e l o p e d d i a b e t e s , h o w e v e r 7 of 16 c o n t r o l s d e v e l o p e d d i a b e t e s two w e e k s a f t e r eyclophosphamide injection (P<0.01). NKactivity of spleen cells from control mice increased significantly after cyelophosphamide injection(P<0.01). In e o n t r a s t ~ N K - a e t i v i t y of s p l e e n c e l l s f r o m A G l - i n j e c t ~ d mice decreased significantly. A l s o , in A G I injected mice, AGI- positive cells disappeared f r o m the s p l e e n as d e m o n s t r a t e d b y immunohistochemieal staining. Flow cytometry analysis of spleen cells from AGI- injected m i c e d i d n o t s h o w the s u p p r e s s i o n of Tlymphoeytes. These results suggest that N K - c e l l s p l a y a r o l e in the p a t h o g e n e s i s of cyclophosphamide-indueed d i a b e t e s in the NOD mice.

A193

PS 28 Cytokines and Immunology in Animals P711

P712

TUMOR NECROSIS FACTOR c~ (TNF) PRODUCTION IN THE BB RAT

ABERRANT BB-RATS

D. Janjic, M. Asfari, J.F. Piguet; Laboratoire Louis Jeantet, Institut de Biochime Clinique, Dpt of Pathology; University of Geneva, Geneva; Switzerland.

TNF~ P R O D U C T I O N

IN D I A B E T E S

PRONE

H.Rothe , K.Fehsel , H.Kolb , Diabetes R e s e a r c h Institute, H e i n r i c h - H e i n e University, DUsseldorf, F.R.G.

Macrophages may play a role in autoimmune destruction of beta-cell. Since Tumor Necrosis Factor (TNFe) is a major macrophage cytokine, its production was studied in vivo and in vitro using prediabetic BB rats as an animal model. 5 weeks old, diabetic-prone (BB/DP) and diabetic resistant (BB/DR) BB rats were challenged i.p with Upopolysacchadde (LPS) 0.05/~g/gr BW and serum TNF measured by bioassay using L929 cells. There was no significant difference between the two groups. Areas under the curve (Uxmin) for a 90 min period (30-120 min); BB/DP (n=5) : 90300_+39750; BB/DR (n=5) : 50100_+14400. TNF release was measured in vitro from both resident peritoneal macrophages (non-elicited) and adherent splenocytes derived from 6 weeks old BB/DP animals with age matched BB/DR and inbred (Wistar/Furth, Lewis, NEDH) rats as control. Adherent splenocyte TNF production was comparable for any groups. For peritoneal macrophages, whereas TNF production was similar to controls at 4 hrs, it was significantly decreased to approximately 20% of controls at 18 hrs. 4 hrs : BB/DP = 112-+ 69, Wistar-Furth = 143 + 82, Lewis = 131 -+92. 18hrs : BB/DP = 11 -+4, Wistar-Furth = 51 _+20 (2p<0.1), Lewis = 41 _+12 (2p<0.05)(n=6 in each group). In summary, prediabetic BB rats did not present a significant alteration in production of LPS-induced TNFc~ activity in vivo. However, peritoneal BB/DP macrophages displayed altered kinetic of TNFe production when assessed in vitro.

Macrophages and t h e i r p r o d u c t s have been i m p l i c a t e d in the p a t h o g e n e s i s of Type I diabetes. Therefore we studied the TNF~ production in diabetes-prone, diabetesresitent BB/WorDand Wistar rats. Following activation in vivo peritoneal macrophages and bone marrow macrophage p r e c u r s o r cells w e r e i s o l a t e d and a d h e r e n t cells s t i m u l a t e d w i t h LPS. TNF~ s e c r e t i o n was d e t e c t e d by a s t a n d a r d b i o a s s a y u s i n g L929 cells. Peritoneal macrophages of d i a b e t e s - p r o n e BB rats s e c r e t e d m u c h h i g h e r amounts of TNF~ (1300 U/106 cells) c o m p a r e d to d i a b e t e s - r e s i s t e n t BB- and W i s t a r - r a t s ( 40 U/106 cells and 52 U/106 cells). A b e r r a n t TNF~ p r o d u c t i o n was seen p r i o r to insulitis. Bone m a r r o w d e r i v e d m a c r o p h a g e s also showed enhanced TNF~ production, indicating an early defect in p r e c u r s o r cells~ Crosses b e t w e e n BB and W i s t a r rats r e v e a l e d that the a b e r r a n t TNF~ p r o d u c t i o n is a recessive trait. The enhanced p r o d u c t i o n of TNF~ m a y affect both, T-cell m a t u r a t i o n and islet inflammation.

P713

P714

in

The P a t o g e n e s i s of Type the NOD/Shi M o u s e : The

1 Diabetes Mel I i t u s L a c k o f TNF~ I n d u c t i o n

K.Nakano, J.Setoguchi, G.Hasegawa, M. Nakai, N. N a k a m u r a , T.Kanatsuna, and M.Kondo. Kyoto Prefectual U n i v e r s i t y of Medicine, Kyoto, Japan. The N O D / S h i m o u s e develops Type 1 d i a b e t e s s p o n t a n e o u s l y (the i n c i d e n c e of diabetes ; 10% of male, 70% of female). To i n v e s t i g a t e the p a t h o g e n e s i s , we m e a s u r e d TNFI a c t i v i t y in the serum o b t a i n e d from 0K432 i.v. mice. The value of NOD (n=35, 17• was significantly lower than that of any other strain ; NON (n=10, 583• and BALB/c (n=9, 184• The value of the female N O D (6.5• was lower than that of the m a l e (21i4.5) (p<0.05). A n d the lack of TNF i n d u c t i o n (<5U/ml) was found in 44% (11/25) of the m a l e NOD and 80% (8/10) of the female, but not found in other strains. These results w o u l d c o r r e s p o n d to the incidence of diabetes. In the other study we m e a s u r e d the level of TNFI a c t i v i t y of peritoneal exudate cells o b t a i n e d from T G C - t r e a t e d mice. A f t e r the i n c u b a t i o n w i t h LPS (ipg/ml), TNF levels of p e r i t o n e a l cells in female NOD (87• and BALB/c (99• w e r e no difference. These results suggest that TNF p r o d u c i n g cells of NOD have normal f u n c t i o n in v~tro,but they are s u p p r e s s e d r e m a r k a b l y in vivo. We c o n c l u d e that this TNFi d e f e c t in vivo w o u l d c o n t r i b u t e to the onset of Type 1 d i a b e t e s m e l l i t u s in NOD mice.

INTERACTION BETWEEN HYDROCORTISONE AND INTERLEUKIN-I ON THE f-CELL FUNCTION IN VITRO. M. Stahl, H. Cheng, S. Linde and J.H. Nielsen. Hagedorn Research Laboratory, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark. Cytokines are supposed to be involved in the pathogenesis of type 1 diabetes, and immunosuppression is implicated in the prevention of the disease. The aim of this study was to see the effect of glucocorticoids on the inhibitory effect of interleukin-i (IL-I) on pancreatic islets in vitro. Since the preculture time is known to influence the bimodal action of IL-I on the insulin release, newborn rat islets were cultured for 1 or 7 days in RPMI 1640 plus 10% newborn calf serum prior to the exposure to 10-6 M hydrocortisone (HC) and/or 500 ng/ml human rIL-l~. The release and biosynthesis of insulin were studied after 7 days in culture. The reduction in insulin release was significantly greater in islets precultured for 7 days than in those only precuitured for 1 day (64% vs. 50% of control islets). The insulin-biosynthesis evaluated by HPLC analysis of 3H-leucine labelled islets after IL-I, HC and IL-I +HC was 56 • 20, 80 • 29, 20 • i0 respectively in % of control islets precultured for 7 days and 117 • 21, 59 • 14 and 21 • 5 in % of control islets precultured for 1 day. In conclusion, the results indicate t h a t the suppression of the ~-cell function by IL-I is augmented by preculture time and by hydrocortisone.

A194

P715

P716

INDEPENDENT EFFECTS OF INTERLEUKIN-I, INTERLEUKIN-6 AND TUMOUR NECROSIS FACTOR ON INSULIN SECRETION.

INTERLEUKIN-I ~ (IL-IB) ACTION ON I S L E T R-CELL FUNCTION MAY BE MEDIATED BY A THIOL PROTEASE

C.Southern, D.Schulster* and l.C.Green; University of Sussex, Brighton, BNI 9QG. * NIBSC,U.K. Interleukin-i and tumour necrosis factor (TNF) impair B-cell function in vitro, they also induce interleukin-6 production from many cell types. We investigated the effect of interleukin-6 alone and in combination with interleukin-i or TNF on insulin secretion in vitro to determine whether interleukia-l's effects are mediated by interleukin-6. Isolated rat islets cultured for 48h were subjected to acute challenges with cytokines (10pM-InM), or cultured with 0.5nM cytokines for 6 or 12h in RPMI-1640 prior to a 20mM glucose challenge. Interleukin-6, unlike interleukin-iB, did not stimulate insulin secretion during a 30min acute exposure in medium containing 8mM glucose. After 6h culture, interleukia-iB inhibited secretion (P<0.01), TNF or interleukin-6 did not potentiate this effect; interleukin-6 alone had no effect. After 12h culture, interleukin-iB and interleukin-6 inhibited secretion [40+5 and 36+5 vs control 77+8 fmol ins~lin/isle~/3Omin, P
N. Welsh, K. Bendtzen and S. Sandler. Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden and Laboratory of Medical Immunology, University Hospital, Copenhagen, Denmark. This study aims to investigate by which intracellular mechanisms IL-1B exerts its action on the B-cell. Isolated rat islets were cultured for 24 h or incubated acutely with human rIL-1B (25 U/ml) in the presence or absence of different protease inhibitors. In acute experiments, 0.1 mmol/l N~-tosyl-L-lysine chloromethyi ketone (TLCK) completely counteracted the stimulatory action of rIL-l{3 on glucose-sensitive insulin release, glucose oxidation and (pro)insulin biosynthesis. TLCK prevented also the rIL-1B induced inhibition of m e d i u m insulin accumulation, glucose-stimulated insulin release, (pro)insulin biosynthesis and glucose oxidation observed after 24 h exposure. The medium insulin secretion was 4100+-710 ng insulin/75 islets x 24 h from control islets; 5400_+910 TLCK; 984_+260 rIL-lg; (P<0.01 vs control) and 3140• rIL-l~ + TLCK (P>0.05). The protective effect of TLCK was not due to a direct interaction with the rIL-IB molcule. The thiol protease inhibitor p-chloromercuriphenyl sulfonic acid (CMPSA; 0.01 mmol/l) partially mimiced the TLCK effect as assessed by glucose oxidation (control: 924-+47, CMPSA: 823_+49, rIL-1B: 246• CMPSA + rIL-IB: 355• pmol glucose/10 islets x 90 rain (P<0.05 vs rIL-l~). Other serine-, metallo- and thiol protease reagents used did not protect against rIL-1B. Nor was rIL-l~ induced [3H]diisopropylfluorophosphate (a serine protease reagent) specific labelling observed. It is concluded that the primary effects of rIL-l~ on S-cells involves activation of protease(s), possibly a thiol protease.

P717

P718

NOT ALL SECRETAGOGUES SENSITIZE PANCREATIC ISLETS TO RECOMBINANT HUMAN INTERLEUKIN-IB. J.Johannesen, S.Helqvist and J.Nerup. Steno Memorial Hospital, Gentofte, Denmark. In IDDM and animal models of IDDM intensive i n s u l i n t r e a t m e n t ( i n d u c i n g " b e t a - c e l l r e s t " ) is protective to beta-cells. We investigated this phenomenon in r e l a t i o n t o t h e b e t a - c e l l toxin interleukin IB (IL-I). Insulin-release and contents of insulin and DNA were measured in isolated rat pancreatic islets exposed to IL-I for 6 d in s t i m u l a t o r y a n d n o n - s t i m u l a t o r y glucose concentrations. Furthermore, IL-I effects were studied during beta-cell stimulation by tolbutamide (250 ~ m o l / l ) , i s o - b u t y l l - m e t h y l - x a n t h i n e (IBMX) (50 ~ m o l / l ) , a n d g l u c a g o n (i0 m g / l ) . A t 3.3 m m o l / l g l u c o s e , 6 0 , 0 0 0 U / I o f I L - I c a u s e d inhibition of insulin-release to 62• of control (p<0.01) from 48h to 6d of exposure, whereas i s l e t D N A c o n t e n t w a s u n a f f e c t e d . A t ii m m o l / l glucose, IL-I inhibited insulin-release to 12• (p<0.05) and decreased DNA content (5.35• versus 8.58• ng/islet, p<0.05). On tolbutamide stimulated beta-cells, IL-I inhibited insulin-release to 36• (p<0.05). In contrast, IL-I effects on beta-cells stimulated with IBMX and glucagon were equivalent to IL-I effects on unstimulated beta-cells. In conclusion, beta-cell stimulation by high glucose or tolbutamide increased beta-cell susceptibility to IL-I, whereas s t i m u l a t i o n b y I B M X a n d g l u c a g o n d i d not.

TOXICITY OF I N T E R L E U K I N - I ~ ON ISLETS AND P R E V E N T I O N BY A R T I F I C I A L M E M B R A N E S

ITS

U . S i e b e r s , T . Z e k o r n , R . G . B r e t z e l , M . R e n a r d y * , H. P l a n c k * , P . Z s c h o c k e * and K . F e d e r l i n . M e d . K l i n i k III U n i v e r s i t ~ t G i e s s e n , ITV D e n k e n d o r f * , FRG I n t e r l e u k i n - i (IL-I) m a y b e an i m p o r t a n t f a c t o r in islet d e s t r u c t i o n in autoimmune diabetes. Immuno isolated islet transplantation inside artificial membranes must p r o t e c t the g r a f t not only from cellular and humoral immuno d e s t r u c t i o n but as well f r o m the toxic effects of c y t o k i n e s , i. 5 0 rat i s l e t s w e r e e x p o s e d to 50 to i000 ng/l r h I L - l ~ . After 2 days a glucose c h a l l e n g e w a s p e r f o r m e d ( 1 6 . T m m o l / l ) and i n s u l i n secretion was d e t e r m i n e d a f t e r 90 minutes. 2. 5 0 i s l e t s w e r e a) k e p t in f r e e f l o a t i n g c u l t u r e or b) encapsulated in Polyetheretherketone hollow fibres (PEEK-HF) and c u l t u r e d for 2 d a y s w i t h or w i t h o u t an IL-I s u p p l e m e n t to the m e d i u m ( l O 0 0 n g / l in RPMII640). Glucose challenge was performed as above. I. After 48h of IL-I exposure glucose stimulated insulin release was significantly suppressed for 5 0 0 and I000 ng/l IL-I c o m p a r e d w i t h c o n t r o l s ( i n s u l i n r e l e a s e per i0 islets: c o n t r o l s 658.0 + 254.3 uU/ml; 500 n g / l 3 3 1 . 8 + 1 5 6 . 6 u U / m l ; i 0 0 0 ng/l 2 1 7 . 5 + 9 9 . e uU/ml; p < 0.05). Low doses of IL-I did not significantly affect insulin secretion. 2. Exposure to i000 ng/l IL-I for 48h did not significantly change insulin release of e n c a p s u l a t e d i s l e t s i n s i d e P E E K - H F ( IL-I 1 0 2 5 . ? + ? ? 2 . 6 u U / m l ; no IL-I 8 2 9 . 2 + 6 7 6 . 8 u U / m l ) . In contrast, a significant suppression of islet function occured w h e r e i s l e t s in free floating c u l t u r e w e r e e x p o s e d to IL-I (IL-I 2 6 5 . 3 + 1 1 2 . 9 uU/ml; no IL-I 1085.? + 465.4 uU/ml). Thus, PEEK-HF effectively p r e v e n t e d II-i t o x i c i t y on i s l e t s in t h e s e in v i t r o s t u d i e s .

A195

P719

P720

INSULIN STIMULATES POLYMORPHONUCLEAR LEUKOCYTES CHEM0KINESIS. F.Cavalot~ M.Trovati~ G.Anfossi, I.Russ% P.Massucco, E.Mularoni, L.Mattiello and G.Emanuelli. Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital, Turin, Italy. Polymorphonuclear leukocytes (PMN) function is impaired in diabetes mellitus. Insulin stimulates glucose utilization, lactate production and glycogen synthesis in these cells. We aimed at evaluating the influence of insulin on PMN chemotaxis in response to formyl-methionylleucyl-phenylalanine (fMLP). PMN were obtained from normal v o l u n t e e r s by d i f f e r e n t i a l sedimentation with 2.5% gelatin.Chemotaxis was evaluated in a 48-well chamber (Neuro Probe) using as a buffer Tris Tyrode containing 0.25% BSA and 5-5 mmol glucose~ and as a chemoattractant 10-9 mol fMLP. The number of cells per high power field (x 400) that migrated through a 5 p pore polycarbonate membrane (Nucleopore) was evaluated. Each insulin concentration was tested in triplicate and 4 fields per well were counted. The values are m+sem of 4 experiments. The number of migrated cells was: at 0 ~U/ml-12.7• at 40 ~U/ml- i~.7• at 400 NU/mI=20.72• .86~ at 4~000 pU/ml=23.2i1.43~ at 40.000 ~U/ml=32.123.75 (ANOVA for repeated measures p (0.O001). The difference between O and 40 NU/ml was significant ( p a i r e d Student's t test: p=O.020). In conclusion, insulin stimulates PMN chemokinesis with a dose-related mechanism. Also physiological insulin concentrations are able to increase PMN activity. Therefore, lack of insulin or decreased insulin action may

Coordinate Expression of 2',5'-oligoadenylate S y n t h e t a s e a n d M H C c l a s s I A n t i g e n s in I n s u l i n producing Cells. V. B o n n e v i e - N i e l s e n , T. D y r b e r g . D e p a r t m e n t of Clinical Chemistry, Odense University Hospital, DK-5000 Odense and Hagedorn Research Laboratory, DK-2820 Gentofte, Denmark T h e i n t e r f e r o n d e p e n d e n t (IFN) 2 ' , 5 ' - o l i g o a d e n y l a tesynthetase (2',5'AS) is a c t i v a t e d in v i r u s inf e c t i o n s . E x p r e s s i o n of t h e 2 ' , 5 ' A S g e n e is highly s e n s i t i v e to ~ - I F N a n d is a c t i v a t e d b y t h e p r e s e n c e o f d s R N A . F r o m A T P t h e e n z y m e p r o d u c e s 2',5' adenine oligomers which inhibit virus replication and cellular protein synthesis. IFN also influences HLA antigen expression on pancreatic B-cells. Thus, a-IFN stimulates class I antigens and u IFN i n c r e a s e s c l a s s II p r e s e n t a t i o n . T h i s d o u b l e effect: 2',5'AS and HLA antigen expression sugg e s t s an i n v o l v e m e n t of t h e 2 ' , 5 ' - I F N s y s t e m in the pathogenesis of IDDM by a changed immune defense against virus associated with an inappropriate HLA antigen presentation. Isolated islets f r o m W i e t a r r a t s o r R I N c e l l s w e r e i n c u b a t e d 18 h w i t h a - I F N (0-i000 U/ml). I s l e t s a n d R I N c e l l s inc r e a s e d t h e i r 2 ' , 5 ' A S a c t i v i t y 4 a n d i0 t i m e s . A 15 f o l d i n c r e a s e w a s i n d u c e d in i s l e t h o m o g e n a t e s b y 200 ~ g / m l p o l y ( I ) : (C). W i t h o u t p o l y ( I ) : (C)the e n z y m e a c t i v i t y w a s u n a f f e c t e d b y i n c r e a s i n g aIFN concentrations. R I N c e l l s e x p o s e d to ~ - I F N (5-200 U/ml) o r y - I F N (i-i00 U/ml) w e r e a n a l y z e d b y F A C S scan. H i g h s e n s i t i v i t y in t h e M H C c l a s s I e x p r e s s i o n o c c u r r e d in r e s p o n s e to y-IFN. A l s o , a - I F N i n d u c e d class I antigen expression but the sensitivity w a s l o w e r as c o m p a r e d to u It is c o n c l u d e d , t h a t t h e I F N - d e p e n d e n t 2 ' , 5 ' A S is e x p r e s s e d c o o r d i n a t e l y w i t h M H C c l a s s I a n t i g e n s in i n s u l i n p r o d u c i n g c e l l s

contribute to the

decreased

PMN

function

in

diabetes

mellitus.

P721 MULTIPLE MEDIATED

P722 L O W D O S E S OF S T R E P T O Z O T O C I N I N D U C E D C E L L IMMUNEREACTION A G A I N S T B - C E L L IN W I S T A R

RATS. ] . I . O s u n a , M. C a s t r o , - P a c h e c o y ]. A n e i r o s .

M.A. M a r t i n e z - O i m o s , M. Garc~a-

W e have studied the effects of combined use of multiple injections of complete F r e u n d adjuvant (0.5 ml) on d a y s i, 8, 15 a n d 22 a n d streptozotocin (STZ) (30 m g / K g ) on d a y s 2, 9, 16 a n d 23, on a u t o i m m u n e

r e s p o n s e a g a i n i s l e t s of Wistar r a t s . The s p l e e n of the d i a b e t i c r a t s w a s d i s s e c t e d a n d e x c i s e d in RPMI 1640. The cell s u s p e n s i o n w a s i n c u b a t e d for 24 h a f t e r the a d d i t i o n g of c o n c a n a v a l i n A (0.5 ug/ml). Between 20-&OxIOv l i v e c e i l s were r e s u s p e n d e d in RPMI 1640 (1 m l ) . When t h e s e c e l l s were i n j e c t e d i n t r a v e n o u s l y in n o r m a l W i s t a r r a t s (n=9), 59.5% (n=5) of then d e v e l o ped d i a b e t e s a r o u n d d a y 20-60, a f t e r s p l e n o c y t e s i n j e c t i o n , By e l e c t r o n m i c r o s c o p y , p l a s m o c y t e a n d l y m p h o c y te i n f i l t r a t i o n w a s o b s e r v e d in p a n c r e a t i c i s l e t s of these rats. Normal p a n c r e a t i c i s I e t s ,~ere "in v i t r o " cultures (24 h) w i t h s p l e n o c y t e s (10 cells/ml) or supernatant (1 ml) of t h e s e c e l I s . Insulin release ( I R I ) w a s m e a s u r e d in c u I t u r e medium. S u p e r n a t a n t i n d u c e a s s i g n i f i c a n t l y IRI i n c r e a s e at 24 h c u l t u r e (60.13+8.1 v s . &0.83+1.4 n g / m l / l O i s l e t s ) w i t h a s i g n i f i cantly-- d e c r e a s e a't 1A h culture (20.51+3.54 vs. 40.83+1.4 n g / m I / l O i s l e t s ) . The p r e s e n c e of s-plenocytes no i~'duce s i g n i f i c a n t l y v a r i a t i o n in IRI d u r i n g 24 h. (56.6~+9.34 v s . 56.20+ 8.10 n g / m l / l O i s l e t s ) . I n c o n c l u sion c-ell-mediated i ~ m u n e r e a c t i o n a g a i n s t B-cells in our experimental model is mediated b y s p l e n o c y t e s release factors ,

PREW]~TION AND TREATMenT OF MULTIPLE LOW-DOSE ALLOXAN-INDUCED A U T O I M M U N E D I A B E T E S W I T H CYCLOSPORIN A N.M.Lalid,P.Dordevid,M.Mostarica-Stojkovid,M.Zamaklar and M.L.Lukid, Institute for Endocrinology and Institute for Microbiology and Immunology, Belgrade Yugoslavia

In this study we analysed the effect of cyclosporin A(CsA) on the induction of autoimmune diabetes with multiple low doses of alloxan (5x25mg/kg bw in Dark August (DA) inbred rats, previously shown to be highinterleukin 2(IL2) - Droc~cers and highly susceptible to the induction of this disease. CsA was given 14 days, 5 or 10 mg/kg bw sc daily, and the treatment was initiated immediately, 7 or 14 days after the induction. In DA rats untreated with CsA, moderate hyperglycemia appeared 1132 days after the induction. In contrast, all DA rats treated with 10mg/kg bw CsA failed to develop the disease and remained normoglycemic+ during the treatment initiated either immediately (8.7-1.2 mmol/l) or 7 days after the induction (7.6t2.1mmol/l). The same treatement with CsA initiated 14 days after the induction did not affect the disease in already hyperglycemic DA rats (14.232.1mmoi/i). Treatment with 5mg/kg bw CsA was found to be ineffective in all DA rats irrespective to the regiment of application. The observed dose-dependent effect of CsA signifies that this type of diabetes could be efficiently prevented and treated with an immunosuppressive agent acting on the level of IL 2 activity, even late after the induction but not after the onset of the disease.

A196

P723 EFFECTS OF CYCLOSPORINE A ON INDUCED HIT CELL /XJ~KALINIZATION. F. Martin a n d L. B e s t . D e p a r t m e n t of Medicine, U n i v e r s i t y of M a n c h e s t e r , M a n c h e s t e r , UK

The mechanisms whereby therapeutical doses of C y c l o s p o r i n e a f f e c t t h e c y t o s o l i c pH ( p H i ) i n a n i n s u l i n s e c r e t i n g cell l i n e ( H I T - T 1 5 ) w e r e s t u d i e d . To m e a s u r e pH i t h e f l u o r e s c e n t d y e BCECF was u s e d . A d d i t i o n of 10 mM g l u c o s e i n d u c e d a t r a n s i e n t a c i d i f i c a t i o n followed b y a r i s e in pH i (0 .1 3 + 0.01 pH u n i t s ; n=6, p< 0 . 0 1 ) , w h i c h was n o t b l o c k e d b y 0.5 ~tg]ml CsA. S i m i l a r l y , 100 nM PMA c a u s e d a p r o g r e s s i v e r i s e i n pH i (0.12 + 0.01 pH u n i t s ; n=6, p< 0 . 0 1 ) . A d d i t i o n of CsA d i d n o t c a u s e d a n y modificatio n of t h e e y t o s o l i c a l k a l i n i z a t i o n o b s e r v e d . In t h e p r e s e n c e of 2 ~M i o n e m y c i n a r a p i d r i s e i n pHi w a s o b s e r v e d (0 .3 3 _+ 0.01 pH u n i t s ; n=6, p< 0 . 0 0 5 ) , w h i c h was s i g n i f i c a n t l y r e d u c e d (50%) w h e n CsA w a s a d d e d 3 rain p r i o r to i o n o m y c i n . More s i g n i f i c a n t r e d u c t i o n (67%) was o b s e r v e d w h e n CsA was a d d e d 45 rain b e f o r e . A d d i t i o n of 40 raM KC1 c a u s e d a n immediate fall in pH i (0.07 + 0.01 pH u n i t s ; n=6, p<0.01) followed b y a n a l k a l i n i z a t i o n (0.12 + 0.01 pH u n i t s ; n=6, p< 0 . 0 1 ) . In t h e p r e s e n c e of CsA no i n t r a c e l l u l a r a l k a l i n i z a t i o n was o b s e r v e d . F i n a l l y in all c o n d i t i o n s a m i l o r i d e c o m p l e t e l y b l o c k e d t h e c y t o s o l i e a l k a l i n i z a t i s n . It is p r o p o s e d t h a t CsA i n h i b i t s t h e Ca2+-dependent p a t h w a y of Na+/H + a n t i p o r t a c t i v a t i o n b u t not p r o t e i n k i n a s e C a c t i v a t i o n i n HIT c e l l s .

PS 29 Epidemiology P724

P725

MORTALITY DURING THE FIRST DECADE OF INSULINTREATED AND INSULIN-DEPENDENT DIABETES MELLITUS WITH ONSET AFTER THE AGE OF THIRTY. A . G . M w l b a k , K. B o r c h - J o h n s e n , B. C h r i s t a u a n d J. Nerup, Steno Memorial Hospital, Gentofte, Denmark. In a historical prospective epidemiological s u r v e y e a r l y m o r t a l i t y a n d c a u s e s o f d e a t h in insulin-treated and insulin-dependent diabetes mellitus were studied. The study population was an incident cohort of all diabetic patients over t h e a g e o f 30 t r e a t e d w i t h i n s u l i n in t h e p e r i o d 1973-77. Classification of the cohort, into insulindependent diabetes mellitus (IDDM), i n s u l i n - t r e a t e d d i a b e t e s m e l l i t u s (ITDM), s h o r t t e r m i n s u l i n - t r e a t e d d i a b e t e s m e l l i t u s (SIDM) w a s performed by use of symptoms, clinical and biochemical signs at the time of diagnosis. The cohort included 1234 persons (650 m a l e s , 584 f e m a l e s ) , 1 6 . 4 % c l a s s i f i e d a s IDDM, 5 5 . 1 % I T D M and 27.5% SIDM. Median diabetes duration within t h e c o h o r t w a s 1 2 . 5 y e a r s a n d 889 p e r s o n s h a d d i e d . T h e o v e r a l l S M R is 377, b u t d e p e n d e n t o n the DM-classification and age at onset. The c a u s e s o f d e a t h s h o w e d a s i m i l a r p a t t e r n in t h e three groups. The three most important causes w e r e c a r d i o v a s c u l a r d i s e a s e s (50%), d i a b e t e s m e l l i t u s (20%), a n d c a n c e r (10%). W h e n c o m p a r e d t o an age- and sex-matched background population there was an excess mortality due to diabetes, but not to cardiovascular disease, within the first years of diabetes.

Sudden unexplained deaths of young people with IDDM. R B Tattersall, G V Gill and W D Alexander. Nottingham, U.K. We have investigated UK reports of an increase in sudden and unexplained (no anatomical cause at autopsy) deaths of young people with IDDM under age 50. Ascertainment was from relatives, physicians and pathologists. Data on circumstances of death~ control and complications, insulin regimen and history of hypoglycaemia was obtained from relatives, clinical records and autopsy reports. After excluding DKA and suicide, there were 2 cases of fatal hypoglycaemic brain damage, 3 in which information was inadequate and 22 in which a person with IDDM was found dead in bed. Ages of the 22 "dead in bed" were 12 to 43 and 15 had been seen to be well within 12 hours of death. 21/22 slept alone and 21 were found in an undisturbed bed. 14 were diabetic less than 15 years and only 2 had severe complications. HbA1 was measured within 6 months of death in 12 and was 6.2 - 12.6%. Drug and alcohol screens were negative in all. 14 of the 22 death certificates included the word hypoglycaemia and all but 2 mentioned IDDM. The cause of death is uncertain but we will compare with other series and discuss difficulties in diagnosing hypoglyeaemia post mortem.

A197

P726

P727

DIABETES MORTALITY IN ENGLAND AND WALES: YEARS

INFANT FEEDING OF FINNISH DIABETIC CHILDREN DIAGNOSED BEFORE THE AGE OF 7 YEARS COMPARED TO CONTROL CHILDREN S.M. Virtanen, L. R[s[nen, A. Aro~ J. Lindstr6m, J. Tuomilehto, H.K. Akerblom and the :~Childhood Diabetes in Finland ~' Study Group. Department of Nutrition and the Children's Hospital~University of Helsinki and National Public Health Institute, Helsinki~ Finland.

TRENDS OVER i0

JM Stephenson and JH Fuller. Department of Medicine, University College London, UK.

Community

There have been major efforts to improve diabetes care over the last decade and in an attempt to evaluate their effectiveness, trends in diabetes mortality from 1975-6 to 1985-6 have been studied for England and Wales using multiple cause coded death certificates. This procedure overcomes many of the defects of routine mortality data based on a single "underlying cause" of death, since all conditions mentioned on the death certificate are analysed. The total number of certificates mentioning diabetes were 18195 (men) and 25141 (women) in 1975-6 compared to 21781 (men) and 25617 (women) in 1985-6. Age-specific rates of mention of diabetes in women showed a significant fall, giving an age-standardised reduction in 1985-6 of 12% (95% CI 10% to 13%), but no change in men. The proportion of certificates with circulatory diseases as underlying cause of death decreased from 51% to 48% in men and from 52% to 48% in women. Mortality (underlying cause) from specific complications is consistently high for ketoacidosis in the elderly, decreasing for diabetic coma but increasing for nephropathy. This data may indicate a notable fall in diabetes mortality in women, but not men, in England and Wales over the last 10 years.

P728 DIFFERENCIES IN EPIDEMIOLOGY OF DIABETES BETWEEN FINNISH MALES AND FEMALES, 0-14 YEARS

The feeding in infancy of 103 newly diagnosed diabetic children and their birthday and sex matched controls randomly selected from Finnish population registry was compared. Practically all diabetic and control children had been breast-fed. The proportion of diabetic children exclusively breast-fed for at least 3 months (75% vs. 89Z~ p<0.04, McNemar test) or 4 months (435{ vs. 61Z, p<0.04)was smaller compared to controls. A smaller proportion of diabetic than control children was breast-fed up to the age os 7 months (54Z vs. 72%, p<0.02). A greaZer proportion of diabetic compared to control children had got supplementary milk feeding before the age of 2 months (15Z vs. 5%~ p<0.005) or 3 months (25% vs. 12%, p<0.05) or 4 months (38% vs. 21%, p<0.03). The proportion of mothers educated more than 13 years was greater (p<0.04) among the controls. There were no differences between cases and controls in the attendance to postnatal ward, in mean birth weight or in mean age of the mothers at the time of the delivery. To conclude~ these results suggest that an early introduction of cow milk might be associated with an increased risk of type I diabetes.

P729 LONG-TERM TREND OF INCIDENCE, PREVALENCE, AND MORTALITY DF

TYPE I DIABETES (INSULIN-DEPENDENT) IN GERMAN CHILDREN AND ADOLESCENTS

J.Tuomilehto, A.Reunanen, R.Lounamaa, M.Rewers, H.K.Akerblom and the DIME Study Group. National Public Health Institute, E l i m ~ e n k a t u 25A, 0 0 5 1 0 H e l s i n k i , F i n l a n d .

O. Michaelis and E. Jutzi, Central Institute of Diabetes, Karlsburg/DOR In order to confirm published rising morbidity of insulin-

During 1987-89 the age adjusted incidence of Type i diabetes was 41.2/100 000/year in m a l e s ( M ) a n d 3 4 . 5 i n f e m a l e s ( F ) u n d e r 15 y e a r s ; M / F r a t i o w a s 1.19. N a t i o n a l D r u g Register data during 1965-68 showed M/F ratio 1 . 0 6 a n d d u r i n g 1 9 7 5 - 7 8 1.09. B e t w e e n 1 9 6 5 - 6 8 and 1987-89 the relative increase in incidence was 63% a n d 46%, a n d t h e a b s o l u t e i n c r e a s e 1 4 / 1 0 0 0 0 a n d I 0 / i 0 0 0 0 0 i n M a n d F, respectively. The relative increase was l a r g e s t i n 0 - 4 y e a r s old: 1 0 3 % i n M a n d 6 3 % i n F, a n d M / F r a t i o c h a n g e d f r o m 0 . 9 2 t o 1.15. The absolute increase was smallest in 10-14 y e a r s old. Thus, t h e i n c i d e n c e r a t i o 1 0 - 1 4 / 0 - 4 y e a r s o l d d e c r e a s e d f r o m 2 . 4 t o 1.9 i n M a n d f r o m 2 . 0 t o 1.5 i n F. T h e r e w e r e p e a k s i n i n c i d e n c e i n 1978, 1 9 8 3 a n d 1 9 8 6 w i t h M / F r a t i o 1.20. T h e i n c r e a s e s i n c e 1 9 6 5 w a s related to a period effect, but not to a birth cohort effect. Non-linear associations between incidence and age was found in both sexes w i t h t h r e e p e a k s w h i c h o c c u r r e d 1-2 y e a r s o f a g e e a r l i e r i n F t h a n i n M. O f 8 4 6 n e w c a s e s registered during 1987-89 5.8% had a father and 2.4% mother with Type i diabetes, similar for male and female cases. In multivariate analyses the age at diagnosis was significantly lower in female than male cases, but it did not depend on either paternal or maternal diabetes.

the annual rates of incidence, prevalence, crude and rela-

dependent childhood diabetes in Northern Europe countries,

tive mortality of Type I diabetics under age of 20 in the population of the GOR were analysed over the time period 1960 - 1989. Oata sources are based on the national diabetes registry, which records all newly diaonosed, known: and deceased diabetics annually with an estimated ascertainment over 90 %. The incidence rates rose from 2,2/105 to 5,5/I05 between 1960 and 1974 in children aged 0 - 9, from 5,6/105 (1960) to 12/105 (1989) in those abed lO - 19 years. The age distribution was bimodal with a main incidence peak at 12 years and a second one at 18 - 19 years. Female sex dominated between ages ii - 15, male Type I diabetics between ages 14 - 19. A North-South difference was not evident. Prevalence data revealed a rise from 8/105(1960) to 22/i05 (1975) in children aged 0 - 9 years, a linear rise from 38/i05 (1960) to 12O/lO 5 (1989) in i0 19 years old children. After an initial decrease the mortality rates remained constant. Conclusion: Dissimilarities in rising trend of Type I diabetes during past 15 years underline the role of pubertal hormonal changes for diabetes onset in childhood.

A198

P731

P730 THE INCIDENCE OF CHILDHOOD DIABETES MELLITUS IN WIELKOPOLSKA,POLAND

1970-1989.

M.Walczak,M.Rewers

and D.Wo~nicka.Department

Pediatrics,Academy

of ~edicine,Pozma~,Poland.

of

This study presents surveillance of Type I /insulin-dependent/ diabetes in childhood in Wislkopolska, /population 3.3 million/ over a twenty year long period 1970-1989.From multiple data sources,721 incident cases of insulin-treated diabetes under the age of 15 years were identified.The overall case ascertainment,estimated by a capture-recapture method,was over 96%. Fewer than 2% of the patients presented with atypical diabetes or imsulin dspendemcy associated with other colditions amd syndromes.The annual incidence r~tes varied considerably from 2.0/100,000 /95%.C.I.-1.13.4/I00,000/ in 1973 to 7.3/I00,000 /95%.C.I.-5.7-9.4/ 100,000/ im 1987 and were among the lowest im Europe. A non-linear temporal pattern was noticed with two major epidemios culminating in 1978 and 1987.The temporal c h a ~ e s affected proportionally both sexes and all age groups.The observed lag time between temporal changes suggests that the incubatiom period of diabetes increases by approximately I year per each 5 years of ags.Am outbreak of childhood diabetes reported from the area in 1982 subsided by 1986.However,inn987 it was followed by a new epidemic.The observed pattern of childhood diabetes in Poland resembles considerably this of an infectious disease.

CONFLICTING EVIOENCE FOR ENTEROVIRUS AS THE CAUSE OF CHILOHO00 OIABETES EPIOEMIC IN PDLANO M. Rewers, M.A. Pallansch, E. 8ogaczy6ska, S.L. Patrick, L.A. Anderson, M. Walczak and R.E. LaPorte. Academy of Medicine, Poznad, POLAND Centers for Oisease Control, Atlanta, CA, and University of Pittsburgh, Pittsburgh, PA, U.S.A. Enteroviruses were evaluated as a plausible cause of a childhood diabetes epidemic in Poland. Here we report ecological association between the epidemic and the incidence of enteroviral meningitis (EVM) and the results of a case-control seroepidemiological study. Outing 1975-89, all incident cases of diabetes (n=256) and EVM (n=859) under age 15 were registered in Pozna6 province (l.3m). Every year, I Y U 2 - ~ , the incidence OE diabetes was elevated by JU-~O~ and EVM risk was increased 3-6-times, compared to the respective means for 1975-81. The two diseases displayed strikingly oarallel seasonal and annual patterns. Oiabetes "epidemics" were bimodal, with first peak overlapping ma~or EVM outbreak and the other following 3-6 months later. Time-space clustering was noted of diabetes and EVM, though no child presented with both. IgM and IBA antibodies for Coxsackie 81-6, ECHO11 and EV71 viruses were determined using monotypic detectors in 98 diabetics diagnosed 3/82-5/86 and 67 controls. During 1983-84, Coxsackie 85 IgM was weakly associated with diabetes in the area (0R=3.2, 95BCI i.i-8.8). No other difference was found in the proportion of raised IgM or IgA titers between cases and controls. It is unlikely that the diabetes epidemic was specifically caused by any of the enteroviral serotypes evaluated.

P732

P733

INFECTIONSMAYBERESPONSIBLEFORTHESEASONALITYOFTYPE1

C L I N I C A L F I N D I N G S SUPPORTING G E N E T I C - L I N K A G E BETWEEN TYPE-1 AND TYPE-2 DIABETES

DIABETES A.O Afoke 1 , J Ludvigsson1, J Hed 2 and B Lindblom 3

Deptsof 1Paediatdcs,2CBnicalImmunology,Link6pingUniversity,3StateInstitute for Blood Group Serology, Link~ping, Sweden.

Type 1 diabetes is diagnosed more often during certain seasons, Patients with onset during incidence peaks seem to loose beta cells rapidly. Not only autoimmunity but also an infectious process might cause this destruction. Our aim was to elucidate this further. Patients & Methods: History of infections before diagnosis of diabetes was ascertained from 36 patients aged 0.8-15.99 years who got diabetes during incidence peaks (epidemic patients) and 56 patients of similar age who were diagnosed in between peaks (non-epidemic). Blood was drawn at diagnosis and at regular intervals for 4 years. HLA-typing was done, lymphocytes were classified in sub-populations and serum immunoglobulins G, M and A were determined. R e s u l t s : More epidemic (50%) than non-epidemic (29%) had infections before diagnosis of diabetes (p<0.01), and epidemic patients had higher IgG (11.28+2.9) and IgM (1.97_+0.77) than the non-epidemic group (9.90+2.30 and 1.31+0.58 resp; p<0.001) both at diagnosis and during the first 9 months. HLA-DR4 was found in 93% of patients with preceeding infections but only in 57% of the others (p<0.05). Conclusion: Infections may contribute to diabetes in children diagnosed during incidence peaks especially in patients with HLA-DR4.

i. Satman, E. Ano~lu, T. Yflmaz, E. Ozer, Y. Altunta~, K. Kar~da~ and S. Devrim. Department of Diabetes, Istanbul Medical School, Istanbul University. Between January 1, 1988 and February 28,1990; 200 Type-1 (82 female, 118 male; mean age:27.1 + 7.0 and mean for duration of diabetes: 7.6 + 7.0 years) and 625 Type-2 ( 343 female, 282 male; mean age: 57.1 + 10.3; mean for duration of diabetes: 7.4 + 7.3 years) cases have been enrolled in the records of our outpatient-clinic. The family history of the cases were questioned. Data from these pedigrees covering 17301 people were categorized using a D-base Ill-plus data-base-system. Different familial occurance ratios were compared trough a xMest. In general 53.5 % of Type-l-diabetics and 59.5 % of Type-2-diabetics had at least one diabeticrelative (p>0.05, no significance between the two ratios). In Type-1 cases, 8.5 % had a diabetic-mother, 9.3 % a diabetic-father, 11.5 % at-least one diabetic-sibling and 1.5 % at-least one diabetic-child. The incidence in these first-degree relatives of Type-2 cases were respectively 21.6 %, 12.8 %,30.1% and 4.0 % (differences in ratios of Type-1 and Type-2 were significant, p< 0.05). As to the incicence in second and third-degreerelatives; the occurance for Type-1 were 29.5 and 19.5 %; whereas, for Type-2 23.7 and 12.5 % ( no statistical significance). When the diabetic relatives of Type-1 patients were classified according to the type of diabetes; 91.1% were Type-2-diabetics. In the families of Type-2mothers, the incidence of Type-l-children was 5.4 %. The incidence of having a Type-2-sibling among Type-l-patients was 6.4 %. Both of these n u m b e r s are significantly higher (0.01
A199

P734

P735

A P R O S P E C T I V E STUDY OF L I P O P R O T E I N S U B F R A C T I O N S A N D C A R D I O V A S C U L A R O U T C O M E IN DIABETES. NJ Morrish; M Mattock, LK Stevens**, JH Fuller**, RJ J a r r e t t & H Keen. Unit for M e t a b o l i c M e d i c i n e & *Dept of C o m m u n i t y Medicine, UMDS (Guy's Campus), & **Dept of C o m m u n i t y Medicine, U n i v e r s i t y College, London, UK. We report on r e l a t i o n s h i p s b e t w e e n b a s e l i n e l i p o - p r o t e i n levels and c a r d i o v a s c u l a r outcome after a m e a n 8.33y follow-up in 122 c a u c a s i a n s w i t h diabetes. Fasting serum was s e p a r a t e d ultra-centrifugally into v e r y low d e n s i t y l i p o p r o t e i n (VLDL), low d e n s i t y l i p o p r o t e i n (LDL) and high d e n s i t y l i p o p r o t e i n (HDL) fractions. In age/sex adjusted Cox r e g r e s s i o n V L D L t r i g l y c e r i d e was a s s o c i a t e d with a l l - c a u s e m o r t a l i t y in Type 2 diabetes (p<0.05) as was total t r i g l y c e r i d e in the whole group (p<0.05). V L D L c h o l e s t e r o l was a s s o c i a t e d w i t h a l l - c a u s e m o r t a l i t y in Type I diabetes (p<0.05). In Type 2 d i a b e t e s H D L c h o l e s t e r o l had an inverse a s s o c i a t i o n (p<0.05) w i t h c a r d i o v a s c u l a r death, a s s o c i a t i o n s w i t h LDL-and total c h o l e s t e r o l w e r e also inverse but w e r e non-significant. No a s s o c i a t i o n w i t h m y o c a r d i a l i n f a r c t i o n or total i s c h a e m i c heart disease reached s i g n i f i c a n c e in logistic regression. For new ischaemic heart d i s e a s e the a s s o c i a t i o n s with total and LDL c h o l e s t e r o l w e r e positive, and inverse for H D L cholesterol. The t o t a l / H D L c h o l e s t e r o l ratio was a b e t t e r p r e d i c t o r of ischaemic heart d i s e a s e than total cholesterol alone. Even t h o u g h small in number, these results suggest that cholesterol s u b f r a c t i o n risk factors in n o n - d i a b e t i c g r o u p s may be the same in diabetes and that t r i g l y c e r i d e rich lipoproteins may be pathogenic in diabetes.

I M P R O V E M E N T IN G L U C O S E T O L E R A N C E A F T E R O N E Y E A R IN A H I N D U C O M M U N I T Y IN D A R ES S A L A A M , TANZANIA: R A N D O M O R R E A L ? KL Ramaiya*, A B M Swai*, DG McLarty* and KGMM Alberti**. *Department of Medicine, University of D a r e s Salaam,Muhimbili Medical Centre, Dares Salaam, Tanzania. **Department of Medicine, University of Newcastle upon Tyne,Newcastle upon Tyne, LrK. Debate has surrounded the diagnostic and prognostic significance of impaired glucose tolerance (IGT). To examine this, an OGTT (75 g) was performed in 218 Hindu subjects with known high prevalence of IGT and Type 2 (non insulin dependent) diabetes in Dar es Salaam. O G T T s were repeated one year later in 184 (84.4%). In the first survey 47 (25.5%) of the 184 subjects had IGT, 21 (11.4%) bad Type 2 diabetes (known and newly-diagnosed) and 116 (53.2%) had normal glucose tolerance (NGT). In the repeat survey only 14 (7.6%) had IGT, 14 (7.6%) had diabetes and 156 (84.8%) NGT. Out of 47 IGT subjects, 37 (78.7%) reverted to NGT and only one (2.1%) progressed to Type 2 diabetes. Of the 21 Type 2 diabetes subjects, 4 (19%) had reverted to IGT and 4(19%) to NGT whilst 13 (61.9%) remained diabetic by the OGTT. However only one (0.9%) of the 116 N G T subjects in the first s u r v e y had p r o g r e s s e d to I G T w h i l e 99.1% remained NGT. There was a significant fall in mean fasting and 2h post glucose blood glucose levels, and in mean systolic and diastolic blood pressure levels, but no significant difference in body mass index (BMI), s e r u m total cholesterol and triglyceride levels b e t w e e n surveys, suggesting little change in diet. Further studies will be needed to establish whether the improvement at 1 year was genuine or just reflects poor reproducibility of the OGTI'.

P736

P737

REPRODUCIBILITY OF GLYCATED HAEMOGLOBIN A 1 C IN A HEATHLY POPULATION

FAT WEIGHT MEASUREDBY BIOELECTRICAL IMPEDANCE IS PREDICTIVE OF IMPAIRED GLUCOSETOLERANCE. A.Mitrakou, G. Raptis, E. Ganotakis and S.A. Raptis. 2nd Dept. Internal Medicine and Propaedeutic, Evangelismos Hospital, University of Athens, Athens, Greece. We investigated the stimulated glucose and insulin responses to a 2h-oral glucose tolerance test (OGTT 75 gr) in 54 individuals with fasting plasma glucose<140 mg/dl. Selection was based on matching for age, body mass index and percent body fat. Body composition was assessed by b i o e l e c t r i c a l impedance with a BES-2OOZ Biological Ohm Meter. The measurement yielded estimates for f a t - f r e e body mass, absolute fat weight (FW) and total body water. The subjects were separated into three groups according to t h e i r response to the OGTT by applying the American Diabetes Association c r i t e r i a . Group A: 14 subjects with normal response (N), Group B: 20 subjects with impaired response (IGT) and 20 subjects with non insulin dependent diabetes (NIDDM). Analysis of variance established a s t a t i s t i c a l l y s i g n i f i c a n t difference in postprandial glucose concentrations expressed as AUC (N:l.665• IGT:2.340• NIDDM:3.095• and in fat weight (N:16.37• kg, IGT:18.44• NIDDM:21.59• among the three groups, regardless the identical percent body fat. Fat weight was highly correlated with postprandial glucose increase in each group and with increased insulin levels in the IGT group (FW vs glucose:r=O.805, p
D. SIMON, C. SENAN, M. SAINT-PAUL AND L. PAPOZ - INSERM U21, 16 avenue Paul Vaillant Couturier, 94807 Villcjuif - INSEtLM U91, 94000 Cr6teil -DASES, Bd Saint-Marcel, 75013 Paris, France. Reproducibility of H b A l c in heathly subjects has only been evaluated by coefficient of variation, a poor tool for reproducibility assessment and never by the best index, namely the intraclass correlation coefficient. Therefore, a study has been performed including 202 non diabetic subjects (42.6% males ; 38.1 + 0.5 years*) examined twice (delay = 167 _+4 days). At first exam, H b A l c and Fasting Plasma Glucose (FPG) were equal to 5.02% + 0.04% and 5.0+_0.03 retool/1. Coefficients of variation between both exams were : 8.2% (HbAlc) vs 5.7% (FPG) while the intraclass correlation coefficient (identical to correlation coefficient when exams once repeated) was low for H b A l c : 0.35 compared to 0.67 for FPG (p<0.0001 each). Within subjects variances and between subjects variances were respectively : 16.7 and 8.8 (HbAlc), 26.6 and 50.0 (FPG). Concerning H b A l c , subjects reexamined within the erythrocyte life span (99 + 3 d a y s ) , and after 4 months (195 + 4 days) showed respectively : intraclass correlation coefficients = 0.60 vs 0.28 ; within subjects variances = 9.3 vs 19.6 ; between subjects variances =13.3 vs 7.26. However H b A l c was not linked to haematologic factors : correlation coefficients with erythrocyte count and mean corpuscular volume respectively equal to 0.03 and 0.02 (non significant). No seasonal variation explained H b A l c variability. The poor reproducibility of H b A l c is related to a regression to the mean process in this heathly population but it could prevent from using H b A l c as a tool for diabetes diagnosis. Nevertheless, by applying a cut-off level at the 97.5 percentiles of H b A l c and FPG values, 2 different subjects had twice and 4 different subjects had once over limit values for each parameter. Therefore, H b A l c variability could be higher than FPG's in normal subjects but identical when glucose tolerance is impaired. * m _+sem for all quantitative parameters

A200

P~8 BODY FAT CONTENT, FAT TOPOGRAPHY AND FASTING I N S U L I N IN 1293 1 8 - Y R O L D MEN. M.Zenere, E.Bonora, P.Branzi, M.Bagnani, L . M a g g i u l l i , P . M o g h e t t i and M . M u g g e o . D e p a r t m e n t of Metabolic Diseases, University of Verona, Italy. Recently, it has been reported that fasting insulin l e v e l s i n c r e a s e as the a m o u n t of trunk fat i n c r e a s e s . A i m of the p r e s e n t s t u d y w a s to correlate fasting insulin with both total body fat c o n t e n t (TBF) and fat t o p o g r a p h y (FT) in a population o f y o u n g men. For t h i s p u r p o s e , in 1293 1 8 - y r old m e n (6B 88 of m e n b o r n in 1 9 7 0 and living in V e r o n a ) the f o l l o w i n g anthropometric parameters were measured: weight, height, TBF (bioelectrical impedance), body circumferences (waist, hips, arm, thigh), and skinfolds (subscapular, tricipital, bicipitai, suprailiac, paraumbiiical). B o d y m a s s i n d e x (BMI), w a i s t to hip c i r c u m f e r e n c e r a t i o (WHR), subscapular to t r i c i p i t a l s k i n f o l d r a t i o (STR) w e r e c a l c u l a t e d . P l a s m a i n s u l i n in the m o r n i n g a f t e r an o v e r n i g h t fast was also measured. Insulin was significantly and e q u a l l y c o r r e l a t e d with BMI and T B F ( r = 0 . 2 4 , p the effect of TBF per se. Conversely, partial correlations between insulin and TBF t a k i n g into a c c o u n t W H R or S T R remained highly significant (r=0.21 ~nd 0.19, respectively, p
P739 THE HYPERINSULINAEMIAOF ASIANS IS PRESENT BY THE AGE OF 25 YEARS D. Simmons, H. Dhar and D.R.R. Williams. Sheikh Rashid Diabetes Unit, Oxford; Dept of Community Medicine, Cambridge University. Asians have a high prevalence of diabetes. The plasma insulin was related to age, ethnic group and body mass index (BMI) in a subgroup from a house-to-house survey of the adult population of F o l e s h i l l , Coventry (4395 Asians, 5504 Europids). 84% Asians and 64% Europids were screened for diabetes. Subjects with a casual glucose ~5.0 mmol/l ~2-hours p o s t p r a n d i a l l y , ~ 6 . 0 mmol/l <2-hours postprandially and 10% of others were i n v i t e d to a glucose tolerance test (response: 73% Asians, 65% Europids). C a p i l l a r y insulin samplings commenced h a l f way through the survey. Small and haemolysed samples were rejected. After excluding diabetic and impaired glucose t o l e r a n t subjects, (237 Europid, 229 Asian) fasting and (212 Europid, 235 Asian) 2-hour samples were a v a i l a b l e . Europids were s i g n i f i c a n t l y older (53• vs 40f13 years) and f a t t e r (26.5• vs 25.5• kg.m -2) with a lower median 2-hour glucose (5.4 vs 5.8 mmol/l). Asians had higher fasting (10.8 vs 8.4 mU/l) and 2-hour insulin (57.5 vs 32.8 mU/l) than Europids (Mann-Whitney's p
P740

P741

E A R L I E R O N S E T IN M A T E R N A L I N H E R I T E D T Y P E 2 (NON INSULIN-DEPENDENT) DIABETES H.H.P.J.Lemkes, F.Ong and D.van der Pol Pep. E n d o c r i n o l o g y , U n i v e r s i t y H o s p i t a l , L e i d e n , The N e t h e r l a n d s

SEX

M O D Y a n d E O D (early o n s e t d i a b e t e s ) r e p r e s e n t e x a m p l e s o f o e n e t i c i n f l u e n c e s o n age at o n s e t i n t y p e 2 (non i n s u l i n - d e p e n d e n t ) d i a b e t e s . B o t h are s e l e c t e d p r i m a r i l y b y a g e at d i a g n o s i s . In the p r e s e n t s t u d y w e e x a m i n e d i n f l u e n c e s o n age at o n s e t in a h o s p i t a l - b a s e d , o t h e r w i s e u n s e l e c t e d t y p e 2 (non i n s u l i n - d e p e n d e n t ) diabetic population. I n f o r m a t i v e p e d e g r e e d a t a w e r e o b t a i n e d f r o m 165 non-related caucasian probands by standard interv i e w . A o e at o n s e t w a s n o t r e l a t e d to i n i t i a l b o d y w e i g h t , C p e p t i d e o r t r e a t m e n t m o d a l i t y in probands. 5 pedegrees fulfilled MODY criteria. Remaining pedegrees were divided into: group 0 (n=53) : n o f a m i l y h i s t o r y , g r o u p P (n=33) : p a t e r n a l i n h e r i t e d , g r o u p M (n=44) : m a t e r n a l i n h e r i t e d ( m o t h e r d i a b e t i c o r m o t h e r c a r r i e r , i.e. m o t h e r n o t d i a b e t i c , b u t d i a b e t e s in h e r f a m i l y ) a n d g r o u p P M (n=30) : d i a b e t e s in b o t h p a r e n t a l lines. M e a n (+SD) p r o b a n d a o e s at o n s e t w e r e : 0 : 5 2 . 7 + 12, P 7 5 0 . 2 + 1 1 , M :42.5+9, P M : 4 4 . 5 + 1 4 y e a r s . -M vs P : p < 0 7 0 2 . The difference remained 8 years when diabetic mothers and carrier mothers were compared with corresponding fathers. This renders an i n t r a u t e r i n e (unrecognized gestational diabetes) f a c t o r i m p r o b a b l e . In c o n c l u s i o n : l i k e i n H u n t i n g t o n s d i s e a s e , t h e r e is a p a r e n t a l g e n d e r e f f e c t o n age at o n s e t of t y p e 2 (non i n s u l i n - d e p e n d e n t ) diabetes. Parental imprinting (sex-linked methylation) of d i a b e t e s - r e l a t e d g e n e s m i g h t p r o v i d e an explanation.

INFLUENCE

MYOCARDIAL A

ON P R O G N O S I S

Rynkiewicz,

P

Wewn~trznych University Data

Sleight,

AM

International were

prognostic

importance

used

patients

(D)

course

infarction

was

D

75.5Z

15.1Z b u t o n l y for

In

ND

for

and

have phase

rate

(during

14 d a y s

among

diabetic

males.

87. IZ males. from

the was

(p<0.001). was

discharge

worse

myocardial

In ND

?. iX a n d from

the

a n d D 9. IZ males.

late m o r t a l i t y

much

the

rate w a s

the D f e m a l e s

for m a l e s

for

D, 19@

Among

In ND g r o u p one and

5.8Z

of

infarcted males.

(ND) males

7037

survival

gZ D f e m a l e s

subjects

females acute

571

81.2Z.

females. A f t e r

males

and

(85Z)

9Z in D m a l e s

died

the

diabetic

i n f a r c t i o n . The

females

early mortality

@.0Z

Infarct

in

88Z

to the CCU)

hospital

sex

year

in D m a l e s

Early mortality

group

with

372

one

survived

admission

of

of

estimate

females

and

the

and

year

Chor6b

Department

in g055 n o n d i a b e t i c

compared

(35Z)

females

Study to

with myocardial

subjects, 2 0 1 8 ( 2 2 Z ) (78Z)

II K l i n i k a

Gda~sk, C a r d i a c

Survivals

females

WITH

of O x f o r d

from

of

IN D I A B E T I C S

INFARCTION

rate was

females. prognosis

5. gZ

Diabetic during

infarction

than

A201

PS 30 Hypertension P742

P743

AMBULATORY BLOOD PRESSURE IN MICROALBUMINURIC TYPE i (INSULIN-DEPENDENT) DIABETIC PATIENTS. K.W. H a n s e n , C.K. C h r i s t e n s e n , P . H . A n d e r s e n , J . S . C h r i s t e n s e n a n d C.E. Mogensen. Medical D e p a r t m e n t M, A a r h u s Kommunehospital a n d Medical D e p a r t m e n t I I I , A a r h u s A m t s s y g e h u s , DK-8000 A a r h u s C, Denmark. T w e n t y - f o u r h o u r amliulatory blood p r e s s u r e (24hBP) was p e r f o r m e d in microalbuminuric p a t i e n t s (microalb) (n=35) with the aim of comparison with a matched g r o u p of n o r m o albuminuric p a t i e n t s (normoalb) (n=29) a n d controls (n=24). 24hBP was m e a s u r e d b y oscillomeiry. P a t i e n t s had n e v e r received ant• t r e a t m e n t . Urine albumin e x c r e tion (UAE) f o r microalb ( geometric mean) was 50.4 ]lg] s i n , UAE normoalb 5.7, UAE controls 4.7. BP ( s y s t o l i c ] diastolic) m e a s u r e d oscillometrically in the clinic was not significant d i f f e r e n t b e t w e e n microalb (132179 mmHg) and normoalb (127176) (p=7%). In c o n t r a s t 24hBP in microalb was h i g h e r (133/79) t h a n f o r normoalb (124174) (p95 mmHg (p<2%). No 24hBP difference b e t w e e n normoalb a n d controls (120173) was f o u n d . S t a n d a r d deviation of day time s y s t o l i c BP was not d i f f e r e n t b e t w e e n the g r o u p s . A u s c u l t a t o r y systolic clinic BP f o r the pooled diabetic g r o u p did b o r d e r l i n e correlate with UAE (r=0.24, p=5.1%) w h e r e a s systolic 24hBP d i s c o v e r e d a clear correlation (r=0.46, p<0.1%). This s t u d y s h o w s t h a t BP in microalb is not more labile, b u t elevated d a y and n i g h t as a parallel s h i f t of 24h BP c u r v e .

COMPARISON

OF

THE AUTONOMIC

THE

BLOOD

PRESSURE

THE

YOUNG

TYPE

AND

FUNCTION

HEART

RATE

TESTS

WITH

VARIABILITY

IN

I DIABETICS

J. F u r m a f i s k i , K . N a r k i e w i c z , A. R y n k i e w i c z , B. K r a p a -Wojciechowska, Wewn~trznych In

13

males

mellitus blood

(age

Kliniks

31•

with

8•

type

years)

were

monitoring

calculated.

standard 75•

Blood

was

m m H g .and H R

was

128•

78•

min

rate

of

the

124!10mmHg,

~ During mmH~

tl41t

index(VI) heart

means

was

78i9

SBP was

and

by

SBP

Index(EI).,

Valsalva

estimated

mmH~,DBP

~.Sleepin~

and

pressure

deviations. 24-h

~ diabetes

(SRP, DBP, S p a c e l a b 5 3 0 0 )

index(DAl)

variability

Chor6b

24-h ambulatory

performed. Expiration-Inspiration

Deacceleration

min

II

Gdafsk

{duration

pressure

was

A. K u b a s i k

AM

the

and

mmH~,DBP

DBP

day HR

SBP 82•

88•

mmHg

_I

and

HR

positive

correlation

difference also

~, W e h a v e

65•

awake

correlated

(p<0.05).

SD of

found

between

HR-sleep with

the

SD

of

awakening

Ambulatory

BP d i d

laborator Z

autonomic

tests.

that

only

that

heart on

the

BP variability

noninvasive

DAI

and

HR(p<0.05).

EI(p
estimated

a significant

HR HN

the

DAI

during

was

sleep

correlated

with

correlate

with

not Our

data

suggest

rate

variability

could

be

basis

of a u t o n o m i c

tests

and

should

BP m o n i t o r i n ~

be

estimated

in t y p e

by

24-h

1 diabetics.

P744

P745

R O L E OF E S S E N T I A L HYPERTENSION IN U R I N A R Y A L B U M I N E X C R E T I O N OF T Y P E 2, N O N INSULIN-DEPENDENT DIABETICS.

THE BLOOD PRESSURERESPONSETO EXERCISE IN UNCOMPLICATED TYPE I (INSULIN-DEPENDENT) DIABETES MELLITUS. D.A. Cavan, M.J. O'Donnell, N. Lawson, H. Lewis, A Parkes, and A.H. Barnett. Depts. of Medicine and Pharmacology, U n i v e r s i t y of Birmingham and East Birmingham Hospital, UK.

M. Marre, S. Suraniti, M. Hallab, J.D. Page, A. Billiard and Ph. Fressinaud - M~decine B - C.H.R.U. 49033 A N G E R S C E D E X - F R A N C E The role of blood pressure on Urinary Albumin Excretion (UAE) is debated in diabetics. Elevated UAE can occur in essential hypertension. We compared the consequences of elevated blood pressure on kidney and heart, by studying Systolic Blood Pressure (SBP), UAE (three 24 b-urines), and left ventricular hypertrophy (Sokolow index on ECG) in 64 non-diabetic essential hypertensives (Group I), 174 type 1, insulin-dependent diabetics (Group II), and 222 type 2, non-insulin-dependent diabetics (Group III), comprising 122 hypertensives (Group III a) and 100 non-hypertensives (Group III b). In group I, SBP (x) was related to UAE (Yl ; logl0-transformed ; r = .424 ; p = 10-4, Yl = .016 x - 1.042) and Sokolow (Y2 ; r = .491 ; p = 10-4 ; Y2 = .3 x - 19), and UAE to Sokolow (r = .483 ; p = 10-4). In group II, SBP was related to UAE (r = .384 ; p = 10-4 ; Yl = .015 x .6) and Sokolow (r = .294 ; p = 10-4 ; Y2 = .131 x + 5.7), but UAE was independent from Sokolow (r = .122 ; ns). Variance and coveriance analysis indicated higher UAE for equal SBP in Group 1] than in Group I (p = 10-3). In Group Ifl a, SBP was related to UAE (r = .279 ; p = .0035 ; Yl = .012 x - .31) and Sokolow (r = .299 ; p = .0008 ; Y2 = .167 x + 2.2), and UAE to Sokolow (r = .177 ; p = .0285). The regression lines between SBP, UAE and Sokolow were superimposable for Group I and Group II a. No relationship was observed between these variables in Group III b. In type 2, noninsulin-dependent diabetics with hypertension, levels of UAE can be explained solely by the concomitant essential hypertension

An exaggerated blood pressure (BP) response to exercise has been reported and may be related to the degree of albuminuria. Abnormalities of BP and plasma renin activity (PRA) are associated with microalbuminuria and a t r i a l peptide (AP) is increased during poor d i a b e t i c control. We investigated the extent of abnormalities in the BP, PRA, AP and noradrenaline responses to exercise in 8 uncomplicated Type I ( i n s u l i n dependent) patients with normoalbuminuria (mean age 28, duration 14 yrs) compared with 8 controls. Mean resting d i a s t o l i c BP was higher in the patients (82 v 68 mmHg, p
A202

P746 TWENTY-FOUR

HOUR

URINARY

EXCRETION

DIGITALIS-LIKE FACTOR (DLF) I N T Y P E DIABETICS: A W I N D O W ON H Y P E R T E N S I O N ?

OF 1

G. Penno, G. Gregori, S. Bertoli, M.G. Del Chicca, A. Lucchetti, L. Cruschelli, M. Nannipieri, L. Rizzo, M. Cecere, R. Navalesi, A. Clerico and O. Giampietro. Cattedra di Malattie del Metabolismo, Istituto di Clinica Medica II, Istituto di Fisiologia Clinica del C.N.R., Universit~di Pisa, Italy. An endogenous digitalis-like factor (DLF) with inhibitory action on NK-ATPase is elicited by extracellular volume expansion and probably plays a role in hypertension. Thirty-eight normotensive normoalbuminuric type 1 (insulin-dependent) subjects (30.5+10 age years; H b A l c 8.4+1.8%; daily insulin 52+17 IU, serum creatinine 0.8_+0.2 mg/dl, mean~.+sd) and 9 age-matched healthy controls collected urines in several portions (sample 1, waking-lunch; sample 2, lunch-dinner; sample 3, dinner-bedtime; sample 4, overnight. Urines 1 to 4 were pooled yielding the 24 hour sample, 5). In each urine, we measured the excretion rate of DLF (RIA), albumin (RIA), electrolytes (Na +, K+), creatinine. In normals, the mean DLF differed significantly in the 24 hours (F=6.45, p<0.002, ANOVA); the overnight D L F (36.4+14.3 pg/min) was lower (p<0.01) than all others (sample h 46.4+17; 2: 49.7+18; 3: 59.1+20.4; 5:44.4.+15 pg/min). In diabetics there was no significant 24h DLF rhythm (from 50.3+27.1, sample 4, to 63.6+48.9 pg/min, sample 1; F=1.42, p=0.24). Urinary DLF in diabetics exceeded that of normals by 25-30% in all samples. Both in normals and diabetics, urinary creatinine, Na + and K + showed a significant 24h rhythm with overnight nadir. Kaliuresis was higher in diabetics compared to controls (p<0.02 for all samples). By multiple regression, only creatinine and K + significantly explained DLF variability in each sample, both in normals and diabetics. Twenty-four urinary DLF changes significantly in normals, not in diabetics, where it is supranormal and fixed. In type 1 patients, peripheral hyperinsulinemia by exogenous insulin stimulates renal tubule Na-KATPase, enhances tubular Na + reabsorption and kalium urinary output, leading to extracellular volume expansion followed by a fixed DLF hypersecretion as a compensatory phenomenon. Raised DLF could trigger hypertension in genetically susceptible type 1 diabetics.

PS 31 Lipids and Lipoproteins P748

P747 THE PREVALENCE OF HYPERLIPIDEMIA D E N T DIABETIC PATIENTS.

IN INSULINDEPEN-

A B Haaber, T Deckert and T Jensen. Steno Memorial Hospital, DK-2820 Gentofte, Denmark. To assess the prevalence of hyperlipidemia and its relationship to glycaemic control and albuminuria we screened 2073 insulin-dependent diabetic patients ( onset before age 40 years ), aged 15 to 60 years. None received lipid-lowering drugs. Hyperlipidemia, defined as total-cholesterol higher than 6.4 mmol/l, was found in 269 patients ( 13% ). The frequency of hyperlipidemia in females was 15.2 % (140/921) and in males 11.2 % (129/1152). Compared with the normolipidemic patients, the patients in the hyperlipidemio group were older (48.0 years versus 38.2 years ) and they had higher glycosylated HbAIr (9.0 % versus 8.3 %). Linear regression analysis revealed that only 8 % (r=0.28) of the variation in total-cholesterol was explained by differences in metabolic control, suggesting that elevated blood pressure and increased urinary albumin excretion may be even more important as predictors of hyperlipidemia. Thus, the prevalence of hyperlipidemia in a large unselected group of insulin-dependent diabetic patients seems not to be higher than in the background population. However, patients with albuminuria and elevated blood pressure constitute a subgroup of patients with a higher frequency of hypercholesterolemia.

ASSSOCIATION O F P A R A M E T E R S OF GLYCEMIC CONTROL O F LIPID TRANSPORT IN DIA]~ETES MELLITUS T Y P E I.

AND

A. Dzien, M. Lechleitner, Th. Hopferwieser, H. Drexel, and J. R. Patsch. Division of Clinical Att(ero~c!erosls Research, Department of Medicine, U n i v e r s i t y ~of Innsbruck, A - 6 0 2 0 Innsbruck, Austria. Hyperlipidemia s e v e r e l y a u g m e n t s t h e overall risk for CAD in DM t y p e I. High glucose l e v e l s may lead to e l e v a t i o n of plasma triglycer2des and cholesterol v i a a number of m e c h a n i s m s including i n c r e a s e d h e p a t i c production of v e r y low d e n s i t y lipoproteins (VLDL) and glycation of c e r t a i n a p o l i p o p o t ~ n s . To t e s t t h i s notion we h a v e compared p a r a m e t e r s of glycemic control with t h o s e of plasma lipid t r a n s p o r t in 15 o u t - p a t i e n t s with DM type I. Regression a n a l y s e s were performed to compare f a s t i n g plasma glucose, HbAlc and f r u c t o s a m i n e each with total cholesterol, triglycerides, low density lipoprotein-cholesterol (LDLcholesterol), high density lipoprotein-cholesterol (HDLcholeaSerol), HDL2- and HDL3-cholesterol, as well as with plasma levels of apolipoproteins (apo) A - I and B. Plasma glucose levels showed no s i g n i f i c a n t correlation with any plasma lipids or apos, b u t HbAlc correlated with t o t a l cholesterol (p<0,01) and triglycerides (p<0,05), and "fructosamine correlated significantly not only with cholesterol (p<0,01) and triglycerides (p<0,05) b u t also with LDL-cholesterol (p<0,01) and apo B levels (p<0,05). Our d a t a indicate t h a t of t h e c u r r e n t l y used p a r a m e t e r s of glycemic control, f r u c t o s a m i n e is t h e most s e n s i t i v e in predicting a rise in a t h e r o g e n i c lipid and apolipoprotein fractions. The d a t a l u t h e r s u g g e s t t h a t glycemic control is i m p o r t a n t for improving lipid t r a n s p o r t and in t h i s way reducing t h e risk for CAD in DM type I.

A203

P749

P750

POSTPRANDIAL L I P E M I A A N D CORONARY ARTERY DISEASE (CAD) IN TYPE 2 (NON-INSULIN DEPENDENT) DIABETES M.-R. Tasklnen, H. Hilden a n d M. STyX.he, First and Second Departments of Hedicine, University of Helsinkl, Helsinki, Finland Since remnants of chylomicron catabolism are potentially atherogenic we followed lipid and retinylpalmitate (RP) responses to vitamin A fat load in 12 Type 2 diabetic men (age 55!6 yrs, BMI 27.9+2.9 kg/m, HbA 1 7.5+1.6% , mean• and in 7 age and BNI matched nondiabe~ic men with CAD verified angiographically. Samples were taken before and regularly for 24 hrs after fat load. Plasma was separated into 3 chylomicron fractions (Sf>3200, Sf>ll00 and Sf>400), VLDLI(Sf 60-400) and VLDLp(Sf 20-60) by cumulative ultrac~ntrifugation. Fastin~ triglycerides (TG) averaged 2.0!0.8 and 1.7!0.5 mmol/l, respectively. During first 4 hrs both TG and RP in chylomicron fractions parallelled plasma TG and RP. Thereafter RP decreased in chylol and 2 fractions but increased in chylo3, VLDL 1 and VLDL 2. Chylo5 RP area and peak were higher in diabetic than in nondiabetic men (+87%, p<0.05 and +57%, p
THE DECREASED HDL2-CHOLESTEROL LEVEL AS A MARKER OF INSULIN RESISTANCE IN OBESITY AND TYPE II DIABETES ? G. VAILLANT 1. B. VERGES 1, Ph. GAMBERT2, C. LALLEMANT2, J.M. BRUNT;lDepartment of Endocrino/ogy and 2Department of Biochemistry, CHU de Dijon, BP 1542 - 21 034 DIJON CEDEX- FRANCE

We have previously shown that diminution of high density lipoprotein subfraction 2 (HDL2) cholesterol, in type II diabetes was only related to hypertriglyceridemia and overweight, both acting as independent and additional factors. To point out the role of these two factors, we evaluated triglycerides (TG), HDL Cholesterol (HDL-C) and HDL subfractions (HDL2, HDL3) using a polyacrylamide gel in 3 different groups;group h 30 age matched control normalweighted women, group II : 60~non diabetic obese women (Body Man Index : 36,6 -+ 7,2 kg/m ~) and group ILL,: 49 type II diabetic obese women (BMI : 37,1 -+ 5,7 kg/m~).As expected, TG were significantly different between the 3 groups (group I : 66,9 -+ 24,9 vs group II : 98,2 -+ 49,9 vs group III 9 193,5 + 130 mg/dl; p = 0,O01).HDL-C level was significantly higher in group I than in the two other groups (group I 9 61,13 -+ 14,25 vs group II : 48,16 -+ 12,55 vs group III : 43,77 -+ 13.27 mg/dl ; p<0,001). HDL-C level was not significantly different between group II and III. HDL2-C was significantly different between the 3 groups : (group I : 27,03 -+ 13,9 vs group II 15,03 -+ 10,67 vs group Ill : 6,97 -+ 6,71 mg/dl, p < 0,001). After adjustment for triglyceride level the difference for HDL2-C between group II and III persisted (group II 9 13,6 -+ 1,26 vs group Ill: 8,47 -+ 1,28 mg/dl; p = 0,008). Hyperinsulinemia and insulinresistance are commun features in obesity and type II diabetes and are suspected to explain low HDL2-C in these patients. In obese type II diabetic patients, the imbalance between hyperinsulinemia and insulinresistance is more obvious and could explain their lower HDL2-C level than in non diabetic obese patients.

P751

P752

LOW HIGH DENSITY LIPOPROTEIN C H A R A ~ I Z E S CORONARY ARTERY DISEASE (CAD) IN TYPE 2 DIABETIC MEN M. Syv~'tne, M. S. Nieminen and M.-FL Tasldnen, Fust and Second Deparbnen~ of Medicine, University of Helsinki, Helsinki, Finland To define discriminants for CAD in Type 2 diabetes we measured concentrations of lipoproteins (VLDL, IDL, LDL, HDL and its subfractions) separated by ultracentrifugation, apo A-I, A-II and apo B in 22 Type 2 diabetic men (age 54.5-+6.2 yrs, BMI 29.0-+3.6 kg/m ~, HbA~ 7.5-+1.3%, mean -+SD) and in 11 age and BMI matched nondiabetic men with angiographically verified CAD (significant narrowings of 50% or more in 1 - 3 of the major branches of coronary arteries). The concentrations of serum, IDL- and LDL-cholesterol and apo B were similar in the two groups (5.3-+0.9 vs 5.6-+0.7, 0.26-+0.11 vs 0.25-+0.14, 3.4-+0.8 vs 3.5-+0.6 mmol/I, 116-+19 vs 112-+9 mg/dl). Diabetic men showed elevations of serum and VLDL-triglyceride (2.3-+1.2 vs 1.7-+0.5, NS and 1.5-+0.9 vs 1.0-+0.4 mmol/I, NS) compared to nondiabetic men but the concentrations of IDL- and LDLtriglyceride were not different between the two groups. Diabetic men with CAD had significantly lower concentration of HDLcholesterol than nondiabetic men (1.07-+0.16 vs 1.30-+0.25 mmol/I, p<0.01) due to reduction of HD~-cholesterol (0.50+0.14 vs 0.71 -+0.23 mmol/I, p<0.01). In contrast HDL3-cholesterol was similar in the two groups. The concentrations of both apo A - I and A-II were significantly lower in diabetic men with CAD than in nondiabetic men (99-+10 vs 110-+15 mg/dl, p<0.05 and 27-+5 vs 31-+6 mg/dl, p<0.05). In conclusion low HDL~-cholesterol emerges as a major risk factor for CAD in Type 2 diabetic men.

TRIGLYCERIDES DO NOT INFLUENCE LOW DENSITY LIPOPROTEIN COMPOSITION IN DIABETIC OR NON-DIABETIC PATIENTS Owens D, Johnson A, Collins P and Tomkin GH. Royal College of surgeons in Ireland and the Adelaide Hospital,Dublin. This study examines the effect of serum triglyceride (TG) on LDL composition and cellular cholesterol regulation in diabetic and non-diabetic subjects. Patients examined were; non-diabetics with high TG (4.5• mmol/l) and normal TG (l.5• levels and diabetics with high TG (6.5• and normal TG (2.0• Patients (N=I0 for each group) were matched for age sex and serum cholesterol levels. The ester• cholesterol ratios of LDL from the respective groups were; 2.9• 2.7• 2.8• and 2.6• and the LDL TG/protein ratios were 0.32• 0.29• 0.23• and 0.26• respectively. There was no significant difference in the suppressi?~ of cellular cholesterol synthesis, measured by [~C]-acetate incorporation into cholesterol, by LDL from the various groups, the degree of suppression being 39• 45• 37• 43• This study demonstrates a lack of correlation between serum TG and LDL TG or between serum TG and the LDL esterified/free cholesterol ratio. The HbA I level in diabetic patients correlated positively wlth serum TG but there was no correlation between serum or LDL triglyceride and the ability of LDL, from diabetic or non-diabetic patients, to regulate cellular cholesterologenesis. These results may help to explain the lack of association between diabetic control and large vesel disease in the hypercholesterolaemic diabetic patient.

A204

P753

P7B4

Cholesterylester transfer protein activity is elevated in cigarette-smoking Type i (insulin-dependent) diabetic men.

B I N D I N G 0F I N S U L I N W I T H V E R Y L O W D E N S I T Y L I P O P R O T E I N S IN D I A B E T E S N E L L T U S G. V. D u b r o v s k a y a , V.A. N a l y z h e v and A.S. Yefimov, I n s t i t u t e of cardiology, Kiev, U S S R

RPF Dullaart, JEM Groener, LD Dikkeschei, DW Erkelens and H Doorenbos. University Hospital Groningen, Utrecht and Erasmus University Rotterdam, The Netherlands. Cholesterylester transfer protein (CETP) is instrumental in the distribution of cholesterylester between lipoproteins. A high activity of CETP has previously been found in hyperlipidaemic subjects and in Type i diabetic patients with micro- and maerovascular complications. Cigarette smoking lowers cholesterol in high density lipoproteins (HDL) and raises cholesterol in very low and low density lipoproteins (VLDL and LDL). Serum lipids and CETP activity, assayed independently of endogenous lipoproteins, was determined in 9 cigarette-smoking and in 12 non-smoking Type 1 diabetic men with microvascular complications and in 12 non-smoking male control subjects. The HDL/VLDL+LDL cholesterol ratio was lower in the smoking diabetic patients (0.23!0.07 mmol/mmol, mean ! SD) than in the non-smoking diabetic patients (0.31• mmol/mmol, p<0.05) and in the control subjects (0.34!0.11 , p<0.05). CETP activity was 70% higher in the smoking diabetic patients than in the non-smoking control subjects (p<0.01) and 30 % higher than in the non-smoking diabetic patients (p<0.05). The HDL/VLDL+LDL cholesterol ratio and the Apolipoprotein AI/B ratio were inversely correlated to CETP activity in the diabetic patients (Rs -0.52, p<0.02 and R s -0.45, p<0.05, respectively). It is suggested that a high CETP activity is involved in the unfavourable serum lipid parameters in cigarette-smoking Type 1 diabetic men with microvascular complications.

P o s s i b l e w a y s of i n s u l i n - a n t i i n s u l i n immune c o m p l e x e s p e n e t r a t i o n into the v a s c u l a r w a l l w e r e i n v e s t i g a t e d in d i a b e t e s m e l l i t u s . A t t e n t i o n w a s g r a n t e d to v e r y l o w d e n s i t y l i p o p r o t e ins (VLDL) and t h e i r i n s u l i n - b i n d i n g ability, b e c a u s e these p r o t e i d s are c a p a b l e of n o n - r e c e p t o r y p e n e t r a t i o n into the v a s c u l a r w a l l in c e r t a i n c o n d i t i o n s and m a y p l a y a role in immune c o m p l e x t r a n s p o r t a t i o n . Using disc-electrop h o r e s i s we f o u n d a s i g n i f i c a n t i n c r e a s e in V L D L content in d i a b e t e s m e l l i t u s p a t i e t s and p a r t i c u l a r l y in d i a b e t i c angiopathy. S i m u l t a n e o u s l y a c o n s i d e r a b l e rise u• -~ ~ S j -'ns I U i"in b'nl ding w i t h V L D L w a s observed, its level in diabetic p a t i e n t s ( 1 5 6 0 , 5 3 + 5 5 , 3 mkIU/ml) e x c e e d i n g that in h e a l t h y donors [81,55+7,76 mkiU/ml) 19 times (Pi 0,05). E l e v a t e d i n s U l i n - b i n d i n g abil i t y of V L D L w a s also m a r k e d in p a t i e n t s w i t h newly-discovered insulin-dependent diabetes m e l l i t u s n o t t r e a t e d by e x o g e n o u s insulin. Immunoelectrophoretic a n a l y s i s h a s shown that lip o p r o t e i d b o u n d i n s u l i n r e t a i n s its a n t i g e n i c i ty and m a y be l i n k e d to s p e c i f i c antibodies. It is s u g g e s t e d that c o m p l e x c o m p o u n d s are f o r m e d in d i a b e t e s m e l l i t u s , c o n s i s t i n g of VLDL, insulin and a n t i i n s u l i n a n t i b o d i e s , w h i c h are capable of p e n e t r a t i n g into v a s c u l a r w a l l s and p l a y i n g an e s s e n t i a l role in d i a b e t i c a n g i o p a t h y initiation.

P755

P756

APOLIPOPROTEIN E POLYMORPHISM: GENE AND PHENOTYPE FREQUENCIES IN AN ITALIAN DIABETIC POPULATION M.Boemi,R.W.Jamas*,F.Romagnoli, P.Gerber*, D.Pometta* and P.Fumelli. Division of DiabetologLv and Metabolic Diseases, INRCA, Ancona-ltaly; *Division de Diabetologie, Cantonal Hospital, Geneva-Switzerland. Aim of this study was to determine the phenotype distribution and gene frequencies for Apoprotein E (ApoE) in an italian diabetic population. A group of 309 (258 Type 2, 51 Type l; 162 females, 147 males) diabetic outpatients were investigated. Subjects were enrolled randomly. ApoE phenotyping was carried out by isoelectric focusing. Frequencies of APOE epsilon 2,3 and 4 alleles were respectively 0.069_+0.010, 0.856+ 0.026 and 0.074+0.024. Excess of epsilon 3 allele was recorded in both Type l (0.955 vs 0.889) and Type 2 (0.876 vs 0.806) females co,Tparedto males together with corresponding lower frequencies of epsilon 2 and 4, Type 2 diabetics showed a significantly higher (p(O.05) frequence of epsilon 2 (0.075) and 3 (0.084) alleles than Type l subjects (0.039, 0.029). Phenotype distribution agree with that predictable by HardyWeinberg equilibrium:74.4% of patients were E3/3, I1.3~/~E3/2, II% E4/3, 1.3% E4/2 or E4/4 and 0.6% E2/2. E3/2 and E4/3 heterozygotes were more comTon (12.6%, 14.1%) among Type 2 than among Type l (7.3%, 5.4%) diabetics (p(O.05). Lowest frequencies of these phenotypes were observed among females (Type 2: 10.9% 10.%; Type l: 2.9% 5.7%). Our results differ from those reported in other diabetic populations; unlike patterns related to gender are evi dent.

HYPERTENSION AND HYPERLIPOPROTEIN~MIA IN TYPE II DIABETICS: RESULTS OF THE DIABETES INTERVENTION STUDY (DIS) S.Fischer, M.Hanefeld, U.Julius end U. Schwanebeck Clinic of Internal Medicine, Medical Academy Dresden, GDR We investigated 1139 patients (aged 30-55 years) with newly manifested, dietetically compensated Type II (non insulin dependent ) diabetes. The patients were randomly allotted to 2 intervention groups and 1 control group. The intervention program included the following measures: lipid lowering diet, weight reduction, physical endurance training, intensive treatment of hypertension, clofibric acid 1.6g/d or (double blind manner) placebo. Initially we found in 53 % of the patients a hypertension (women 62.8 %, men 46 %). A primary hyperlipoproteinemia was detectable in 17.6 % (mostly hypertriglyceridemia 1q.3 %). Comparing with the control group, after 5 years both systolic and diastolic ~ood pressure were significantly lower in the interventio~ groups (143/87 and 145/85 vs 154/92 Torr; p
A205 P758

P757 I M P R O V E M E N T O F P L A S M A LIPIDS A N D OPTIMIZED INSULIN T H E R A P Y

GLYCEMIC

CONTROL

BY

M. Lechleitner, A. Dzien, Th. Hopferwieser, H. Drexel and J. R. Patsch Division of Clinical Atherosclerosis Research, Departement of Medicine, University of Innsbruck, A-6020 Innsbruck, Austria. To evaluate the effect of optimized insulin treatment (OIT) not only on glycemic control but also on plasma lipids, 24 p a t i e n t s with DM type I (19 men and 5 women, 18 to 61 yrs.) were switched from a s t a n d a r d insulin t h e r a p y (SIT) to a regimen of OIT for a period of 9 yrs. (group I) and c o m p a r e d t o a c o n t r o l g r o u p o f p a t i e n t s (7 m e n , 4 w o m e n , 25 to 60 y r s . ) r e m a i n i n g on SIT ( g r o u p 2). A f t e r 1 y e a r on OIT, g r o u p 1 e x h i b i t e d a r e d u c t i o n o f H b A l c v a l u e s f r o m 10,2% t o 7,5% (p<0,01), accompanied by am increase in HDLc h o l e s t e r o l f r o m 52 t o 67 m g / d l ( p < 0 , 0 5 ) a n d a d e c r e a s e o f t r i g l y c e r i d e l e v e l s f r o m 3 1 9 t o 67 m g / d l ( p < 0 , 0 0 1 ) . G r o u p 2 s h o w e d n e i t h e r s i g n i f i c a n t c h a n g e s in H b A l c n o r in l i p i d p a r a m e t e r s . A t t h e e n d o f t h e s e c o n d y e a r on OIT, s o m e patients exhibited a reversal of t h e f a v o u r a b l e trend in HbAlc and lipid values. Therefore, intensified instruction r e g a r d i n g t h e i m p l e m e n t a t i o n of OIT w a s r e p e a t e d in t h o s e s u b j e c t s a n d r e s u l t e d in a r e n e w e d i m p r o v e m e n t o f o v e r a l l TIbAlc, H D L - c h o l e s t e r o l and triglyceride l e v e l s t o 6,43%, 67mg/dl, and 78 mg/dl, respectively. Our findings underline t h e v a l u e of OIT n o t o n l y r e g a r d i n g g l y c e m i c c o n t r o l b u t also control of plasma lipids considered major risk factors for CAD. T h e f i n d i n g s d e m o n s t r a t e further that rigorous and repeated instructions may be necessary to m a i n t a i n s u c h favourable results.

P759 D I F F E R E N T I A L EFFECTS OF OMEGA-3 FATTY ACIDS ON INSULIN B I N D I N G AND A C T I O N IN STZ D I A B E T I C RATS I. Klimes, M. Fickov~, L. Kazdov~ I, E. Sv~bovi, P Bohov 2, A. Vr~na I and L. Macho. Inst. Exp. Endocrin., Slov. Acad. Sci., Bratislava; i) Inst. Clin. Exp. Med., P r a g u e and 2) Res. Inst. Gerontol., Malacky; Czechoslovakia. To t h r o w light on m e c h a n i s m s u n d e r l y i n g the c o n t r a d i c t o r y effects of omega-3 (n-3) fatty acids (FA) in diabetes, insulin b i n d i n g (IB) and action (IA) were studied in s t r e p t o z o t o c i n diabetic (DM) rats (50 m g STZ per kg b.w., i.v.) s u p p l e m e n t e d for 24 days with n-3 FA (i ml fish oil /=FO/ daily, MARTENSR; 46 wt% of n-3 FA). FO neither w o r s e n e d f a s t i n g glycemia (DM: 19.33+ 2.97 vs DM+FO: 17.02+2.18 mmol.l-1), nor lowered serum t r i g l y c e r i d e s (C: 1.4+0.2 vs DM: 1.7+0.2 or DM+FO: 1.8• mmol.l-1). FO raised the s u m of n-3 FA in isolated h e p a t o c y t e m e m b r a n e s (HEPM) (14.52+0.91 wt%) as c o m p a r e d to C (7.61• p<0.002) or DM (10.17• p<0.05) rats implicating a raise in m e m b r a n e fluidity. IB to HEPM (C: 113 3+10.7 vs DM: 176.5+18.9 f m o l . m g -I p
M E T F O R M I N - I N D U C E D M O D U L A T I O N O F S E R U M LIPIDS IS N O T R E F L E C T E D IN L E U C O Y T E C H O L E S T E R O G E N I C RATES. U.M. Moore, D.Lyons, O.RTighe, A . H . J o h n s o n , G.H.Tomkin and P.B.Collins. R o y a l College o f S u r g e o n s in Ireland, and Adelaide Hospital, Dublin Ireland.

The anti-hyperglycaemic agent metformin improves both glycaemic control and lipoprotein metabolism. However the mechanism of action responsible for both these effects is not apparent. The aim of this study was to determine if the improvement in lipoprotein metabolism is mediated through regulation of de novo cholesterol biosynthesis. Lipid metabolism was studied in normocholesterolaemic Type 2(non-insulin-dependent diabetic)patients (n=14) and hypercholesterolaemic Type 2 (non-insulin -dependent diabetic) patients(n=10) after a two week treatment with metformin(1.5g/day).De novo cholesterogenesis in isolated leucocytes was estimated by incorporation of 14C-acetate in vitro into 14C-cholesterol.Therapy resulted in a significant fall in blood glucose, serum cholesterol and triglyceride levels (p<0.05) with a corresponding increase in serum HDL levels (p<0.05). No significant change was detected in de novo cholesterogenesis. This would indicate that although improvement in lipoprotein metabolism occured over a two week period with metformin, its action was not accompanied by alteration in the rate of cellular cholesterol synthesis as reflected by acetate incorporation into leucocyte cholesterol.

P760 LIPID INTOLERANCE IN CONTROLLED TYPE II DIABETIC PATIENTS. NORMALIZATION A F T E R GEMFIBROZIL TREATEMENT. E. Cavallero*, D. Neufcour*, C. Dachet*, G.Girardot** and B. Jacotot* * INSERM U.32 - H6pital Henri Mender - 94000 CRETEIL ** Lab. P A R K E - D A V I S - 11 av. D u b o n n e t - 92407 C O U R B E V O I E High t r i g l y c e r i d e a n d low H D L - C h o l l e v e l is t h e c h a r a c t e r i s t i c lipid pattern in Type II diabetes and triglyeeride (TG) rich lipoproteins clearing capacity may be the link between both abnormalities. We studied lipoprotein composition after a fat load in a group of controlled (Hb AiC<7.5%)normocholesterolemic type II diabetics (n=11) compared to a control group matched in age, sex, and BMI (n=11). Determinations were made hourly up to the 4th hour a f t e r the test meal which contained 32.5 g/lipids/m2 body surface with 45% lipids, 20% proteins, 35% carbohydrates. Lipoproteins were separated by ultracentrifugation glucagon and C-peptide values were determined by R.LA. Findings d e a r l y allowed us to identify in diabetic patients two subgroups (I and If) according to the level of basal TG (1.2 g/I) and to the post-prandial (PP) responses. Subgroup I had PP TG increase. HDL-C and HDL2C levels were identical to that of controls. Subgroup II showed higher PP TG area and higher increase in the chylomicron fraction together with low HDL2 levels. CE/TG ratio in HDL2 particles was lower than in controls and decreased even more a f t e r the fat load (p=0.056 and 0.001 respectively). Subgroup I and II differed only in the lower glucagon/Cpept. PP ratio found in the latter group. We treated subgroup II patients with Gemfibrozil (900mg/day). After 3 months of therapy we observed a significant reduction in total TG values with normalisation of PP triglyceridemic response and PP chylomicron. Fasting levels and PP VLDL lipid levels were significantly reduced but remained still higher than control values. HDL-C increased (35 vs 39 mg/dl) and its TG content decreased (19 vs 15 mg/dl p=0.18) mainly for the PP values (23 vs 16 mg p=0.006). Glycemia or Hb AIC values were not changed during this period. In conclusion fat tolerance test showed to amplify lipid abnormalities in diabetic patients and allowed to evidence early changes after Gemfibrozil treatment.

A206

P761

P762

DISPARATE EFFECT OF Cs ON GI/]COSE METABOLISM IN PATIKNTS WITH NON-INSULIN DEPENDENT DIABETES (NIDDM). DC Shen, MT Fuh, SM Shieh, BB Varasteh and GM Reaven, Tri-Service General Hospital, Taipei, Taiwan, ROC., Stanford, CA. USA. This study was initiated to: i) assess gemfibrozil's (GEM) ability to lower plasma triglyceride (TG) in NIDDM patients; 2) to see if this effect was associated with glucose metabolism. Measurements of hourly plasma glucose (G,mg/dl), free fatty acid (FFA,umol/L) from 1200-1600 h after a test meal, hepatic glucose production (HGP,mg/kg/min), insulin-stimulated glucose uptake during a hyperinsulinemic glucose clamp, (~_1% ml/kg/min), fasting (F) plasma TG (mg/dl) in 12 NIDDM patients, before and 3 months after GEM. Although F TG fell (417~43 to 167~16), G, FFA, HGP, and MCR did not change. However, patients in "fair" control (FPG>I50 mg/dl,n=6) had decreased (P<0.05) in G (272131 to 227 + 17), FFA (652+150 to 504+76), HGP (1.71~0.07 to 1.46~ 0.07) and an increase in MCR (2.63~0.49 to 3.72~0.54) with a fall in FI~ from 399+67 to 155+21. Despite a comparable fall in FfG (437~57 to 180~26), neither FFA, HGP, or MCR changed in "good" control (fasting G <150 mg/dl, n=6), and G increased somewhat (166~12 to 211~26, P<0.05). Conclusion: i) TG levels decreased in GF~4-treated NIDDM patients; 2) changes in glycemic control were unrelated to the TG-lowering effect of GEM, confined to patients in "fair" control, and consisted of a decrease in FFA and HGP and an increase in insulin-stimulated glucose uptake; 3) the fall in FTG seen in C~94-treated patients with NIDDM is independent of baseline G concentration, and is associated with an improvement in glycemic control in only a subset of this population.

DIFFERENT EFFECTS OF EXOGENOUS AND ENDOGENOUS H Y P E R I N S U L I N E M I A ON APO AI CONCENTRATIONS A. Scoppola, A. Lala, A. Giaccari~, F. Leonetti~, P. Sbraccia~, G. Tamburrano~ and G. Menzinget - Malattie del Ricambio and ~ I Cattedra di E n d o c r i n o l o g i a - University of Rome - Italy. It was the aim of the present work to investigate the d i f f e r e n t effects of endogenous and exogenous h y p e r i n s u l i n e m i a on apolipoprotein

P763 HEPATIC CHOLESTEROLOGENESIS IN N O N - I N S U L I N D E P E N D E N T DIABETES: S T U D I E S IN db/db M I C E I. G. O b r o s o v a , V . L. T s i r u k , F . S. Larin,E. I. B a b i c h e v a , I n s t i t u t e of E n d o c r i n o l o g y a n d Z e t a b o l i s m , V i s h g o r o d s k a y a str. 69, 2 5 2 1 1 4 Kiev, U S S R The d e v e l o p m e n t of a t h e r o s c l e r o s i s in Type 2 d i a b e t e s m e l l i t u s is o f t e n a s s o c i a t e d w i t h e l e v a t e d s e r u m c h o l e s t e r o l (Ch) levels. We s t u d i e d hepatic cholesterologenesis in db/db mice -a m o d e l of T y p ~ A 2 diabetes. C h b i o s y n t h e s i s was measured by~?C i n c o r p o r a t i o n f r o m in v i v o administered " C-labelled p r e c u r s o r s into h e p a t i c lipids with subsequent separation and quantit a t i o n of r a d i o a c t i v e Ch by t h i n l a y e r c h r o m a tography a n d l i q u i d s c i n t i l l a t i o n counting. Results indicated increased Ch biosynthesis f r o m p y r u v a t e and, to m u c h l e s s e r extent, f r o m acetate a n d m e v a l o n a t e in db/db mice as comp a r e d to c o n t r o l (p / 0.05). 1 4 - d a y h y d r o x y t h i a mine a ~ i n i s t r a t i o n ~ 1 2 . 5 m g / k g b o d y weight, i.m. daily) to diabetic mice l e d to d r a m a t i c fall of l i v e r p y r u v a t s d e h y d r o g e n a s e a c t i v i t y , m a r k e d i n h i b i t i o n of c h o l e s t e r o l o g e n e s i s from p y r u v a t e as w e l l as the s i g n i f i c a n t decrease of C h levels in l i v e r a n d s e r u m ( p / 0.05). At the same time the rates of c h o l e s t e r o l o g e n e s i s from m e v a l o n a t e were not a f f e c t e d w h i l e Ch b i o s y n t h e sis f r o m a c e t a t e s i g n i f i c a n t l y increased. The c o n c l u s i o n c a n be d r a w n that the i n c r e a s e of c a r b o n f l u x t h r o u g h p y r u v a t e d e h y d r o g e n a s e comp l e x is of k e y i m p o r t a n c e in the c h a i n of e v e n t s p r o m o t i n g i n c r e a s e d Ch b i o s y n t h e s i s a n d hypercholesterolemia in db/db mice. The increased cytgsolic phosphate potential(ATP/ADP. P~ free N A D ( P T ) / N A D ( P ) H a n d C o A / a c e t y l - C o A ratios-" were i d e n t i f i e d a m o n g the i n t r a c e l l u l a r f a c t o r s providing elevated hepatic cholesterologenesis in n o n - i n s u l i n - d e p e n d e n t diabetes.

AI (apo A I ) c o n c e n t r a t i o n s in human beings. First, we performed a 120 minute hyperglycemic clamp s t u d y i n 5 h e a l t h y males. Under c o n d i t i o n s of endogenous h y p e r i n s u l i n e m i a (IRI=409 pM; C-

peptide=2nM) apo AI levels (124!5 mg/dl at baseline) decreased by about 15% at 40, 60, 80~ 100 and 120 min (p<0.05 at least). We therefore performed a euglycemic hyperinsulinemic clamp study in another group of normal subjects comparable

for

age

and

sex.

Under

these

conditions of peripheral hyperinsulinemia (IRI=770 pM) with suppressed endogenous insulin secretion ( C-peptide levels below 0.16 nM) baseline ape AI levels (9625 mg/dl) increased by i0% at least at 40, 60, 8(] and iO0 minutes ( p< 0.05 or less). Finally~ the euglycemic clamp studies were performed in 5 insulinoma patients, whose endogenous insulin secretion from tumoral tissue was not inhibited by the exogenous insulin infusion. Under conditions of peripheral plus poFtal hyperinsulinemia (IRI=789 DM; Cpeptide >1.5 nM) apo AI levels were lower than normal in the baseline (70~i0 mg/dl) and didn't show any s i g n i f i c a n t changes. In c o n c l u s i o n , our study provides evidence that endogenous hyperinsulinemia acutely decreases while exogenous h y p e r i n s u l i n e m i a a c u t e l y increases apo AI c o n c e n t r a t i o n s in humans.

A207

PS 32 Metabolism in Type 1 Diabetes P764

P765

EFFECT OF INTRAPERITONEAL OR SUBCUTANEOUS OVERNIGHT INSULIN INFUSION ON GLUCOSE TURNOVER IN TYPE I DIABETICS. M. S c a v i n i , C. C r u c i a n i , A. P i n c e l l i , E. O r s • A. C a u m o , P. M i c o s s i , a n d G. Pozza. H. S. R a f f a e l e , M i l a n o , I t a l y .

LEUCINE METABOLISM IN TYPE I(INSULIN DEPENDENT) DIABETIC URAEMIC PATIENTS. A Battezzati,L Luzi,F Facchini,G Perseghin,A Secchi,D Spo! ti,V Di Carlo, G Pozza. Departments of Medicine and S u r g e ry, Istituto Seientifico San Raffaele, University of Milan. Fifteen Type l(insulin dependent)diabetic uraemie patients (IDDUP),5 patients with isolated kidney transplant (KTx), and i0 patients after combined kidney pancreas transplant (K~Tx)were studied along with 9 controls (CON)by means of i - C leucine infusion,indirect calorimetry and euglycaemic insulin clamp(l mU/kg.min). KTx and KPTx were on prednisone (lOmg/day),cyclosporin(5mg/kg.day),azatbioprine(img/kg.day~ In the basal state IDDUP had increased plasma glucose (8.9 mmol/l), HbAic(g!l%),free IRI(16!2 ~U/ml), IRG (65!5 pmo~l) while decreased pH(7.29!O.O2U)and LEU(85L4pmoI/I).KTx norn~_ lized only pH and IRG;KPTx normalized also plasma G,MbAlc , LEU. in the insulin stimulated state IDDUP bad defective suppression of plasma LEU(25),branched chain AA(62~mol/l), Endogenous LEU flux~6il), non oxidative LEU disposal(5!l) and LEU OX(I ~mol/m-min), while defective increase of tissue glucose disposal (M=3.9~0.3 mg/kg.min) with respect to CON. KTx normalized the insulin stimulated suppression of ELF anJ NOLD;KTx had increased basal ELF (50+4) and LEU Ox (8+ifamo~ 2 m min) with respect to CON. KPTx completely normalized the basal and insulin stimulated metabolic rates of protein turn over, while the insulin stimulated glucose metabolism was still impaired (6.5~0.4 vs 7.9!0.5 mg/kg.min). In conclusi~ the present data suggest the hypothesis that the combination of Type i diabetes and uraemia induce a defect in insulin mediated protein metabolism. Such a defect can be reverted restoring renal function.

Aim of o u r s t u d y w a s t o c o m p a r e t h e e f f e c t of intraperitoneal (IP) o r s u b c u t a n e o u s (SC) o v e r night continuous insulin infusion on glucose turnover in Type 1 diabetics. Seven patients were studied twice, a f t e r 2 m o n t h s of C S I I or continuous IP insulin therapy through an implantable pump. Insulin was infused overnight at c o n s t a n t r a t e ( 0 . 3 ! . 0 7 m U / k g / m i n ) e i t h e r IP or SC. B l o o d g l u c o s e w a s m a i n t a i n e d c o n s t a n t b y intravenous glucose infusion. Steady normoinsulinemia (10• ~ U / m l I P a n d 9i3 ~ U / m l SC, NS) and e u g l y c a e m i a (88• mg/dl IP and 85• mg/dl SC, NS) w e r e m a i n t a i n e d t h r o u g h o u t the study. In the morning, glucose turnover was measured in steady-state conditions using a 150' prime-continuous 3-H3-glucose infusion. P l a s m a c l e a r a n c e r a t e (PCR) a n d h e p a t i c g l u c o s e production (HGP) w e r e n o t d i f f e r e n t in I P and SC. PCR was in the normal range ( 2.8• m l / k g / m i n IP a n d 2 . 5 • m l / k g / m i n SC, N S ) ; HGP was unexpectedly suppressed (1.3• mg/kg/min IP a n d 1 . 3 • m g / k g / m i n SC, N S ) . N E F A , l a c t a t e , piruvate, alanine, cortisol and GH circulating levels were normal and not different. Plasma IRG was 69f20 pg/ml IP and 89• pg/ml SC (p<0.05). In conclusion, IP a n d S C overnight continuous insulin infusions, achieving normoinsulinemic levels, are equally effective in n o r m a l i z i n g P C R a n d s u p p r e s s i n g HGP.

P766 SUBSTRATE METABOLISM DURING ISOLATED HYPERKETONAEMIA TYPE 1 (INSULIN-DEPENDENT) DIABETIC SUBJECTS.

P767 IN

N. M~ller, N. P ~ r k s e n a n d O. Schmitz. Medical D e p a r t m e n t M (Diabetes ~ E n d o c r i n o l o g y ) , A a r h u s Kommunehospital a n d Medical D e p a r t m e n t I I I , A a r h u s A m t s s y g e h u s , DK-8000 A a r h u s C, D e n m a r k . To a s s e s s the metabolic effects of moderate h y p e r k e t o n a e m i a 6 y o u n g T y p e 1 diabetic p a t i e n t s r e c e i v e d a 200 min i . v . i n f u s i o n of 1) 0.9 mmol 3 - h y d r o x y b u t y r a t e ( 3 - O H B ) ] k g / h and 2) saline. To e n s u r e comparable metabolic conditions a low dose h y p e r i n s u l i n a e m i c euglycemic clamp was p e r f o r m e d from 5h p r i o r to a n d t h r o u g h o u t 3-OHB/saline i n f u s i o n s . The f o r e a r m t e c h n i q u e was employed to estimate s u b s t r a t e f l u x e s . I n f u s i o n of 3-OHB c a u s e d : 1) I n c r e a s e s (p<0.05) in circulating levels of 3-OHB (from 112 + 73 ~unol/l to 825 + 111 pmol/l) and forearm a/v differences of 3-OHB (from 19 _+ 10 bunol/1 to 145 + 46 p~nol]l) and an 8-fold increase of plasma acetoacetate. 2) Decreased (p<0.05) levels of nonester• fatty acids (from 466 _+ 85 bunol]l to 201 _+ 15 ~tmol/l) and glycerol (from 39 -+ 7 plnol/1 to 11 -+ 4 p~mol]l) and decreased (p<0.05) v / a differences of glycerol (from 16 _+8 gunol/l to 3 + 2 p_mol/]). 3) Increased (p<0.05) levels of serum growth hormone (GH) (from 4.1 +- 1.5 ~tg/l to 15.9 -+ 8.0 ~tg]l). No change was recorded in concentrations of free insulin, glucagon, glucose, lactate or alanine. Nor were a / v balances of these metabolites, isotopieally determined glucose turnover or amounts of exogenously administered glucose affected. Conclusions: In Type 1 diabetic man the main regulatory effect of isolated hyperketonaemia appear to be a direct negative feedback inhibition of lipolysis. The following decrease in NEFA concentration may stimulate GH secretion and obscure any possible role for ketone bodies in "Randle cycle" substrate competition.

Hyperinsulinemiaprevents the Ketogenic Response to Epinephrine in Insulin-DependentDiabetic Subjects. A.Avogaro, R.Nosadini,L.Gnudi,A.Valerio,A.Maran,E.Iori,E.Duner, A.Doria,C.Marescotti, and A.Tiengo. Padova,ltaly. The effects of different insulin (I) concentrations (18,32, and 70 mU/l) on Epinephrine (E) induced lipolysis and ketogenesis were assessed in 5 normals (N) and 5 insulin-dependentdiabetics, in separate occasions, while euglycemic. Indirect beta and alfa-adrenergic effects were avoided by somatostatin infused along with I, glucagon and growth hormone. In each study, after a baseline period, E was administered (18 ng/kg/min). During E, in diabetics the FFA increase was 546+310 (p =0.01) (201+26 in N), 146+71 (p=0.01) (31+13 in N), and 52+30 ]_M/1(p<0.05) (I+_3in N), at 0.2,0.4, and 0.8 mU/Kg/min I in{us• respectively. In diabetics the raise of FFA rate of production (Ra) was 7.16+0.84 tmol/kg/min (p<0.01) (8.48+1.28 in N), 3.76+0.45, (3.88+0.44-in N) and 2.96+0.34 (0.08+ 0.02 i-n N). In diabetics Ketones (~IB) increase was 97~26 UM/I(p~ 0.01) (39+11 in N), 24_+17(p<0.05) (27_+8 in N), and 8+5 (7+5 in N). In Diabetics KB Ra increase was 2.65+0.9 ~mol/kg/min(p< 0.01) (1.22+0.7 in N), 0.39+0.16 (p< II05) (0.43+0.21 in N), and 0.06+0.02 (0.00-_+0.01 in N). Atlow insulin levels, euglycemic Type 1 diabetics showed increased lipolysis and ketogenesis during E. High insulin levels completely restrained the ketogenetic response to E. The ketogenetic response to E is dependent upon plasma FFA, excluding significant direct hepatic ketogenic action.

A208

P768

P769

BODY COMPOSITION, SUBSTRATE OXIDATION AND RESTING METABOLIC RATE BEFORE AND AFTER A 6 WEEKS PERIOD OF BETTER METABOLIC CONTROL IN INSULINDEPENDENTDIABETIC PATIENTS, I. De Leeuw, G. Vansant, K. Van Acker and L. Van Gaal.

INFLUENCE OF PREPRANDIAL BLOOD GLUCOSE VALUES ON GLYCEMIC RESPONSES IN INSULIN-DEPENDENT DIABETES MELLITUS (IDDM) AT CONSTANT INSULINEMIA.

Dept. of Endocrinology, Metabolismand Clinical Nutrition, University of Antwerp (UIA), Antwerp, Belgium. Improvement of metabolic control in insulindependent diabetic patients is generally accompanied by a decrease of energy expenditure but most studies do not give data on possible changes in body composition or substrate oxidation. Sixteen C-peptide negative insulindependent diabet i c patients (4 men, 12 women; mean age 26.4, SD 5; mean duration of diabetes 11 y, SD 6) showed a significant decrease of mean total HbAI (from 10.6%, SD I.g to 8.7%, SD 1.3; p < 0.001) after a 6 weeks period of intensified insulin therapy (ICTT). Body weight and fatfree mass, measured by impedance, did not change s i g n i f i c a n t l y . Daily urinary nitrogen excretion decreased significantly (p < 0.01). Resting metabolic rate (RMR) measured by indirect calorimetry decreased from 4.08 kJ/min, SD 0.71 to 3.60 kJ/min~ SD 0.66 (p < 0.001) and remained significantly different after correction for the small changes in fatfree mass. Substrate balances were measured using the tUSK-constants. Carefull dietetic energy intake control did not show significant changes. Protein (P)-, fat (F)and carbohydrate (KH) oxidation decreased in absolute terms but the basal RQ (from 0.79, SD O.Ol to 0.82, SD 0.06) and mean energypercentages(from KH 27.5 to 31.8, P: 22.5 to 21.7, F: 50 to 46.8) did not change. Conclusion: After a period of 6 weeks ICTT, RMR decreased with 690 kJ/day and is accompaniedby lesser oxidation of all substrates and no change in body composition.

O Rasmussenl,K Hermansen1'2 Sec. University Clinic of Internal Medicine~, Aarhus Kommunehospital, Medical Department2, Faaborg Hospital, Denmark. The influence of the premeal blood glucose level on the glycemic responses was studied in seven insulin-dependent diabetic subjects, who by means of the artificial pancreas had achived premeal blood glucose concentrations of 5, 9 and 17 mM. A test meal of 50 g parboiled rice was given at the three different occasions and a constant insulin infusions was provided during the observation periods. The postprandial blood glucose response area (above basal) differed significantly at the three blood glucose levels of 6,9 and 17 mM attaining 1371 -+ 201 mmol/I x 240 min, 621 -+138mmol/I x 240 rain and 179 -+ 74 mmol/I x 240 rain , respectively (P < 0.01) . A negative correlation between the preprandial blood glucose levels and the glycemic responses to the test meal was found (R =o.94; p<0.01). The amount of glucose spilled in the urine was higher at the preprandial blood glucose of 17 mM as compared to the lower preprandial glucose concentrations of 6 and 9 mM (P < 0.01). The insulin levels varied beween 15 and 30 mU/I . In conclusion the glycemic responses to a carbohydrate meal is inversly correlated to the evel of preprandial blood glucose in insulin-dependent diabetic subjects.

P770

P771

Serial a s s e s s m e n t of s e n s i t i v i t y to insulin in t y p e 1 d i a b e t i c p a t i e n t s a t o n s e t , d u r i n g a g g r e s s i v e t r e a t m e n t , and during r e m i s s i o n s f r o m and r e l a p s e s to i n s u l i n - d e p e n d e n c y .

RELATIONSHIP BETWEEN STIMULATED C-PEPTIDE RESPONSES AND INSULIN SENSITIVITY IN INSULIN-DEPENDENT DIABETICS TREATED WITH CYCLOSPORIN D.T. Finegood, I.M. Hramiak, and J. Dupre, The Univers i t y o f A l b e r t a , Edmonton, A l b e r t a and The U n i v e r s i t y o f Western Ontario, London, Ontario, CANADA In a 12 month prospective study o f newly diagnosed insulin-dependent d i a b e t i c p a t i e n t s (IDDM) t r e a t e d with c y c l o s p o r i n , we found no c o r r e l a t i o n o f changes in i n s u l i n s e n s i t i v i t y (ST) with basal or glucagon-stimul a t e d C-peptide levels~ In the present study, we i n v e s t i g a t e d the r e l a t i o n s h i p between SI and the plasma C-peptide response to intravenous glucose. Sx was determined using the minimal-model method applied to the plasma i n s u l i n and glucose dynamics from an i n s u l i n modified (4 mU/min per kg from 20-25 min) f r e q u e n t l y sampled intravenous glucose (1.67 mmol/kg at 0 min) t o l e r a n c e t e s t (FSIGT). The stimulated plasma Cpeptide response (SCP) was the mean incremental plasma C-peptide from 0 to 19 min f o l l o w i n g the glucose bolus. ST increased s i g n i f i c a n t l y from 0 to 3 mo (18• to 59• m i n - I / ( n m o l / m l ) , P0.31). CONCLUSION: Although glucagon was a more potent secretagogue than glucose in these r e c e n t l y diagnosed IDDM, the incremental C-peptide response to glucose c o r r e l a t e s more c l o s e l y with i n s u l i n s e n s i t i v i t y and remission s t a t u s . (Funded by Juveni l e Diabetes Foundation and the A l b e r t a Heritage Foundation f o r Medical Research).

R . B U R C E L I N 1, R.ASSAN 1.

M.EDDOUKS I,

M.BEYLOT 2,

J.P.

RIOU 2,

1 D i a b e t e s Dpt B i c h a t Hospital, P a r i s , F r a n c e 2 INSERM, U 197, Lyon, F r a n c e

The e u g l y c e m i c h y p e r i n s u l i n e m i c c l a m p t e c h n i q u e , c o u p l e d w i t h s t a b l e i s o t o p e - l a b e l l e d g l u c o s e infusion, was used to a s s e s s s e n s i t i v i t y to insulin in r e c e n t o n s e t t y p e 1 d i a b e t i c s u b j e c t s a t o n s e t (n=17), during t r e a t m e n t w i t h e i t h e r insulin + c y c l o s p o r i n A (CyA ; n = l l ) or insulin a l o n e (n=6), d u r i n g r e m i s s i o n s ( i n s u l i n - f r e e , CyA + : n = l ] ) and r e l a p s e s (n=3). Recent onset patients were insulin-resistant : Km=88• pU/ml; Vmax=13.2~+0.9 m g / K g . m i n -1) ( c o n t r o l s Km :39+6 p U / m l ; Vmax=18.5-+0.6 m g / K g . m i n -1 ; n=9). Their h e p a t i c g l u c o s e p r o d u c t i o n , i n i t i a l l y 3 . 6 / 0 . 8 mg/Kg.mir~ I, was r a p i d l y s u p p r e s s e d during t e s t s . S e n s i t i v i t y i m p r o v e d r a p i d l y under t r e a t m e n t and was n e a r - n o r m a l a t 4 rth w e e k ( w h e t h e r CyA was used or not). It was n o r m a l in p a t i e n t s w i t h r e m i s s i o n s (a m e a n of 29-+3 too) : Km = 28-+9 p U / m l ; Vmax =18.5-+1.3 m g / K g . m i f f 1 (n=ll). Resistance reappeared progressively during relapse to insulin-dependency with suboptimal metabolic control, a l t h o u g h p r e c e s s i o n of one e v e n t on the o t h e r was d i f f i c u l t t o d e t e r m i n e . P a t i e n t s ' age, body m a s s index, p l a s m a l e v e l s of 3 - h y d r o x y b u t y r a t e , NEFA, c o r t i s o l w e r e "similar, w h e n s t a r t i n g the t e s t s , in a l l groups. H b A l c was higher, and C p e p t i d e l e v e l s l o w e r a t o n s e t and d u r i n g r e l a p s e s t h a n during r e m i s s i o n s . C o n c l u s i o n s : 1) Suppression of t h e r e s i s t a n c e t o insulin, which is p r e s e n t a t o n s e t and s u p p r e s s e d by t r e a t m e n t , m a y c o n t r i b u t e to r e m i s s i o n s ; 2) F a c t o r s of r e s i s t a n c e , i t s s u p p r e s s i o n and reappearance remain unclear.

A209

P772

PZ73

INFLUENCE OF A N ORAL PROTEIN LOAD ON THE P O S T P R A N D I A L (PP) B L O O D G L U C O S E HOMEOSTASIS IN TYPE-I-DIABETICS

Effect of insulin deprivation and replacement on human adipose tissue metabolism in vivo. E. Hagstrb'm-Toft, P. Arner, B. Ndslund, U. Ungerstedt and J. Bolinder. Department of Medicine and the Research Center at Huddinge Hospital and Department of Pharmacology, Karolinska Institute, Stockholm, Sweden.

M.Frank, A.M~ller, G.Biro and K.F.Weinges Protein (P) intake is not considered for variable insulin dosages in diabetic diet. 7-C-peptid neg., normal weight IDDM, aged 2953 y. HbAI within the normal range were studied on 4 consecutive occasions in randomised order. Insulin was infused i.v. in a forearm vein using an insulin pump for basal- and prandial insulin need. The usual diabetic diet was given except the study period overnight. The right chosen basal rate should constantly hold blood glucose (BG) levels between 5,0-6,7 mmol/l tinder fasting conditions in the afternoon and was intraindividually always identical. At 7 p.m. a standardized dinner containing 48 g Carbohydrates (C) or 0,25; -0,5;-1,0 g P/kg body weight ( B W ) plus 48 g C were eaten. BG, NEFA, C o r t i s o l , H G H and Glucagon were analysed overnight. Results are: i. At 7 p.m. BG-levels were measured between 5,2-7,7 mmol/l (u:5,Smmol/l). 2. Normalised that BG data to 100 % the exclusive C intake showed a nadir at 2 a.m. (78• up to 7 a.m. (86~14%).Addition of 0,25 g P/kg BW resulted in the lowest value 4 hours later (60+7%, p< 0,05) and at 7 p.m. 99• The ingestion of 0,5 (I,0) g P/kg BW caused a BG increase 3 h pp, maximtLm 4-5 (7) h with 142~12% (194~ 34%, p<0,05).3.NEFA (0,05 • mmol/l),Triglycerides (i18~0,18 mmol/l), H G H (652 uE/ml), Glucagon (58~19 ng/ml) showed no significant change (7 p.m.-7 a.m.). Cortisol increased from 2,1• (7 p.m.) to 14 T3ug/ml (7 a.m.). It is concluded that the protein content of diabetic diet worsens the pp BG-levels dose dependent inducing some brittleness in TYPe I-diabetics.

The effect of hypa/hyperinsulinemia on in vivo adipose tissue metabolism was studied using microdialysis in nine Type I diabetics, without residual insulin production. Dialysis probes, implanted in the subcutaneous adipose tissue were continuasly perfused and tissue dialysate metabalites were determined. Following withdrawal of an overnight intravenous insulin infusion plasma free insulin levels fell from 20 to i0 mull and there was a parallel increase in tissue and plasma glucose although the relative increase was 25% greater in adipose tissue (p<0.025). After insulin withdrawal the increase in adipose tissue glycerol (lipolysis index) was smaller than in plasma (p<0.025). Insulin replacement (plasma free insulin 100 mull) resulted in rapidly decreased glycerol and glucose levels in plasma and tissue. Addition of the adrene~'gic blockers phentolamine and propanolol to the tissue perfusate did not influence the glycerol levels. Adipose tissue and plasma lactate and pyruvate remained unchanged during insulin deficiency, but increased transiently (in plasma only lactate) following insulin replacement possibly reflecting local adipacyte metabolife production. In conclusion, insulin-induced changes in circulating metabolites only partly reflect metabolism in adipose tissue, a target tissue for insulin. In addition adrenergic mechanisms seem not to be involved in changes in lipolysis rate induced by hypo/hyperinsulinemia.

PS 33 Metabolism in Type 2 Diabetes P774

P775

DEFECTIVE NON OXIDATIVE GLUCOSE DISPOSAL IN NORMAL OFFSPRINGOF-RNONON-INSULIN-DEPENDENTDIABETICPARENTS G. Gulli, S. Haffner, E. Ferrannini, R.A. DeFronzo Diabetes Division, University of Texas at San Antonio

A DEFECT OF INSULIN SENSITIVITY AND ANTILIPOLYTIC ACTION IS PRESENT IN FIRST DEGREE RELATIVES OF TYPE 2 (NON-INSULINDEPENDENT) DIABETIC PATIENTS P.M. Piatti, M.G. Perfetti, L.D. Monti, M~ Nonato, E. Contrino, E. Sandoli, A.E. Pontiroli and G. Pozza. Istituto San Raffaele, Clinica Medica VII, Universita' di Milano, Milano, Italy. The aim of this study was to identify early metabolic defects in non diabetic first degree relatives (FDR) of subjects with type 2 (noninsulin-dependent) diabetes mellitus. Following an oral glucose tolerance test (75gr) performed in 37 FDR of diabetic patients, 13 subjects (overweight or obese) were classified as impaired glucose tolerance (IGT) and 24 (9 overweight or obese) as normal glucose tolerance (NGT). Insulin release was significantly higher in IGT than in NGT, independently of body weight. To further investigate insulin action in these subjects, 8 IGT and 8 NGT underwent a continuous insulin (25 mU/kg/h) glucose (4 mg/kg/min) infusion for 150 minutes. IGT subjects showed higher insulin levels at baseline and during the last 30 minutes of steady state (12.3~i.0 vs 8.3~0.9 mU/l, 55.4+4.9 vs 35.1~3.5 mU/l, p<0.005 respectively~, and higher blood glucose levels during the last 30 minutes (127~9.0 vs 91~5.1 mg/dl, p<0.005) and a significantly reduced suppression of plasma FFA levels (at 60 min, 0.30+0.04 vs 0.17+0.01 mM, p<0.01, at 150 min: 0.22~0.03 vs 0713+0.01 raM, p<0.05). In conclusion: i) IGT --is frequent among FDR of patients with type 2 (non-insulin-dependent) diabetes mellitus; 2) IGT subjects are hyperinsulinemic and insulin resistant, and

To investigate the metabolic defect inherited in Type 2 (noninsulin-dependent) diabetes (NIDD), we studied 12 MexicanAmericans (MA) (age 38+4 yrs, BMI 27+1 ) with normal OGTT and two NIDD parents. Ten matched MA without family history of diabetes (FHD) served as controls. Plasma insulin during OGTT (64+5 vs 45+4 #U/ml) and hyperglycemic (+100 mg/dl) clamp (HC) (78+3 vs 59+3 I~U/ml) was increased in MA with FHD while glucose uptake during HC was 25% reduced (7.45+1.14 vs 9.85+.87 mg/min-kg free fat mass (FFM)). In the basal state of a two step (20 and 40 mU/min-m 2) eugtycemic hyperinsulinemic clamp (EC) with3H-6-glucose (matching specific activity) and indirect calorimetry, hepatic glucose production (HGP), glucose oxidation (COX) and non-oxidative glucose disposal (NOGD) were similar in both groups. During 20 mU/min-m 2 EC, HGP suppressed by > 90% but glucose uptake (Rd) (3.30_+.46 vs 4.76+.4t mg/min'kg FFM ) was reduced in MA wifh FH, due to a decreased NOGD (.19+.27 vs 1.65+.50 mg/min.kg FFM ); GOX was normal. During 40 mU/min 9 m2 EC, Rd (8.35+.84 vs 11.072:.72 mg/min'kg FFM ) and NOGD (4.48+.66 vs 7.08 _+.68 mg/min-kg FFM ) were reduced (p < 0.01) while GOX (4.11+.39 vs 4.64+.41 mg/min-kg FFM ) was normal. CONCLUSION: in normal glucose tolerant MA at high risk of NIDDM, (1) insulin resistance is a characteristic feature; (2) it specifically involves the pathway of NOGD; (3) plasma insulin response to glucose is enhanced.

A210

P776

P777

IMPAIRED AEROBIC FUNCTION DURING EXERCISE IN MEN WITH RECENTLY DIAGNOSED TYPE 2 DIABETES E.Vanninen 1, M.Uusitupa 2, J.Remes 3, O.Siitonen3, J.Jormanainen 2, E.L/insimies1 and K.Py6r/il/i s. Departments of Clinical Physiology 1, Clinical Nutrition 2 and Medicine3, University Central Hospital, Kuopio, Finland The aim of the study was to assess the main determinants of the aerobic function during exercise in middle-aged men with recently diagnosed type 2 diabetes. 19 diabetic men without cardiovasoalar diseases (DM group), 20 diabetic men with coronary heart disease or hypertension (CHM group) and 19 healthy men (HM group) were studied. Maximum oxygen uptake (VOenax) and anaerobic

THE MYTH OF INSULIN RESISTANCE ASSOCIATED WITH AGING

threshold (VO2at) were measured with direct respiratory gas analysis during maximal incremental bicycle exercise test. Aerobic function declined similarly in all groups by aging. Adjusted for age, both VOzmax (ml/min, mean -+ SE) and VO2at (ml/min) were lower in the DM group than in the HM group (VO2max 2061 -+ 140 vs 2943 -+ 121, p=0.001, and VOzat 1252 -+ 61 vs 1738 -+ 74, p=0.012). VOzmax and Vozat were in the CHM group lower than in the DM group. Among diabetic men, the impaired VOzmaxwas associated with low insulin response during the oral glucose tolerance test accompanied by hyperglycemia. In addition, smoking more than 20 years was associated with lowered VO~max. To conclude, the lowered VO~ax and VO2at in patients with type 2 diabetes are associated to metabolic changes known to result in impaired glucose utilization.

A Franssila-Kallunki, C Schalin, A Ekstrand, C Saloranta, J Eriksson and L Groop. Fourth Department of Medicine, Helsinki University Hospital, Helsinki, Finland To examine the effect of aging on glucose and lipid metabolism we studied 8 young (age 22• years, BMI 21.8!0.8 kg/m2), I0 middle-aged (age 43+3 years, BMI 24.2+0.9 kg/m 2) and 8 ,,old', (age 68+2 years, BMI 23.9+0.9 kg/m 2) healthy subjects with a + 80 uU/ml insulin clam2 in combination with indirect calorimetry and =M-3glucose. Lean body mass (measured with 3H20) was lower in old (44+3 kg) compared with young (53+6 kg) and middle-aged (54+3 kg) subjects (both p<0.05), resulting in higher fat percentage in old subjects (35% vs 24% and 27%). Fat percentage and basal FFA correlated positively with age (R=0.54 and R=0.64; both p<0.01). Basal rate of glucose oxidation was lower in old compared with young subjects (1.71+0.14 vs 2.49+0.38 mg/kgLBM.min; p<0.05) and inversely correlated with age (R=-0.64; p<0.001). Insulin-stimulated glucose oxidation and storage did not differ between young (3.85+0.49 and 5.57!0.61 mg/kgLBM.min), middleaged (4.38+0.51 and 4.94+0.62 mg/kgLBM.min) and old (4.04+0.21 and 5.14+0.76 mg/kgLBM.min respectively) subjects. Conclusion: Both the oxidative and nonoxidative pathways of glucose metabolism are normally sensitive to insulin in aging. However, aging is associated with impaired basal oxidation of glucose, which is unrelated to the increased fat mass.

PTT8

P779

RELATIVEIMPORTANCEOF INSULINSENSITIVITYANDB-CELLRESPONSIVITYTO IMPAIREDGLUCOSETOLERANCEWITHAGEANDDIABETES G.L. Viviani, G. Pacini, M.G. Borgoglio, C. Cobelli, and L. Adezati. ISMI,

COMPARISON OF ELDERLY DIABETES AND MATURITY DIABETES: SIMILARITIES AND DIFFERENCES. V.Cacciatori, E.Bonora, M.G.Zenti, M.Zenere, F.Saggiani, P.Moghetti and M . M u g g e o . D e p a r t m e n t of Metabolic Diseases, University of Verona, Italy. Aim of the p r e s e n t s t u d y w a s to compare two groups of non-insulin-dependent diabetic subjects, one i n c l u d i n g 89 p a t i e n t s w i t h o n s e t of the disease after the age of 65 (elderly diabetics=ED), and the other including 89 patients w i t h o n s e t of t h e d i s e a s e between 40 and 55 yr (maturity diabetics=MD). The two g r o u p s w e r e i n d e n t i c a l f o r sex, a n d s i m i l a r for duration of disease. E D and M D d i d not differ in body mass index, skinfold-estimated body composition, and fat t o p o g r a p h y . Also plasma glucose, HbAlc, total cholesterol and triglycerides w e r e s i m i l a r in the two g r o u p s . In ED H D L - c h o l e s t e r o l w a s lower, while fasting Cpeptide was higher. ED had a less frequent family history of diabetes (21 vs. 56%, p
University of Genova and LADSEB-CNR, Padova, Italy.

The present study evaluated the effect of age and diabetes on reduced glucose tolerance, assessing individual contributions of insulin sensitivity and B-cell responsiveness. Subjects: all non-obese, selected by 75g-OGTT area (GA): Controls: 11 young (C, age=25!2[SE] yrs, weight=72+4 kg, GA=11205:56 mM.3h), 6 elderly (E, 73+3, 63+4, 1288.+.56); Type-II Diabetics: 14 young (N, 26+3, 58-+3, 1624+56), 11 elderly (D, 675:2, 64+2, 2184+112). Tes~: 0.3g/kg-IVGTT with minimal modeling analysis which provides: insulin sensitivity (SI, 10-4mina/~U/ml)), dynamic insulin-independent fractional glucose disappearance (So,10-3/min), pancreatic first (Ol,102min(pM)/(mM)) and second ((I)2,10-1min2(pM)/(mM)) phase responsivity. Results: tolerance index K o was C=1.750.3 (min-1), E=1.550.2, N=l.050.1, D=0.6+0.1, negatively correlated to GA (r=-0.611, p<0.0001). The other parameters were: SI: C=4.4+0.6, E=3.5+0.3, N=1.85:0.2, D=3.6+0.4; SO: C=25+4, E=20+3, N=25+3, D=10+I; (I)1: C=5.1+1.3, E=3.8+1.3, N=5.1-+1.3, D=0.8+0.3; ~2: C=2.0+0.3, E=1.3+0.1, N=1.2+0.3, D--0.8+0.3. SI was reduced by 59% (p<0.0005) with diabetes in young, while was not diminished in D, who however exhibited lower So (D vs C p<0.005, D vs E p<0.0005) and markedly reduced ~1 (p<0.0005). Major defects were in 9 2 reduced by 38% (p<0.05) with age, by 44% (p<0.025) with diabetes, by 63% (p<0.005) with both. Synergistic effect of aging and diabetes induces reduction of insulin release which is unable to counterbalance diminished insulin sensitivity.

A211

P780

P781

INCREASED ARACHIDONIC ACID IN ADIPOSE TISSUE FROM PATIENTS WITH TYPE 2 (NON-INSULIN-DEPENDENT) DIABETES

Proglucagon products in plasma of non-insulin dependent diabetics and non diabetic controls.

C.Popp-Snijders and M.C. Blonk, Free University Hospital, Amsterdam, The Netherlands.

C. ~rskov, J. Jeppesen, S. Madsbad, and J.J. Holst Dept. of Clinical Chemistry, Rigshospitalet, and Institute of Medical Physiology C, Panum Institute, UniVersity of Copenhagen, Copenhagen, Dermmrk, and Dept. of Medicine F, Herlev Hospital, Herlev, ~ k .

The insulin resistant state is associated with a decreased conversion of linoleic into arachidonic acid. In experimental diabetes a lower arachidonic acid content of adipose tissue is seen. As adipose tissue is a source of fatty acids for the de novo synthesis of phospholipids, altered levels of arachidonic acid might have consequences for membrane lipid composition, especially in periods of negative energy balance. We analysed, using capillary gas chromatography, the fatty acid composition of adipose tissue from three groups (mean • SD): subjects with type 2 (non-insulindependent) diabetes (D) (n = 59, age 55.8 i 9.3 yr, body mass index (BMI) 32.0 +- 4.0 kg/m2); subjects with intermittent claudication (CI) (n = 37, age 66.1 i 8.2 yr, BMI 26.7 i 3.1 kg/m2); healthy subjects (H) (n = 26, age 33.7 i 5.2 yr, BMI 23.2 i 2.0 kg/m2). Arachidonic acid (mol%) was significantly higher in the group with diabetes: (D 0.50 • 0.ii; CI 0.36 i 0.13"; H 0.24 +- 0.07*, *P<0.001). Linoleic acid content (mol%) in the three groups did not differ: (D 14.0 +- 3.2; CI 14.9 i 5.3; H 14.8 • 2.6), indicating a similar dietary intake of linoleic acid. The results suggest that in type 2 (non-insulin-dependent) diabetes the metabolic conversion of linoleie into arachidonic acid is increased rather than decreased~ We conclude that insulin resistance resulting in glucose intolerance does not necessarily imply impaired fatty acid metabolism.

We investigated the major proglucagon (FG) products in plasma in the fasting state, following intravenous arginine and after an oral glucose load in non-insulin dependent diabetics (NIDDM) and weight matched controls using specific radioinmmnoassays and analytical gel filtration. In the fasting state the glucagon-like peptide-i (GLP-I) ~ o r e a c t i v i t y was significantly elevated in the NIDDM group ~ e d to the control group. Both following intravenous arginine and after oral glucose load a rise in the plasma concentrations of all ~ e a c t i v e moieties measured was seen. Furthermore, all integrated incremental responses following intravenous arginine were identical in the two groups. Following oral glucose the insulin concentrations in plasma were lower and the concentrations of all proglucagon products were higher in the NIDDM group cfmi0ared to the control group. Ehe gel filtration analysis showed that arginine sti~mlated the secretion of pancreatic glucagon (PG 33-61), major proglucagon fragment (PG 72-158) and probably GLP-I (PG 72-108) in both groups, whereas oral glucose stimulated the secretion of glioentin (PG 1-69) and intestinal GLP-I (PG 78-107 amide), an insulinotropic hormone.

P782

P783

GLUCOSE AND GLUCONEOGENIC SUBSTRATE EXCHANGE BY THE FOREARM SKELETAL MUSCLE IN H Y P E R G L Y C E M I C AND INSULIN-TREATED T Y P E II D I A B E T I C P A T I E N T S . B C a p a l d o , R N a p o l i , P Di B o n i t o , G A l b a n o a n d L S a c c ~ - Dept~ of I n t e r n a l M e d i c i n e N a p l e s To determine the contribution of skeletal muscle to f a s t i n g hyperglycemia in t y p e II (non-insulin-dependent) diabetes, the forearm balance of g l u c o s e , lactate, and alanine was quantitated in 25 control subjects, 21 hyperglycemic (blood g l u c o s e : 11.6 m m o l / L ) a n d 19 insulin-treated patients with type II diabetes (blood glucose: 5.8 mmol/L). Forearm glucose uptake was similar in controls (4.6• ~mol/liter/min) and in hyperglycemic diabetic patients (4.5• ~mol/liter/min). D e s p i t e this, in diabetic patients lactate (5.1• ~mol/liter/min) and alanine (2.6• release by t h e forearm was five- and two-fold higher t h a n in the control group (lactate: 1 . 1 • p<0.005; and alanine: 1.3• p<0.05, respectively). The percentage of t h e g l u c o s e t a k e n u p b y t h e m u s c l e c o n v e r t e d to l a c t a t e w a s 57% and 12% in diabetic and control subjects, respectively (p<0.01). Insulin administration did not a f f e c t either g l u c o s e u p t a k e or t h e r e l e a s e of g l u c o n e o g e n i c substrates by the f o r e a r m . It is concluded that: i) In fasting patients with t y p e II diabetes, g l u c o s e is t a k e n u p by the s k e l e t a l m u s c l e in normal amounts but preferentially utilized non-oxidatively with lactate formation. This suggests that, although the m u s c l e d o e s not c o n t r i b u t e d i r e c t l y to f a s t i n g hyperglycemia, it may play an i n d i r e c t role through an increased delivery of glucose precursors; and 2) insulin-induced normoglycemia is m a i n t a i n e d by m e c h a n i s m s t h a t do not i n v o l v e the exchange of g l u c o s e and gluconeogenic substrates by the skeletal muscle.

INHIBITION OF GLUCONEOGENESIS DOES NOT REDUCE HEPATIC GLUCOSE OUTPUT IN TYPE 2 DIABETIC PATIENTS I. Puhakainen, V. Koivisto and H. Yid-J~Mnen, Second Department of Medicine, Helsinld University, Helsinld, Finland increased gluconeogenesis has been suggested to account for all of the increase in basal hepatic glucose output (HGO) in type 2 diabetes. We studied the effect of inhibition of gluconeogenesis with ethanol on total HGO in type 2 diabetes. 4- years, fasting glucose 11.4_0.8 4Fourteen patients (age 61-2 raM, body mass index 27+1 kg/m2) were studied twice after an overnight fast, once during a 300 rain ethanol (blood ethanol 13+2 raM) and once during saline infusion. Total HGO was measured using tritiated glucose and inhibition of gluconeogenesis was followed using (U-l'C)lactate (n=8) and (U-l'C)glycerol (n=6). 3H and '4C-glucose, glycerol and lactate specific activities were determined with HPLC. Ethanol inhibited gluconeogenesis from lactate by 71+-5% (1.1+-0.4 vs. 3.7+-0.4 pmol/kg.min, 240-300 min, ethanol vs. saline, p<0.001), and from glycerol by 65+-6% (1.6+-0.6 vs. 4.6+1.2 pmol/kg.min, p<0.001, respectively). Total HGO remained unchanged and averaged 12.8+-1.8 in the saline and 11.8-+2.1 /~mol/kg.min in the ethanol study (NS). We conclude that a "70% inhibition of gluconeogenesis by ethanol does not decrease total glucose production in type 2 diabetic patients. These results suggest the existence of an intrahepatic mechanism that maintains total HGO constant despite inhibition of gluconeogenesis. Therefore therapeutic means aimed at inhibition of HGO should either inhibit this compensatory mechanism, or inhibit HGO at sites where gluconeogenesis and glycogenolysis converge.

A212

P784 QUANTIFICATION OF THE CONVERSION TO CIRCULATING GLUCOSE OF FRUCTOSE INGESTED DURING EXERCISE IN MAN. B.J. 3andrain, S. Normand, A.3. 5cheen, F. Pirnay, M. Lacroix, F. Mosora, N. Paiiikarakis, O.P. Riou and P.O. Lef~bvre. University of Li@ge, Belgium and University of Lyon, France. We investigated the metabolic fate of fructose ingested during prolonged-duration moderate-intensity exercise in man, and evaluated fructose conversion to plasma glucose. Five healthy male volunteers exsrcized for 5 hours on a eadmill at 45 % of their V02 . "Naturally labeled" ~C-fructose was given orally ~ 0 g, i.e. 25 g every 30 min). Substrate utilization was evaluated by indirect calorimetry, fructose oxidation by isotope ratio mass spectrometry (IRMS) on expired C02, and fructose conversion to plasma glucose was measured combining gas chromatography and IRMS. Throughout exercise, blood glucose plateaued at about 4.0 mmol/l, blood fructose levels reached about 0.4 mmol/l, plasma insulin and C-peptide levels regularly decreased, plasma NEFA and glycerol levels increased continuously. Total fructose utilization (oxidation of fructose plus oxidation of glucose resulting from hepatic fructose conversion) reached 62 + 7 g/3 h and covered 15.5 + 0.8 % of the total 6.5 MO ene?gy need, of which total ca?bohydrstes and lipids respectively accounted for 44.7 Z 2.9 and 53.8 Z 3.0 %. During the last 2 h of exercise, 55 to 60 ~ of plasma glucose was issued from ingested fructose. In conclusion, fructose ingested during prolonged exercise maintains normoglycaemia and significantly contributes to the energy demand ; at the end of the exercise bout its hepatic conversion to glucose accounts for more thsn half of the circulating glucose.

~

PS 34 Obesity and Insulin Resistant States P785

P786

TOTAL AND R E G I O N A L BODY FAT: R E L A T I O N S H I P S WITH THE MAIN METABOLIC PARAMETERS IN TYPE II DIABETES MELLITUS. F.Tosi, E.Bonora, V.Cacciatori, M.Zenere, P.Branzi, D.Travia and M.Muggeo. D e p a r t m e n t of M e t a b o l i c Diseases, U n i v e r s i t y of Verona, Italy. Recent studies have indicated that in the general population body fat t o p o g r a p h y is an important determinant of several metabolic parameters, including plasma g l u c o s e and lipids. Aim of the present study was to investigate the impact of total body fat content and fat topography on several m e t a b o l i c parameters in 300 randomly selected p o s t m e n o p a u s a l type II diabetic women. Age was 66.5• yr (mean• duration of d i a b e t e s was 9.520.44 yr, body mass index was 2 7 . 7 • Kg/m2, skinfoldestimated total body fat content averaged 2 5 . 4 2 0 . 4 3 Kg. Body fat t o p o g r a p h y was e v a l u a t e d from the analysis of waist to hip c i r c u m f e r e n c e ratio (WHR, 0.9420.005) and subscapular to tricipital skinfold ratio (STR, 1.38+0.05). Total body fat content was significantly correlated with fasting glucose (r=O.18, p, triglycerides (r=0.2~ p<0.22, p
P L A S M A P R O I N S U L I N IN O B E S I T Y A N D T Y P E 2 (NONINSULIN-DEPENDENT) DIABETES MELLITUS. S. Madsbad, B. Tronier, A. D e j g a a r d and T. Andersen. Hvid0re Hospital and NOVO R e s e a r c h Institute, Copenhagen, Denmark. P r o i n s u l i n s e c r e t i o n was s t u d i e d in 8 lean and 7 obese(85%) n o r m a l subjects and 6 lean and 7 obese(53%) Type 2 p a t i e n t s d u r i n g f a s t i n g and after 25 g of oral glucose. In lean normal subjects fasting proinsulin was 3.8 +/-0.6 pmol/l. A f t e r oral g l u c o s e the p r o i n s u l i n secretion e v a l u a t e d by the area u n d e r the curve (AUC) was 1.5 nmol/i/min. The f a s t i n g m o l a r ratio(%) b e t w e e n p r o i n s u l i n and c - p e p t i d e was 0.84 +/0.15 and i n c r e a s e d to 1.2 +/- 0.2 d u r i n g oral glucose. Obese vs lean n o r m a l s d i s p l a y e d a 4-5 fold increase in p r o i n s u l i n levels (fasting 20.3 +/- 7.3 p m o l / l , p < O . 0 5 , AUC: 6.0 +/- 2.0 n m o l / i / min, p<0.05), and a two fold increase in f a s t i n g p r o i n s u l i n / c - p e p t i d e ratio (1.6 +/- 0.5, p:NS). This ratio did not increase after oral glucose. In Type 2 p a t i e n t s fasting p r o i n s u l i n levels (lean: 7.0 +/- 1.7 pmol/l, obese: 14.8 +/-3.6 pmol/l) and s t i m u l a t e d s e c r e t i o n (lean: 2.1 +/0.3 nmol/i/min, obese: 4.0 +/- 1.2 nmol/i/min) were not s i g n i f i c a n t l y d i f f e r e n t from those of m a t c h e d normal subjects. The p r o i n s u l i n / c - p e p t i d e ratios w e r e the same in Type 2 p a t i e n t s and normal subjects. Conclusion: Normal p r o i n s u l i n s e c r e t i o n was d e m o n s t r a t e d in m i l d Type 2 diabetes. O b e s i t y per se is a s s o c i a t e d w i t h e l e v a t e d plasma p r o i n s u l i n in normal s u b j e c t s as well as in Type 2 d i a b e t i c patients.

A213

P787

P788

NORMALIZATION OF BODY krEZGHT DOES NOT CO~:LECT THE EXAGGERATED INSULIN RESPONSE TO BETA-ENDOP,2H~',I IN HUMAN OBESE'r~" Giugliano D, Cozzolino D, Saecomanno F, Torella R, Lefebvre PJ and D'Onofrio F University of Naples, Italy and University of Liege, Belgium. Beta-endorphin (B-endo) inhibits insulin secretion both in the basal state and in response to nutrients in normal man. By contrast, B-endo induces hyperinsulinemia in obese subjects which suggests a role for the peptide in some metabolic events associated with obesity. We studied the hormonal and metabolic responses to an infusion of human synthetic B-endo (0.5 mg/kg) in 10 formerly obese (FO) subjects who had lost 30 kg gr more by dieting and compared with those of 10 normal weight (NW) subjects matched for age, sex and weight (BMI <25). In NW subjects, B~endo produced concomitant and significant increases of plasma glucose and glucagon without changing insulin or C-peptide; in F0 subjects, the same infusion rate of B-endo significantly increased both insulin and C-peptide plasma levels which restrained the hyperglycemic effect of the opioid. Basal plasma B-endo concentrations were significantly higher (p <0.01) in FO subjects respect to NW subjects. These results demontrate that FO subjects behave like obese subjects in their metabolic and hormonal responses to B-endo and suggest some role for the opioid peptide in the disregulation of B-cells function in obesity and in the predisposition to weight gain.

DORELATIVES OF TYPE 2 DIABETICS DEVELOPDIABETES BECAUSETHEY ARE OBESE OR DO THEY DEVELOPOBESITYBECAUSETHEY ARE RELATIVESOF TYPE2 DIABETICS? d,Eriksson, A.Ekst,rand, A.Franssila-Kallunki, C.Saloranta, C,Schalin, E.WidBn and L. Groop. IV Department of Medicine, Helsinki University Hospital, Helsinki, Finland.

P789

P790

IMPAIRMENT OF GLYCOLYTIC RATE, GLUCOSE OXIDATION, AND GLYCOGEN SYNTHESIS IN OBESE TYPE 2 DIABETICS. S. Del Prato, E. Bonora, G. Gulli, R_A. De Fronzo, and R.C. Bonadonna. University of Texas, San Antonio, Texas, Usa

IMPAIRED ACTIVATION OF SKELETAL MUSCLE GLYCOGEN SYNTHASE (GS) IN TYPE 2 (NON-INSULIN DEPENDENT) DIABETES IS UNRELATED TO THE DEGREE OF OBESITY. AB Johnson, M Argyraki, J Thow and R Taylor. Department of Medicine, University of Newcastle upon Tyne, UK.

The metabolic pathways of glucose disposal were examined in 9 obeseType 2.diabetics (Ft~=10.7+1.0 retool/I; age=-56.~ y;, BM.I=28+~) after correction of glucose uptake with hyperglycemia/hyperinsulinemia during three clamps plus [3-~-IJ-and [U-t~-q-glucose and indirect calorimetry. Study 1:20 mU/m2/min euglycemic insulin clamp; Study 2: hyperglycemic (15.7+1.4 retool/l) insulin (20mU/m=/min) clamp; Study 3: euglycemic hyperinsulinemic (92.~.16mU/m:/min)clamp. Study 1 was performed in 7 controls. In Study I, glucose uptake (GU) was 55% lower in diabetics (18.3+12.2 vs 40.5_+6.1 I.tmoI/kgFFM/min); glycolysis ( ~ O generation; GR= 12.2A:1.1 vs 22.2.+_2.2),glycogen synthesis (GU-GR--GS=6.1+I.7 vs 18.9-k-6.1),glucose oxidation (t4C"Ozproduction; GOX--8.9+l. 1 vs 19.4+1.7) were also lower (p<0.01). Lipid oxidation was higher in diabetics (LOX=5.6__+0.6 vs 1.I+0.6). In Studies 2 & 3 GU was equal to controls (40.0-L-_1.7and 41.7+-1.7). However, in Study 2, GR (15.5+1.1) and GOX (I2.7+1. I) remained lower (p
The contribution of obesity to peripheral insulin resistance in Type 2 (non-insulin dependent) diabetes remains uncertain. To examine this 25 newly presenting, untreated, Type 2 diabetic subjects were studied. They were allocated to 3 groups according to their body mass index (BMI), [lean BMI<25.0, n=9; overweight 25.0-30.0, n=6; obese >30.0kg/m 2, n=10]. All 3 groups exhibited equivalent hyperglycaemia. Fasting serum insulin increased with BMI (4.6-+1.3 vs 8.5-+1.4 vs ll.l-+l.8mU/l respectively), the difference between lean and obese being significant (p<0.02). 11 normal controls were also studied. Muscle biopsies for estimation of GS activity were taken prior to and during a 240min hyperinsulinaemic euglycaemic clamp (100mU/kg/hr). Fractional GS activity did not increase (insulinstimulated minus basal) in the lean (change -0.9• the overweight (-1.9-+2.7%) or the obese (+2.2_+1.6%) Type 2 diabetic subjects during the insulin infusion and was markedly decreased compared to the controls (change +14.6• all p<0.001). Glucose disposal (M) was also significantly decreased in all 3 diabetic groups (103_+23 vs 81-+14 vs 52_+14mg/m2/min respectively compared to the controls (319_+18mg/m2/min, all p<0.001). Stimulated GS activity correlated with M (r=0.50, p<0.001). Thus these studies demonstrate the failure of insulin to activate skeletal muscle glycogen synthase in Type 2 diabetic patients and that this was independent of the degree of obesity. This defect may play a major role in the development of peripheral insulin resistance in these patients.

To address the above question we studied the thermogenic response to insulin in 18 controls (CON; age 47+3 y; BMI 23.7+0.6 kg/m2), 20 Type 2 diabetics (Type 2; age 59+t y; BMI 26.9+0.9 kg/m 2) and 44 of their first-degree relatives (REL; age 46+2 y: BMI 26.0+0.5 kg/m 2) using the hyperinsulinemic euglycemic insulin damp technique combined with indirect calorimetry. 61ucose disposal was impaired in Type 2 and REL (52.1• and 55.5• vs CON 47.7• pmoltkgLBM.min; p
vs CON 5.3•

kJlkgLBM,mtn; p
Conclusions: Both Type 2 diabeLics and their relatives have impaired glucose storage which correlates wtt,h impaired thermogenesis, While the diabetics show enhanced BEE and LOX their relatives have normal BEE and LOX, These findings suggest that relatives or Type 2 diabetics show defects in their energy metabolism which may result, in maintenance of the obese state.

A214

P791

P792

DIMINISHED HEPATIC INSULIN EXTRACTION IS NOT RESPONSIBLE FOR HYPERINSULINAEMIA IN OBESITY A.Kautzky, G.Pacini, E.Brauner, G.Schernthaner and R.Prager Department of Medicine II, University of Vienna, Garnisongasse 13, A-1090 Wien & LADSEB, Padua, Italy & Rudolfstiftung Hospital, Department of Medicine I, Juchgasse 25, A-1030 Wien

DIFFERENT ETIOLOGY FOR NON-INSULIN DEPENDENT DIABETES IN OBESE AND NON-OBESE SUBJECTS

A diminished hepatic insulin extraction could be responsible for peripheral hyperinsulinemiS in obesity. To t e s t %his hypothesis, we investigated insulin sensitivity and hepatic insulin extraction in 18 nondiabetic obese (BMI: 39+1.3 kg/m 2) patients (O) in comparison to 18 healthy lean (BMI: 21.3+0.7 kg/m 2) control subjects (C) by using a noninvasive mathematical model (Diabetes 37: 223, 1988). In addition, body f a t distribution was calculated by the waist to hip girth ratio (WHR). A highly reduced tissue insulin sensitivity (24+0.5 vs. 9.5+1.5 min-2/(pU/ml); p<0.0005) and glucose e f f e c t i v e n e s s (0.016~.003 vs. 0.03+ 0.003 rain -1, p<0.005) was found in 0 compared to C. The basal and total prehepatic insulin secretion (37848_+5562 vs. 16864+ 1850) as well as t h e basal and total posthepatic insulin delivery (8285+2009 vs. 2840+210 pM) were significantly higher in 0 compared with C, whe{eas t h e hepatic insulin extraction did not differ (77.8_+2.6 vs. 79.5_+2.6). However, we found a significant inverse correlation between hepatic insulin extraction and WHR (p<0.04; R=0.5). A diminished hepatic insulin extraction was only found in patients with an extraordinary central body f a t distribution (WHR>0.95; n=4) in comparison to C (60.3_+2 vs. 79.5_+2.6). In summary, hyperinsulinemia in obesity is not caused by diminished hepatic insulin extraction, but is simply due to pancreatic 8-cell hypersecretion.

P, Arner, T. Pollare and H. Lithelh Huddinge and Kungsg~rdets Hospital, Karolinska Institute and Uppsale University, Sweden. The aim was to evaluate insulin secretion and peripheral insulin sensitivity in subjects with newly diagnosed untreated noninsulin-dependent diabetes, The insulin response to intravenous glucose and total glucose disposal during an eugfycemie hyperinsulinemlc ('mean ]01] mU/I) clamp were investigated in 12 non-obese and ]B obese 65 year-old male diabetics, qq body weight matched 195-year old healthy male subjects with normal qlucose tolerance served as control. In obese non-dlabetics insulin sensitivity [~lucose disposal/plasma insulin during clamp) was decreased by 1/3 ('p
P793

P794

OF INSULIN RELEASE IN EXPERIMENTAL OBESITY. B. Balkan, J.B. Strubbe, and A.B. Steffens. Department of Animal Physiology, University of Groningen, Haren, The Netherlands.

INFLUENCE OF BODY FAT TOPOGRAPHY IN GLUCOSE AND SERUM LIPIDS HOMEOSTASIS. J.Anselmo, F.Vaz, L.G.Correia, E.Pereira, F.L.Silva, M.T.Pires and J.C.Nunes-Correa. Endocrinology Unit, Curry Cabral Hospital, Lisbon, Portugal. Predominant fat distribution in the upper body segment evaluated by waist to hip circunference ratio (WH ratio) has been associated with diabetes and cardiovascular morbidity excess. To investigate metabolic alterations underlying this risk excess, we selected 2 groups of i0 obese women without history of hipertension, menstrual irregularities or oral contraceptives, matched according to age (average~SD:30.5+5.3 vs 30.6• range 18-38 years) and BMI (35.5+6.5 vs 35.7+6.7, range 30.5-53 Kg/m2). Each matched pair had a difference in WH ratio superior to 0.15 (0.83J0.04 vs 1.02~0.05). Insulinaemia and C peptide were determined during an oral glucose tolerance testa (75g). Statistical analysis was by the t test with 9 degrees of freedom. At 30, 60, 90 and 120 minutes differences were significant for glycaemia (p<0.05,~O.01,p~0.02 and p < O . 0 2 ) , insulinaemia (p~=0.02,p~0.02,p~10.01 and p ~ 0 . 0 5 ) and C peptide (p ~ 0 . 0 5 , p ~ = 0 . O 5 , p ~ O . O l and p~O.01). Fasting triglycerides were 1.13+0.53 mmol/l in the lower WH ratio group vs 1.81+0.73 (p
DETERMINANTS

Hyperinsulinemia and insulin insensitivity ~re characteristics of obesity as well as type 2 diabetes. To investigate the regulation of insulin secretion in obesity, rats rendered obese by electrolytical lesioning of the ventromedial hypothalamus, were subjected to insulin release stimulating and suppressing situations. Male Wistar rats were provided with permanent cardiac catheters for blood sampling. During physical exercise (15min swimming) adrenal epinephrine is suppressing plasma insulin levels more in obese (120•177 than in lean (44•177 animals (p<0.01). Relative to basal concentrations the changes are, however, identical (-38• vs -41• This indicates that insulin insensitivity is compensated for by a stronger sympathetic suppression. Intracardial infusion of glucose (150mg/15min) in resting animals resulted in higher rises in plasma insulin in obese than in leans (128•177 vs 50•177 mU/l, p<0.05), while relative responses were lower in the obese animals (99• vs 183• %, p
A 215

P812

P811 IN VIVO FUNCTIONAL MORPHOLOGY

OF BROWN ADIPOSE TISSUE BY

MAGNETIC RESONANCE IMAGING C.Zancanarol,A.Sbarbatil,F.Leclercq~and F.0sculati I. Inst! tute of Human Anatomy and Histology, University of Verons, Italy I and Institut de recherches scientifiques sur le can

cer du CNRS, Villejuif,

France 2.

We used high-resolution magnetic resonance imaging (MRI, spin-echo pulse sequence TE=800 ms,TR=30 ms) to investigate in vivo the interscapular brown adipose tissue (IBAT) of laboratory rodents. The IBAT of mice at different ages was visualized, giving insight into the BAT content of intact animals; regions of prevailing multivacuolar or monovacular adipocytes, identified by subsequent ultrastructural examination, were defined by MRI. MRI easily identified the IBAT of newborn mice because it emitted a very weak signal, probably because of the low lipid content during active thermogenesis in the early post-natal period and the abundance of iron-containing mitochondrial inclusions. MRI detected changes in the volume and adipoeyte composition of the IBAT after rats underwent cold exposure and/or dietary manipulation .The reverse phenomena upon re-acclimatation to ambient temperature were also visualized. Results show that MRI performs efficient investigation of the functional morphology of IBAT in vivo; repeated examinations are feasible; IBAT can in principle be quantified by means of multislice MRI and morphometry; the response of BAT to physiological (and pharmacological) stimuli can be evaluated. These data are potentially relevant to the study of obesity and Type 2 (non insulin-dependent) diabetes in animal models and, possibly, in man.

ACETAMINOPHEN GLUCURONIDATION ACCURATELY REFLECTS GLUCONEOGENESIS IN FASTED DOGS. W.F. Schwenk, Mayo Graduate School of Medicine, Rochester, MN. An in vivo technique for sampling the intrahepatic UDP-glucose pool recently has been reported using the urinary glucuronide of acetaminophen. However, hepatic metabolic zonation may exist, with periportal gluconeogenesis and glucose release along with perivenous glucose uptake and glucuronidation. If such zonation exists, use of the glucuronide may not correctly indicate the relative importance of gluconeogenesis to glycogen synthesis. To assess whether the acetaminophen glucuronide accurately reflects UDP-glucose derived from gluceneogenesis during fasting, on 2 occasions mongrel dogs received steady state infusions of [UJ4C]lactateand [1-13C]galactose followed by acetaminophen. The mean plasma [14C]glucose specific activity was similar to the mean urinary acetaminophen glucuronide specific activity both after fasting overnight (299 + 19 vs 296 + 14 dpm/nmol, respectively) and after 2 additional days of fasting (511 _+ 8 vs 562 + 32 dpm/nmol, respectively). In contrast, the mean plasma [13C]glucese enrichment was lower than the mean urinary acetaminophen glucuronide specific activity (25.7 _+ 4.4% vs 28.5 _+ 7.7% for overnight plus 2 additional days of fasting, respectively). In summary, the acetaminophen glucuronide specific activity derived from labeled lactate, but not labeled galactose, accurately reflected the plasma glucose SA in overnight and 289 fasted dogs. In conclusion, in fasted dogs, plasma glucose and UDP-glucese as sampled by the glucuronidation of acetaminophen equally reflect gluconeogenesis.

P813

P814

AN APPROXIMATE METHOD FOR NON-STEADY-STATE TRACER ANALYSIS

A NEW MINIMAL MODEL ALLOWS TO MEASURE HEPATIC GLUCOSE PRODUCTION DURING THE LABELLED IVGTT. Andrea Caumo and Claudio Cobelli* S. Raffaele Scientific Institute, University of Milan, Milan. *Department of Electronics and Informatics, University of Padua, Padua.

A. Mari, CNR Institute for System Dynamics and Bioen~neering (LADSEB), Padova, Italy Non-steady-state tracer analysis is difficult because mathematical modelling is inevitable. Steele equation is not always

a reliable solution, while the more complex models proposed as alternative pose several difficulties or limitations. This study presents a new equation for the rate of appearance (Ra), based on a two-comparmaent model. The equation parameters are the initial and total distribution volume, and the time constant of the non-accessible compartment. Calculations require

only a spreadsheet on a personal computer. The method applies to every experimental condition. It yields Ra and an error bound for it, which can be calculated individually using estimates of the errors of the equation parameters. Data from hyperinsulinemic (1 mU/min/kg) euglyc3emic clamp experiments (n=5) with primed-constant [3- H]glucose infusion have been analyzed. Calculated glucose production fell progressively to nonnegafive values in 120 rain (12.35+0.11, 6.09+1.36, 0.17i-0.75 ~tmol/min/kg, mean.-I:se, at 0, 50-70,

100-120 min). Steele equation predicted total suppression in 30 rain and negative values in the last hour (-2.62i0.72 Imaol/min/kg, 100-120 rain). The pattern of glucose production suppression is comparable to that obtained in similar conditions with more sophisticated modelling or experiment design. In conclusion, the method is simple to implement and is a valid ahemative to Steele equation when an effective specific activity clamp is impossible.

Measuring hepatic glucose production (HGP) during labelled intravenous glucose tolerance test (LIVGTT) is difficult since the system is in non-steady-state. HGP can be derived from the two classical minimal models describing unlabelled and labelled glucose kinetics during LIVGTT, but the predicted profile is physiologically absurd. In order to develop a new minimal model able to yield a physiological HGP, we analyzed stable-label IVGTT studies (lasting 240 mins) in 5 normals. First, we derived evidence that the classical minimal models perform poorly because they hinge on a monocompartmental description of glucose kinetics, which has well-known limitations in nonsteady-state. Subsequently, we developed a new minimal model describing labelled glucose kinetics by a twocompartment structure with insulin action taking place in the slowly equilibrating compartment. HGP from the new model resulted physiologically plausible: it reached nadir (17% of basal, [range 0-30%]) within 20 mins from the injection; then, returned to the basal level within ii0 mins [range 100-120 mins]; finally, showed a transient overshoot (16% of basal, [range 124%]). In conclusion, the two-compartment minimal model yields a physiological HGP, thus overcoming the inadequacy of the classical minimal models. This model-based measurement of HGP enhances the ability of the minimal model method to characterize glucose metabolism in vivo.

A216

P795

P796

INSULIN SECRETION, METABOLISM AND SENSITIVITY BEFORE AND AFTER A PROTEIN-SPARING MODIFIED FAST IN OBESE SUBJECTS. N. Paquot, A.J. Scheen, J. Juchmes, P. Gerard, C. $aminet and P.J. Lef~bvre. Division of Diabetes, Nutrition and Metabolic Disorders, CHU Liege, Sart Tilman, B-4000 Liege, Belgium.

INSULIN RESISTANCE IS CORRELATED WITH SKIN IRON CONTENT IN f~-THALASSEMIA MAJOR. B. Glaser, G. Admon, R. Gorodetsky, A. W. Goldfarb, E. A. Rachmilewitz and E. Cerasi. Departments of Endocrinology and Metabolism and of Hematology, Hebrew University Hadassah Medical Center, Jerusalem, Israel.

To investigate the influence of acute weight loss on insulin secretion rate (ISR), metabolic clearance rate (MCR-I) and sensitivity (Sz), ten overnight fasted obese subjects (42.9 i 2.2 kg/m ~) underwent an intravenous glucose tolerance test (25 g) just before and after a 13day protein-sparing modified fast (PSMF : 80 g protein = 320 Kcal/day). ISR was derived by deconvolution of plasma C-peptide levels; MCR-I was obtained by dividing the area under the curve (AUC) of ISR by the AUC of plasma insulin levels (IRI); Sz was calculated from the plasma glucose and IRI versus time curves according to Bergman's minimal model. After PSME inducing a 7.9 i i.i kg weight reduction, AUC-ISR moderately increased from 58745 • 5537 to 66208 ~ 4846 pmol/180 min (NS) while AUC-IRI slightly decreased from 7914 • 739 to 7519 • 804 mU.min/l (NS); consequently, MCR-I significantly increased : from 1063 i 82 to 1344 i 149 ml/min (p < 0.05), from 491 ~ 45 to 648 • 81 ml/min.m z corporeal surface (p < 0.05) and from 9.5 i i.I to 13.1 ~ 2.1 ml/min.kg (p < 0.02). Sz averaged 1.7 0.2 10 -~ min-x/mU.l-z before and 2.1 • 0.6 i0 -~ minz/mU.l -~ after PSMF (NS). In conclusion, reduced plasma insulin levels observed just after a PSMF in obese subjects are better explained by an enhanced insulin metabolism than by a change in insulin secretion; insulin sensitivity is not significantly improved immediately after PSMF despite marked weight loss.

Thalassemia major is associated with a high incidence of diabetes mellitus, and both t~-cell damage and insulin resistance have been implicated. We examined the insulin and glucose responses to intravenous glucose (0.5 g / k g IVGTT) in 22 patients (11 M, 11 F, aged 11-30 years, BMI 19.5+0.6) who had no evidence of glucose intolerance. All but one received deferoxamine (median 16, range 0-49 m g / k g / d ) . Four patients had first degree relatives with Type 2 diabetes. Skin iron content was determined non-invasively by diagnostic x-ray spectrometry. Fasting plasma glucose, glycosylated serum protein and glucose clearance (Kg) were normal in all patients (mean Kg = 2.1+0.1, range 1.3-3.8, normal >1). Phase 1 insulin response (incremental response at 2+5 minutes) was positively correlated with dermal (p=0.01) and epidermal (p=0.05) iron content and with serum ferritin (p=0.01). Insulin action (Kg/insulin response) was negatively correlated with dermal (p=0.01) and epidermal (p=0.02) iron but not with ferritin (p=0.16). Insulin/C-peptide ratios were significantly correlated to epidermal (p=0.005) but not to dermal (p=0.08) iron. No significant correlations were found between insulin secretion or action and liver enzymes, age, transfusion number, or deferoxamine dose. These findings suggest that decreased insulin clearance and insulin resistance exist in non-diabetic thalassemics and correlate with skin iron deposition. Presumably concomitant ~-cell damage is necessary to precipitate glucose intolerance or diabetes.

P797

P798

INSUL..IN RES ISI'PINCE AND HYF'ERINSUL. I N E M I A IN [5"'HOMOZYGOUSE I'I'~I...ASSEMI(~ ([5FIT> r utti F., F'a,:.'.:i.t',:i. G..~, S;.:~c,~l",et'ti L . , , I::'iga ~ , , B,e s s o n e A . , Cer, dib Q,.x..~, Cavalle-F:'ePir, F','~'~ I r,stit,.rte o'f F'e d i a t r, i ,.?.s.-..LJr, :i. v e P s .i. t y o f T,.JV i r, (I't'aly) ; .x.LAI)SE:B CNR, F'ad,.J;!~ (Italy) ; 9x..x.Ir,s % i % u t e e~: IT',tePl',;al He<:lieir,e-Lh',ivePsity o'f'

INSULIN RESISTANCE IN CONGESTIVE HEART FAILURE:ROLE OF PLASMA NOREPINEPHRINE LEVELS G.Marrazzo,G.Paolisso,S.De Riu,G.Pizza,N.Passariello , M. Varricchio and F.D'Onofrio -Institute of General Medecine and Metabolic Diseases - Institute of Geriatric Medecine University of Naples ,Naples,Italy

"T'uPi n ( I t a l y ) ImpaiPed g].ucese 'toler';~mce~ .r.l,.le t o ir~sulir, de~;icieI',,.'.'y a n d / o P : i r r t $ , J l i r l r'e!~istanee h a v e beer~ Pel:)ovted

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4.1",, :t[.'~, 8 gr'/m z IUGI'T ~,~i't'h p l a s m a glueo.ge, ir,sulir, ar,d C " ' p e l : ~ ' t i d e m e a s u v e m e n t ~ analyzed by m i r, i m.'..Kt mode. 1 i n g "t.'e,::r, i q u e ~.R',i "t",.-I", e v a I uai'es Jb-.,;'e].]. s e c t r ' e t i e r , , :i.nsuliT'I sel'isitivity (51) a n d hep;ati~: :Lr,s , J l : i . n ~L'tr';a,:.'.t:i.or, (l"l) , F ; a s t i r , g ;al',d ~fi'er" ].o~ld g l u , 2 o s e ;..~n,:J i r l s i . . l l i n pa%ter'ris w e P e s].igl"rtly higher" i n [bH'l', SI was t,9% l o w e r i n BI-IT ' t h a n i t , C5 P e s p e , 2 i i v e l y :3,5!(3,6 [ 1 0 ~' m i r ; z /(pUlml)] vs 6 , 9 : L I , 0 : I:)=:,0:I.#, -, " l ' o ' t a l a m o u r , t o f l : ) P e - " h e p a t i , : ~ i r , s,..ll:i.r, :i.n ,2.4.0 m i r l , Was $ i m i ] . ; . l r El.M-IT 1 2 ' . , 1 : s v s (2S : 1 . 3 . 8 : s - 3 pM) as w e l l a s hepat:i.,-; :i.rtsu].ir~ e v ' t p a , : : ' t ' i e n EISl-I'l" 70,',:.".:L3,8 vs C.S 74,2:[:4,7 (%) ] , We ,:.:an ,:'or,,z 1,..tde that ado le~cen't r~on d i a b e ' t i,:: JbI-i T exl"~ibit rlor'ma 1 I3"',::c§ "Fun,:~ticm bt.lt Pedu,.'.'e
To study the possible presence of insulin resistance in heart failure,8 patients suffering from Congestive Heart Failure (CHF) and i0 healthy,age- and body mass index matched subjects conducing sedentary life were submitted to 2 x 2 h euglycemic hyperinsulinemic (50 and i00 mU/kg x h successively)glucose clamp. A priming-constant 3-H-glucose infusion allowed determinations of glucose kinetic. In basal conditions, patients vs controls had similar plasma glucose (5.2+0.1 vs 4.9+0.3 mmol/l p=NS) but higher plasma norepinep~rine (5.4+0.i vs 1.5+0.2 nmol/l p<0.001), plasma insulin (125+9 vs 35+3 pmol/l p<0.01) and free fatty acids (927+79 vs 792+88 umol/l p
A217 P799

P800

Insulin Resistance in the Metabolic Acidotic Rats: Assessment by Glucose Clamp Technique. H.SASAKI, M. MATSUMOTO, H. EGUCHI, A. SEKIKAWA, M. TOMINAGA Third Dept. of Int. Med., Yamagata University, Japan To determine which is responsible for insulin resistance in metabolic ketoacidosis, ketosis or acidosis, euglycemic insulin clamp study was performed in adult male Wister rats. Metabolic acidosis and ketosis were induced by continuous i.v. infusion of ammonium chloride and 3-B hydroxybutyrate, respectively. Insulin was i n f u s e d at the r a t e of 3 m U / k g / m i n and 20 m U / k g / m i n d u r i n g the g l u c o s e c l a m p study. Insulin sensitivity was assessed by steady state glucose infusion rate (GIR), mean GIR during the last 20 min of the clamp period. Hepatic glucose output (HGO) and glucose di$~appearance rate (Gd) were measured by using 3-JH-glucose. Steady state arterial blood pH was 6.8 in the acidotic rats and 7.3 in the ketotin rats. Steady state GIR and Gd of the acidotic rats were 2.65+0.25 and 3.91+--0.46 mg/kg/min with low dose insulin and 7.22+--0.42 and 4.75+-0.29 mg/kg/min with high dose insulin and significantly low compared with control and ketotic rats. Steady state HGO of acidotic rats was not suppressed by the low dose insulin infusion, b u t it w a s c o m p l e t e l y suppressed in the high dose study, and in both c o n t r o l and k e t o s i s s t u d i e s . T h e s e data suggested that metabolic acidosis but not ketosis c a u s e d i n s u l i n r e s i s t a n c e in the l i v e r and peripheral tissues.

INSULIN RESISTANCE IN CARBOHYDRATE AND FAT METABOLISM IN MOTOR IqE~ROIqEDISEASE

A.J.Krentz, A.C.Williams and M.Nattrass. The General Hospital and Queen Elizabeth Hospitals, Birmingham, UK Insulin resistance has been implicated in the aetiology of the glucose intolerance commonly associated with motor neurone disease (MND) although the available evidence is conflicting. In this study the response of circulating intermediary metabolites to a low-dose incremental insulin infusion (basal, 0.005, 0.01 and 0.05 U/kg/h) were examined in 7 subjects with MND and 7 normal controls of similar age (55+--8vs 52+8y) and body mass index (24~4 vs 25+--3kg/m2). 0ral glucose tolerance (75g) was impaired (WHO 1985) in 5 of the subjects with MND. During the incremental insulin infusion blood lactate (F:5.29,p<0.05) and pyruvate (F:15.61,p<0.01) were significantly higher in the subjects with MND. Analysis of variance confirmed significant (p<0.01) linear dose response relationships for plasma insulin (log) and glucose, glycerol and non-esterified fatty acids (NEFA). In the subjects with MND the group dose-response regression lines for glucose (p<0.001)~glycerol (p<0.001) and NEFA (p<0.001) were displaced to the right of those for the normal controls indicating insulin resistance in the regulation of these metabolites. In conclusion, insulin resistance is a feature of both carbohydrate and fat metabolism in MND and may contribute to the glucose intolerance commonly associated with this disorder.

PS 35 Metabolism in Vivo P801 CHARACTERIZATION

P802 OF T I M E

AND DOSE DEPENDENT

INS~

LIN-ANTAGONISTIC EFFECT OF GROWTH HORMONE IN MAN J. F o w e l i n , S. A t t v a l l , H. y o n S c h e n c k , U. S m i t h a n d I. L a g e r , D e p t . o f M e d . II, U n i v . of G o t h e n b u r g a n d D e p t . of C l i n . C h e m . , U n i v . o f Link~ping, Sweden. E l e v a t e d l e v e l s o f G r o w t h H o r m o n e (GH) a r e o f t e n f o u n d in t y p e I d i a b e t e s . To c h a r a c t e r i z e t h e insulin-antagonistic effect of GH this hormone was i n f u s e d d u r i n g I h o u r to i m i t a t e t h e l e v e l s f o u n d during a pronounced, moderate or mild hypoglyc a e m i a o r d u r i n g p o o r m e t a b o l i c c o n t r o l (24, 12, 6 m U / k g ' h ) in 11 h e a l t h y s u b j e c t s . T h e i n s u l i n effect was measured with the euglycaemic clamp technique and D-(3-3H)-glucose w a s u s e d to e v a luate glucose turn-over. Comparisons were made to a c o n t r o l s t u d y (C) w i t h o u t GH. T h e i n s u l i n l e v e l s w e r e s i m i l a r d u r i n g a l l c l a m p s (31+3 mU/l) T h e G H l e v e l s i n c r e a s e d to I04211 m U / l d u r i n g the h i g h i n f u s i o n . T h e i n s u l i n - a n t a g o n i s t i c e f f e c t of G H s t a r t e d a f t e r 2 h, w a s m a x i m a l 5 to 6 h a f t e r s t a r t o f t h e i n f u s i o n ( d i f f e r e n c e in g l u c o s e i n f u s i o n r a t e , GIR, b e t w e e n C a n d G H 2 . 6 ~ 0 . 4 m g / k g - m i n 10<0.01) a n d r e m a i n e d d u r i n g 5 h. T h e r e s i s t a n c e w a s d u e t o a l e s s p r o n o u n c e d i n s u l i n e f f e c t b o t h t o i n h i b i t Ra a n d t o s t i m u l a t e Rd. I n f u s i o n o f 12 m U / k g ' h i n d u c e d a s i m i l a r i n s u l i n a n t a g o n i s t i c e f f e c t as t h e h i g h e r i n f u s i o n r a t e b e t h r e g a r d i n g m a x i m a l e f f e c t (GIR C - G H 2.4+0.5 mg/kg.min p<0.01) and duration whereas 6 m U / k g ' m i n i n d u c e d a l e s s r e s p o n s e (GIR C - G H 1 . 6 ~ 0 . 4 m g / k g ' m i n p < 0 . 0 2 ) w i t h a d u r a t i o n of 2 h. The i n s u l i n - a n t a g o n i s t i c e f f e c t o f G H is t h u s r e l a t e d t o t h e p l a s m a l e v e l s w i t h r e g a r d s to d u ration and response. The ~ng duration supports t h e c o n c e p t t h a t G H is i m p o r t a n t for t h e r e g u l a t i o n of g l u c o s e m e t a b o l i s m in m a n .

PARTIAL PURIFICATION OF TWO INHIBITORS OF SERUM SOMATOMEDIN BIOACTIVITY FROM SERA OF ADOLESCENT INSULIN DEPENDENT DIABETICS. 1 2 A.M. Taylor, R.A. Brow~, M.A. Preece & D.B. Dunger. Institute of C~ild Health, Institute of Orthopaedics, University of London & ~The John Radcliffe Hospital, University of Oxford, U.K.

We recently reported that the growth-hormone independent binding protein (IGBP-I) for the insulin-like growth factors (IGFs) or somatomedins is associated with serum IGF inhibitory bioactivity in adolescent Type-I diabetics. IGBP-I is not the only inhibitory factor present in diabetic serum. We report here the partial purification of two inhibitory factors of IGF bioactivity from diabetic serum, as measured by 35S sulphate incorporation by cartilage. Initial liquid-gel chromatography of diabetic sera on an HW55 size-gel indicated the presence of two factors with molecular weights of 170KDa and 40KDa. Further purification of diabetic serum by ion exchange, using DEAE-52 cellulose and eluted with 0.25M NaCI yielded an inhibitory fraction containing at least 10% of the total serum protein. This fraction was further purified by HPLC using a TSK-3000 size-gel column. Two inhibitory fractions eluted with molecular weights of 169KDa and 39KDa, similar to the molecular weights of the IGF GH-dependent binding protein (IGBP-3) and IGBP-I respectively. These fractions consistently inhibited serum IGF bioactivity on cartilage. It remains to be determined as to whether these fractions are related structurally and functionally to the IGF binding proteins, and whether they have a role in growth retardation and delayed puberty often observed in adolescent diabetics.

A218

P803

P804

METABOLIC EFFECTS OF SUBCUTANEOUS CONTINUOUS INFUSION OF SOMATOSTATIN ANALOG IN TYPE 1 DIABETES D. Bruttomesso, C. Fongher, S. Barberin, B. Silvestri, A. Tiengo, and S. Del Prato. Cattedra di Malattie del Ricambio, University of Padova, Italy

EXAGGERATED GROWTH HORMONE RELEASE BY PYRIDOSTIC~INE IN INSULIN-DEPENDENT DIABETES. F. Caviezel, A. Tus M. Bombonato, L.Morricone and M. Greco Istituto di Scienze Medico-Chirtmgiche S. Donato, University os Milarlo, Milano, Italy. Growth hormone (GH) secretion is s abnormal in Type I (insulin-dependent) diabetes. Since cholinergic system participates as regulatory mechanism os GH secretion, we studied GH response to 60 mg p.o. os pyridostigmine, a cholinesterase inhibitor, in 6 non-insulin-dependent diabetics (4M/2F, aged 46-55, mean BMI 24.7)(group i), 8 insulin-dependent diabetics (SM/3F, aged 19-44, mean BMI 22.2) (group B), all with variable metabolic control (%HbAI 8.0514), and 9 matched healthy controls (group C). All subjects randomly received pyridostigmine or placebo. Plasma GH was measured at -15, O, 30, 60, 90, 120 and 180 min. GH peaks after pyridostig~ine in group A, B and C were 6.3 +_2.3(SEM), 16.1 +4.4 ~naximum 39.9), and 6.1 +I ng/ml, respectively (p
The effect of 8 d,3.ycontinuous sc infusion of SMS 201-995 (200 Ixg/day) was evaluated in 6 insulin-pump treated Type 1 diabetics (age--45+_2yrs; BMI=26.2_+0.4 kg,/(m)2;HbA~c=7.gio.4%). A 24hr metabolic and hormonal profile, and an euglycemic (5.5 retool/l) hyperinsulinemic clamp (0.25, 0.5, 1.0 mU/kg/min) clamp, associated to 3I-I-glucose infusion and indirect calorimetry., were performed before and after 8day SMS infusion. Mean 24hr plasma glucose was similar before and after SMS (9.5_+0.9vs 9.3+1,2 mmoF1) but insulin requirement dropped by 42_%5% (51+__5vs 28+_2 U/day; p<0.001). Both 24hr plasma hGH and glueagon were suppressed by SMS (1.85_+0.35 vs 0.52--+0.04ng/ml, and 145+_23vs 122.%.14 pg/ml, respectively; p<0.5-0.05).'Glucose utilization increased after SMS (lmU clamp=28.2_%4.9 vs 38.1+_5.5 Izmol/kg/min; p<0.01) and hepatic production was similarly suppressed. The change in glucose disposal rate and insulin requirement were correlated (r=0.74). Glucose oxidation was not affected by SMS (10.7+1.8 vs 11.3+1.8 Ilmol/1), while the improvement in glucose storage (17.2.%-3.8vs 26.8+4.6; p<0.001) entirely accounted for the increase in glucose disposal. Energy expenditure declined after SMS (1.22-+0.07 vs 1.06_+0.07;p<0.05). Conclusions: Continuous sc SMS infusion 1) suppresses counterregulatory hormones 2) increases insulinmediated glucose metabolism by enhancing glucose storage 3) reduces energy expenditure. These results support a role for cotmterregulatory hormones in the genesis of insulin resistance and catabolic state of Type 1 diabetes.

P805

P806

EFFECTS OF GROWTH HORMONE TREATMENT ON GLUCOSE METABOLISM IN GROWTH HORMONE DEFICIENCY. F. Salomon~ R.C. Cuneo 9 M.A. Umpleby and P.H. SOnksen. D e p a r t m e n t o f M e d i c i n e and Chemlcml Pathology, UMD5, S t . Thomas's H o s p i t a l , London, UK. We i n v e s t i g a t e d the iongterm effects of recombin a n t human g r o w t h hormone (rhGH) t r e a t m e n t on g l u c o s e m e t a b o l i s m i n 24 a d u l t s w i t h g r o w t h h o r mone d e f i c i e n c y (GHD) i n a d a u b l e - b l i n d ~ placebo controlled s t u d y . B a s a l g l u c o s e m e t a b o l i s m ~as measured using a bolus injection of 3-3H glucose before and after 1 and 6 months of treatment. Fasting plasma glucose increased in the rhGH group from 4.6• (baseline) to 5.5• month) to 5.1• mmol/1 (6 months) vs 4.9• (bl) 4.8Q 0.i (1 m) 4.9• mmol/1 (6 m) in the placebo group (ANOVA p
RENAL CATABOLISM [7-36] A M I D E .

OF GLUCAGON-LIKE

PEPTIDE-I

C. Ruiz-Grande, C. A l a r c d n and I. Valverde. Fundacidn Jim~nez Diaz, Centro Asociado C.S.I.C., Madrid, Spain. The g l u c a g o n - l i k e p e p t i d e - i [7-36] amide, also denominated truncated GLP-I (tGLP-I), is a naturally ocurring peptide corresponding to proglucagon [78-107] amide that d i s p l a y s high i n s u l i n o t r o D i c activity. The d i s a p p e a r a n c e from p l a s m a of [~SI]tGLP-I after a single intravenous i n j e c t i o n was studied in normal (C), b i l a t e r a l l y ureteral ligated (BUL) and b i n e f r e c t o m i z e d (BNX) rats. Blood samples were obtained at d i f f e r e n t intervals d u r i n g 60 min after injection, and total and T C A - p r e c i p i t a b l e radioactivity were measured in p l a s m a and urine samples. The l o g a r i t h m of T C A - p r e c i p i t a b l e counts p r e s e n t in p l a s m a from rats injected w i t h the r a d i o a c t i v e p e p t i d e followed a s t r a i g h t - l i n e b e t w e e n 5 and 15 m i n after injection. The tl/2 of [125I]tGLP-I was 4.5• m i n (mean• n=8) in C, 6.2• m i n in BUL (n=5, p<0.01 vs C), and 6.3• min in BNX (n=6, p<0.001 vs C, p>0.05 vs BUL). Gel f i l t r a t i o n of plasma samples showed m a i n l y two r a d i o a c t i v e peaks, one in the elution v o l u m e of the p e p t i d e and the other in that of the salts, in good c o r r e s p o n d e n c e to T C A - p r e c i p i t a b l e and TCA-soluble radioactivity respectively. The r a d i o a c t i v i t y of the urine samples from C rats injected with the peptide was not TCAprecipitable. In conclusion: i) tGLP-I is removed from circulation with a tl/2 more p r o l o n g e d than glucagon, but s h o r t e r that other GLPs and ii) the k i d n e y plays a role in the c a t a b o l i s m of this p e p t i d e a p p a r e n t l y only via g l o m e r u l a r filtration.

A219

P807

P808

S K E L E T A L MUSCLE G L U C O S E U T I L I Z A T I O N IN R E L A T I O N TO D E C R E A S E D INSULIN C O N C E N T R A T I O N S IN FASTING M.C. Sugden, Y.-L. Lui and M.J. Holuess, Department of Biochemistry, London Hospital Medical College (University of London), Turner Street, London E1 2AD, U.K. The aim was to determine the relative importance of decreased insulin and elevated lipid (fatty acid; ketone body) concentrations in mediating the progressive effects of starvation on glucose utilization in three representative working muscles. Glucose utilization was estimated in v i v o in resting, conscious rats using 2-[3H]deoxygincose. A moderate decline in glucose utilization was observed in diaphragm (27%), soleus (54%) and adductor longus (36%) after starvation for 12h. A further steady decline was observed over the subsequent 36h of starvation, but rates remained substantial (38-59% of the initial value) even after starvation for 24h. Overall mean suppression of glucose utilization in these muscles was ca. 27% over the period f r o m 0-12h of starvation, during which time insulin concentrations declined but only small increases in lipid fuel concentrations were observed. Mean suppression of glucose utilization was ca. 70% over the period f r o m 24-48h of starvation, where sustained increases in fatty acids and ketone body concentrations were observed but insulin concentrations were little further diminished. The results indicate a role for insulin in mediating the acute (0-12h) response to starvation, but support the proposal that further major suppression of muscle glucose utilization in prolonged starvation may be largely secondary to increased lipid fuel oxidation.

EFFECTS OF HYPERCORTICISM ON THE REGULATIONOF SKELETAL MUSCLE GLYCOGENMETABOLISMBY EPINEPHRINE. J.-L. Chiasson, L. Coderre, and A.K. Srivastava, Clinical Research I n s t i t u t e of Montreal, Canada, H2W 1R7. Using the hindlimb perfusion technique we have studied the effects of epinephrine (10"~ M) on the regulation of skeletal muscle glycogen metabolism in rats treated either with saline or dexamethasone (DEX) for 15 days. Basal glycogen phosphorylase a c t i v i t y was 0.16 in the control rats and 0.15 in the DEX treated rats and increased to 0.78 and 0.80 respectively after epinephrine treatment. Basal glycogen synthase a c t i v i t y was 0.10 in the control rats and 0.07 in the DEX treated rats and decreased to 0.06 in both groups after epinephrine. Muscle glycogen content was 33.1 • 0.4 ~mol/g in the control rats and 41.7 • 1.5 #mol/g ~ < 0.05) in the DEX treated rats and epinephrine treatment was associated with a decrease in glycogen to the control rats (23.6 • i . i pmol/g) but not in the DEX treated rats (43.1 • 2.7 ~mol/g). We have measured a number of i n h i b i t o r s of phosphorylase a; among these a l l were normal except f o r the s k e l e t a l muscle adenosine which was increased 30 f o l d in the DEX treated rats ( i . 0 nmol/g versus 0.03 nmol/g; p < 0.01). In conclusion, hypercorticism is associated with an absence of glycogenolysis in response to epinephrine despite the activation of phosphorylase and the i n a c t i v a t i o n glycogen synthase. I t is suggested that this absence of glycogenolysis in response to epinephrine could be due to inhibition of phosphorylase by increased adenosine in skeletal muscle due to hypercorticism.

P809

P810

REGIONAL VARIATION IN THEANTILIPOLYTIC EFFECT OF INSULIN IN HUMAN BEINGS. B. Richelsen, S.B. Pedersen, T. ~k~ller-Pedersen, and J.F. Bak. University Clinic of Internal Medicine, Aarhus amtssygehus, DK-8000 Denmark

THE M Y O C A R D I A L M E T A B O L I S M AND I~GNCTION IN DOGS WITH E X P E R I M E N T A L DIABETES

Recent studies have shown that abdominal adiposity is of particular health risk. Abnormalities of the lipolytic regulation have been suggested to play a role for the health implications of the intraabdontinal fat depot. In the present study we have compared the regulation of triglycerid breakdown in adipocytes obtained from three different regions. Intraabdominal adipose tissue (omental - O) (n=7) and subcutaneous abdominal (SA) adipose tissue were obtained from the same individual undergoing abdominal surgery, in addition, adipocytes from the subcutaneous gluteal (SG) region were investigated (n=9). The antilipolytic effect was investigated on the isoproterenol(50 nM)-stimulated lipolysis. The maximal antilipolytic effect of insulin (i nM) was an inhibition of 23% in O adipocytes and of 52% in SA adipocytes (P < 0.001), respectively, cor~pared to an inhibition by 73% in SG adipocytes. The insulin sensitivity was, however, similar in all regions (ED,^: 133 - 272 I~M). In contrast to insulin another antili~ytie compound, PGE2, had similar maximal effects in all regions since llpolysis was inhibited by 85 - 91%, however, with different sensitivity (ED : 2.8 nM(O), 2.7 nM (SAI, 1.2 nM (SG)). In conclusion, 50eat regional variations in the antilipolytic effect of insulin exist in hi,an adipose tissue. particularily, the impaired antilipolytic activity of insulin in omental adipocytes as compared with s.c. adipocytes raight be a factor for the enhanced health risk of abdominal obesity.

k.I. Khomazjuk, L.N. Glebova, E.L. Gapitch and A.P. Nescheret. Institute of E n d o c r i n o l o g y & Metabolism, Vishgorodskaya 69,252114,Kiev,USSR. The a i m of the present study was to assess m e t a b o l i s m and f u n c t i o n of m y o c a r d i u m in dogs with e x p e r i m e n t a l diabetes. Experiments were performed using the catheterization, extracorpetal perfusion(programmed) of the coronary artery, catheterization and continuous drenage of the coronary sinus. Cardiohemodynam~c p a r a m ~ r ~ heart blopotentials, coronary vessels resistance, respiration, blood oxygen saturation were recorded and the levels of Poo,Pcog, glucose, lactate, pyruvate and F2A in'arterial and venous heart blood were determined. In ~ogs w i t h m i l d and moderate diabetes(hyperglycemia to 9.3+ 0.44 mmol/1) adrenergic reactivity of the heart and coronary vessels was enhanced but heart performance was not significantly changed. In dogs with severe diabetes(hyperglycemia to 15,4+ 0.88 mmol/1) cardiac output and contractility were reduced, coronary f l o w in the left circumflex artery was diminished by 31%. Arterial b l o o d level of Flea was e l e v a t e d (from 1.19+ 0.10to 3.42+ 0.50 r m o l / l ) , c o r o n a r y arteriovenSus differenc~(AVD) of ~ A showed 4 fold inorease(1.23+ 0.40 mmol/1). AVD of glucose was u m c h a n g e d ( 0 _ 4 5 + 0.17 mmol/1) but extraction ratio was ~epres~ed and pyruvate was excrated into coronary sinus blood. AVD of oxygen saturatiom enhanced f r o m 55.6_+ 176 to 7 3 + 1.10~ The myocardial ensuring alteration have shown to be one of the reasons of myocardial f~metion disturbances.

A220

PS 36 Carbohydrate and Protein Metabolism P815

P816

ALTERED RATIO BETWEEN D-[2-3H]GLUCOSE AND D-[5-3~GLUCOSE CONVERSION TO 3HOH IN ERYTHROCYTES OF DIABETIC RATS V. Liemans, D. Z~hner, F. Malaisse-Lagae and W.J. Malaisse. Laboratory of Experimental Medicine, Brussels Free Univers i t y , Brussels, Belgium.

EFFECT OF FRUCTOSE/FRUCIOSE-6-PHOSPHATE CYCLING (FPC) ON MEASUREMENT OF GLUCOSE FLUX IN MAN H. Katz, M. Homan, P. Butler, and R. Rizza, Endocrine Research Unit, Mayo Clinic, Rochester, MN, USA [3-3H]glucose is used to measure glucose turnover in man. To determine whether FPC results in sufficient cycling of tracer to cause an overestimate of irreversible glucose uptake, GU was measured in nondiabetic subjects with a simultaneous infusion of [6-14C]glucose (a tracer minimally influenced by FPC), and [3-3H]glucose. So as to evaluate turnover when glycolytic flux has been stimulated to different degrees and in different directions, GU was measured a) before, b) during low ( - 2 0 0 pmol), and c) high ( - 7 0 0 pmol) insulin, the latter in the presence of low ( - 1 0 0 pg/ml) and high ( - 7 0 0 pg/ml) glucagon. Glucose was - 5 mmol throughout. GU was comparable with [6-z4C]- and [3-3H]glucose respectively, before (11.7 + 0.4 vs 10.9 + 0.4/~mol/kg/min) insulin, during low (30.0 + 3.4 vs 29.2 + 3.4/~mol/kg/min) and high (56.0 + 3.4 vs 53.4 + 3.3 ~.mol/kg/min) insulin and during high glucagon (56.1 + 2.9 vs 52.4 + 2.4 #mol/kg/min). Rate and absolute degree of suppression of hepatic glucose release also were comparable with both tracers. Neither tracer resulted in "negative" glucose production rates. We conclude that since [3-3H]- and [6-z4C]glucose yield comparable rates of glucose turnover over a wide range of insulin and glucagon concentrations, FPC is unlikely to influence measurement of glucose turnover under physiologic conditions in man.

In e r y t h r o c y t e s from normal human subjects or r a t s , the generation of 3HOH from D-[2-3H]glucose only represents 75 % of t h a t from D-[5-3H]glucose. This d i f f e r e n c e is mainly a t t r i b u t a b l e to the i n t r a m o l e c u l a r 3H t r a n s f e r between D-[2-3H]glucose 6-phosphate and D-[1-3H]fructose 6-phosphate and to i s o t o p i c d i s c r i m i n a t i o n towards the t r i t i a t e d esters in the r e a c t i o n catalyzed by phosphogluco4.somerase. In monodirectional r e a c t i o n s , the i n t r a m o l e cular t r a n s f e r (50-54 %) and i s o t o p i c d i s c r i m i n a t i o n (ZH/ IH = 0.3) d i s p l a y comparable magnitudes with p u r i f i e d yeast and r a t l i v e r phosphoglucoisomerase, and are unaff e c t e d by the ex vivo non-enzymatic g l y c a t i o n o f the enzyme. Nevertheless, in d i a b e t i c r a t s examined 5 days a f t e r a d m i n i s t r a t i o n of s t r e p t o z o t o c i n , and even in animals rend e r e d m o d e r a t e l y hyperglycemic by diazoxide (3 x 50 mg/ day) f o r only 48 hours, the r a t i o between D-[2-3H]/[5-~H] glucose conversion to 3HOH by e r y t h r o c y t e s is f u r t h e r decreased. Since the process of 3H i n t r a m o l e c u l a r t r a n s f e r i.s affected, b~ ~everal f a c t o r s , i n c l u d i n g the concentrat i o n of inorganic phosphate, the a l t e r e d r a t i o in i n s u l i nopenia is probably a t t r i b u t a b l e to an environmental change w i t h i n the e r y t h r o c y t e s . These data not s o l e l y i n d i c a t e t h a t the generation of 3HOH from D-[2-3H]glucose underestimates the r a t e o f glucose phosphorylation , but also reveal t h a t the r e l a t i v e e x t e n t of such an underestimation is i t s e l f v a r i a b l e in d i s t i n c t metabolic s i t u a tions.

P817

P818

INCREASED INSULIN-INSENSITIVE GLUCOSE TRANSPORT IN POLYMORPHONUCLEAR LEUKOCYTES FROM TYPE 2 (NON-INSULINDEPENDENT] DIABETIC PATI~qTS Y.Okuno, Y.Nishizawa and H.Morii, Second Department of Internal Medicine, Osaka City University, Abeno, Osaka, Japan 545 We studied the transport rate of a non-metabolizable hexose analogue, 3-0-methyl-D-glucose (30MG), in polymorphonuclear leukocytes (Pb/~Ls) from patients with Type 2 (non-insulin-dependent) diabetes mellitus to examine whether or not insulin-insensitive glucose transport is affected by abnormalities in glucose metabolism. P ~ s were prepared using Mono-Poly resolving medium.Transport assay was carried out at 37~ pH 7.4 as reported elsewhere /FEBS 195, 1986). The mean t r a n s p o r t r a t e of 0.05 mM 30MG measured at 5 sec was s i g n i f i c a n t l y e l e v a t e d in the 23 d i a b e t i c p a t i e n t s campared with 29 h e a l t h y c o n t r o l s [13.3• vs 10.4~2.5 fl / c e l l - s e c , meanmSD). In the d i a b e t i c s u b j e c t s , glucose t r a n s p o r t r a t e s were c o r r e l a t e d w i t h HbAlc l e v e l s , but had no r e l a t i o n s w i t h ambient plasma glucose concent r a t i o n s . S h o r t - t e r m incubation with 20 mM D-glucose had no e f f e c t on glucose t r a n s p o r t in those c e i l s . When the degree of d i a b e t i c derangement of the metabolism in some p a t i e n t s improved a f t e r h o s p i t a l i z a t i o n , t r a n s p o r t r a t e s reduced in p a r a l l e l w i t h the decrease of I-IbAlc l e v e l s . We conclude that i n s u l i n - i n s e n s i t i v e glucose t r a n s p o r t in human PMNLs, u n l i k e i n s u l i n - s e n s i t i v e glucose t r a n s p o r t , i s increased in p a t i e n t s w i t h Type 2 ( n o n - i n s u l i n - d e p e n d e n t ) d i a b e t e s and long-term, not s h o r t - t e r m , derangement of glucose metabolism may be a s s o c i a t e d with increased glucose t r a n s p o r t found in those p a t i e n t s .

A G L U C O K I N A S E - L I K E ENZYME P H O S P H O R I L A T E S GLUCOSE IN CAPILLARIES. R STUDY IN THE EEL. F. Bekfiore, S. I a n n e L L o , R. Campione and G. VoLpiceLli. University of Catania Catania, Italy. GLucose (G) p h o s p h o r y L a t i o n was s t u d i e d in a pure capillary preparation ("rete mirabiLe" of e e l s w i m b L a d d e r ) . I n 3000 g s u p e r n a t a n t s of eel capillary homogenates (n=lO, 400-500g BW, 67• mg/dL mean2SEM g L y c e m i a ) , G p h o s p h o r y Lating activity d i d n o t r e a c h t h e maximum at Low G (lmM), as it occurs in most tissues, but increased with the increase in G concentration, approaching the plateau at very high (300mM) G Cmean• values: 5.8520.34 nmoL/ min/mg prot and 1 3 . 9 7 2 1 . 8 3 a t lmM and 300mM, respectively). W i t h G c o n c e n t r a t i o n s above 300 mM, t h e activity f a L L s s h a r p l y C3.67~1,20 and 2,86• at 400 and 500mM). These kinetic characteristics are similar to those of the enzyme gLucokinase (GK), which is present in tlTe Liver (maximum activity at pH 7.8-8.6, similarly to the pH n p t ~ m u m for hepatic GK). This new enzyme did not p h o s p h o r y L a t e fructose and was not inhibited but stimulated by N-acetyL-giucosamine. Thus, capillaries phosphoryLate G in a concentration-dependent manner through a GKLike enzyme; this would suggest that hyperglycemia in diabetes may be associated with increased G utilization in capillaries, which may play a role in the development of mioroangiopathy.

A221

P819

P820

ON THE ROLE OF INSULIN IN REGULATION OF ADENOSINE

MODULATION OF CALCIUM-DEPENDENT GLYCOGENOLYSIS BY ENDOTHELIN-I IN THE ISOLATED, PERFUSEDRAT LIVER H. Vierhapper, M. Roden, I. Kothbauer and W.~]dh~usl Dept. of Endocrino]., I.Med.Univ.Klinik, Vienna, Austria

OEAMINASE (ADA) ACTIVITY IN SKELETAL MUSCLES OF THE RAT. J.Rutkiewicz and J.G6rski, Department of Physiology, Medical School, Bia~ystok, Poland. Adenosine decreases the insulin-stimulated glucose transport in skeletal muscles. This effect is prevented by adding of adenosine deaminase (adenosine degrading enzyme). The aim of the present study was to examine whether insulin may effect adenosine metabolism in the muscles. ADA activity was determined in the soleus, plantaris, gastrocnemius (white and red parts) muscles of the following groups of rats: 1-control, 2-streptozotocin-diabetic (SD), and ]-treated with insulin in a dose of 0.5 IU/kg. Half of the dose was administered subcutaneously and the other half intraperitoneally. The latter group was divided into two subgroups: fed ad libitum and having the food withrawn after administration of the hormone. The muscle samples were taken in 48h after administration of streptozotocin or in ]h after administration of insulin. In SO rats activity of AOA increased by about 50% in muscles with high oxidative capacity and by ]OZ in those with low oxidative potential. In insulin-treated rats AOA activity decreased by about 40Z in each muscle studied. These results suggest that insulin may play an important role in adenosine metabolism in skeletal muscles by inhibiting activity of AOA.

Hepatic glycoqenolysis by peptide hormones (angiotensin II, vasopressin, activin A) and ~ -sympathomimeties is due to stimulation of the phosphatidy]-inositol pathway resulting in an elevation of the intracel]ular Ca++-concentration. The recently discovered, vasoactive peptide, endothelin-I (ET-I), acts via the same cellular mechanism in its target tissues. However, the potential impact of ET-I on hepatic carbohydrate metabolism has not yet been investigated. Thus, we measured glucose output of isolated rat livers, perfused with an oxygenated Krebs-Rinqer buffer and dialyzed erythrocytes in a non-recirculating system, in presence of ET-] or buffer (control). Continuous infusion of 10E-9 M ET1 resulted in a transient increase of the glucose production rate (tmlo]/(min x 100g b.w.)) reaching a maximuma of 7.56+1.04 versus ].32+0.13 in control experiments. The h~pattc glucose release, calculated frc~n the AUC (umol x 96min/ 100g b.w.), showed corrcsfonding enhancements (383.1+77.3 at 10E-9 M ET-I and 279.4+22.9 at 10E-8 M ET-I versus -72.2+21.0 in control experiments). However, endothelin-i ~ad no effect on hepatic lactate release within 96min: 0.25+0.12, -0.18+019, -0.01+0.20 umol/100g b.w.. In the ab-sence of Ca$+ in the p~rfusate ET-I was not capable of stimulating hepatic glucose ouput. Continuous infusion of insulin (0.I, 0.3, 1 U/l) decreased the ET-l-stimulated glucose release (351.8~ 103.6, 267.0~51.9, 211.8+--65.7). ET-I stimulates Ca++-dependent hepatic glucose production without affecting lactate release, indicating a glycogenolyttc action. The ET-l-sttmulated glucose release is sensitive to dose-dependent inhibition by insulin.

P821

P822

INCREASE IN MUSCLE GLYCOGEN PHOSPHORYLASE (GP) ACTIVITY F O L L O W I N G G L U C O S E - I N S U L I N INFUSION IN MAN. CORRELATION WITH GLYCOGEN CONCENTRATION. J.P. Felber, H. Kleiber, R. Munger, E. Temler, A. Golay, P. Frascarolo, and E. J6quier. Division of Endocrinology, University Hospital, and Institute of Physiology, Lausanne, Switzerland.

GLYCOLYSIS AS A SOURCE FOR AI_ANINE AND LACTATE FORMATION IN NORMAL SUBJECTS A. Virkam~ and H. Y I d - ~ n e n , Second Department of Medicine, Helsinld University, Finland Although indirect evidence has suggested operation of a glucose-alanine cycle in humans, the existence of this cycle and its regulation by insulin has not been directly verified in man. To determine the glycolytic origin of plasma lactate and alanine in humans, 6 normal males (age 27-+3 years, body mass index 22-+1 kg/m ~) received after an overnight fast a primedcontinuous infusion of [6-1"C]glucose, for 240 rain in the basal state, and for 120 rain during hyperinsulinemia (240-360 rain, serum insulin "30 mU/I). Blood samples were taken for the determination of glucose, lactate and alanine specific activities using combined ion-exchange and HPLC-chromatography. Basally, the percent of lactate originating from plasma glucose was 49-+6% while that of alanine was 45-+6%. During hyperinsulinemia, the percent of lactate and alanine originating from plasma glucose were 81+-10% (p<0.01 vs basal) and 82-+20% (p<0.05 vs basal). These data indicate that plasma glucose is an equally important source for both lactate and alanine formation in the basal state and during hyperinsulinemia. Since only -50% of both alanine and lactate are derived from plasma glucose in the basal state, other carbon sources, most likely glycogenolysis, contribute to operation of both Curl and glucose-alanine cycles. During hyperinsulinemia, plasma glucose is the main source of both lactate and alanine formation.

The purpose of the work was to study changes in GP activity in response to glucose-insulin infusions. 16 normal subjects participated in the study which was divided into 3 successive periods : A) basal; B) 120 min preclamp with infusion of either saline or tLpyridyl-carbinol (~-PC) to lower plasma FFA; C) Euglycemic hyperinsulinemic (89-94 ]xU/ml) clamp. Continuous indirect calorimetry was used throughout. Muscle biopsies were obtained in 9 subjects in vastus lateralis at the end of each period for measurement of GP activity and glycogen concentration. Glucose oxidation rose during tLPC infusion, before the clamp (from 2.24 _+ 0.24 to 2.98 + 0.18 mg/kg.min, p <0.001) and glucose storage that followed was higher compared to control (4.29 + 0.31 vs 2.99 + 0.53 mg/kg.min, p <0.05). GP activity in muscle biopsies rose at the end of the clamp following both saline (153 + 20 vs basal 71 + 21 n m o l / m i n . m g protein, p <0.05) and ~-PC infusions (160 _+ 17 vs 70 + 15 n m o l / m i n . m g protein, p <0.005). GP activity was positively correlated with glycogen concentration following clamp, both after saline (r=0.80, p <0.01) and tLPC (r=0.61, p <0.05). These results suggest that the increase in muscle glycogen concentration due to glycogen synthesis during the clamps leads to a stimulation of GP activity, thus promoting glycogenolysis subsequently.

A222 P823

P824

N O R M A L I S A T I O N OF THE C A P A C I T Y OF U R E A S Y N T H E S I S IN D I A B E T I C RATS BY G L U C A G O N I M M U N O N E U T R A L I Z A T I O N REDUCES N I T R O G E N LOSSES

T I M E - D E P E N D E N T E F F E C T S OF G L U C A G O N ON R N A A N D P R O T E I N B I O S Y N T H E S I S IN RAT L I V E R M I T O C H O N D R I A

T Almdal 1'2, JJ Holst 3 & H V i l s t r u p ]' Dept of m e d i c i n e A, R i g s h o s p i t a l e t (I), Steno M e m o r i a l Hospital (2), Institute of P h y s i o l o g y C (3), C o p e n h a g e n Denmark.

A.G.Minchenko I n s t i t u t e of E n d o c r i n o l o g y K i e v 252114, Ukraine, U S S R

P l a s m a g l u c a g o n c o n c e n t r a t i o n is i n c r e a s e d in diabetes. This a c c e l e r a t e s the hepatic conversion of amino n i t r o g e n to urea nitrogen, and may be one r e a s o n for n i t r o g e n w a s t i n g in diabetes. We studied the e f f e c t of g l u c a g o n i m m u n o n e u t r a l i zation on these phenomenas. Rats of 225 g were fed ad lib. D i a b e t e s was induced w i t h s t r e p t o z o t o c i n 75 mg/kg i.v. Controls r e c e i v e d the vehicle. The rats were d i v i d e d into 2 groups and t r e a t e d for 4 weeks. DM + ser (n=6) : were given 1 ml of n o n i m m u n e serum i.p. every week. DM + g l u c A b (n=6) : were given I ml of glucagon a n t i b o d i e s every week. Plasma samples from the DM + glucAb rats had high c a p a c i t y for binding of g l u c a g o n t h r o u g h o u t the e x p e r i m e n t a l period. T h r o u g h o u t the 4 weeks the n i t r o g e n balance was more p o s i t i v e in DM + glucAb than in DM + ser. After 4 weeks the C a p a c i t y of the Urea-N Synthesis (umol/min 100g BW) d e t e r m i n e d in d u r i n g i.v. alanine loading were in controls: 8 + I, in DM + ser: 21 + 2 and in DM + glucAb: 9 + ~ (p< 0.01 versus D M + set). After dissection--the N contents of the total m u s c l e mass (ml~ol) was: Controls: 319 + 6, DM + ser: 168 + 8 and in DM + GlucAb: 200 + 8 (p<0.05). This d e m o n s t r a t e s that the h y p e r g l u c a g o n a e m i a of diabetes is of p r i m a r y i m p o r t a n c e for the n i t r o gen loss of diabetes.

and

Metabolism,

RNA b i o s y n t h e s i s in rat liver m i t o c h o n dria starts i n c r e a s i n g 15 min after g l u c a g o n a d m i n i s t r a t i o n to animals. A b i p h a s i c effect of g l u c a g o n on the i n d u c t i o n of rat liver mit o c h o n d r i a l RNA b i o s y n t h e s i s was o b s e r v e d w i t h m a x i m a in m i n u t e s 45 and 105. The second p h a s e of the g l u c a g o n - i n d u c e d m i t o c h o n d r i a l R N A biosynthesis is p r o l o n g e d and more p r o n o u n c e d than the first one. As shown by e l e c t r o p h o r e tic analysis of p o l y ( A ) - c o n t a i n i n g RNA from rat liver m i t o c h o n d r i a and by m o l e c u l a r hybrid i z a t i o n of RNA w i t h c l o n e d f r a g m e n t s of rat m i t o c h o n d r i a l DNA, the m e c h a n i s m of g l u c a g o n i n d u c e d increase of m i t o c h o n d r i a l RNA b i o s y n thesis is not s e l e c t i v e i n d u c t i o n of individual gene t r a n s c r i p t i o n . I n c r e a s e d b i o s y n t h e sis of m i t o c h o n d r i a l R N A was o b s e r v e d w i t h a gradual i n c r e a s e of the pool of l a b e l l e d precursors in the m i t o c h o n d r i a ; in the cytoplasm, the pool of p r e c u r s o r s i n c r e a s e d less. This m a y suggest that the i n t r a m i t o c h o n d r i a l and the c y t o p l a s m i c pools are p a r t l y independent. G l u c a g o n e n h a n c e d the b i o s y n t h e s i s of m i t o c h o n d r i a l m a t r i x proteins, and of outer and inner m i t o c h o n d r i a l membrane proteins synt h e s i z e d on m i t o c h o n d r i a l and c y t o p l a s m i c polysomes. It m a y be c o n c l u d e d that g l u c a g o n enhances the f u n c t i o n a l a c t i v i t y of the mitoc h o n d r i a l genome in the liver.

P825

P826

LEUCINE AND PHENYLALANINE TURNOVER IN LIVER CIRRHOSIS. G. Biolo, S. Inchiostro, L. Sabadin, M. Vettore, M.C. Marescotti, R. Orlando, A. Tiengo, and P. Tessari. University of Padova, Italy. Liver cirrhosis is characterized by decreased sensitivity to insulin of glucose metabolism. To assess whether such an impairment is extended also to amino acids, leucine and phenylalanine turnover, and KIC oxidation, were studied in 11 cirrhotic patients with normal glucose tolerance and 11 controls, both in the post-absorptive state and following an euglycaemic-hyperinsulinaemic (~70 mU/L) clamp, with and without a simultaneus exogenous amino acid infusion. In the post-absorptive state, cirrhotic patients, with respect to controls, showed normal leucine rate of appearance (Ra) (1.72_+0.17 vs 2.19_+0.17 ~mol/kg.min), normal phenylalanine Ra (0.85-+0.17 vs 0.68+-0.07 ,# mol/kg.min), slightly greater (p<0.05) KIC oxidation (0.29-+0.08 vs 0.16-+0.03umol/kg.min), and slightly reduced non-oxidative leucine disposal (1.65-+0.11 vs 2.00-+0.13 v mol/kg.min). Following euglycemic hyperinsulinemia, the relative decrements vs basal of all kinetic parameters were comparable or even greater than in controls, despite a decreased (p<0.01) insulin-mediated glucose disposal in the cirrhotics. The simultaneous infusion of insulin and amino acids resulted in normal suppression of endogenous leucine and phenylalanine Ra and in comparable increases in KIC oxidation and non-oxidative leucine disposal. Thus, insulin-resistance of liver cirrhosis appears to be present as regards glucose but not amino acid metabolism, since leucine and phenylalanine turnover are normal in the basal state, and are normally or even more responsive to insulin, with or without accompanying hyperaminoacidemia.

GLUCOSE AND AMINO ACID METABOLISM IN UREMIA. EFFECTS OF INSULIN AND AMINO ACIDS. A. Solini, L. Luzi, A. Pctrides, R.A. DeFronzo and P. Castellino. Catholic University, Rome, Italy and University of Texas, U.S.A. To investigate the ability of insulin and amino acids to promote protein anabolism in Chronie Renal Failure (CRF) we studied 16 controls (age=47+_2, IBW=113• and 13 CRF (age=49+_3, IBW=112• serum creadnine--4.0-k0.4 rag%), participating in two protocols. Study One: Euglyeemic insulin clamp (70 mU/1) and Study Two: Amino acids infusion, both with indirect calorimetry and [l-14C]-leucine infusion. Basal leucine flux and oxidation, calculated from plasma KIC specific activity, were reduced in CRF (1.50i-O.06 vs 1.75_+0.06 and 0.25_+0.02 vs 0.30-20.02, gmol/Kg/min, p<0.05).Glueose oxidation was normal (8.7+1.0 vs 7.6+1.0 gmol/Kg]min). In Study One, leucine flux (1.06_+0.07 vs 0.98+0.09) and oxidation (0.17_+0.02 vs 0.17_+0.02) were similar in both groups. Insulin-mediated glucose uptake was reduced in C R F (23.94-_3.3vs 37.8•

gmol/Kg/min, p
dccreascd non-oxidative glucosc disposal (9.4•

vs 23.9•

In

Study Two, Icucinc flux was similar in both groups (0.88!-0.06 vs 0.84_+0.04), but non-oxidative Icucine disposal was stimulated and nct leucine balance into protein was reduced in C R F

(1.54_+0.11 vs

2.1OAO.lOp.mol/Kg/min and 0.29-!-0.05 vs 0.41_+0.05, both p<0.01). Conclusions: in C R F basal leucine flux and oxidation arc reduced; insulin-mediated supprcssion of protein degradation is normal; amino acid-inducedstimulationof proteinsynthcsisisimpaired.

A223 P827

P828

INSULIN ACUTELY REDUCES WHOLE-BODY PROTEOLYSIS ACTING AT EXTRAMUSCULAR SITES.

EPINEPHRINE INCREASES PERIPHERAL RATHER THAN S P L A N C H N I C D I S P O S A L OF E X O G E N O U S A M I N O A C I D S IN H E A L T H Y MAN. W.Waldh~usl, K.Ratheiser, S.Gasic, M.Komjati, and P.Bratusch-Marrain, ist M e d i c a l Department, Univ. of V i e n n a , A u s t r i a .

S. Inchiostro, G. Biolo, L. Sabadin, E. Vincenti, M.G. Fratton, M. Vettore, A. Tiengo, E. Duner and P. Tessari. University of Padova, Italy. To investigate the hypoaminoacidemie and the antiproteolytic effect of insulin in the whole-body as well as at the forearm level, we have evaluated leucine and phenylalanine kinetics, in normal volunteers, before and following systemic hyperinsulinemia (~80 mU/L), with the forearm arterial-deep venous catheterization combined with L-[1-14C[-leucine and L- [2,6-3HI-phenylalanine infusions. Post-absorptive arterial leucine concentrations were significantly greater (p<0.01) than in the deep-vein (140-+4 vs 145-+3 mol/1, respectively), thus resulting in forearm leucine release (0.771-+0.154 nmol/min.ml of forearm) greater (p<0.01) than uptake (0.676-+0.157). Arterial phenylalanine concentrations (44/5 tool/l) tended also to be greater (t = 2.07, p<0.07) than in the deep-vein (48-+5), since release (0.291-+0.064 nmol/min.ml of forearm) was slightly even if not significantly greater than uptake (0.197-+0.068). Insulin infusion decreased (p<0.01) arterial leucine and phenylalanine concentrations to 95_+4 mol/l and to 30-+3 mol/I respectively. Systemic leucine and phenylalanine Ra were also reduced by 12-15% (p<0.01). Surprisingly, the reduction in the systemic concentrations of both leucine and phenylalanine were accompanied by net release (p<0.05) instead that by uptake of these substrates by the forearm. By the end of insulin infusion, isotopically-deter mined uptake and release of leucine (0.602-+0.109 and 0.564-+0.072 nmol/min.ml of forearm) and of phenylalanine (0.178-+0.046 and 0.226+0.043 nmol/min.ml of forearm) were not statistically different from basal values. We conclude, that skeletal muscle is not the immediate target site of the hypoaminoacidaemic and antiproteolytic effect of systemically-induced hyperinsulinemia.

To d e t e r m i n e t h e i m p a c t of s t r e s s h o r m o n e s o n amino acid (AA) d i s p o s a l healthy men (25~4 yrs) were infused with an AA-mixture (VAL, MET, ILE, LEU, PHE, LYS, HIS) w i t h o u t (group I: N=6) and with concomitant epinephrine (EPI,II: 6 u g / m i n ; t = - 7 5 to 120 min; N=6) o r dexamethasone (DEX, III: 6 mg/d, N=7) e x p o sure. Employing hepatic venous catheterization both transsplanchnic b a l a n c e of g l u c o s e a n d A A as w e l l as t h e i r m e t a b o l i c clearance r a t e (MCR) w e r e d e t e r m i n e d . O n l y i n t h e b a s a l s t a t e a n i n c r e a s e b y EPI & D E X of s p l a n c h n i c AA uptake was observed. MCR (i/min; range: 0 , 3 5 to 0 , 6 5 i) of i n f u s e d A A w a s e l e v a t e d b y 40% (MET) t o 85% (LEU) vs. control studies f o l l o w i n g EPI (p <0.005) b u t u n c h a n g e d a f t e r DEX. T o t a l s p l a n c h n i c A A u p t a k e w a s i n c r e a s e d after AA infusion vs. basal (5,446~ 3,635 n m o l / k g . m i n . I: 8 , 5 7 7 • II: 8 , 9 5 7 • 3,714; III: I 0 . 7 5 7 Z 2 , 6 8 9 ) as w a s s p l a n c h n i c g l u t a m i n e u p t a k e (+140%), b o t h of w h i c h w e r e u n i n f l u e n c e d b y e i t h e r EPI o r DEX. W e c o n c l u d e t h a t (a) epinephrine and dexamethasone augment provision of gluconeogenic precursors to the liver in the basal state; (b) epinephrine f a i l s t o i n c r e a s e s p l a n c h n i o u p t a k e of i n f u s e d AA, but (c) elevates their total MCR suggesting an epinephrine dependent alternate s u b s t r a t e s u p p l y for p e r i p h e r a l t i s s u e s .

P829

P830

OVERESTIMATION OF ENDOGENOUS PROTEOLYSIS BY THE "PRIMARY POOL" MODEL OF LEUCINE. L. Sabadin, S. Inchiostro, G. Biolo, and P. Tessari. University of Padova, Italy. During exogenous amino acid and/or insulin infusion, endogenous amino acid rate of appearance (Ra) is estimated by subtracting exogenous amino acid infusion rates from total rates of appearance (Ra). However, it is not clear which model has to be used to calculate total amino acid Ra. We have re-examined this issue by measuring simultaneously leucine, 0(-ketoisoeaproate (KIC) and phenylalanine kinetics in the postabsorptive state and following a combined insulin plus amino acid (I+AA) infusion with the euglycaemic clamp. The infused amino acid solution was rich in the branched-chain amino acids and poor in phenylalanine, in order to challenge the various models at different plasma leucine and phenylalanine levels. During the combined I+AA infusion, the percent suppression of endogenous leucine Ra, calculated with the "primary-pool" model, was 2-fold greater (p<0.01) than that obtained with either the "reciprocal-pool" model or with phenylalanine Ra. Thus, the extent of suppression of endogenous proteolysis is greately dependent upon the model used, and ~ppears to be largely overestimated, as regards leucine, by the "primary" pool model.

LEUCINE METABOLISM IN TOTAL L!POATROPHIC DIABETES D Spotti, L Luzi, A Battezzati, F Facchini, G Perseghin,and G Pozza. Department of Medicine, H San Raffaele, Mil~no. Total acquired lipoatrophic diabetes, or Lawrence Syndro~ (LS) is characterized by insulin resistance with respect to glucose and lipid metabolism, non ketotic hyperglycaemia, and muscle mass hypertrophy. To assess whether the defect in insulin action extends to leucine/prctein metabolism, a female subject (age=SSy; BW=44kg; HbAlc=9.5% ; i.m. insulin dose:f60 U/day; FPG:lOmmol/l; TG=850ng/dl; free IRI=I5 ~U/ ml; GH=5 ng/ml; Somatomedins:O$SC~)with LS was studied by means of a S step euglycaemic insulin clamp (0.3, 2.0, 5.0 mU/kg.min),oombined with 3 ~ H glucose, 1 I--4Cleucine infu sion and indirect calorimetry. Five controls were also stu died. In the basal state LS had plasma LEU(125 ~mol/l),ILE (68),VAL(236),GLU(S86) and PHE(48) comparable to those of CON. Basal hepatic glucose production (2.6 vs 2.0+0.1 mg/k~ min), Endogenous LEU flux(S0 vs 40~i ~mol/m 2 .min), LEU OX (i0 vs 6~i), non oxidative LEU disposal(40 vs Sahl), were increased in LS with respect to CON. Following the 3 step insulin infusion, the insulin stimulated glucose metabol~m was defective in LS with respect to CON (GOx:50%,65%and80% of CON; non oxidative glucose disposal=50%,55% and 60% of CON at 0.3, 2 and 5 mU/kg.min respectively). The decrease of LEU, ILE, VAL, PHE as well as the suppression of ELF,LO and NOLD was defective in LS vs CON at each insulin step. Conclusions: a)LS demonstrate both a reduced insulin sensi tivity and maximal insulin response common to glucose and protein metabolism; b)the present data support the hypoth[ sis that in LS the defect in insulin action is both at the receptor and post receptorial sites.

A224

PS 37 Glucose Transport P831

P832

INSULIN SENSITIVE GLUCOSE TRANSPORTER, OBESITY AND INSULIN RESISTANCE. G. A. HITMAN, J. U. WEAVER and P. G. KOPELMAN. Medical Unit,The London Hospital, London, U.K. Recently it has been shown that changes in insulin sensitive

IN VIVO ~2-ADRENERGI C STIMULATION REGULATES GLUCOSE TRANSPORTER mRNA LEVELS

glucose transporter (Glut 4) gene

expression are associated

with impaired insulin action and insulin resistance (IR).We have previously reported lack of association of Kpn I polymorphism of Glut 4 gene with Type 2 (non-insulin-dependent) diabetes mellitus and obesity. In order to determine whether this resulted from diversity of IR in these 2 groups we studied this genetic polymorphism in

54 well characterised obese non-diabetic,

insulin resistant

premenopausal women (Body Mass Index

>30). IR was measured using

HOMA analysis

of fasting

glucose and insulin. Glut 4 polymorphism was studied

by

extraction of DNA from the lymphocytes, digestion with Kpn I, Southern blot hybridisation with 32p labelled Glut 4 probe.Two types of alleles have been identified, sized 5.8 and 6.6 kb and identified genotypes were 5.8,6.6, 5.8 homozygous and 6.6 homozygous. The allelic and genotype frequencies in the obese women were comparable to our previous observations. Moreover analysis of variance did not demonstrate any significant correlation between genotypes or alleles and insulin

DIFFERENTIALLY IN RAT TISSUES

R.J. Moss, S.J. Koopmans, H.C.M. Sips, J.A. Maassen and H.M.J. Krans. Department of Endocrinology, University Hospital, Leiden, The Netherlands Insulin resistance is often accompanied by low sympathetic tone. Increasing sympathetic tone might therefore influence insulin action. The ~2-adrenergic agonist Fenoterol (F) stimulates hexose uptake and affects glucose transporter gene expression and number in 3T3-L I adipocytes. We studied the effect of F infusion in rats on Brain (GLUT1) and Muscle (G~OT4) Glucose transporter mRNA levels. F(bromide) was infused at 0.12 m g . k g l . h "I in freely moving male wistar rats. After 96 hours the rats were decapitated, mRNA was isolated from soleus muscle, liver and epididymal fat. GLUT1 and GLUT4 mRNA levels were measured using Northern blotting. GLUT1 and GLUT4 mRNA levels in fat did not change after F infusion compared to controls. GLUT1 and GLUT4 mRNA were not detectable in liver. In muscle from F infused rats GLUT4 mRNA levels were increased (i0 fold), GLUT1 mRNA levels did not show a major change. We conclude that ~2-adrenergic stimulation causes a differential regulation of GLUT1 and GLUT4 mRNA levels in the rat and that the regulation is tissue specific.

resistance. We conclude that these findings confirm that Kpn I polymorphism of Glut 4 gene is not a useful marker for insulin resistance.

P833

P834

GLUCOSE INDUCES REDISTRIBUTIONOF GLUCOSE TRANSPORTERS IN SKELETAL MUSCLE CELLS S. Sasson, R. Greco-Perotto, E. Wertheimer, N. Reiss, B. Jeanrenaud and E. Cerasi. Departments of Pharmacology and Endocrinology & Metabolism, Hebrew University Hadassah Medical Center, Jerusalem, Israel, and Laboratoires de Recherches Metaboliques, Faculty and Department of Medicine, University of Geneva, Geneva, Switzerland.

The Phorbol Ester TPA Induces Translocation of the Glucose Transporter Subtype GLUT4 but not of GLUT1 in Rat Adipocytes B. Vogt, J. Nushack, E. Seffer, H.U. H~ring, Munich, FRG

Glucose transport in skeletal muscle is regulated by glucose itself: low glucose increases, while high glucose deceases Vmax of the transport without affecting Kin. The major glucose transporter in L8 myocytes is GLUT-1. Here we demonstrate that the changes in Vmax of hexose transport, induced by glucose concentrations between 1-20 mmol/1 are accompanied by similar changes in the number of GLUT-1 in L8 myocyte plasma membranes. Following cell fractionation, [3H]cytochalasin B binding and Western blot analyses of the resulting plasma and microsomal membranes indicated that upregulation of transport is associated with increased number of transporters in the plasma membrane. Upon downregulation, plasma and microsomal membranes contained equal quantities of transporters. These changes were fully reversible and quantitatively similar to the changes in hexose transport. Therefore, the intrinsic activity of the transporter is not modified by glucose. We conclude that the autoregulation of glucose transport results from redistribution of glucose transporters between the internal pool and the plasma membrane of skeletal muscle cells.

I n s u l i n regulates the glucose transport in i s o l a t e d f a t c e l l s by a c t i v a t i o n of glucose transporters in the plasma membranes and through t r a n s l o c a t i o n of the glucose transporter subtypas GLUT4 and GLUT1. Protein kinase C s~imulat i n 9 phorbol esters are able to mimick p a r t i a l l y the insul i n e f f e c t on glucose t r a n s p o r t . In order to study whether t h i s phorbol ester e f f e c t occurs by t r a n s l o c a t i o n of GLUT1 or GLUT4 the s u b c e l l u l a r d i s t r i b u t i o n of these transporters was determined by western b l o t using s p e c i f i c a n t i bodies directed against GLU~I or GLUT4 r e s p e c t i v e l y . Isolated r a t f a t c e l l s (4 x 10~ c e l l s / m l ) were stimulated for 10 min with i n s u l i n (3009PU/ml) or TPA (12-O-tetradeeanoylphorbol-13-acetate) ( 1 0 - ) . 3-O-Nethylglucose t r a n s p o r t was determined and plasma membranes and low density microsomes were prepared f o r western b l o t t i n g . I n s u l i n stimulated 3-O-Hethylglueose transport 8-9 f o l d , TPA 4-5 f o l d (Basal: 4.2 + 2.5 %, I n s u l i n : 36 + 5.6 %, TPA: 22 + 6 %, n=5). TPA is--able to increase the--amount of GLUT4 Tn the plasma membrane f r a c t i o n about 2.5 f o l d (2.5 ~ 0.9, n=5) while i n s u l i n stimulates 4.4 fold (4.4 ~ 1.7, n=5), par a l l e l e d by a corresponding decrease in the low density microsomes ( I n s u l i n : decrease 1.9 + 0.3 f o l d , TPA: 1.5 + 0.2 f o l d , n=G). While TPA r e g u l a t e ~ t h e t r a n s l o c a t i o n of GLUT4 i t has no e f f e c t on GLUT1 in the same c e l l f r a c t i o n s . These data are in favour of a r o l e of protein k i nasa C in the r e g u l a t i o n of GLUT4 but not of GLUT1 in f a t ceils.

A225

P835

P836

INSULIN RESISTANCE AND mRNA EXPRESSION OF THE INSULINRESPONSIVE GLUCOSE TRANSPORTER IN ISOLATED CARDIAC MYOCYTES.

Glucose transporter number is decreased in brown adipose tissue from obese mice, a defect which disappears after BRL 26830A treatment.

J. Eckel, M. RuB, S. Petersen, L. Herberg and H. Reinauer, Diabetes Research Institute, DUsseldorf, FRG The role of glucose transporter gene expression in the pathogenesis of insulin resistance in muscle tissue is presently unknown. Cardiomyoeytes from insulin resistant rats (streptozotocin-diabetie, obese Zueker rat) have been used to compare basal and insulin-stimulated glucose transport activities to the mRNA level of the insulin-responsive glucose transporter (IRGT), as determined by Northern blot analysis. In streptozotocin-diabetes basal glucose transport decreased by 29% with a reduction of insulin responsiveness by 64%. The mRNA level of IRGT (2.5 • 0.i kb transcript) was found to decrease by 87 • 4 %. The specificity of this effect was demonstrated by an unaltered Gsalpha mRNA (1.99 kb transcript) expression. In obese Zueker rats basal transport was reduced by about 50% with a concomittant decrease in the maximal response to insulin by 40%. Under these conditions IRGT mRNA content even increased by about 30%. This increase was also observed after incubation of cardiomyocytes from normal rats for 4 hours. In conclusion, these studies show that insulin regulates expression of the IRGT gene in cardiac myocytes. A largely reduced expression of this transporter species may explain cardiac insulin resistance in insulin deficient states. In obesity additional post-transcriptional events appear to be involved.

Y . Le Marehand-Brustel, T. Grdmeaux, J.F. Tanti, N. Rochet and E. Van Obberghen. INSERM U145, Facult6 de Mddecine, Avenue de Valombrose, 06034 Nice Cddex, France.

Glucose transport is decreased in brown adipose tissue (BAT) of obese, hyperglycaemic, insulin-resistant animals. Our aims were to characterize the glucose transporter(s) in BAT from lean and obese animal~, aad to study the effect of a treatment with the thermogenie agent BRL 26830A (Beecham laboratories). Glucose transporters were characterized in crude BAT membrane fractions by Western blot analysis, using antipeptides specific for the erythroid (EGT) or muscle/fat (MGT) glucose transporter. In BAT membranes from lean mice, only MGT, considered as the insulin regulatable transporter, was present in significant amount. In membranes from obese animals, MGT number was decreased by 40% + 4% (mean + SEM of 9 preparations, P< 0.001), expressed per total tissue or per mg protein. Following a three week-treatment with BRL 26830A (1 or 2 mg/kg/day), glycaemia o f obese mice was normalized while insulinemia remained elevated. Total BAT glucose transporter number returned to normal at 1 mg or increased by 63% + 14% at 2 mg. When expressed per mg of membrane protein, MGT number was similar in lean and 1 or 2 mg BRL26830A-treated obese mice. These results suggest that the defect in glucose transporter number could participate in the hyperglycaemic syndrome, since treatment with the thermogenic agent normalizes in parallel glucose transporter number and glycemia.

P837

P838

IN VIVO METFORNIN-TREATMENT OF OBESE (fa/fa) ZUCKER RATS: EFFECT ON INSULIN BINDING, GLUCOSE TRANSPORT, ACTIVATION OF INSULIN RECEPTOR KINASE IN INTACT CELLS, AND SUBCELLULAR DISTRIBUTION OF GLUT-1 AND GLUT-4 GLUCOSE TRANSPORTERS IN ISOLATED ADIPOCYTES .

INCREASED MUSCLE HYPERTHYROIDISM.

S.Matthaei J.P.Reibold* H.H.Klein *~ H.Benecke* A.Hamann*, and H.Greten*. Dept.of Medicine,University of Hamburg and ~ 2000 Hamburg-20, FRG. To examine the mechanism of the antihyperglycemic action of the biguanide metformin (M) in vivo, we studied the effect of metformin-treatment (3 weeks, 250ug/kg/day, metformin-plasma levels after 3 weeks treatment were determined by HPLC and were in the therapeutical range) on (i) various blood parameters, (2) basal and insulinstimulated glucose transport,(3) insulin binding,(4) activation of insulin receptor kinase in intact cells, and (5) subcellular distribution of GLUT-I and GLUT-4 glucose transporters. The following metabolic paramters were obtained: (mean Z S.E.M., N=50) -metformin +metformin P Bodyweight (g), 535.6+6.3 530.9+6.3 ns Plasma glucose (mg/dl) 129.4+3.6 i19.8+3.0 <.05 Plasma insulin (uU/ml) 405 +23 223 +12 <.001 Triglycerides,Cholesterol,LDL,VLDL,HDL,Laqtate: ns Metformin-treatment had neither a significant effect on insulin binding (0.45~• (-M) vs 0.49!0.i0 (+M) B/F * i00), insulin receptor kinase activity in intact cells (basal: 15.8• (-M) vs 20.3• (+M); insulin: 166.4• (-M) 158.8+13.8 (+M) fmol P/pmol binding * min) nor on basal glucose transport (6.2+ 1.8 (-M) vs 7.5+2.5 (+M) fmol/5 9 i04 cells * se c). In contrast, metformin increased insulin-stimulated glucose transport 2.6 fold (20.4• (-M) vs 53.8• 12.2 (+M) fmol/5*104cells). Immunodetection using anti-peptide antibodies against GLUT-I and GLUT-4 revealed, that this metformin effect is due to a potentiation of insulin-induced translocation from the intracellular pool to the plasma membrane, predominantly mediated by the GLUT-4-system, while the GLUT-I transporters are far less abundant in these membranes.

GLUCOSE

TRANSPORTERS

IN

A. C a s l a , A. R o v i r a a n d G.L. Dohm*. F u n d a c i 6 n Jim~nez Diaz, Univ. Autonoma de Madrid, Spain. "School o f M e d i c i n e , E.C.U., Greenville N.C., U.S.A. To study the role of muscle in t h e a l t e r e d carbohydrate metabolism found in hyperthyroidism, glucose uptake and glucose transporter protein were measured in resting skeletal muscle from male wistar rats (2OO-230g, w e i g h t ) t r e a t e d w i t h L - t h y r o x i n e (375 ~ g / k g / d a y ) i.p.for i0 (HTI0, n=10) a n d 30 d a y s (HT30, n=lO). Hindquarters of hyperthyroid and control r a t s (n=20) w e r e p e r f u s e d w i t h 0, 1 0 g m o l / l a n d 107mol/l insulin and glucose uptake was measured. Gastrocnemius muscles were quickly r e m o v e d a n d g l u c o s e t r a n s p o r t e r p r o t e i n (GLUT4) was determined by Western analysis. Basal glucose uptake was increased in both HTIO (3.6•176 vs 2.3• ~mol/g/h p
A226

P839 EFFECTS OF ML-9 ON I N S U L I N - S T I M U L A T E D T R A N S P O R T IN 3T3-LI A D I P O C Y T E S

P840 GLUCOSE

G. Inoue, H. Kuzuya, A. Kosaki, I. Maeda, M. Okamoto, M. Okamoto, S. Kono, M. Kubota, S. Kato, and H. Imura. Second Division, D e p a r t m e n t of Medicine, Kyoto U n i v e r s i t y School of Medicine, Kyoto, Japan We studied effects of ML-9, a m y o s i n light chain kinase inhibitor, on the g l u c o s e t r a n s p o r t system in 3T3-LI adipocytes. ML-9 i n h i b i t e d the insulin s t i m u l a t i o n of 2 - d e o x y g l u c o s e t r a n s p o r t (Max;80%, IC50;25~M), but did not affect the basal activity. The i n h i b i t i o n was o b s e r v e d m a x i m a l l y at 10min after the addition, independent of e x t r a c e l l u l a r Ca 2+ c o n c e n t r a t i o n and reversible. Next we e x a m i n e d changes of insulin - r e s p o n s i v e g l u c o s e t r a n s p o r t e r (Glut4) in the s u b c e l l u l a r f r a c t i o n s of 3T3-LI a d i p o c y t e s by W e s t e r n b l o t t i n g w i t h a n t i - C - t e r m i n u s of Glut4 antibody. In the fraction which was d e p l e t e d of 5 ' - n u c l e o t i d a s e a c t i v i t y and e n r i c h e d with UDP -galactose:N-acetylglucosamine galactosyltransferase activity, Glut4 was d e c r e a s e d by insulin, and ML-9 i n h i S i t e d the i n s u l i n - i n d u c e d d e c r e a s e dose-dependently. By W e s t e r n b l o t t i n g with anti - p h o s p h o t y r o s i n e antibody, ML-9 did not inhibit p h o s p h o r y l a t i o n of 95kDa p r o t e i n in response to insulin in the W G A f r a c t i o n of 3T3-LI a d i p o c y t e s and of 95kDa and 160kDa p r o t e i n s in the whole cells. These results suggest that ML-9 a f f e c t s the signal t r a n s d u c t i o n of insulin to the glucose t r a n s p o r t system b e y o n d i n s u l i n r e c e p t o r t y r o s i n e kinase.

C - P E P T I D E S T I M U L A T E S G L U C O S E T R A N S P O R T IN TYPE 1 D I A B E T I C S K E L E T A L MUSCLE. J R Zierath, A Th6rne, B-L J o h a n s s o n and H Wallberg-Henriksson. Department of Clinical Physiology, Karolinska Hospital, Stockholm, Sweden C - P e p t i d e (CP) is p r o d u c e d w h e n p r o - i n s u l i n is c o n v e r t e d to i n s u l i n and is l i b e r a t e d from the p a n c r e a t i c B - c e l l s in c o n c e n t r a t i o n s e q u i m o l a r to insulin. Recently, we p r o v i d e d e v i d e n c e for a s t i m u l a t o r y effect of CP on g l u c o s e t r a n s p o r t in n o n - d i a b e t i c muscle. In the p r e s e n t study, we investigated the effect of a physiological concentration of CP on g l u c o s e t r a n s p o r t in skeletal m u s c l e from type 1 d i a b e t i c patients. Using a newly developed open m u s c l e biopsy technique, m u s c l e s p e c i m e n s w e r e o b t a i n e d from the v a s t u s l a t e r a l i s p o r t i o n of the q u a d r i c e p s m u s c l e from 6 type 1 d i a b e t i c p a t i e n t s (age 24• d i s e a s e d u r a t i o n 11• yrs). M u s c l e strips (20-25 mg) w e r e p r e p a r e d for in v i t r o i n c u b a t i o n s and 3 - 0 - m e t h y l g l u c o s e t r a n s p o r t m e a s u r e m e n t s w e r e performed. The m u s c l e strips m a i n t a i n e d a d e q u a t e e n e r g y levels t h r o u g h o u t the 60 m i n i n c u b a t i o n p e r i o d as m e a s u r e d by u n c h a n g e d ATP, l a c t a t e and g l y c o g e n c o n c e n t r a t i o n s . E x p o s u r e to CP at a conc e n t r a t i o n e q u i m o l a r to i00 ~U/ml i n s u l i n (0.6 nmol/l CP) i n c r e a s e d basal g l u c o s e t r a n s p o r t from 0.88• to 1.15• ~mol x m l " x hr "I (p<0.05) and insulin stimulated (i00 ~U/ml) g l u c o s e t r a n s p o r t from 1.20+0.12 to 1.53+0.17 #mol x ml "I x hr "I (p<0.05).--In conclusion, Cp e p t i d e is a b i o l o g i c a l l y a c t i v e s u b s t a n c e w h i c h i n c r e a s e s g l u c o s e t r a n s p o r t in type 1 d i a b e t i c muscle. However, the clinical r e l e v a n c e for the s t i m u l a t o r y e f f e c t of C - p e p t i d e on g l u c o s e transport in d i a b e t i c skeletal m u s c l e r e m a i n s to be evaluated.

PS 38 Insulin Receptors and Action P841

P842

MOLECULAR BASIS OF THE KINETIC DIFFERENCES BETWEEN THE INSULIN AND IGF-I RECEPTORS. C.M. Rotella, N . S . Gonzales, R.M. Shymko, Y. FujitaYamaguchi, J. ten Hoeve, N. Heisterkamp, J. Groffen, H. Kadowaki, T. Kadowaki, S.I. Taylor and P. De Meyts, City of Hope Medical Center and Beckman Research Institute, Duarte, CA. USA; Children's Hospital of Los Angeles, USA, and Diabetes Branch, NIH, Bethesda, USA.

INSULIN INCREASES SITES - A MECHANISM

Despite their similar structures, the insulin receptor and the IGF I receptors have markedly different binding kinetics. In the present work, we studied in detail the kinetic properties of insulin and IGF I receptors on a variety of cells, as well as of purified receptors and cells transfected with recombinant receptors. We analyzed insulin and IGF I equilibrium and kinetic binding data with a simple computer model that allows for negative cooperativity and transition to a tight binding (Ksuper) state. Compared with the insulin receptor, the IGF I receptor is striking by its much higher affinity (2.7 x I0 I0 M -I ) and lack of negative cooperativity at pH 7.6 as well as by a shift in the pH optimum from 7.6 to 8.4. The insulin receptor acquired properties similar to those of IGF I receptors, upon site-directed mutagenesis of Lys 460 to Arg, a residue positioned between the 435 and 468 disulfides recently shown to link the two ~ subunits of the receptor. This demostrates that this region plays a key role in controlling the espression of site-site interactions. These models will enable us to evaluate the physiological relevance of negatively cooperative binding on metabolic and mitogenic responses to insulin and growth factors.

THE

NUMBER REGULATING

OF

INSULIN INSULIN

BINDING SENSITIVITY

U. Smith, P. Lbnnroth and J. Eriksson. Department of Medicine II, SahIgren's Hospital, 413 45 GSteborg, Sweden. To assess receptor regulation by insulin without interference by receptor internalization and recycling, rat adipocytes were incubated with or without insulin (1000!JU/ml) for 20 min at 37~ Subsequently, the cells were energy-depleted with KCN, insulin removed and 1251-insulin binding measured to intact cells or after solubilization with Triton-X 100. Insulin exposure rapidly (<5 min) increased cell surface insulin binding -5-fold (p<0.001, n=7) without changing receptor affinity. Insulin also increased cell-associated 1251-insulin remaining after trypsin treatment (p<0.05, n=7), which indicates a conformational change of the insulin receptor. Total insulin binding to solubilized cells was not changed by insulin, whereas insulin increased the binding to solubilized cells after trypsin treatment (p<0.01, n=10), indicating receptor internalization. The increase in binding following insulin exposure was blunted in cells from several insulin-resistant states (cells from aged obese rats, cells treated with cAMP analogues or with amiloride). The data indicate that, 1) insulin rapidly uncovers insulin binding sites probably through a conformational change in the receptor itself and; 2) this process is an early and important step in the regulation of insulin sensitivity in rat adipocytes.

A227

P843

P844

RECEPTOR NEGATIVE COOPERATIVITY AND INSULIN DIMERIZATION: NEW INSIGHTS FROM MONOMERIC INSULINS. P. De Meyts, N. Gonzales, R.M. Shymko, K. Drejer, L. Sch~ffer, J. Markussen and J. Brange, City of Hope Medical Center, Duarte, CA, USA and NOVO Research I n s t i t u t e , Bagsvaerd, Denmark.

PATIENT WITH TYPE A INSULIN RESISTANCE ASSOCIATED WITH A

A variety of new insulin analogs prepared by sitedirected mutagenesis has allowed us to resolve some controversies regarding the nature of site-site interactions at the insulin receptor 9 The dissociation of 12sI-insulin from i t s receptors is markedly accelerated by unlabeled insulin at IO-I~ to IO-7M, r e f l e c t i n g the negative cooperativity in binding; this effect is reversed as the insulin concentration is increased from 10 7M to I0-5M, a phenomenon previously attributed to insulin dimerization. We have now studied twenty new analogs: a series of analogs with absent or reduced capacity to dimerize ("monomeric insulins") and several soluble, prolongedacting insulin derivatives. All "monomeric" insulins (except B25 Asp) accelerated insulin dissociation from IM9 lymphocytes in proportion to t h e i r a f f i n i t y for the receptor 9 Surprisingly, the disappearance of negative cooperativity at higher concentrations was observed with most monemeric insulins, showing that this is unrelated to insulin dimerization. The only exceptions were analogs substituted at AI3 and B]7, which map at the insulin surface involved in hexamerization. These data indicate that a so far unsuspected interaction of the hexamerization surface of the unlabeled insulin s t a b i l i z e s at high concentrations the t i g h t l y bound ("Ksuper") state of the 12~l-insulin-receptor complex and abolishes the negative cooperativity.

DELETION OF EXON 14 OF THE INSULIN RECEPTOR GENE F.Shimada, H.Makino, M.Taira, Y.Suzuki, N.Hashimoto, O.Nozaki and S.Yoshida. Second Department of Internal Medicine, Chiba University School of Medicine, Chiba, Japan We examined the insulin receptor gene in a teen-age Japanese girl with Type A insulin resistance and her family. The proband presented with acanthosis nigricans in the axillary area and on the neck. Cloning of her mutant insulin receptor gene from the genomic library revealed that one allele of her insulin receptor gene contained a 1.2-kilo base pair (kb) deletion arising from a recombination between two Alu elements and removed the 14th exon in the ~ subunit. We also synthesized cDNA and amplified the region correspond to the deletion joint using polymerase chain reaction. Analysis of amplified products indicated that the deletion does not affect the level of transcription and the splicing pattern of insulin receptor mRNA and altered the reading frame which caused a stop codon after amino acid 867, thereby producing a truncated insulin receptor without a transmembrane region and cytoplasmic domain. Four other members of her family were heterozygotes for the same mutant allele, in all there was a decrease in insulin binding and slight abnormalities in the oral glucose tolerance test. These data suggest that this deletion in the insulin receptor gene result in a decreased insulin binding and a mild insulin resistance.

P845

P846

PHORBOL ESTER TPA INHIBITS AN INSULIN DEPENDENT P H O S P H O L I P A S E C (PLC) IN F A T C E L L M E M B R A N E S

IN VIVO PHOSPHORYLATION OF ANNEXINS BY INSULIN AND pp60 c-src TYROSINE KINASES IN XENOPUS OOCYTE.

B. O b e r m a i e r - K u s s e r , M. K e l l e r e r , J. M u s h a c k , E. S e f f e r a n d H.U. H ~ r i n g Institut fur Diabetesforschung, M~nchen, FRG

F. Grigorescu, A. Laurent, A. Chavanien, F. Martin and J.P. Capony. INSERM U249, Montpellier, France.

There is indirect evidence that the intrinsic g l u c o s e c a r r i e r a c t i v i t y in fat cells is r e g u l a t e d b y a G - P r o t e i n a n d P L C ( O b e r m a i e r - K u s s e r et al. (1988) B i o c h e m . J. 256: 5 1 5 - 5 2 0 ) . AICI~ (i0 pmole/l) stimulates 3-0-methylglucose transport either through a G-protein or a PLC. T h i s e f f e c t is suppressed by p h o r b o l e s t e r T P A (I n m o l / l ) , however, not by combination of TPA and protein kinase C (PKC) inhibitor H7 (i0 n m o l / l ) (3-0m e t h y l g l u c o s e u p t a k e , e x p r e s s e d as 9 of e q u i l i b rium per 4 s: basal 5.1• AICI3 2 2 . 8 • AICI3 + T P A 1 2 . 4 • AICIs + T P A + H7 1 9 . 5 • In o r d e r to e l u c i d a t e w h e t h e r P K C i n h i b i t s a PLC i n v o l v e d in i n s u l i n s i g n a l l i n g , we characterized the PLC a c t i v i t y of f a t c e l l m e m b r a n e s . [ 3 H ] p h o s phatidylinositol 4-phosphate hydrolysis was measured after in v i t r o s t i m u l a t i o n w i t h a n d w i t h o u t i n s u l i n (I00 n m o l e / l ) . W e f o u n d t h a t 12.7% • of total PLC-activity is i n s u l i n s t i m u l a t a b l e . When plasma membranes from T P A - t r e a t e d fat c e l l s were used, the insulin stimulated PLC-activity was suppressed by 87.4% (n=4), provided, membranes have been prepared in a buffer containing s e r i n e p h o s p h a t a s e i n h i b i t o r s . In c o n c l u s i o n : T h e insulin dependent PLC of fat c e l l s is i n h i b i t e d b y PKC m o s t likely via serine phosphorylation. This suggests that PKC inhibits insulin signal transduction not only at the level of insulin r e c e p t o r k i n a s e b u t a l s o at the l e v e l o f PLC.

Activation of insulin receptor, and tyrosine phosphorylatlon of substrates are important steps in insulin signalling. Annexins (or lipocortins) have been recently recognized as substrates of tyrosine kinases. In order to elucidate their functional role, we have studied in vivo phosphorylation of annexins in X.laevis oocytes during insulin stimulation with or without prior microinjection of pp60 c-src kinase or treatment with metformin. Insulin induced progression through the ceil cycle from G 2 phase to M phase (oocyte maturation). Microinjection of pp60 c-src kinase potentiated both the rate and the level of insulin-induced oocyte maturation. Metformin potentiated the insulin- but not the progesterone~duced maturation. In oocytes prelabelled with P-orthophosphate, insulin stimulated lO-fold the phosphorylation of the p67, p45 and p36 annexins. The phosphorylation of these proteins was also stimulated 9 c-src by mzcroinjected pp60 but the effect was not additive. Treatment with metformin alone did not affect the phosphorylation of annexins but potentiated two-fold the insulin-stimulated phosphorylation of the p45 annexin. We conclude that members of annexin family ire common substrates of insulin receptor and pp60 c-src kinases and that their phosphorylation correlates with the biological effect of insulin alone or in conjuction with an antidiabetic drug treatment.

A228

P847

P848

INSULIN AND GLICLAZIDE INCREASE GLUCOSE UTILIZATION BY DIABETIC RAT INTESTINE. M.A.Tormo,N.A.G.Zubeldia,F.Ropero,and J.E.CampiIlo. Departamento de Fisiologia. Facultad de Medicina. Universidad de Extremadura. Badajoz. Spain. The effect of insulin (dO ~U/ml) and glielazide (200 pg/ml) on intestinal glucose metabolism was investigated by using an in vitro perfused intestinepancreas preparation isolated from normal and streptozotocin-diabetic rats (n=3-4). Glucose, lactate, alanine and pyruvate were measured enzymatically in the portal effluent (mean• At ii.i mmol/l arterial glucose, the glucose utilization (mmol/h) was reduced (p< 0.05) in diabetic (0.46• versus control (0.68• rats. Portal lactate and pyruvate levels were not modified but alanine (~mol/h) levels were increased (p< 0.05) in diabetic (31.3• versus control (18.8• rats. In diabetic rats the glucose utilization was increased (p
EFFECTS OF CHRONIC INSULIN-LIKE GROWTHFACTOR I THERAPYON NITROGEN BALANCEAND GLYCEMIC CONTROLIN DIABETIC RATS. R.J. Jacob, G.I. Shulman, W.V. Tamborlane, L. Asmundson, and R.S. Sherwin. Division of Endocrinology, Yale University School of Medicine, USA. I t is not known whether chronic insulin-like growth factor I (IGF-I) administration can reverse nitrogen wasting or correct hyperglycemia in poorly controlled diabetic rats. To examine these questions we studied spontaneously diabetic BB animals, housed in metabolic cages for 2wk. During the f i r s t week animals were fed ad libitum and treated with insulin (1-3U/day) to maintain body weight and allow hyperglycemia. During the second week an osmotic minipump was implanted subcutaneously for delivery of IGF-I (Img/day). Insulin therapy was continued and animals were pair-fed amounts ingested before IGF-I. IGF-I significantly reduced hyperglycemia (from 23.0• to 12.6• p
P849

P850

SEPARATION O F IM-9 LYMPHOBLASTOID CELLS I N T O T W O P O P U L A T I O N S W I T H H O M O G E N O U S INSULIN BINDING SITES OF H I G H OR L O W AFFINITY AND LINEAR SCATCHARD ANALYSIS.

SOMATOSTATIN ANTAGONIZES THE INSULIN EFFECT UPON L I P O G E N E S l S AND L I P O L Y S I S IN RAT ADIPOCYTES. L.T.Ho, C.Y.Cbeng, M.T.Gao, J.C.Perng, T.Ho and M . S . S h i a o . Department of Medicine, Veterans General Hospital, Taipei. T a i w a n , ROC.

P.M.Jehle, R.D.Fussg~inger, *I.Melzner, *O.Haferkamp, and H.Ditschuneit. Internal Medicine II and *Dep. of Pathology University of Ulm, FP,G. Recent studies isolating two different mRNAs have suggested the presence of two types of insulin receptors in IM-9 lymphocytes. By means of a new type of high resolution laser-scan-microscopy (LSM-Zeiss) we were able to image binding of FITC-insulin at the cell surface of IM-9. Unequal distribution of high and low capacity binding sites was observed within the mitosis and the following cell cycle. Consequendy we separated these two populations by fluorescence activated cell sorting (FACS-Coulter). Competition-inhibition binding studies under equilibrium conditions with 125I-insulin were performed at 15 ~ following acidic wash-procedure (PBS, pH 6) of the preloaded F1TC-insulin. Despite considerable down regulation following this treatment, controls exhibited still curvilinear scatchard analysis, whereas the two separated populations (each 107 cells) displayed distinct differences: one subpopulation with high capacity, 430.000 sites/cell, had low alTmity of Kd - 5,8 nM, in comparison to a low capacity subpopulation with only 16.200 sites/cell, but high affinity of Kd = 0,34 ruM. Both binding sites caused straight linear scatchard analysis. DNA-flowcytometry revealed the high capacity population as the more proliferative one (preponderance of G2/M-phase). In conclusion 1M-9 lymphocytes constitute a heterogenous mixture of cells with two distinct insulin receptors, homogenously distributed to different populations.

Rat adipocytes were used to study the effects of somatostatin ISRIF) and s a n d o s t a t i n [SAND) u p o n glucose and lipid metabolism in vitro. Adipocytes were isolated from 8-week-old male Sprague-Dowley rats. Lipogenesis was m e a s u r e d as t h e i n c o r p o r a t i o n ratio of the radioactivity of U-C-14 glucose into lipids. The s t i m u l a t e d lipolysis was m e a s u r e d u n d e r 10 - 1 0 t o 10 - 4 M o f isopreterenol ( I S O } and t h e r e l e a s e d free fatty acids were detected by calorimetry as an i n d e x of lipolysis. The r e s u l t s showed : (1) Both SRIF a n d SAND p e r se have no effect on glucose transport, glucose oxidation, I ipogenesis or ISO-stimulated lipolysis. ( 2 ) B o t h S R I F and SAND {10-9M} decrease the insulin binding ( control : 13.0+0.9xi05, SRIF : 3.4+0.5xi05, SAND : 2.8+~3xI05 per cell}. (3} Both 5 R I F and SAND decrease the insulin effect on [ i p o g e n e s i s IVmax of incorporation ratio : 4.3~1.9% vs. 2.2~1.7%, p<0.05). [4} Both S R I F a n d SAND d e c r e a s e the ant i-I ipolyt ic effect of insulin (Vmax of supFression : 58~2% v s . 70~19%, p<0.01). In conclusion, S R I F a n d SAND m a y a n t a g o n i z e the insulin's effect upon glucose mediated lipogenesis a n d u p o n ISO s t i m u l a t e d lipolysis in adipocytes, probably via a reduction in i n s u l i n receptor numbers.

A229

P851 LOW I N S U L I N B I N D I N G TO A D I P O C Y T E - D E R I V E D M O R B I D L Y OBESE P A T I E N T S W I T H AND W I T H O U T

P852 CELLS IN DIABETES

B. Casanova, F.J. Arrieta and A. Rovira. Fundacion Jimenez Diaz. Universidad Autonoma, Madrid. Spain.

INSULIN RESISTANCE IN THE GK RAT, A SPONTANEOUS NON OBESE M O D E L F O R N O N - I N S U L I N - D E P E N D E N T DIABETES. B. PORTHA, D. BAILBE, P. SERRADAS, O. BLONDEL, M.-H. GIROIX.

Cultured mature fat cells loose their lipidic content and revert into fibroblast-like cells. Insulin binding to adipocytes from morbidly obese patients with or without diabetes is impaired. In order to know if this alteration exists in adipocyte-derived cells, we have studied insulin binding in both cell types. A s u b c u t a n e o u s fat biopsy from gluteal region was performed in 3 women: a non-obese (BMI 20.7 kg/m 2, NO), a morbidly obese (BMI 46 kg/m 2, MO) and a non-insulin-dependent diabetic morbidly obese (BMI 46 Kg/m ~, NIDD-MO). The percent of maximal [1~5I]insulin binding (104~ to isolated adipocytes (Sx10 ~ cells/0.25 ml) was: 9.1 in NO, 8.2 in MO and 5.4 in NIDD-MO. An aliquot of freshly isolated adipocytes was suspended in DMEM containing glutamine (2mmol/l), 10% fetal bovine serum, penicillin (i00 U/ml) and streptomicyn (100~g/ml), and mantained at 37~ and 5% CO~. Glutamine (2mmol/l) was added weekly to the medium. Three weeks later, an homogeneous population of fibroblast-like cells was obtained. The percent of maximal [~I]insulin binding (101~mol/l) to 5x10 s cells/ml was: 22.7 in NO, 16.0 in MO and 1.3 in NIDD-MO. Conolusion: a d i p o c y t e - d e r i v e d cells show a higher expression of the insulin binding deflect observed in their corresponding mature cells.

Using our own colony of GK rats started with progenitors issued from 9 m 9 the original colony (Tohoku University, Sendal, the 35 th generauon Japan), we have found that the GK rats showed as early as weaning (lmonth) significantly higher basal plasma glucose (9 mmolA) and insulin levels (twice increased) as compared to Wistar controls, altered glucose tolerance (i.v. glucose) and a very poor insulin secretory response to glucose in vivo and in vitro. Insulin action was assessed in adult GK females at basal, submaximal and maximal (euglycemic clamp) insulin levels. Hepatic glucose production (GP), overall glucose utilization (OGU) and individual tissue glucose utilization (GU) were determined from combined [3-3H]glucose infusion and [114C]-2-deoxyglucose bolus injection. The basal GP and OGU were significantly higher (p
P853

P854

EFFECT OF VANADATE AND H202-VANADATE ON GLUCOSE TRANSPORT AND LIPOLYSIS IN HUMAN ADIPOCYTES P. Lbnnroth, J. Eriksson and U. Smith. Department of Medicine If, Sahlgren's Hospital, 413 45 Gbteborg, Sweden Vanadate and H202-Va were recently reported to exert maximal (Va) or even supramaximal (H202-Va) insulin-like effects in r a t adipocytes. To evaluate the response in human cells isolated human adipocytes were exposed (60 rain, 37oc) to insulin (10001~U/ml) or Va (0-i0 raM) or H202-Va (0-2 mM). [14C-U] glucose (50 nM) uptake by the cells and glycerol release to the medium were measured. No effect of any concentration of Va tested was seen on glucose transport, whereas 0.5 mM H202-Va exerted a full insulin-like response (p<0.05) but never exceeded the effect of insulin. Neither Va nor H202-Va exerted an effect additive to that of insulin. 2 mM H202-Va was as effective as insulin to inhibit isoproterenol-stimulated lipolysis. Neither H202-Va nor insulin inhibited lipolysis stimulated by N6_monobutyry] cAMP, an analogue which is not hydrolyzed by the cAMP-phosphodiesterase. Thus, high concentrations of H202-Va, but not Va, are effective in exerting a full insulin-like response in human adipocytes. However, the effect of H202-Va is not additive to that of insulin suggesting a similar mechanism of action. It is concluded that human f a t cells in contrast to r a t adipocytes are not responsive to Va while H202-Va exerts an insulin-like effect.

Laboratoire de Physiologie du Dtveloppement, CNRS URA 307, Universit6 Paris 7, F-75251 Paris, France.

I N S U L I N R E s I ~ r A N c E IN R A T S W I T H N O N - I N ~ U L I N DEPENDENT DIABETES INDUCED BY NEONATAL ~rREPTOZOTOCIN :EYIDENCE FOR REVERSAL FOLLOWING BENFLUOREX TREATMENT O. BLONDEL, P. SERRADAS, D. BAILBE, B. PORTHA Lab.Physiol.D~v. -CNRS U R A ~ 7 -Universi~Pare7 -F75251 PARIS We have e~s~i~,4 ~e effectof chrome (20 d.) oral benfluomx t~a~nt (35 mg/kg) on tissuesensitivity~o insulinin ~dult ra~s with non-insulindependent diabe~s induced by suep~zozocm (80 mg/~) given 5 day~ afar birth(nS-~'Z). Inulin ec~on ~ ~ in vWo ~ ~ ~ submexir~l (er~lycem~ c ~ p ) ir~su~ levels. Hepe~c g~ose produc~n (OP), overall glucose util=a~n (OOU) and individualtissueG U were de.mined from combined [3-3H] glucose inf~ion and [l-14C]-2-d~gluco~ bolus h ~ n In the unused diabetics the besei o p ~ (oou) ~ ~re~sed @<0.02) ~ d Curi~ hy~m~u~m~ ~ livur and peripheraltissues revealed insulin ~-~ance. In the benfluomx - ~ t e d diabeticspos~bsorptivepksz~ glucose levelswere decr-v~,d(7.9~0.2 mmo~l ~s competed ~o 17.2• hi the unt~aed diabeticsand 6.7~.2 in the benflumex cona~l ra~s) ~ the ~ and the glucose-s~ula~,d (IVGTD pl~ama levels veto ~t impmved~ In the benfluomx tm~ed diabeticsthe b~sal GP and O G U veto n o ~ . Followh~ h y p e ~ i m u ~ . ~ , OP ~ l e d nonn~dly suppressed 'whileOOU ~ not s~if~mtly improved.Thesedaa demoratrazethat "oenSuomx 1) ~ effec~,e m ~n~nng h y p ~ g ~ m ~ m n~STZ d ~ mu 2) D~ally reversed the impaired suppression of hepa~ output by ~ in ~ese 2 ~ . It is sugges~M that benflum~ counteracts insulin msis~ace vm a predominant effect on the liver.

A230 P856

P855 RU 486 GIVEN GLYCOGENESIS

TO RATS IMPROVES THE RESPONSE OF T O I N S U L I N IN M U S C L E IN V I T R O

C. DaCosta, E.A. Foot and E. Leighton. Department of Biochemistry, University of Oxford, South Parks Road, Oxford, UK Chronically high blood levels of glucocorticoids cause insulin r e s i s t a n c e in skeletal muscle. Whether physiological c h a n g e s in blood g l u c o c o r t i c o i d s levels alter muscle insulin sensitivity has not been established. To e l u c i d a t e this we a d m i n i s t e r e d the potent s t e r o i d r e c e p t o r a n t a g o n i s t RU 486 (50 mg/kg) to rats 2 h before their f e e d i n g cycle (1800 h) and isolated m u s c l e s at the end of this cycle (0900 h). Control and t r e a t e d a n i m a l s ate the same amount of food. Soleus m u s c l e s w e r e isolated from c o n t r o l and RU 486t r e a t e d rats and i n c u b a t e d w i t h v a r i o u s levels of insulin (i, I0, I00, I000 or I0000 pU/ml). We m e a s u r e d the effects of insulin on g l y c o g e n s y n t h e s i s and lactate f o r m a t i o n (i.e. g l u c o s e transport). The rate of lactate f o r m a t i o n was not a l t e r e d in m u s c l e s isolated from RU-486 t r e a t e d rats. In m a r k e d c o n t r a s t insulin at i, i0 or i00 pU/ml i n c r e a s e d the r a t e of g l y c o g e n s y n t h e s i s by 175 • 24%, 68 • 15% and 36 • 11%, respectively. The increased responsiveness of glycogenesis to p h y s i o l o g i c a l c o n c e n t r a t i o n s of insulin was not c a u s e d by c h a n g e s in the c o n t e n t of m u s c l e glycogen. We p r o v i d e e v i d e n c e that a n t a g o n i s m of g l u o o c o r t i c o i d s action in v i v o a p p e a r s to h~aprocedure for a c u t e l y reversing insulin r e s i s t a n c e in skeletal m u s c l e in vitro.

ETOMOXIR PREVENTS THE INHIBITORY EFFECTS OF FFA ON INSULIN BINDING AND ACTION IN ISOLATED RAT HEPATOCYTES. J. Svedberg, P. L6nnroth, P. Bj6rntorp, U. Smith. The Wallenberg Laboratory and Department of Medicine, Sahlgrens Hospital, 413 45 GSteborg, Sweden. We recently demonstrated that FFA (free fatty acids) at high physiological concentrations dose-dependantly inhibit insulin binding, degradation and action in isolated rat hepatocytes through an energy-requiring process. To further elucidate the mechanism for this, male Sprague-Dawley rats (200 g) were treated with Etomoxir (50 mg/kg BW/b.i.d.)-a carn6 tine acyi transferase inhibitor, which blocks the fatty acid transport into the mitochondria, for 3 days before isolation of the hepatocytes. In hepatocytes from control animals, the binding of (125 I)-insulin and insulin stimulated uptake of (14 C)-AIB (aminoisobutyric acid), measured at 37o C after 45 min incubation, was decreased (30-50 %) in the presence of oleic acid (0.4 mM), whereas in cells from the Etomoxir-treated animals 0.4 mM oleic acid was ineffective. The doseresponse-curve for the inhibitory effect of oleic acid on insulin binding was shifted 2-3 fold to the right and the maximal inhibitory effect was reduced with 50 % in hepatocytes from Etomoxir-treated rats. Conclusion : Etomoxir prevents the inhibitory effect of FFA on insulin binding and action in isolated rat hepatocytes. Thus, this effect of FFA seems to be dependant on their mitochondrial oxidation.