Diabetologia

Diabetologia (1987) 30:492 A-600 A

9 Springer-Verlag 1987

23rd Annual Meeting of the European Association for the Study of Diabetes Leipzig, German Democratic Republic, 15-19 September 1987 Abstracts 1. The amperometric estimation of interstitial glucose concentration: how to interprete the electrode signal P.Abel, U. Fischer, E. Salzsieder and E. Brunstein. Central Institute of Diabetes "Gerhardt Katsch", Karlsburg, G D R Continuous glucose measurement for monitoring and control purposes needs a sensing technique which is applicable by the patient himself, e.g. by subcutaneous (SC) puncture. This study employs in dogs a miniaturised sensor with an in vitro response time of approximately 1 min and a linear calibration over 30 mmol/1; it measures the H202 from enzymatic glucose oxidation and needs no sample dilution. After in vitro calibration, the in vivo SC signal during steady states or smooth fluctuations is only _<80% of plasma glucose. However, kinetic modelling of interstitial glucose concentration on the basis of an established structural model of the glucose/insulin control system revealed the sensor signal to represent the interstitial glucose concentration with a physiological delay in relation to glycaemia. During steady states the two variables are identical or close to each other, which was also confirmed by an independent analytical reference. Thus, calibration of SC sensor signal according to glycaemia is necessary. On this basis the SC sensor signal appears suitable as an input for the artificial B cell.

2. Activation of cAMP-phosphodiesterase activity may be involved in the glycogenic effect of insulin but is not involved in its antikitogenic and antigluconeogenic effects. L. Agius and M. H. Chowdhury. Department of Medicine, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, U K Insulin suppresses the cAMP response to glucagon in liver by activation of cAMP-PDE. The importance of this mechanism in mediating the metabolic effects of insulin in liver is not clear. The aim of this study was to investigate whether inhibition of cAMP-PDE with isobutylmethyl xanthine (IBMX) influences the effects of insulin on carbohydrate and lipid metabolism. Rat parenchymal hepatocytes were cultured with insulin (0.1-100 nmol/1) in the absence or presence of concentrations of IBMX (0.1-1 mmol/1) which increase the cAMP response to glucagon and abolish the suppression by insulin. In the absence of insulin, 0.5 mmol/1-IBMX (like cAMP analogues) increased ketogenesis (471+26 vs 388+30) and gluconeogenesis (348 _+24 vs 256 _+26) but suppressed glycogen synthesis (0.37 + 0.10 vs 0.68 + 0.21, nmol/h per mg protein, mean_+ SEM, n = 6). Insulin (10 nmol/1) inhibited ketogenesis by 20% and 30% and gluconeogenesis by 33% and 50% in the absence and presence, respectively, of 0.5 mmol/1-IBMX, suggesting that activation of cAMP-PDE is not involved in the antiketogenic and antigluconeogenic action. Insulin increased glycogen synthesis in both the absence and the presence of IBMX (10- and 8-fold, respectively), however the maximum rate of glycogen synthesis was lower in the presence of IBMX (2.9 _+0.9 vs 6.4 + 1.7 nmol/h per mg protein. Activation of cAMP-PDE may potentiate the effect of insulin on glycogen synthesis but is unlikely to be the sole mechanism.

3. Extraction of adrenaline and noradrenaline by the dog pancreas in vivo B. Ahr6n, B.E. Dunning, P.J. Havel, R.C. Veith and G.J. Taborsky Jr. Departments of Surgery and Pharmacology, Lund University, Lund, Sweden, and VA Medical Center and University of Washington, Seattle, WA, USA A correct interpretation of studies on the effects of catecholamines on islet function requires more knowledge of pancreatic catecholamine metabolism. We determined the fractional extraction of adrenaline and noradrenaline by the in situ dog pancreas. The arteriovenous concentration difference of adrenaline across the pancreas in the basal state (arterial levels 380+ 123 pg/ml) and during low and high rates of intravenous adrenaline infusion (arterial levels 896 + 123 pg/ml and 2956 _+414 pg/ml) revealed a pancreatic adrenaline extraction of 73 _+5%, 76 +4% and 84+ 1% respectively. This indicates a high and non-saturable extraction process. When noradrenaline was infused intravenously at a high rate (arterial levels 107+28 ng/ml), the arterio-venous concentration difference revealed a pancreatic noradrenaline extraction of 65 _+7%. To calculate pancreatic noradrenaline extraction during the basal state, the arterio-venous concentration difference of intravenously infused 3H-noradrenaline was measured: the pancreatic extraction was 74 + 4%. We also studied whether pancreatic catecholamine extraction is altered by a- or fl-adrenoceptor antagonists: phenoxybenzamine thereby reduced the extraction of both adrenaline (p< 0.001) and noradrenaline (p<0.05), whereas propranolol did not. We conclude that 60-80% of adrenaline or noradrenaline is extracted in one pass by the dog pancreas by a non-saturable process that is impaired by phenoxybenzamine but not affected by propranolol.

4. Simplified simultaneous estimation of insulin sensitivity and glucose mediated glucose disposal in Type 1 (insulin-dependent) diabetic patients F. P. Alford, G. Ward, K. Weber, B. Lee, R. Boston and J. Best. Endocrine Unit and Department of Medicine, University of Melbourne, St.Vincent's Hospital, Fitzroy, Australia Impairment of glucose utilisation in Type 1 (insulin-dependent) diabetes may be due to reductions in insulin sensitivity (SI) and glucose-mediated glucose disposal (SG). Simultaneous estimates of SG and SI, and insulin secretion are obtained from the intravenous glucose tolerance test (IVGTI') "minimal model" of Bergman, but the model cannot be used in Type 1 diabetic patients. The pattern of insulin infusion required to simulate normal endogenous free insulin responses during an IVGTT was determined in 8 fasting normoglycaemic Type 1 diabetic 1 patients with insulin antibodies ( 2 - 4 m i n = 1 4 m U . k g - .min- ; 6-16 m i n = l m U - k g - l . m i n - 1 ; 17-50 min = 0.5 mU. kg -1. min-1). Bergman's model was modified, (eliminating the endogenous insulin loop and substituting the exogenous insulin infusion as separate input) and data fitted using simulation analysis and modelling program, SAAM. Plasma free insulin

Abstracts

493 A

levels were 45% above those of matched normal subjects (lst phase response 62 + 9; 2nd phase response 34 +_9 mU/1) with KG values in diabetic patients (n=8) of 1.3+0.3% min -1. Good resolution of model parameters was achieved (SI 2.5 _+0.6 x 10 -4 m i n - 1 / m U per 1; SG 1.6_+0.5x ]0 -2 min-1), and these were significantly (p<0.05) below those from normaly subjects (n=]7) (SI 8.3_+1.5x 10 -4 m i n - 1 / m U per 1; SG 2.6_+ 0.2 x 10 -2. min-1). In conclusion: (1) application of our modified Bergman model in Type I diabetic patients is practical for simultaneous measurement of SI and SG; (2) reduced SI and SG contribute to the insulin resistance of Type 1 diabetes.

betic group between the first and second trimester as it did in nondiabetic subjects. A good metabolic control in diabetic patients was attested by repeated determination of glycosylated haemoglobin: 5.0+-1.1%. In the 3 diabetic women with high but stable UAE (40, 80, 80 gg/ml) no abnormal parameter was found, the outcome of pregnancy was normal as in all others. In conclusion, UAE is not enhanced by pregnancy in normal or diabetic women. Elevated UAE at start has no pejorative predicting value. Lack of adequate intravascular volume expansion may be present in diabetic women but pregnancy in this group did not markedly impair the renal function.

5. Hyperglucagonaemia in diabetic rats results in increased hepatic amino-nitrogen conversion and loss of nitrogen from muscles T.Almdal and H.Vilstrup. Division of Hepatology, Rigshospitalet Copenhagen, Denmark In diabetes muscle nitrogen (N) is wasted, the hepatic amino-N conversion is accelerated, and the concentration of glucagon is more or less increased. This work studies the relation between these phenomenon in rats. Rats of 225 g were fed ad libitum. Diabetes was induced with streptozotocin 75 mg/kg intravenously. Controls received the vehicle. The rats were divided into 3 groups and treated for 4 weeks. Diabetes I (n=6): no insulin; Diabetes II (n=6): one daily injection of insulin dosed to glucosuria below 4 retool/24 h; Diabetes III (n= 5): Insulin as group II plus Zink Protamin Insulin 60 ~tg 3 times daily. The energy balance remained constant. After 4 weeks the fasting glucagon (ng/1) was, Controls: 50+8 (mean+SEM); Diabetes II 250+25; Diabetes II 60+8; Diabetes III: 600_+40. The capacity of urea-N (~tmol/min 100 g BW) determined during intravenous alanine loading was, Controls: 9 + ] ; Diabetes I: 22 + 2; Diabetes II: 8 _+] ; Diabetes III: 20 + 2. After dissection the N-content of the total muscle mass (mmol) was (% of controls in parentheses): Controls 356+7 (100%); Diabetes I: 235-+6 (66%); Diabetes II: 360+8 (102%); Diabetes III: 300+7 (84%). Thus the hepatic and muscular N wasting of untreated diabetes was abolished by insulin that normalised glucagon, but was recreated by exogenous glucagon despite insulin treatment.

8. A phospho-oligosaccharide mimicks insulin action in glycogen phosphorylase a and pyruvate kinase activities in isolated rat hepatocytes J. F. Alvarez~, M.A. Cabello 2, K. L. Kelly3, J. M. Mato 1 and J. E. Feliu 2. qnstituto de Investigaciones Biolrgicas del C.S.I.C., Fundaci6n Jimrnez Diaz. 2Clinica Puerta de Hierro, Universidad Aut6noma de Madrid, Madrid, Spain; 3University of Pennsylvania, Philadelphia, USA A novel mechanism that seems to mediate some of the actions of insulin has been described. It consists of the hydrolysis of a glycophospholipid precursor, by activation of a specific insulin-sensitive phospholipase C, which generates a water soluble phospho-oligosaccharide (POS). We have investigated the insulin-like effects of a preparation of POS on both glycogen phosphorylase a and pyruvate kinase activities. POS was obtained by phosphatidylinositol-specific phospholipase C (S. aureus) treatment of a glycophospholipid purified from rat liver membranes. Hepatocytes were isolated from livers of fed Wistar rats by collagenase digestion. As expected, glucagon (0.1 nmol/1) activated glycogen phosphorylase a (2.2_+ 0.8 vs 9.6 + 1.6 U / g of cells at 3 min; n=4, mean+ SEM) and inactivated pyruvate kinase (21.3+4.8 vs 9.9___2.3 U / g of cells at 3 min; n=4). POS mimicked insulin action antagonising either the stimulatory effect of glucagon on glycogen phosphorylase a (19.1 + 1.4% and 13.7 +_4% inhibition of glucagon-stimulated phosphorylase activity, p<0.01 and p=0.07 for insulin and POS respectively; n=4), as well as the inactivation of pyruvate kinase (43.7 + 6.7% and 26.1 + 3.2% inhibition of pyruvate kinase inactivation, p = 0.02 and p = 0.0] for insulin and POS respectively; n=3). These results support the hypothesis that POS is implicated in mediating insulin action at the cellular level.

6. Molecular cloning of a membrane antigen c-DNA from a pancreatic rat islet c-DNA library by using a BB/H rat monoclonal autoantibody M. Altieri, G. Contreas and W. H. Kastern. Hagedorn Research Laboratory, Gentofte, Denmark In order to study possible islet cell autoantigenes related to Type 1 (insulin-dependent) diabetes, we have produced several monoclonal autoantibodies from unimmunised prediabetic BB/H rats. Immunoblots of membrane preparations from N E D H rat tissues and 7 different rat transformed cell lines were probed with EA512, one of the monoclonal autoantibodies, of the IgM class. A protein band of (Mr=22,000) was detected in most of the tissues, except skeletal muscle, and in all the cell lines, while an additional higher band of Mr = ]00,000 was found only in 5 different insulinoma cell lines, but not in a fibroblastoid line and in a pituitary tumour line. A 2~gtll cDNA library from rat pancreatic islets was screened with EA512 autoantibody and 6 independent clones were isolated. The largest clone, a c-DNA 2000 bp long, hybridised to the other 5. A computerassisted analysis revealed a strong homology to mammalian Alu type repeated sequence, probably lying on the 3'-nontranslated sequence of the c-DNA, while a sequence thought to be in the coding frame did not show any homology to any known sequence. The results show the identification and molecular cloning of a new membrane antigen which is the target of a natural BB/H rat autoantibody. 7. Microalbuminuria in normal and diabetic pregnancy J.J.Altman, M.Boillot and C.Tchobroutsky. Department of Diabetology, Nephrology and Obstetrics, Centre Pasteur-Vallery-Radot, H6tel-Dieu and Port-Royal, Paris, France In normal pregnancy glomerular filtration rate increases to levels about 50% above non-pregnant: it may hasten the decline in renal function of diabetic women. Urinary albumin excretion (UAE) was determined by radioimmunoassay before, during (monthly) and after pregnancy in 21 diabetic women (mean__. SD, age = 30.6 + 5 years, duration of diabetes = 12 -+ 5 years) and 25 paired non-diabetic pregnant women. UAE was, and remained, normal in 18 diabetic women: 9.2_+4.9 ~tg/ml and 25 control subjects: 8.1 _+4.8 gg/ml. In both groups, blood pressure 114_+10/67_+6 and 107_+16/65+11mm Hg, serum creatinine 68 _+17 and 59 +- 12 lxmol/1, uricaemia 167 _+59 and 152+59 ~tmol/l, were in the normal range during the whole length of pregnancy and did not physiologically decrease in the dia-

9. Presence of group B-streptococci in diabetic patients G. Amisano 1, S. Caramello 1, D. Nespoli 2, A. Moiraghi-Ruggenini 1 and Q. Carta.2 1 Institute of Hygiene, University of Turin, and z Centro Antidiabetico, San Giovanni Hospital, Turin, Italy Group B-Streptococci (GBS) have been recognised and mentioned as significant agents in many human pathological conditions, particularly in perinatal diseases and in patients with underlying illnesses. In several studies diabetic patients appeared to be at increased risk for GBS infections inside peripheral gangrenous lesions and the urinary tract. However, the prevalence of GBS infections is different in several countries. From November 1985 to January 1987 we looked for GBS in urine and in peripheral gangrenous lesions of diabetic patients. Six hundred thirty-four samples of urine and 30 swabs from foot-lesions were randomly taken and examined. Urine of 634 normal subjects was also cultured. We have worked on Blood-agar containing 16 g/ml gentamycin and identified GBS by using CAMP-test and serological typing, while such an identification is not possible by means of the usual laboratory methods (Uroslides). 15.14% of urines and 6.6% of gangrenous lesions were GBS positive. Only 2% of the controls' urine was positive. The results demonstrate that diabetic patients have higher GBS carriage rates than non-diabetic subjects. Therefore, it is important to evaluate the factors which can cause these higher carriage rates in diabetic patients and to estimate how frequently carriage rates may cause infections. 10. A prozone phenomenon interferes in islet cell antibody detection: a study in subjects at high risk to develop diabetes E. Anastasi, U. Di Mario, F. Dotta, A. Rabizadeh, P.G. Colman and G. S. Eisenbarth. Department of Endocrinology, University of Rome, Italy; Joslin Diabetes Center, Boston, Mass, USA In the search for more reliable islet cell antibody (ICA) methods, different techniques have been compared in sera from 26 prospectively evaluated high risk patients, who subsequently either developed diabetes or showed an impaired insulin response, and in 12 normal control subjects. ICA have been detected by either fluorescein-labelled protein A (ICA-pA) or fluoresceinated anti-IgG (ICA-IgG) using the

494 A same substrate pancreas, fluorescence microscope and observers. End-point titres for ICA positive sera were ten-fold greater using the ICA-IgG than the ICA-pA assay. Despite greater end-point titres, the ICA-IgG assay failed to detect more "prediabetics" and showed a significant prozone. Fourteen subjects were positive at a 1 : 2 dilution using the ICA-pA assay. Only 10 of these 14 were positive at a 1:2 dilution using the ICA-IgG assay but all became positive at greater sera dilutions. A similar prozone was observed with anti-islet monoclonal antibodies A2B5 and 4F2. Thus in individuals developing Type 1 (insulin-dependent) diabetes, ICA-IgG did not detect more positive individuals than ICA-pA assay and, on the contrary, showed an interfering prozone effect. Both diluted and undiluted sera should be assayed in order to avoid false negative using either assay. 11, Different sensitivity of stimulatory receptor (Rs)- and inhibitory receptor (R.O-mediated changes in cAMP production to thioloxidants in rat islets of Langerhans M. I. Anazodo and H.P.T. Ammon. Department of Pharmacology, Institute of Pharmaceutical Sciences, University of Ttibingen, FRG In the adenylate cyclase system, the stimulatory and inhibitory guanine nucleotide-binding regulatory proteins (Ns and Ni) possess functional thiol groups. In collagenase isolated pancreatic islets, the effects of glucagon (5 p.g/ml), isoprenaline (1 ~mol/1), a ~-receptor agonist, and glucagon plus clonidine (10 ~tmol/1), an ~z2-receptor agonist, on the cAMP accumulation in the absence/presence of the thioloxidants diamide and N-ethylmaleimide was studied. Ten islets each were incubated for 2.5 min in 2ml Krebs-Ringer-HEPES (20 mmol/1)-albumine (2%)-solution pH 7.4, and assayed for cAMP after acetylation by means of radioimmunoassay. In the presence of glucose (5.6 mmol/1), glucagon significantly increased the cAMPlevel above the basal value from 81 +_7 to 108 + 9 fmol/10 islets (p< 0.05). Clonidine (10-7-10-4mol/1) had no effect on the basal cAMP-level, but inhibited glucagon-mediated effect from 108_+ 9 to 74+8fmol/10islets (p<0.05). Diamide and N-ethytmaleimide (10-6-10 -4 mol/1) affected neither the basal nor glucagon/isoprenaline-mediated effects, but abolished the inhibitory action of clonidine. It is suggested that this selective effect may be due to the differences in the sensitivity of the sulphydryl groups of N-proteins to thioloxidation, i.e. Ni being more sensitive than N~. 12. Activation of protein kinase C is probably not essential for the action of insulin in rat adipocytes P. H. Andersen, B. Richelsen and H. Juhl. Medical Department III, Arhus Amtssygehus, Denmark Recently it has been suggested that protein kinase C may play a role in the mechanism whereby insulin regulates glucose metabolism in rat adipocytes. To investigate this hypothesis, rat adipocyte protein kinase C was activated by the phorbol esther, phorbol 12-myristate 13-acetate (PMA). 2-deoxy-lnc-glucose transport, lipogenesis (from 14C-glucose), glucose oxidation (from 14C-glucose), and lipolysis (activated by adenosine deaminase) were assessed in the presence of insulin and PMA, separately and combined PMA had no effect on insulin binding. The maximal effect of insulin (12.5 ng/ml) and PMA (70 ng/ml) on 2-deoxyglucose transport were 234% and 140% of control respectively. Combined, the effect of insulin and PMA were additive (291% of control). Corresponding values for lipogenesis and glucose oxidation were 219%/203% (insulin alone), 150%/145% (PMA alone) and 294%/289% (insulin plus PMA) of control. The antilipolytic action of insulin in adipocytes treated by adenosine deaminase was unaffected by PMA. In contrast to insulin, PMA had a small stimulatory effect on lipolysis. Conclusions: 1. PMA had stimulatory effects on glucose transport and glucose metabolism in rat adipocytes. 2. The additive effect of PMA on the insulin stimulated 2-deoxyglucose transport, lipogenesis and glucose oxidation suggest that a direct activation of protein kinase C cannot explain the action of insulin in rat adipocytes. 3. PMA and insulin had opposite effects on lipolysis indicating that protein kinase C may not be involved in the antilipolytic action of insulin. 13. Effectiveness of recombinant human proinsulin in patients transferred from NPH insulins J. H. Anderson, C.T. Spradlin and J.A. Galloway. Lilly Laboratory for Clinical Research, Eli Lilly and Company, Indianapolis, Ind, USA To elucidate possible clinical benefits of human proinsulin (HPI), transfer studies were conducted in 130 patients maintained on hu-

Abstracts man insulin (HI) for seven months then randomised to HPI (64) or continued on NPH (66). An additional 129 patients on mixed beefpork insulin (MBP) were randomised to HPI (66) and animal NPH (63). In both studies the amount of HPI required for control of both Type 1 (insulin-dependent) and Type 2 (non-insulin-dependent) diabetic patients was significantly less than HI NPH or MBP NPH. Mean doses at last visit were: 30.8 U HPI vs 44.0 U HI NPH, p=0.0001; 28.9 U HPI vs 41.6 U MBP NPH, p<0.0001. Consistent with earlier pharmacodynamic observations, HPI appeared to be most effective in Type 2 diabetic patients. During the study glycohaemoglobins fell in the Type 2 diabetic patients from a baseline of 6.3% to an average of 5.7% in the HPI group but only from 5.9% to 5.8% in the HI NPH group, p<0.0359. Fasting glycaemia at last visit in the Type 2 patients on the MBP study were 8.77 mmol/1 HPI vs 10.54 mmol/1 MBP NPH, p = 0.047. HPI appears to exert greater hypoglycaemic effect per unit than NPH insulins, especially in Type 2 diabetic patients. 14. The kinase activity of the insulin receptor is modified during ageing A. Andres, J.A. Pulido, J. Satrustegui and J. M. Carrascosa. Departamento de Biologia Molecular, Centro de Biologia, Universidad Autonoma de Madrid, Spain It is generally believed that the insulin resistance of ageing is associated to a post-binding defect. It has been suggested that insulin stimulated receptor autophosphorylation activates the kinase activity of the receptor and serves to couple insulin binding to some or all of insulin's biological effects. We have studied the tyrosine kinase activity of the insulin receptor during ageing in order to establish whether a modification of this activity could be related to the well known insulin resistance of old age. We have measured the receptor kinase activity using insulin receptors from solubilised extracts of adipocyte plasma membranes from 3- and 24-month old rats. Kinetic studies did not show any differences in the Km for ATP in the two age groups. However, we have found a decrease in the Vmax of the insulin receptor autophosphorylation during ageing. A similar decrease in the phosphorylation of the synthetic polypeptide G l u 4 : T y r l , used as exogenous substrate, has also been observed. These results suggest that the tyrosine kinase activity of the insulin receptor is modified during ageing and could be involved in the insulin resistance of old age. 15. Insulin-induced hypoglycaemia increases platelet sensitivity to collagen and Na Arachidonate G.Anfossi, M.Trovati, F.Cavalot, P. Massucco, E. Mularoni and G.Emanuelli. Cattedra di Clinica Medica Generale III, Ospedale San Luigi Gonzaga, University of Turin, Italy Previous studies have shown that insulin-induced hypoglycaemia increases platelet sensitivity to ADP, platelet activating factor and thrombin. The present study aims at investigating whether a similar pheonomenon occurs for agents influencing platelet function by different mechanisms, as collagen and Na Arachidonate. Hypoglycaemia was induced by intravenous bolus of human regular insulin (3.8 U / m 2) in 8 healthy male volunteers, 26+_2 years, BMI 23+_2. Throughout the study we measured plasma glucose, insulin, counterregulatory hormones and platelet sensitivity to collagen and Na Arachidonate, determining the ED50 that is the dose of each agent necessary to induce a maximal aggregation of 50%. Data (mean_+ SEM) are expressed as % of basal values. Platelet response to both collagen and Na Arachidonate significantly increased at the hypoglycaemic nadir, as indicated by the reduction of ED50 values: collagen ED50 was 87_+2% (p<0.025), Na Arachidonate ED50 was 59 +_11% (p< 0.025). A return to the basal sensitivity to both aggregating agents was observed in the recovery phase. The temporal correlation in vivo and studies in vitro demonstrated that the adrenergic response to hypoglycaemia accounts for the increased sensitivity of platelets to collagen and Na Arachidonate, as previously shown for ADP, platelet activating factor and thrombin. 16. Receptors and biological effects of insulin and insulin-like growth factor I in cultured glomerular mesangial ceils H. J. Arnqvist, B.J. Ballermann and G.L. King. Research Division, Joslin Diabetes Center and Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass, USA Glomerular mesangial cells probably have an important role in the development of diabetic nephropathy. In this investigation, we studied receptors and biological effects of insulin and insulin-like growth factor I (IGF-I) in cultured mesangial cells. The cells were grown

Abstracts from glomeruli isolated from male Sprague-Dawley rats and cultured in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum. Confluent cells between passages 3 and 10 were used. Specific binding of 125I-IGF-I was 5.7% and of 125I-insulin 0.02% per 0.2 mg cell protein. 125I-IGF-I binding was inhibited 50% by 10 ng unlabelled IGF-I and 100 ng insulin-like growth factor II and was also inhibited by insulin 1000 ng/ml (-25%). Specific insulin binding was displaced to 50% by insulin 20 ng/ml. IGF-I receptor could be demonstrated in cross-linking experiments. IGF-I and insulin stimulated 3H-thymidine incorporation into D N A with halfmaximal effect obtained at about 10 mg for IGF-I and 100 ng for insulin. 14C-glucose accumulation was stimulated by insulin at low concentrations. In conclusion, kidney mesangial cells were found to have abundant IGF-I receptors and very little insulin receptors. Both IGF-I and insulin produced biological effects and may, therefore, be of importance in the development of diabetic nephropathy.

17. Further studies of ATP-modulation of K-channels in rat isolated pancreatic B cells F. M. Ashcroft and M. Kakei. University Laboratory of Physiology, Oxford, U K The initiation of glucose-stimulated insulin release involves the closing of ATP-sensitive K-channels in the B-cell membrane. The mechanism by which glucose metabolism leads to channel closure is controversial, however, since the channel's sensitivity to ATP in the intact cell is very much less than that found for the excised patch. We have investigated the relative ability of ATP4- and MgATP to inhibit ATP-sensitive K-channels using inside-out membrane patches excised from isolated rat B cells and standard patch-clamp methods. The relationship between channel activity and ATP 4- (in Mg-free solution) was well fitted with a Hill coefficient of 1 and a Ki of 1.5 l~mol/1 ATP4- (4 ~Lmo1/1total ATP). When plotted as a function of total ATP the addition of 2 retool/1 Mg 2+ shifted Ki from 4 I~mol/1 to 26 ~Lmo1/l. However, no significant shift was found when the dose-response curves were replotted as a function of ATP4-. These results suggest that ATP4 is a more potent channel inhibitor than MgATP and that the main effect of Mg 2§ is to reduce ATP 4-. 18. Male/female differences in the changes in activity of the hypothalamic pulse generator in Type I (insulin-dependent) diabetes C. M. Asplin, W.S. Evans, E. Christiansen, A.C.S. Faria and E. Parish. Department of Internal Medicine, University of Virginia, Charlottesville, Va, USA Type I (insulin-dependent) diabetes is associated with abnormalities of growth hormone (GH) secretion and menstrual dysfunction, which could both arise from changes in the putative hypothalamic pulse generator. Generator activity can be assessed by frequent pituitary target hormone measurements and rigorous objective pulse detection. Thus, GH and luteinizing hormone (LH) levels, measured every 10 rain for 24 h, 7 female (with menstrual dysfunction) diabetic patients with non-diabetic control subjects. Pulse characteristics were similar in non-diabetic males and females. Diabetic females, compared to control subjects, had lower LH pulse frequency (10 vs 14/24 h, p < 0.05) and wider pulses (79 vs 62 rain, p < 0.05) but similar inter-pulse-valley (5.4 vs 4.5 mIU/ml), peak amplitude (7.8 vs 7.4 mIU/ml), pulse areas and total LH areas. Male diabetic patients were similar to control subjects. Female diabetic patients had more frequent GH pulses (9.5 vs 5; p < 0.02) and greater inter-pulse-valleys (4.8 vs 1.6 ng/ml; p < 0.05) with similar pulse width and area, giving rise to greater total GH secretion (7570 vs 4397 ng/ml, p<0.01). Male diabetic patients also had greater total GH (7093 vs 3882 ng/ ml, p < 0.05) due to greater pulse width (131 vs 90 rain, p < 0.01) and area (1191 vs 45 n g . m l - l . m i n -1, p<0.05), with similar frequency and inter-pulse-valley. These data suggest male/female differences in the changes in the hypothalamic pulse generator seen in Type 1 diabetes. 19. Rapid short-term decline of cardiovascular autonomic function tests in patients with diabetic peripheral neuropathy J-Ph. Assal, C. Liniger, A. Pernet and J-F. Moody. Diabetes Treatment and Teaching Unit, Electromyography Unit, University Cantonal Hospital, Geneva, Switzerland The evolution of cardiovascular autonomic function was studied in 15 diabetic patients (median age 51, diabetes duration 22 years) with peripheral neuropathy and moderately controlled diabetes (HbA1 c: 9.4 + 1.6%, mean _+SD), each of whom was reassessed after a median

495 A of 10 months. Heart rate variability (HRV) during deep breathing fell from 14.1 to 10.2 bpm (p=0.005), Valsalva ratio (VR) from 1.62 to 1.45 (p=0.030), and 30/15 ratio from 1.10 to 1.05 (p=0.042). 53% of individual tests were normal, 17% borderline and 30% abnormal at the outset, compared with 31% normal, 22% borderline and 47% abnormal at the latest assessment. 69% of results worsened, 11% were unchanged and 19% improved. HbA1 c had not changed at the final assessment (9.2_+1.3%). Peroneal nerve motor conduction velocity (CV) and sural nerve sensory CV were determined at the same timepoints as autonomic function in 11 of these patients and did not change: motor CV 37.7+3.9m/s vs 37.5+_2.6m/s; sensory CV 42.7 + 4.6 vs 41.5 + 4.9 m/s. In this subgroup HRV, VR and 30/15 ratio declined from 15.2 to 10.8 bpm (p=0.016), 1.62 to 1.5 (p=0.030), and 1.14 to 1.07 (p=0.031) respectively. These results suggest that cardiovascular autonomic function may worsen rapidly in patients with peripheral neuropathy, independently of CV or change in glycaemic control.

20. Renal effects of nicardipine in hypertensive Type 2 (non-insulin-dependent) diabetic patients with nephropathy T. Baba, S.Murabayashi0 K.Aoyagi and K.Takebe. Third Department of Internal Medicine Hirosaki University School of Medicine, Hirosaki, Japan We studied the renal effects of nicardipine, a calcium antagonist, administrated in a 4-week oral dosage (60 to 120 mg/day) in 7 hypertensive Type 2 (non-insulin-dependent) diabetic patients with nephropathy (albuminuria 50-500 ug/min, serum ereatinine level 0.8-1.4 mg/dl). Glomerular filtration rate (GFR) and renal blood flow (RBF) were measured by means of thiosulfate and para-aminohippurate respectively. Nicardipine increased RBF (control phase vs drug phase: 752.8 + 40.2 vs 841.0 + 66.2 ml/min, p < 0.05) and decreased total renal vascular resistance (11251+625 vs 8445_+ 729dyne.s.cm 5, p<0.05) and albuminuria (172.4_+67.9 vs 119.2 _+71.3 pg/min, p < 0.05) with a significant reduction in systolic and diastolic blood pressures (166.0/99.6 +_3.5/2.2 mmHg) as compared to the control values (138.2/83.7 ___3.6/1.7 mmHg), while GFR remained statistically unchanged (93.7 + 4.8 vs 102.4_+ 8.7 ml/min, p < 0.1). Heart rate, plasma renin activity, plasma aldosterone concentration, plasma osmotic pressure and HbAlc did remain unaltered by nicardipine. The results indicate that nicardipine has favorable renal effects with a concomitant hypotensive action and that the agent can be useful for treatment of hypertension in Type 2 diabetic patients with nephropathy. 21. Decayed missing filled teeth (DMFr) status in diabetic patients M. Bali6, D. Plan~ak, I. Ciglar, M. Grani~ 2. 1Faculty of Stomatology, University of Zagreb, 2Vuk Vrhovac Institute of Diabetes, Zagreb School of Medicine, University of Zagreb, Zagreb, Yugoslavia Since the prevalence of caries in diabetic patients is still a controversial issue, the frequency of caries in a group of Type 1 (insulin-dependent) diabetic patients (n= 109), a group of Type 2 (non-insulindependent) diabetic patients (n=113) and a group of control subjects (n= 189) was investigated using the DMFT index according to WHO criteria. The relationship between diabetic complications (neuropathy and retinopathy), duration of diabetes mellitus, the level of diabetic control and the DMFT index was also studied. There was no difference in the prevalence of caries between the diabetic patients and the control group, although total DMFT index was higher in the diabetic patients due to a greater number of extracted teeth (7.1 vs 12.3). Also, there was no significant difference between Type 1 and Type 2 diabetic patients (DMFT=I7.02 vs 18.4). Differences due to the level of diabetic control or diabetic complications were not found either. As caries is more frequent in young people, the examinees were divided into two groups: under 34 and over 35. Although in diabetic patients under 34 the DMFT index was slightly above that of the control subjects of the same age (13.9 vs 12.1) the differences were not statistically significant. It has been concluded that there are no statistically significant differences between diabetic patients and control subjects, although the diabetic patients have a slightly higher DMFT index. 22. The Mr64000 human islet cell autoantigen in Type I (insulin-dependent) diabetes: evidence for charge heterogenicity and disulphide bridging of two chains S. Beekkeskov, M. Christie, R.V. Rajotte and G. Warnock. Hagedorn Research Laboratory, Gentofte, Denmark, and Surgical Medical Research Institute, University of Alberta, Edmonton, Canada

496 A Antibodies in sera from newly-diagnosed Type I (insulin-dependent) diabetic patients are directed to a human islet cell membrane protein of Mr64000. Antibodies to this protein can be detected up to several years before the clinical onset of Type 1 diabetes indicating that this protein is a primary target of an autoimmune agression to B cells. To study the structure of this protein we have analysed the purified Mr64000 membrane protein using two dimensional gelelectrophoretic analyses. Diagonal two dimensional gelelectrophoresis using nonreducing SDS-PAGE in the first dimension and reducing SDSPAGE in the second dimension show that this protein has a Mr of 128 000 under non-reducing conditions. Two dimensional gelelectrophoretic analyses using separation by isoelectric focusing in the first dimension and SDS-PAGE in the second dimension show a basic Mr64000 component of pI > 8 and an acidic component which displays extensive charge heterogenicity (pI 6.5-7.5). The results indicate that the human islet membrane autoantigen in Type I diabetes is a disulphide-linked complex of two chains of similar molecular weight but different charge. 23. Overnight metabolic regulation in Type I (insulin-dependent) diabetes: further support for bedtime injection M.Bahnsen, H. Sengelov, N. Hoost and C. KOhl. Hvidore Hospital, DK-2930 Klampenborg, Denmark The aim of our study was to evaluate the effect of postponing p.m. injection of human intermediate-acting insulin in 12 C-peptide negative Type 1 (insulin-dependent) diabetic patients selected at random from the out-patient clinic. The overnight metabolic profile was studied in the hospital while on the usual twice daily regime and compared with the effect of bedtime injection. On conventional treatment, the blood glucose nadir (mean 5.4+SEM 0.4 retool/l) was reached at 03.00 hours, whereas nadir after bedtime injection (7.2_+ 0.9 mmol/1) was at 05.00 hours. The blood glucose level from 03.00-05.00 hours was significantly lower 07< 0.05, Wilcoxon) on the conventional regime, but rose sharply hereafter to a fasting value of 11.6 + 0.9 mmol/l and 15.9 + 1.0 mmol/1 after breakfast. With bedtime injection both fasting (8.6 _ 1.2 mmol/1) and postprandial blood glucose (10.5_+1.3 retool/l) were significantly lower 07<0.05 and p < 0.005 respectively). Also, 3-OH-buturate concentrations were significantly lower from 05.00-10.00 hours after bedtime injection. Fasting free insulin concentrations were higher at 07.00 hours when using bedtime injection (100.8 + 19.1 vs 72.1 + 15.6 pmol/1, p < 0.05). In conclusion: Bedtime human intermediate-acting insulin improved morning glycaemia without increasing the risk of nocturnal hypoglycaemia. 24. Metformin-insulin synergism in the control of hepatic gluconeogenesis C. J. Bailey and N. Wollen. Department of Molecular Sciences, Aston University, Birmingham, U K Metformin (dimethylbiguanide) directly inhibits hepatic gluconeogenesis at concentrations above the therapeutic range (up to 5 x 10 .5 mol/1) and in the absence of insulin. Since metformin increases hepatocyte insulin receptor binding and enhances insulin action in other tissues, this study investigates the interaction of metforrain and insulin in the control of hepatic gluconeogenesis. Glucose production from collagenase-isolated hepatocytes of starved rats was determined during incubations with 10- 2 mol/1-1actate-10- 3 mol/1 pyruvate, 10- 2 mol/1-alanine, 10- 2 mol/1 glutamine, and 5 x 10- 3 tool/I-glycerol. In the absence of insulin, high concentrations (10- 3 and 10- 2 mol/1) of metformin reduced ( > 35 0V0) glucose production. Lower concentrations of metformin were not effective. However, a therapeutic concentration (10 .5 tool/l) of metformin acted synergistically with insulin (10 -s mol/1) to suppress gluconeogenesis from each substrate by an additional 10-14% compared with insulin alone. This was achieved without alteration of the ATP, NADH or NAD + contents of digitonin-separated cytosolic and mitochondrial-rich hepatocyte fractions. However, 10 .2 mol/1 metformin alone increased the cytosolic N A D H : N A D § ratio. Thus high concentrations of metformin may reduce gluconeogenesis via changes in redox state, but therapeutic concentrations of metformin potentiate the antigluconeogenic effect of insulin without altering either energy status or redox state. 25. Effects on prolonged sucrose intake on plasma lipids, lipoproteins, platelet functions and metabolic control in Type 1 (insulin-dependent) diabetic patients J. Bak, U. Gerdes, S. E. Husted, H. K. Nielsen and O. Pedersen. Medical Department I and III, Aarhus Amtssygehus, Denmark

Abstracts Reports on the effects of short-term use of sucrose on glycaemia suggest that intake of modest amounts of sucrose by diabetic patients may be acceptable. In an attempt to further evaluate the prolonged effects of sucrose we fed 8 Type I (insulin-dependent) diabetic patients (mean age 26 + 2 years, mean duration of diabetes 11 + 3 years, mean daily insulin dosage 55 units) isocaloric diets (diets A and B), each containing 50% carbohydrates, 32% fat and 18% protein. Diet B was identical to diet A except 50 g starch was replaced by 50 g sucrose. Sucrose was given as part of the meals. The diabetic patients were examined for 4 months using a randomised cross-over design. Intake of diet B caused no significant changes in the total plasma concentrations of cholesterol and triglycerides or lipids in VLDL, LDL, HDL2 and HDL3-fractions or plasma apolipoprotein A-I, A-II and B. Similarly, no significant differences between the diet groups could be shown in platelet aggregation, platelet 14C-serotonine release, plasma antithrombin III or serum thromboxane B2 concentrations. Neither did diet B cause any alterations in daily insulin requirement, HbAlc, fasting or postprandial plasma levels of insulin, glucose, FFA or ketone bodies. In conclusion: Prolonged, moderate sucrose consumption in Type 1 diabetic patients does not affect plasma lipids, lipoproteins, platelet functions or metabolic control. 26. Effect of tolbutamide, gliclazide and glipizide on the ventricular ectopic beats and fibrillation caused by coronary occlusion in rats G. Ballagi-Pordfiny, A. Krszeghy-Gesztesi, M.Z. Koltai and G. Pogfitsa. Research Department, National Institute of Cardiology, Budapest, Hungary Cardiovascular side effects of hypoglycaemic sulphonylureas have been studied by numerous authors following the U G D P study. It is well known that glibenclamide (a second generation sulphonylurea) differs from tolbutamide and chlorpropamide (first generation sulphonylureas) in influencing polyuria in diabetes insipidus. This phenomenon prompted us to investigate whether, similarly to this, there is any different effect between the first generation tolbutamide and gliclazide as well as second generation glipizide on the cardiac electrical activity of ischaemic rat heart. It was found that tolbutamide and gliclazide (0.01-1000.0 p~mol/kg) increased, while glipizide (0.01-10.0 lzmol/kg) decreased both the number of ventricular ectopic beats and duration of transitional ventricular fibrillation in a dose dependent manner during the investigation period (30 min after coronary artery ligation). According to this finding the dose-response curves of tolbutamide and glipizide as well as gliclazide and glipizide differed significantly. The hypoglycaemic effect of sulphonylureas was prevented by glucose administration (2.5 mmol/kg). Substitution of potassium did not prove to be necessary. The results suggest that the arrhythmogenic property of investigated first generation sulphonylureas and antiarrhythmic effect of second generation glipizide might be a group-specific property based on the chemical structure. It is concluded that the second generation sulphonylureas should be preferred in the therapy of Type 2 (non-insulin-dependent) diabetes mellitus that is accompanied by ischaemic heart disease. 27. Follow up of serum fructosamine levels during pregnancy in healthy and Type I (insulin-dependent) diabetic women E. Baranyi, G.Y. Csfik/my, A. Ferencz, G.Y. Tamfis jr. and J. Simon. Postgraduate Medical University and 1st Department of Medicine Semmelweis University, Budapest, Hungary Serum fructosamine (SeFA) may be an indicator for previous glycaemia. Recently our diabetic pregnancy care program has included SeFA measurements (fortnightly, La-Roche Test, normal nonpregnant value 2.52 + 0.26 mmol/1, n= 100). Sequential investigations have been made in 16 healthy pregnant women (Group I) and in diabetic (White B-F) pregnant women: 10 being cared for preconceptionally (Group lI) and another 10 from the first trimester (Group III). The mean values in Group I showed minimal decrease (1st trimester: 2.2+0.16 2nd: 2.2+0.28 3rd: 2.1_+0.14retool/I); in Group II a gradual decrease (1st trimester: 2.8 _-4-0.402rid: 2.7 _+0.45 3rd: 2.5__.0.36 mmol/1; 1st vs 3rd, p<0.001); and in Group I I I a pronounced fall by the 2nd trimester (1st: 3.2_+0.47 2nd: 2.6_+0.40 3rd: 2.4 + 0.34 mmol/1; 1st vs 2rid, p < 0.001). The values of healthy and diabetic pregnant women differed significantly 07< 0.001) in all periods but Group II vs III only in the first trimester 07<0.001). SeFA gives clinically useful information on maternal glycaemia. Although efforts at maximising metabolic control led to a continuous fall in SeFA of pregnant diabetic women, their values were higher than of healthy pregnant women, being lower than of nonpregnant control subjects. Preconceptional care is decisive for the metabolic state in the first trimester.

Abstracts 28. Intraplatelet serotonin in diabetes mellitus and peripheral vascular disease M. A. Barradas, D. S. Gill, V. A. Fonseca, D. P. Mikhailidis and P. Dandona. Metabolic Unit, Department of Chemical Pathology & Human Metabolism, Royal Free Hospital and School of Medicine, London, NW3 UK Diabetes mellitus is associated with an increased incidence of atherosclerosis and platelet hyperaggregability. Serotonin (5-HT) is a vasoconstrictor and proaggregator. We have determined (using fluorimetric methods) the platelet content of 5-HT in Type 1 (insulin-dependent) diabetes (n = 23, median age 50, range 23-72 years); Type 2 (non-insulin-dependent) diabetes (n=23, median age 66, range 40-86 years); peripheral vascular disease (PVD) without diabetes (n=29, median age 70, range 48-79 years), and PVD with diabetes (n=9, median age 68, range 48-74 years). We compared their 5-HT content with that found in healthy control subjects (n= 16, median age 72, range 56-80 years). The median and (range) 5-HT content (nmol/109platelets) was: 3.01 (1.26-6.34) in Type1 and 2.46 (1.74-5.83) in Type 2 diabetic patients; 2.42 (0.94-4.98) in PVD patients and 3.87 (2.8-6.0) in healthy control subjects. Type 1 diabetes, Type 2 diabetes and PVD platelets contained significantly less 5-HT compared with healthy control subjects of similar age (Type 1 diabetic patients vs control subjects, p<0.004; Type 2 diabetic patients vs control subjects, p<0.002; PVD vs control subjects, p<0.002). Platelets from PVD-diabetes patients had 5-HT content similar to that of PVD and Type 2 diabetic patients. Diminished 5-HT in platelets may reflect enhanced release by hyperactive platelets, which may contribute to the pathogenesis of atherosclerosis. 29. Some psychological factors which influence the position of diabetic children in society L. Barta, M.Anster and M.Barta. First Department of Paediatrics, Research and Training Group for Medical Psychology Semmelweis University Medical School and Hygienic and Epidemiologic Station of Budapest, Hungary The aim of the study was to clarify some factors which might be helpful for the social integration of diabetic children. Applying questionaires, projective tests and interviews we have studied the reactions of 10-14-year-old diabetic children and their parents to diabetes. The examined groups - compared with adequate controls included 50, 100 and 150 children. In comparison to controls, the relation of diabetic children to parents and siblings is significantly more often inadequate. Their opportunities for sports and exercise are limited. Good diabetic education exerts a decisive influence on treatment as well as on school achievement. Parents often judge the diabetic state more severely than children do. Children are extremely attached to their parents. Sickness consciousness is not in correlation with the quality of metabolic control. The Rorschach test has revealed intact personality in most children. Thus diabetes has not caused personality disturbances yet at this age, but symptoms that are precursors of the later formation of such disturbances are already present. This is why the psychological status of both parents and children must be considered in the early phase of diabetes care in order to prevent neurotic disturbances. 30. Insulin pump in the therapy of pancreas graft rejection V. Bartog, M. Dryfikovfi, I.Vanrk, F. Saudek and K.Vondra. Institute for Clinical and Experimental Medicine, Prague, Czechoslovakia In 18 Type 1 (insulin-dependent) diabetic patients segmental pancreas and kidney transplantations have been performed. In 12 patients endocrine pancreatic and in 14 renal function developed. During rejection episodes of the renal graft, probable pancreas graft rejection because of hyperglycaemia and a decrease in the serum Cpeptide level was diagnosed in four recipients. In three of them, antirejection therapy was supported by continuous subcutaneous insulin infusion (CSII). While original renal function reappeared after 6 to 10 days, pancreas graft function continued to be decreased. In two recipients insulin independence reappeared after 40 and 86 days of application of CSII. Normoglycaemia and high levels of serum Cpeptide are being maintained in both patients. In one recipient CSII continues but daily insulin decreased to one-third of the original dose. Rejection of the pancreatic graft may be reversible even after a long period of time and normoglycaemia without insulin therapy may be restored. It is speculated that B cell regeneration may be achieved with the help of long-term CSII application.

497 A 31. Rhinopathy - is this a specific complication of diabetes mellitus? H. Baumann and B. Schulz. Ears, Nose and Throat Clinic, ArndtUniversity of Greifswald, GDR

Functional and morphological changes of the nose are not well studied in Type 1 (insulin-dependent) diabetic patients. Therefore, we carried out rhinological and smell examinations, rhinomanometry, determination of nasal protein secretion, light and transmission electron microscopic examinations in 436 Type 1 diabetic patients. A disturbed function of the nose was found in 20%. Patients with a relatively short duration of diabetes exhibited nasal mucous membrane swelling (in 26%), dryness (in 47%) and crusts (29%). Fifty-seven percent of all diabetic patients demonstrated olfactory losses for mixed olfactory irritants. Nasal obstructions were observed in 43% and correlated to the duration of diabetes, quality of metabolic control and frequency as well as severity of vascular complications. There was an increased permeability of the nasal capillaries to proteins. Morphologically, basement membrane thickening, pericyte degeneration and endothelial cell swelling were found. We conclude that the high percentage of electronmicroscopic and functional changes of the nose in Type 1 diabetes justifies the term diabetic rhinopathy for this microangiopathy. 32. Stimulation of human skeletal muscle glycogen synthase and pyruvate dehydrogenase by insulin: regulation of oxidative and monoxidative glucose metabolism H. Beck-Nielsen, L.Verity, K. Wright, O. Kolterman and L. Mandarino. Hvidrre Hospital, Klampenborg, Denmark, Department of Medicine, University of California, San Diego and Department of Physical Education, San Diego State University, San Diego, California, USA

Glycogen synthase (GS) and pyruvate dehydrogenase (PDH) catalyse key steps for glucose flux into nonoxidative (NONOX) and oxidative (OX) pathways. Thus, activation of muscle GS and PDH by I may be regulatory for intracellular glucose metabolism. To test this, 8nondiabetic subjects (aged 26+2years; BMI, 25.5+1.6) were studied in the basal state and during euglycaemic hyperinsulinaemic clamp. Glucose disposal (Re) was determined isotopically; OX by indirect calorimetry; and N O N O X = Ra-OX. GS and PDH were assayed in vastus lateralis muscle biopsies. Mean serum I levels were 7+1, 22+2, 49_+3 and 631_+49 lxU/ml. Rd was 2.37+--0.11, 3.15+ 0.19, 6.71 +0.44 and 11.7+ 1.73 mg/kg-min, while OX was 1.96+ 0.18, 2.81 +-0.28, 4.43 _+0.32, and 4.85 _+0.46, respectively. The proportion of active GS was 0.174 _+0.20, 0.273 + 0.29, 0.347 +-0.016, and 0.545+0.031; that of active PDH was 0.575_+0.07, 0.654+0.101, 0.801 +0.068, and 0.852+-0.089 at the four I levels. PDH activation correlated with activation of OX (r= 0.68, p < 0.01); GS activation correlated with activation of NONOX (r=0.75, p<0.01). Conclusions: (1) Re is primarily due to OX at physiologic I but NONOX predominates at maximally effective I. (2)Activation of skeletal muscle GS correlates with overall NONOX and PDH activation correlates with overall OX. (3) Therefore, these enzymes may regulate intracellular I-stimulated glucose metabolism. 33. Increased hexokinase/glucose-6-phosphatase ratio in the diabetic kidney as index of glucose over-utilisation F. Belfiore, S. Iannello, A.M. Rabuazzo and R.Campione. Chair of Pathophysiology of Metabolism, Istituto Clinica Medica III, University of Catania, Ospedale Garibaldi, Catania, Italy

The ratio between hexokinase (HK, E.C. 2.7.1.1) and glucose6-phosphatase (G6Pase, E.C. 3. I. 3.9) was studied in the kidney cortex of streptozotocin-diabetic mice. This ratio reflects glucose uptake and release (thus indicating glucose utilisation) and is unaffected by the reference basis used to express enzyme activity. In 3-day diabetic mice, as compared to control mice, the HK/G6Pase ratio in the kidney was enhanced by 52% (mean + SEM: 0.40 + 0.04 versus 0.26 + 0.03, n=5, p<0.02), as result of a 25% increase of HK (16.68+ 0.93 n m o l . m i n - l - m g -1 protein versus 13.31+1.04, p=0.05) and a 17% decrease of G6Pase (42.51 +2.75 versus 51.25 +1.89, p<0.05). In contrast, as expected, in the liver the corresponding ratio, i.e. the ratio between HK+glucokinase (GK, E.C. 2.7.1.2) and G6Pase, was reduced by 87% (0.02 + 0.004 versus 0.18 + 0.05, p < 0.05), as result of a 65% decrease of GK (1.71 _+0.31 versus 4.85 + 0.65, p < 0.02) and a 155% increase of G6Pase (84.21 +7.22 versus 33.01 _+5.73, p < 0.01). In 9-day diabetic mice ( n = 7), the kidney enzyme changes were much smaller, in agreement with the gradual attenuation previously reported for other metabolic changes. The enhanced HK/G6Pase ratio in the diabetic kidney suggests an increase in glucose utilisation.

498 A This may contribute to the increased synthesis of glycogen, glycoproteins (including basement membrane) and RNA (via provision of ribose-phosphate) occurring in the diabetic kidney and supports the view that the kidney (as opposed to other tissues) shows an "anabolic response" to diabetes. 34. Assessment of postabsorptive and postprandial futile cycling and cycling through glycogen in Type 2 (uon-insulin-dependen0 diabetes mellitus P.M.BelP, R.G.Firth2 and tLA.Rizza2. 1Royal Victoria Hospital, Grosvenor Road, Belfast BT12 63A, Northern Ireland, and 2Endocrine Research Unit, Mayo Clinic, Rochester, Minnesota, 55905, USA Futile cycling and cycling through glycogen may both occur during the transition from the fasting to fed state. To quantify effects of these processes on diabetes mellitus, glucose turnover was measured simultaneously with (23H)glucose (23H) (an isotope that loses label during glucose/glucose-6P (G6P) futile cycling but does not cycle through glycogen) and (33H)glucose (33H) (an isotope that does not G6P futile cycle but does cycle through glycogen) before and after ingestion of an isotopically labeled ((6a4C)glucose) carbohydrate meal in Type 2 (non-insulin-dependent) diabetic patients (D) and nondiabetic (N) subjects. D had higher (p< 0.01) glucose concentrations before (9.8+0.7 vs 5.2_+0.1 mmol/1) and for 7 h after glucose ingestion (4489_+261 vs 2456_+67 min-mmol/1). (23H) provided higher (p< 0.05-0.005) estimates than did (33H) of meal glucose entry (3.4+0.3 vs 2.6_+0.2 mmol/kg.7 h) hepatic glucose release (3.8 _+0.3 vs 2.8 _+0.3 rnmol/kg. 7 h) and total glucose uptake (7.3 _+ 0.5 vs 5.4 _+0.3 mmol/kg. 7 h) in N as well as in D (3.3 _+0.3 vs 2.9 _+ 0.3, 4.4_+0.7 vs 3.5_+0.4 and 8.0_+0.9 vs 6.7+0.6 mmol/kg.7 h respectively). The difference between turnover with (23H) and (33H) was equal in N and D indicating comparable futile cycling and cycling through glycogen. Both isotopes demonstrated that fasting and postprandial hyperglycaemia in D was due to excessive hepatic glucose release. We conclude that futile cycling and cycling through glycogen are equal in nondiabetic subjects and Type 2 diabetic patients both in postabsorptive and postprandial states. Both (23H) and (33H) glucose indicate excessive hepatic glucose release in the cause of pOstprandial hyperglycaemia in Type 2 diabetes mellitus. 35. Dietary protein restriction delays the progression of diabetic renal failure J.J.Bending, R.S.Doods, J.D.Walker, G.C.Viberti and H.Keen. Unit for Metabolic Medicine, UMDS (Guys Campus), London, UK The long-term effect of dietary protein on the progression of diabetic nephropathy (DN) is unknown. We studied serially the effect of dietary protein restriction in 23 Type 1 (insulin-dependent)diabetic patients with DN [17 M/6 F; age 46 (29-67) years; duration of diabetes 27 (15-43) years; duration of clinical proteinuria 7 (2-16) years] during a 44 (13-89) month period on usual protein diet (UPD) and a mean 21 (12-39) months after a prescribed 40 g low protein diet (LPD). A fall in protein intake, mainly of animal origin, on LPD by 45% to 45 _+6 g/day (p< 0.0001) from 82 _+19 g/day on UPD was reflected by a fall in plasma urea concentration (UPD 10.3_+ 5.1 mmol/1 to LPD 8.5 _+5.3 mmol/1, p < 0.02) and 24 h urinary urea excretion (UPD 353 _+125 mmol/24 h to LPD 219 _+67 mmol/24 h, p<0.001). Mean body Weight fell from 72_+12 kg to 70_+12 kg (p< 0.025) but mid-arm muscle circumference remained unchanged on LPD. Urinary total protein excretion fell from 1.8 g/24 h (UPD) to 0.8 g/24 h (LPD) (p< 0.005) paralleled by a fall in albumin excretion rate in the majority of patients. Plasma albumin and glycaemia were unchanged. Arterial pressure was maintained by treatment. The mean rate of slCr EDTA glomerular filtration rate was significantly reduced from 0.7ml.min-l.month-1 (UPD) to 0.4ml.min -1. month-1 (LPD) (p < 0.01). Thus dietary protein restriction appears to delay the progressive decline in glomerular function in patients with diabetic nephropathy. 36. Abnormal disposal of an oral glucose load in Type 1 (insulin-dependent) diabetic patients J. J. Benn, S.J. Bozzard, D. Kelley, A. Mitrakou, T.T. A0ki, J. Sorensen, J.Gerich, M. Berger and P.H. Sonksen. UMDS, St. Thomas's Hospital, London; Rochester, MN; Pittsburgh, PA; Diisseldorf, FRG and Sacramento CA To determine the cause of persisting early postprandial hyperglycaemia in insulin treated diabetic patients we studied 12 normal volunteers (NV) and 10 non-obese Type 1 (insulin-dependent) diabetic

Abstracts patients (DM) following a 1 g/kg oral glucose load using a double isotope and forearm technique and indirect calorimetry. The diabetic patients were given 0.15 ~t/kg insulin subcutaneously 30 min before the glucose. Despite similar fasting concentrations (5.5 + 0.2 (DM) vs 5.4+0.1 (NV) mmol/l mean_+SEM) the diabetic patients had a higher peak arterial glucose (14.3+ 1.2 vs 10.0_+0.7 mmol/1, p< 0.01). The two groups had similar basal values of endogenous glucose. ]?roduction (129+09. _ . . (DM). vs 11.5+04._. (NV) ~tmol.kg-1 mm -1) but by 90 min the diabetic patients showed less suppression (to 6.2+1.2 (DM) vs 2.4+1.0 (NV) ~tmol.kg -1 .min -1, p<0.05) and at 120 min greater appearance rates of oral glucose (23.6_+2.3 (DM) vs 18.5 -+1.1 (NV) lxmol,kg -1. rain -1, p < 0.05). Total glucose disposal over 210 min was similar despite higher glucose concentrations in the diabetic patients (57.7+5.8 g (DM) vs 55.0+3.9 g (NV)) as was forearm uptake (61.7_+9.0 (DM) vs 78.0+ 8.5 (NV) mg/100 ml forearm) but glucose oxidation was considerably reduced (17.3+3.2 g (DM) vs 29.9+2.9 g (NV), p<0.01). The diabetic patients lost 3.5-+ 0.9 g glucose in their urine. We conclude that hyperglycaemia in insulin treated diabetic patients resulted from residual abnormalities of the magnitude and timecourse of glucose appearance and the routes of glucose disposal. 37. Metabolism of albumin and fibrogen in Type I (insulin-dependent) diabetes mellitus L. Bent-Hansen and T. Deckert. Steno Memorial Hospital, Gentofte, Denmark The metabolism of albumin and fibrinogen was studied in 25 longterm Type 1 (insulin-dependent) diabetic patients, using a double tracer technique. Group 1 (n=8) had normal albumin excretion (<30 mg/24 h), Group 2 (n=9) had persistent microalbuminuria (30-300 mg/24 h), and Group3 (n=8) had clinical nephropathy (>300 rag/24 h). Eight normal persons served as control subjects. Except for slightly lower distribution fractions of both proteins (intravascular mass/total mass), Group 1 did not differ from normal subjects. In Groups 2 and 3 the relative catabolism of albumin was increased, as denoted by an increase in the :fractional catabolic rate. Albumin synthesis was unaltered, resulting in lower plasma concentrations and lower intravascular and total body masses of albumin. Oppositely, fibrinogen synthesis was augmented, leading to an increase in plasma fibrinogen concentration and total fibrinogen body mass, in spite of increased catabolism. The fractional catabolic rates were unaltered. The study demonstrates that long-term diabetic patients with normal urinary albumin excretion had normal albumin and fibrinogen metabolism; but that grave alterations in plasma protein metabolism are present in patients with only slightly increased urinary albumin excretion. The association of increased fibrinogen turnover and microalbuminuria suggests a pathogenetic role of fibrinogen in the development of diabetic complications. 38. Evidence for progressive impairment of insulin processing in monocytes from obese and obese Type 2 (non-insulin-dependent) diabetic patients L. Benzi, A.M.Ciccarone, P.Cecchetti, V.Trischitta, G.Di Cianni, A. Masoni, R. Vigneri and R. Navalesi. Cattedra di Malattie del Ricambio, Pisa; Istituto di Fisiologia Clinica, del CNR, Pisa; Cattedra di Endocrinologia, Catania, Italy A14-125 I-Insulin (A14-I) binding, internalisationand degradation at 37 ~ were studied in circulating monocytes from 8 obese (O) and obese untreated Type 2 (non-insulin-dependent)diabetic (O-Type 2) patients and from 7 matched control subjects (N). Total cell associated radioactivity was lower in O-Type 2 in respect to N (2.87_+1.47% vs 4.98 + 1.8%, p < 0.05) but not in O (3.68 + 0.91%). The percentage of A14-I that was internalised, measured by an acid-washing procedure, was progressively reduced (ANOVA: p < 0.01) in O (54 + 10%) and in O-Type 2 (44+13%) as compared to N (60+12%). Internalised radioactivity was characterised by gel-permeation and reversedphase HPLC after monocytes solubilisation. Three classes of A14-I derivates were identified: a)radioactivity associated with high molecular weight material (MW 300.000), probably representing the insulin receptor complex internalised, b) intact A14-I, c) radioactivity corresponding to 12sI-tyrosine and to three intermediate degradative compounds. A progressive increase of intact A14-I was observed comparing the three groups (N 4 + 2%; O 19 + 8%; O-Type 2 32 + 10%; ANOVA, p<0.01). Conversely insulin degradation products (~2sI-tyrosineplus intermediate compounds) were significantly higher in N in respect to O and O-Type 2. These data show a progressive impairment of internalisation and degradation of insulin in monocytes from O and O-Type 2 and suggest that a worsening in insulin handling may parallel the insulin resistance of these patients.

Abstracts 39. Clonidine modifications of glucose effects on B cell cytoplasmic Ca z+ and insulin release

P.Bersten. Department of Medical Cell Biology, Biomedicum, Uppsala, Sweden Glucose has been found to have a dual effect on cytoplasmic Ca 2+ resulting in stimulation or inhibition of insulin release. It was tested how activation of aa-adrenoceptors affected the glucose action on the Ca 2+ balance by measuring its cytoplasmic concentration and insulin release using B-cell-rich pancreatic islets isolated from ob/obmice. Increasing the glucose concentration from 3 to 20 retool/1 resulted in elevated cytoplasmic Ca 2+ after an initial drop in cells loaded with the fluorescent indicator fura-2. In the presence of 1 Ixmol/l clonidine only a decrease was seen when raising the glucose concentration. After introductory culture in a cae+-deficient medium 6mmol/1 glucose inhibited insulin release whereas 20 retool/1 of the sugar was stimulatory. When 1 p,mol/1 clonidine was added also 20 mmol/l glucose inhibited insulin release, an effect reversed by simultaneous addition of the a2-adrenergic antagonist idazoxan. In the presence of 1 mmol/l theophylline glucose stimulated insulin release even in the presence of 6 mmol/1 of the sugar. The findings provide further support for a dual action of glucose on insulin release, suggesting that stimulation of cAMP formation by glucose is a factor usually obscuring its inhibition of insulin release. 40. Cross-correlation between heart rate and respiration versus deep breath: comparative evaluation of a new test of cardiac autonomic function in diabetes L. Bernardi, M, Rossi, F. Soffiantino, L. Ricordi, G. Marti, A. Giardullo, G. Finardi and R Fratino. Department of Internal Medicine, University of Pavia, Italy

Cross-correlation is a mathematical function using spectral analysis for describing the relationship between heart-rate fluctuations (256 RR intervals) and respiration (simultaneously obtained by pneumotachograph). To assess its usefulness for testing autonomic integrity, cross-correlation and deep-breath were compared in 53 diabetic patients (age 48 _+14 years) and in 53 age-matched control subjects. To characterise patients, Valsalva and 30:15 ratios, tilt and handgrip had been performed: 19 (Group-A) had 2 or more abnormal tests, 9 (Group-B) had 1, 25 had none (Group-C). Sensitivity to parasympathetic withdrawal was compared in 9 normal subjects (age 26 + 4 years) and 6 Type 1 (insulin-dependent) diabetic patients (age 30 + 16 years) by 4 subsequent 0.01 mg/kg intravenous atropine administrations. Reproducibility was compared in 11 normal subjects (age 25 +_2 years) by repeating the tests 4 times on 2 consecutive days. Cross-correlation and deep-breath were below 2 standard deviations of the mean of normal subjects, respectively, in 28 and 17 diabetic patients (Group-A: 19 and 13; Group-B: 3 and 1; Group-C: 6 and 3). Cross-correlation had better reproducibility than deep-breath (coefficient of variations: 10.3% vs 30.6%) and greater sensitivity to atropine: F-ratio/normals (repeated-measures analysis of variance) was 37.4 vs 17.8, p<0.001 ; F-ratio/diabetic patients was 19.8 vs 14.7, p < 0.001). These data support the use of cross-correlation to improve the diagnostic approach to cardiac autonomic damage.

499 A tive MSA in response to feeding may contribute to the association of over-feeding with hypertension. 42. Operation of Randle's cycle in Type 2 (non-insulin-dependent) diabetes S. Bevilacqua, G. Buzzigoli, M.A. Giorico, D. Del Chiaro, C. Boni, D. Ciociaro, R. Bonadonna, M. Oleggini, L.S. Brandi and E. Ferrannini. C. N. R. Institute of Clinical Physiology, and 2rid Medical Clinic, University of Pisa, Pisa, Italy

We tested the hypothesis that the glucose-FFA (Randle's) cycle is operative in Type 2 (non-insulin-dependent) diabetes. Ten nonobese, mildly hyperglycaemic (fasting plasma glucose=7.3_+ 0.5 mmol/1) diabetic patients received a 2 h, isoglycaemic insulin (40 mU/min, m 2) clamp during concomitant infusion of saline (control study, C) or Intralipid (3 m l / m i n ) + heparin (0.4 U/min-kg) (lipid study, L) on separate days. At comparable hyperinsulinaemaa (90+4 vs 94+7 mU/1), plasma FFA levels were suppressed in C (0.56 + 0.07 to 2.43 + 0.19 mmol/1, p < 0.001). Blood glycerol and flOH-butyrate, and serum triglyceride levels changed accordingly. The blood lactate/pyruvate ratio remained at baseline in C (10+ 1 to 12 + 1, p = NS), but rose significantly in L (10 + 1 to 15 _+2, p < 0.02). By continuous indirect calorimetry, lipid oxidation was inhibited in C (0.90 + 0.12 to 0.39 + 0.09 gmol/min, kg, p < 0.001), and stimulated in L (0.96 + 0.08 to 1.09 + 0.07 gmol/min- kg, p < 0.01). Glucose oxidation doubled in C (7.3 + 1.3 to 14.1 + 1.8 pomol/min- kg, p < 0.001), but failed to rise in L (7.2 + 0.8 to 7.9 _+1.3, p = NS). Energy expenditure showed no change in C (1.14+0.07 to 1.18_+0.08 kcal/min, p = NS), but was enhanced in L (1.21 + 0.08 to 1.33 + 0.09 kcal/min, p < 0.01). We conclude that, in Type 2 diabetes increased supply of fat substrates impairs glucose oxidation, i.e. Randle's cycle is acutely inducible. 43. Chronic implantation of glucose sensors in dogs E.Biermann, J.C. Armour, B.D. McKean, J.Y. Lucisano and D. A. Gough. Institut for Diabetesforschung, Mfinchen, FRG, Bioengineering Group, University of California, San Diego, Calif, USA An enzyme sensor for glucose monitoring can be fabricated in a miniaturised form of a catheter (Q 2 ram). It is based on the amprometric measurement of oxygen not being consumed in an enzyme reaction with glucose. With respect to the sensitivity range for glucose, the stability of the central oxygen sensor and the enzyme lifetime of several months in vitro, the sensor is suitable for implantation. Intravenous chronic implant studies were performed to get a preliminary assessment of the enzyme inactivation in vivo and to test the biocompatibility of the applied materials. The sensors were inserted into the jugular veins of mongrel dogs and an implanted telemetry system was used for signal transmission of glucose- and oxygen sensor currents. The sensor output followed glucose excursions closely, initiated by intravenous glucose injections. Oxygen tensions were quite stable at about 40 mmHg. Enzyme inactivation in vivo and in vitro was comparable. Because of mechanical lead failure two experiments were terminated by explanation after 2 and 6 weeks respectively. These preliminary results indicate that longer operation of sensors in vivo may be feasible.

41. Muscle nerve sympathetic outflow increases in response to oral glucose C. Berne and J. Fagius. Departments of Internal Medicine, Neurology and Clinical Neurophysiology, University Hospital, Uppsala, Sweden

44. Specificity and sensitivity of an anti-B cell cell-mediated cytotoxicity assay in Type I (insulin-dependent) diabetes B. Bierwolf, J. Krug, E.F. Lampeter and D. Lohmann. City Hospital of Leipzig, G D R

Carbohydrate intake is associated with increased plasma norepinephrine levels, suggesting an enhanced sympathetic nervous system activity. In order to assess directly changes in nerve function the sympathetic discharge in extremity muscle nerves was recorded by microneurography in nine healthy volunteers. The baroreceptor-mediated muscle nerve sympathetic activity (MSA) was measured by a 5 gm tungsten electrode in the fight peroneal nerve below the knee at rest and for up to 90 rain after a 100 g oral glucose load. Blood samples for analysis of glucose and insulin were taken every 15 min. A significant increase of MSA from a basal level of 21+ 1 bursts/min at rest to a peak of 35 + 5 bursts/min at 30 min (p< 0.05) was flollowed by a gradual return to 27+2bursts/min at 90 min. In contrast, the sympathetic activity of the skin composed of sudomotor and vasoconstrictor components, recorded in three subjects, displayed no change after glucose. An association between the initial insulin response and the MSA increase was observed. The present study has identified one major source of the norepinephrine increase after oral glucose. The increase of the mainly vasoconstric-

Despite a number of in vitro observations and investigations on the role of cell-mediated immune processes in the pathogenesis of Type 1 (insulin-dependent) diabetes, their significance is still in question. The main problem is the lack of a specific evaluation of the Bcell damage by cell-mediated reactions. In our method isolated neonatal rat islets were incubated with peripheral blood mononuclear cells (PBMC) from 61 newly diagnosed Type 1 diabetic patients and 37 healthy subjects for 20 h. The difference between unstimulated insulin release after incubation with PBMC and the release after incubation in the buffer medium alone was defined as "cytotoxic effect". This cytotoxic reaction was significantly increased by incubation with PBMC from diabetic patients in comparison to PBMC from healthy subjects (11.2 + 0.9 vs 1.9 _+0.5 ng insulin/islet, mean _+SEM, p < 0.005). The specificity and the sensitivity of this cell mediated cytotoxicity assay was 86.5% and 86.9% respectively. In a group of patients the cytotoxicity was controlled up to one year and remained unchanged. The results indicate that cell-mediated cytotoxicity against B cells with this assay can be determined and cell-mediated

500 A cytotoxicity is present in Type I diabetes at a high frequency of manifestation (53 out of 61 patients) and persists constantly for several months. 45. Abstract withdrawn 46. The development of renal impairment in diabetic nephropathy is dependent upon individual mean glomerular volume R.W.Bilous, M.W. Steffes, F.C.Goetz, D.E.R. Sutherland and S. M. Mauer. Departments of Laboratory Medicine and Pathology, Medicine, Surgery, and Pediatrics; University of Minnesota, Minneapolis, Minnesota USA Available data on structural and functional relationships in diabetic nephropathy have been obtained from patients with differing durations of diabetes. In order to investigate the potential role of glomerular structural factors in the development and progression of nephropathy we studied kidney biopsies from normoalbuminuric (NA) (albuminuria <20 mg/24 h) and proteinuric (P) (albuminuria > 250 mg/24 h) Type i (insulin-dependent) diabetic patients with different durations of diabetes: 14-16years (Group1, n=15) and 24-26 years (Group 2, n=15). There were no significant differences in the age of onset of diabetes and glycosylated haemoglobin levels between NA and P patients within or between Groups 1 and 2. Creatinine clearances were lower in P than in NA patients, but similar in NA and P patients comparing Groups 1 and 2 (.mean + SD, Group 1:113-+12 (NA) vs 75_+27 (P) ml/min/1.73 m~; Group 2: 100 +_19 (NA) vs 75 + 25 (P) ml/min/1.73 m2). P patients in Group 2 compared to Group I had larger mean glomerular volume (2.13 + 0.64 vs 1.41-+0.45 • 10 6 ~tm3, p<0.01) and mesangial volume per glomerulus (0.77+0.22 vs 0.44+0.18 x 106 ~m 3, p<0.01) whereas NA patients in Groups 1 and 2 showed no significant differences in these measurements. Thus, a large glomerular volume is associated with a delayed expression of clinical nephropathy in patients with established mesangial expansion. We propose that, in addition to diabetes duration and glycaemic control, glomerular volume is a modulator of the natural history of diabetic nephropathy. 47. T-lymphocyte activation markers in newly diagnosed Type I (insulin-dependent) diabetic patients and in islet cell antibody-positive nondiabetic siblings J. Binimelis, J. Oriola, M.Codina, B.Arill, F.Carretero, A.Perez, J.Barbosa and A.de Leiva. Servei d'Endocrinologia i Nutricio. Unitat de Recerca Biomedica, Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Spain and Department of Medicine, University of Minnesota, Minneapolis, Minn, USA Some reports have shown an excess of activated T-lymphocytes in early Type 1 (insulin-dependent) diabetic patients, and in first degree relatives, but the prevalence and significance of that alteration still remain unclear. T-lymphocyte activation markers have been studied in 17 Type 1 diabetic patients (symptoms for less than 8 weeks), 5 islet cell antibody-positive non-diabetic siblings (NDS) - 3 with decreased first phase insulin secretion - and 12 healthy control subjects. Using fluorescent monoclonal antibodies and two-color flow cytometry (EPICS-V), peripheral blood mononuclear cells were studied for Interleukin-2 receptor (TAC) expression and T-lymphocyte HLA-DR expression (dual staining with anti CD3/anti HLADR). An increased percentage of HLA-DR positive T-lymphocytes (8.6-31%), more than 2 SD over the mean of control subjects, was observed in 7 Type 1 diabetic patients. Another patient expressed TAC in 18% of T-cells. No increased co-expression of both markers was observed in any patient. None of the NDS showed increased percentages of activated T-lymphocytes. Conclusions: 1) 47% of the early diabetic patients studied showed an excess of activated T-lymphocytes. 2) There is a discrepancy between HLA-DR and TAC expression in early Type 1 diabetes. 3) T-lymphocyte activation is not a sensitive marker of the ongoing autoimmune process in NDS. 48. Effects of insulin plus amino acid infusion on leucine and alphaketo-isocaproate kinetics in Type I (insulin-dependent) diabetes G. Biolo, S. Inchiostro, D. Bruttomesso, E. Iori, A. Valerio, A. Tiengo, G. Crepaldi and P. Tessari. Malattie del Ricambio and Patologia Medica 1, University of Padova, Italy We have previously reported that Type I (insulin-dependent) diabetes is resistant to the suppressive effect of hyperinsulinaernia on endogenous proteolysis. To determine whether this insulin resistance is present also with combined hyperinsulinaemia and hyperaminoacidoemia, six Type 1 diabetic patients, maintained in euglycaemia, and

Abstracts five control subjects were studied before and following an identical infusion of insulin plus amino acids, which raised plasma free-insulin to ~90 mU/1, and plasma amino acids by 50-150%. Basal freeinsulin was greater (p<0.05) in the euglycaemic diabetic patients (15+2 mU/1) than in control subjects (9+1 mU/1), while steadystate plasma leucine (120-130 p~mol/1) and alpha-keto-isocaproate (KIC) concentrations (35-40 gmol/1), as well as leucine, KIC and leucine + KIC rates of appearance (Ra) were comparable in the diabetic patients (1.78 +_0.07, 0.96 _+0.08 and 2.74 + 0.10 p.mob kg -1min -1 respectively) and in control subjects (1.64+0.09, 0.75 +0.05 and 2.39+0.13~tmol.kg-l.min -1 respectively). Following the combined infusion, leucine (--,210-220 p~mol/1) and KIC (--*35-40 ~tmol/1) were similar in both groups. Leucine and KIC Ra increased in both groups. Leucine + KIC Ra was greater (p< 0.02) in the diabetic patients (4.00_+0.16 vs 3.34 + 0.16 lzmol- kg -1. min-1). After subtraction of exogenous leucine infusion, endogenous leucine Ra (1.20_+0.07 l.tmol.kg-l.min -1) and leucine+KIC Ra (2.40_+ 0.11 ~tmol-kg-l-min-1) in the diabetic patients were greater (p< 0.005) than in control subjects (0.69+0.10 and 1.60_+0.13 ~tmol. kg-l.min-1). Thus, increased whole-body leucine+KIC Ra in Type I diabetes, following the combined infusion, was entirely due to defective suppression of endogenous proteolysis, confirming the presence of insulin-resitance with respect to amino acid metabolism. 49. Is cortical bone mass normal in diabetic patients? J.Birtwell, C.Williams, B.Carey, J.Wentworth, P.G.Wiles and J.K.Wales. University Department of Medicine and Radiology, General Infirmary, Leeds, U K Osteopoenia has been said to occur more frequently in diabetic patients. We have therefore compared foot radiographs from the first 195 diabetic patients (median age 65, range 19-87; 99 male, 96 female) attending a screening clinic with radiographs from control subjects matched for left or right foot, age and sex. Cortical bone mass, estimated as the metatarsal index, decreased significantly with age in both diabetic (rs-0.2335, p<0.001) and control (rs-0.2836, p<0.0001) groups, primarily influenced by the results for the females in both groups, but there was no difference between the diabetic patients and control subjects. Medial vascular calcification, periosteal reactions and joint subluxation occurred significantly more frequently among the diabetic patients (p< 0.001). Within the diabetic group, age was highly significant associated with the presence of atheromatous vascular calcification at the ankle (p< 0.0001) but not with the other abnormalities. Duration of diabetes associated significantly (p< 0.01) with medial vascular calcification in the foot but not with joint subluxation, periosteal reactions or atheroma. Independent of fractures or infection (2 patients), there was an association between the presence of periosteal reaction and joint subluxation (p< 0.05). We conclude that, in this group of patients, there was no evidence of increased osteopoenia related to diabetes. 50. Insulin secretory responses of islets of Langerhans following depletion of protein kinase C activity T. Bjaaland, C. S. T. Hii, P. M. Jones and S. L. Howell. Department of Physiology, King's College London, London, UK Prolonged pretreatment with tumour-promoting phorbol esters reduces protein kinase C (PKC) activity in a number of tissues. We have evaluated whether a similar depletion occurs in islets of Langerhans as a way of investigating the role of PKC in the regulation of insulin secretion. Collagenase isolated rat islets were cultured for 24 h in the presence of phorbol 12-myristate 13-acetate (PMA, 200 nmol/l) or the inactive analogue 4a-phorbol 12, 13 didecanoate (PDD 200 nmol/1). Ca2+/phospholipid dependent protein kinase activity in islet homogenates was estimated by measuring 32p incorporation from (~ZP)ATP into histone type IIIs in the presence of phosphatidyl serine, Ca 2 + and PMA. In PDD pretreated islets this produced a stimulation of 86+17% over control (n=5, p<0.05) whereas in PMA pretreated islets no increase in activity was observed (7 • 10%, n = 5, NS). Secretory responses in the pretreated islets were compared during a subsequent 1 h incubation in vitro. In the presence of 5 mmol/1 glucose, PMA pretreated islets did not respond to subsequent exposure to PMA (control 0.58_+0.07; + 10 -6 mol/l PMA, 0.58 _+0.06 ng/islet per h; mean -+ SEM, n = 16) whereas PDD pretreated islets showed a significant increase in insulin secretion (control 0.48 + 0.06; + 10-~ PMA, 1.49 + 0.1, n = 8 , p<0.01). In contrast both PMA and PDD pretreated islets showed a significant secretory response to 20 mmol/l glucose (PMA pretreated, 5 mmol/1 glucose 0.58 • 20 mmol/1 glucose 1.04+ 0.09, n = 16, p < 0.05; PDD pretreated, 5 mmol/1 glucose, 0.48 • 0.06;

Abstracts 20mmol/1 glucose 0.74+0.07; n=18, p<0.05). Thus prolonged treatment with PMA reduces CaZ+/phospholipid dependent protein kinase activity in islets, and PKC may not be essential for insulin secretory response to glucose. 51. First administration of ETOMOXIR to Type 2 (non-insulin-dependent) diabetic patients H. Bliesath, E. Haupt, R. Ltihmann, F.U. Hoppe and H.W. Radtke. Byk Gulden Research Centre, Constance and Saale Klinik der BFA Bad Kissingen, W.-Germany

ETOMOXIR-(2-6-(chlorophenoxyl)hexyl)-oxirane-2-carbonic acid ethylester) is an inhibitor of the mitochondrial carnitine-palmitoyltransferase I (CPT I-EC 2.3.1.21-). It reduces the transport of long chain fatty acids across the inner mitochondrial membrane and consequently, B-oxidation, thereby decreasing ketogenesis, gluconeogenesis and blood glucose levels. In an open pilot study according to the declaration of Helsinki, this potential new antidiabetic drug was first administered to 6 Type 2 (non-insulin-dependent) diabetic patients, age 56.5 -+2.1 years with oral single dosages of 150 mg. The patients were admitted to the hospital and after giving informed consent they received a constant weight maintaining diet for one week. On day 4 and day 7 serum glucose was measured at 07.00 hours (fasting value); the patients had breakfast at 08.00 hours and serum glucose was determined again at 09.00 hours (post prandial value). On day 7 150 mg ETOMOXIR was administered at 07.55 hours. Fasting serum glucose concentrations were comparable (NS, p<0.05) on day 4:7.06+0.68 mol/1 and on day 7:7.45+0.68 mol/l (mean-+ SEM). The postprandial serum glucose levels were clearly higher on day 4:10.51 -+ 0.82 mol/l, while after ETOMOXIR treatment on day 7 serum glucose levels reached only 8.25 -+ 0.80 retool/1. The difference was statistically significant (p<0.05). It was concluded that ETOMOXIR caused an increase in insulin sensitivity and consequently a higher glucose metabolisation. 52. The Swedish childhood diabetes registry - familial disease pattern L.Blom, G.Dahlquist, L. Nystr6m, A.Sandstr6m and S.Wall. Department of Pediatrics, Karolinska Institute, Sachs' Children's Hospital, Stockholm and Department of Epidemiology and Health Care Research, Ume5, Sweden

From 1 July 1977 to 1 July 1986, 3503 incident Type 1 (insulin-dependent) diabetes cases were registered. From this register and from an incident case-referent study we have studied the familial occurrence of Type 1 diabetes, Type 2 (non-insulin-dependent) diabetes, allergy, rheumatism, thyroid, adrenal and coeliac diseases. The mean annual incidence during the 9 year period was 25A/100,000 for boys and 23.5/100,000 for girls. The cumulative incidence per 1000 at 5 years was 0.8, at 10 years 2.1 and at 15 years 3.6. For 8.5% of index cases Type 1 diabetes was present among parents (9.1% and 8.2%, respectively, for boys and girls). Seventy-three percent of parental cases were fathers. The younger the age at onset of index cases the higher was the frequency of parental Type 1 diabetes. There was no difference in season at onset for cases with a parent with Type 1 diabetes. Eighty-four percent of the index had siblings. Of the cases 4.5% had at least one sibling with Type 1 diabetes. In the case-referent study the odds ratio for Type 1 diabetes in first-degree relatives was 7.8. A highly significant odds ratio was found also for Type 2 diabetes among all relatives (3.3). Odds ratios for all relatives were also significant for thyroid disease, rheumatism and allergy but not for coeliac or adrenal disease. The main findings are: 1) an association between parental Type 1 diabetes and age at onset of the index case 2)high odds ratios also for Type 2 diabetes, thyroid disease, rheumatism and allergy among relatives to Type 1 diabetic patients. 53. Changes of insulin action on free fatty acid and glucose fluxes in human aging R. C. Bonadonna 1, L.C. Groop 2, D.C. Simonson 3 and R.A. Defronzo 3. lIstituto di Clinica Medica, Facolta' die Medicina, Genova, Italy; 24th Department of Medicine, School of Medicine, Helsinki, Finland; 3Yale School of Medicine, New Haven, Connect, USA

Alterations of glucose metabolism in the elderly might be mediated through increased outflow of free fatty acids from an enlarged fat mass. To test this hypothesis, we studied 7 young (Y) (21 -+ 1 years; 68 -+ 3 kg) and 7 older (O) (71 -+ 2 years; 70 _+4 kg) healthy subjects using the euglycaemic insulin clamp technique (+10, +25, +50, 3 +100, +250 IxU/ml), with indirect calorimetry, 3-H-glucose and 1-14C-palmitate turnover. Lean body mass measured by 3H20 was 47+3 vs 54-+3 kg, while fat mass was 23 -+3 vs 14+2 kg (p< 0.05) in

501 A O vs Y. Basal free fatty acid concentration (897+106 vs 412_+ 50 umol/1), turnover (530 _+64 vs 273 + 40 p.mol/min) and oxidation (161+31 vs 73-+12 p.mol/min) were all increased in O vs Y (p< 0.05) and failed to suppress to the same degree as in Y at each insulin concentration (all p < 0.05). Glucose turnover was reduced in O vs Y at each insulin infusion (689 vs 756, 872 vs 967, 1639 vs 1928, 2372 vs 3017, 32728 vs 4222 p.mol/min), as well as basal and insulin stimulated glucose oxidation, but oxidation failed to normalise when corrected for lean body mass. In conclusion, in the elderly: 1) Increased fat mass is associated with high plasma free fatty acid concentration, turnover and oxidation; 2) Glucose oxidation in lean tissues is impaired; 3) Substrate competition between free fatty acids and glucose may account for the reduction in glucose oxidation. 54. In vitro modulation of M H C antigen expression on islets from BB/E rats: A role for cytokines? A. J. Bone 1, R. Walker I, A. Cooke 2, B.M. Dean 2, P. van der Meide 3 and J.D. Baird 1. 1Metabolic Unit, Western General Hospital, Edinburgh, UK, 2Dept. of Immunology, Middlesex Hos. Med. Sch., London, UK, 3Primate Centre, TNO, Rijswick, The Netherlands

Possible cytokine involvement leading to autoimmune destruction on pancreatic B cells was suggested by recombinant rat interferon-y (r. IFNy) induction of MHC class II antigen e expression on islet B cells isolated from adult diabetes-prone (DP) BB/E rats. Induction was never observed on glucagon and somatostatin producing cells or on B cells from normal Wistar or low diabetes incidence subline BB/E rats. B cells from 30-day old DP rats also failed to show r. IFN?'-induced class II expression. Class I expression however, was enhanced by r. IFN~/on all cell types from all groups of animals. Tumour Necrosis Factor (TNF), a cytokine produced by activated macrophages, has been implicated in the pathogenesis of human Type 1 (insulin-dependent) diabetes. We have therefore assessed the effects of recombinant human TNF (r. Hu.TNF) in vitro, both with and without r. IFN~/, on MHC antigen expression on islet cells from norreal Wistar rats. r. Hu. TNF, in concentrations above 50 U/ml, had marked cytotoxic effects on isolated islets in culture. These effects were both time and dose dependent. Class I expression was enhanced on all islet cell types by r.Hu.TNF (50 U/ml); addition of r. IFN?" augmented this effect. We failed to induce class II expression on any islet cell types with r.Hu.TNF (10-105 U/ml) or r. Hu. TNF plus r. IFN ?"over a 3-10 day culture period. Thus the precise role of TNF in the pathogenesis of Type 1 diabetes still remains unclear. 55. Poststimulatory activity in oligo A synthetase after immunisation with tetanus or yellow fever V. Bonnevie-Nielsen, T. Kristensen, N.I. Lytken and A. Lernmark. Odense University Hospital, Dpt. of Clinical Chemistry and Immunology and Hagedorn Research Laboratory, Denmark

An increasing amount of evidence demonstrates the importance of interferon (IFN) in immune regulatory processes. Thus, IFN exerts an antiproliferative effect on viruses and different cell types, and will stimulate B-lymphocytes to produce immunoglobulins. Furthermore, IFN induces presentation of type II antigens in activated T-helper cells, which will lead to an activation of monocytes, macrophages and killer cells. Interferon also influences induction of many genes among which is (2'-5') oligo A synthetase, which generates adenine nucleotides from ATE This enzyme is increased in mononuclear cells during viral infections and forms the basis for analysing activity in the IFN-system. In order to elucidate the reactivity in the IFNsystem during controlled stimulation healthy individuals were immunised with tetanus or yellow fever vaccine. On day 8 after immunisation an increase of more than 1000% in oligo A synthetase activity (in units per mg protein) was seen after yellow fever in contrast to a minor decrease after tetanus toxoid. Immunisation of 12 patients with Type 1 (insulin-dependent) diabetes showed a significantly (p< 0.05) lower increase in oligo A synthetase activity on day 8 as compared to control subjects. In conclusion, the immunisation described is capable of detecting differences in immune reactivity, when Type 1 (insulin-dependent) patients are compared with healthy individuals. 56. Estimates of insulin action in healthy subjects with insulin tolerance test and glucose clamp E. Bonora, P. Moghetti, M. Cigolini, C. Zancanaro, M. Querena and M. Muggeo. Chair of Metabolic Diseases and Institute of Clinical Medicine, University of Verona, Italy

502 A In the present study estimates of insulin action derived from insulin tolerance tests (Iqq?) and euglycaemic or hyperglycaemic clamp were correlated in 16 healthy subjects. Eight underwent an ITT and a euglycaemic clamp, and 8 underwent an I T r and a hyperglycaemic clamp. ITT consisted of a bolus intravenous injection of regular insulin (0.1 U/kg BW). Glucose disappearance rate from plasma (Kitt) in the 3-15 rain period following insulin injection was taken as a measure of insulin action. In both euglycaemic and hyperglycaemic clamp the M / I ratio between the amount of glucose infused to maintain glycaemia at the desired level and the mean plasma insulin concentration in the 20-120 min period of the study was used as a measure of insulin action. Kitt averaged 5.67 + 0.44 mg/(dl • min) x 100 (mean_+ SEM). During euglycaemic insulin clamp M / I ratio was 4.8 + 1 mg/(m 2 • min) • ~tU/ml. During hyperglycaemic clamp M / I ratio was 6.8 _+0.8 mg/(m 2 x rain) x fxU/ml. A high correlation was found between Kit t and M / I ratio of either euglycaemic (r=0.858, p < 0.01) or hyperglycaemic (r= 0.968, p < 0.001) clamp. These results suggest that 15 min-ITf could represent a valid approach for a simple and rapid estimation of in vivo insulin action when glucose clamp cannot be used, as in large series of subjects or in serial studies. 57. Streptozotocin diabetes in mice: Study of enzyme activities and mitochondrial changes L.Boquist. Institute of Pathology, University of Ume~t, Ume~t, Sweden According to our hypothesis, mitochondrial changes play a role in the development of spontaneous and experimental diabetes. The aim was to see whether mitochondrial alterations occur in streptozotocin diabetes. In addition to islets, liver and kidneys were studied. An increase in mitochondrial volume was observed by qualitative and quantitative electron microscopy, and a dislocation of Ca2+-containing precipitation from mitochondria to cytosol was demonstrated by combined ultrastructural and roentgenological methods. The transport of CaZ+was studied in mitochondria isolated from mice injected with streptozotocin and in mitochondria which were incubated with streptozotocin in vitro. In both kinds of mitochondria the uptake of Ca 2+ was decreased as to rate and extent, and the release of Ca 2+ was potentiated. Alterations were found in metabolite concentration ratios, which suggested a relative oxidation of the mitochondrial pyridine nucleotides and a relative reduction of the pyridine nucleotides in the cytosol of diabetic mice. The activities of succinyl-CoA synthetase and citrate synthetase were decreased in diabetic mice. The data suggest a role of inhibited citric acid cycle enzyme activity and altered mitochondrial Ca 2+ transport in streptozotocin diabetes. 58. Receptors and metabolic effects of insulin-like growth factor-I in vascular smooth muscle K. E. Bornfeldt, H.J. Arnqvist, A. Skottner and J. E. S. Wikberg. Dept. of Pharmacology, Link6ping University, Link6ping; Kabi Vitrum Peptide Hormones, Stockholm; and Dept. of Pharmacology, University of Gothenburg, Gothenburg, Sweden

Insulin-like growth factors are affected by the diabetic state and may be of importance to the development of vascular disorders. In this study we investigated receptors for insulin-like growth factor-I (IGFI) in plasma membranes from bovine mesenteric arteries (BMA), and metabolic effects of IGF-I in intima-media preparations of BMA. Specific binding of 125I-IGF-I to plasma membranes was 7.4_+ 1.7%, -.~ and a 500~ displacement of 125 I-IGF-I with 29.2_ 4.1 0F0. The molecular weight of the IGF-I receptor was determined using SDS-polyacrylamide gel electrophoresis. Covalent cross-linking of a25I-IGF-I to the receptor showed a molecular weight of 146,000 dalton, probably corresponding to the a-subunit of the IGF-I receptor. Binding of a2sI-IGF-I was almost completely displaced by IGF-I (1 ~mol/1), and to some extent by 1 gmol/1 insulin. IGF-I (2.2 nmol/1) was found to increase glucose accumulation, amino acid uptake and protein synthesis after 4 h incubation (p<0.05), while 22 nmol/1 increased glucose accumulation even after 2 h (p<0.05). In conclusion, these results suggest that arterial smooth muscle cells are abundant in receptors for IGF-I, and that IGF-t influences the metabolism of vascular smooth muscle. Furthermore, insulin was found to interact with the IGF-I receptor. 59. Metabolic effects of long-term administration of high carbohydrate-high fiber diet in diabetic patients O. Bosetlo, F. Ferrari, A. Paroli, L. Cominadni, I. Zocca, E. Cavallo and T.Todesco. Clinica Medica, Universitd di Verona, Italy

Abstracts The effect of a high carbohydrate, naturally high fiber diet (HCHOHF diet) was studied over a long-term period on Type i (insulin-dependent) and Type 2 (non-insulin-dependent) diabetic patients. Twenty-eight patients (16 Type 1 and 12 Type 2) were changed from their usual diet (CHO 44%:lipid 37%:protein 19%:fiber 19 g) to a HCHO-HF isocaloric diet (CHO 62% : lipid 21% : protein 17% : fiber 60 g). Blood glucose (BG) behaviour during the day (samples: fasting, two h after breakfast and two h after lunch), total cholesterol (CH), HDL-CH, triglycerides (TG), glycosylated haemoglobin A1 c, fructosamine test were evaluated before and after a 3 month period. Body weight did not change. Fasting BG significantly decreased (9.56+2.57 mmol/1 to 6.94+ 1.51 mmol/l, p<0.01) as well as mean BG profile (from 8.9+2.43 mmol/1 to 6.91+1.55 mmol/1, p<0.01) and fructosamine level (from 2.6 _+0.6 to 2.0_+0.3%, p < 0.01). While the decrease in H b A I c was relevant but not significant (from 12.8 _+8.7 to 7.5 + 1.5%, NS). CH significantly reduced (from 216 + 42 to 173 + 19 mg/dl, p < 0.001) while the decrease in TG was not statistically significant (from 150 _+64 to 123 _+38 mg/dl, NS). HDLCH showed a significant increase (from 31.7+5.3 to 38.1+ 3.9 mg/dl, p<0.01). The data demonstrate that HCHO-HF diet improves glycaemic control and reduces lipid levels over a long-term period in diabetic patients. 60. Pre-Type I (insulin-dependen0 diabetes in autoimmune polyendocrine patients with islet cell antibodies E.Bosi, F.Becker, A.C.Tarn, G.Schwarz, L.A1-Sakkaf, D.Overkamp, E.A.M. Gale and G. F. Bottazzo. Dept. of Immunology, Middlesex Hospital and Dept. of Diabetes & Immunogenetics, St. Bartholomew's Hospital, London, UK One hundred forty-seven ICA + (120 Complement-Fixing) non-diabetic patients with associated endocrine/organ-specific autoimmunity were identified and enrolled in a prospective study: age 49.8 +_ 14.4years, 80% females; associated diseases: thyroid 56%, pernicious anaemia 18%, vitiligo 12%, Addison 11%, infertility 4%; associated autoantibodies: thyroglobulin 41%, thyroid microsomal 73%, gastric parietal cell 73%, intrinsic factor 22%, adrenal 16%, steroid cell 11%. Over a maximal follow-up of 10.5 years, 16 patients (11%, all CF-ICA +) became diabetic, 11 (7%) died non-diabetic, while 120 (82%, 98 initially CF-ICA +) are still non-diabetic: of them, 20 (14%, 9 initially CF-ICA +) lost ICA during the follow-up, 76 (52%) are persistently ICA + (69 CF-ICA +) and in the remaining 24 (16%, 20 initially CF-ICA +) the current ICA status is not known. Cumulative survival without diabetes after the first ICA detection was 96, 91, 86, 81 and 72% at 2, 4, 6, 8 and 10 years respectively. When considering the 94 persistently CF-ICA +, patients B cell survival was 94, 85, 78, 70 and 53% at 2, 4, 6, 8 and 10 years respectively. Conclusions: in adult ICA + polyendocrine patients persistent CF-ICA has the best predictive value; progression to diabetes is slower than in first degree relatives of diabetic children (36% non-diabetic at 8 years in those CF-ICA+). These data extend the concept of heterogeneity of Type 1 (insulin-dependent) diabetes to the pre-clinical period. 6t. Reduced hyperaemic response under the diabetic foot: an additional aetiological mechanism for ulceration? A.J.M.Boulton, P.G.Newrick, T.Cochrane and R.P.Betts. Royal Hallamshire Hospital, Sheffield, UK We previously reported that diabetic neuropathic ulcers occur at high pressure sites under the foot. In this study, recovery of blood flow under the metatarsal heads after standing has been measured in three matched groups of 11 subjects without vascular disease: (A) Diabetic neuropathy with abnormal foot pressures; (B) non-neuropathic diabetic patients, normal foot pressures and (C) non-diabetic control subjects. Resting baseline erythrocyte flux was measured at the highest pressure point under the metatarsal heads (confirmed by the optical pedobarograph) using laser doppler flowmetry. The hyperaemic response was assessed at the same site immediately after standing for 3 rain. Peak erythrocyte flux was significantly reduced in Group A (mean 2.4___1.4volts, SD) compared with Group C (4.0+2.0volts, p<0.05) with an intermediate result in Group B (3.2_+ 1.5 volts; A vs B, B vs C, both p = NS). Similarly, the time for blood flow to return to baseline was significantly delayed in Group A (163 _+73 s) compared to Group C (92 _+18 s, p < 0.01), with significant delay also seen in Group B (143 _+36 s; B vs C, p < 0.001; A vs B p = NS). Thus although the most impaired blood flow response was seen in Group A subjects, significant abnormalities were also seen in non-neuropathic subjects. We conclude that impaired microvascular function together with high foot pressure during

Abstracts standing or walking may together contribute to the aetiology of diabetic foot ulcers. 62. Glucose and leucine turnover are reduced in Cushing's syndrome

S. B. Bowes, I.N. ScoNe, J.J. Benn, A.M. Umpleby, C. Lowy and P. H. S6nksen. Department of Medicine United Medical and Dental Schools of Guy's and St.Thomas' Hospitals, St.Thomas' Hospital, London, U K Glucose intolerance, severe enough to produce frank diabetes, and proximal muscle wasting are frequent features of Cushing's syndrome. The primary cause of diabetes (glucose overproduction or underutilisation) and myopathy (reduced protein synthesis or increased degradation) is unclear. We therefore studied protein and glucose turnover in 7 patients with Cushing's syndrome and 7 normal subjects using an intravenous bolus of 25 ~tCi of 1-14C leucine and 75 ~tCi of 3-~H glucose. Plasma glucose concentrations were similar in Cushing's patients (5.0_+ 0.2 mmol/1) (mean + SEM) and normal subjects (5.1 + 0.2). However, both glucose metabolic clearance rate (MCR) (1.75 _+0.06 ml. min- 1. kg-I vs 2.12 + 0.14, p < 0.05) and glucose turnover rate (8.84_+ 0.37 lxmol, rain -1. kg -1 vs 10.79_+ 0.66, p < 0.05) were significantly reduced in Cushing's patients compared to normal subjects. In the Cushing's patients, plasma leucine concentration (98 _+7 .umol/1 vs 131 _+6, p < 0.005), leucine turnover (1.05_+0.09 lxmol.min-a.kg -1 vs 1.65_+0.15, p<0.01) and leucine incorporation into protein (0.83 + 0.08 ~tmol. min -a. kg -a vs 1.46 + 0.13, p < 0.005) were significantly reduced. Leucine MCR (10.97_+ 1.23ml.min-l.kg -1 vs 12.35_+1.35) and oxidation rate (0.21_+ 0.03 gmol. rain -1- kg -1 vs 0.18 + 0.03) were similar in both groups. Mean insulin concentrations were 9.1 +4.2 mU/1 in the Cushing's patients and 5.2 + 1.0 in the normal subjects (NS). We conclude that the impaired glucose tolerance found in Cushing's syndrome is primarily due to decreased glucose utilisation and the muscle wasting is due to reduced protein synthesis. 63. The membrane potential of mouse B cells: a third level of control by glucose? M. Bozem and J. C. Henquin. I Physiologisches Institut, University of Saarland, Homburg/Saar, FRG

Low glucose concentrations (3-7 retool/l) depolarize the B-cell membrane from a resting to a threshold potential, at which slow waves (SW) with spikes superimposed on the plateau appear. As glucose levels are raised further (7-25 mmol/1), SW become longer and the membrane eventually remains depolarized at the plateau potential. The number of spikes also increases, but this increase could simply be due to the SW lengthening. We thus investigated whether glucose can influence spike activity when no SW occur. Persistent depolarization to the plateau potential was achieved by different means in B cells perifused with 10 mmol/l glucose: ouabain (blockade of electrogenic Na-pump), arginine (positively charged), tolbutamide (blocker of ATP-dependent K channels), and low Ca 2+ (1ess activation of Ca-dependent K channels). Under all these conditions, raising glucose to 15-25 mmol/1 increased spike frequency and amplitude. Other experiments showed that the large spikes originating from the threshold potential in the presence of tetraethylammonium also increased in frequency and amplitude as the concentration of glucose was raised. In conclusion, the evidence that glucose may affect spike activity more directly than by changing SW duration raises the possibility that the channels involved in spike generation are under metabolic control. 64. Na,K-ATPase-dependent rubidium uptake on human lymphocytes in insulin-resistance: role in spontaneous obesity C. Bozzo, M. Goria, S. Marena, S. Avagnina, C. Marengo, V. Tagliaferro and G. Pagano. Institute of Internal Medicine, University of Turin, Italy

The role of Na,K-ATPase in human obesity is still controversial. Some reports obtained on human erythrncytes show higher, lower or similar activity to that of lean control subjects. In order to clarify this problem, we studied 20 obese patients (44 + 11 years, body mass index 37.5 _+6.2) and 20 matched control subjects as follows: oral glucose tolerance test, intravenous insulin test (0.05 U/kg), 86RbC1 uptake (30 ~tmol/l) by isolated lymphocytes with and without ouabain (10 -5 tool/l). Vmax and Km were evaluated on kinetic curves, obtained in 0-10 mmol/1 ranged of RbC1 concentration. Na, K-ATPase-dependent 8~ uptake (32.7 _+8.6 vs 34.7 + 13.5 nmol in con0 ouabain-inhibited uptake (IDso=9.2,+ trol subjects, NS), 50Y0 4.8 • 10 -8 tool/1 vs 8.9_+3.7 in control subjects, NS), Vmax (1111 _+

503 A 384 pmol vs 1207 + 261, NS) and Km (1.64+ 0.29 mmol/l vs 1.63 + 0.59, NS) were similar in obese and control group. In all subjects (obese and control) a negative correlation ( r = - 0 . 4 2 , p < 0.05) between body mass index and 86Rb uptake and a positive one (r=0.74, p<0.001) between insulin sensitivity index (Conard's K) and 86Rb uptake were obtained. This suggests that Na, K-ATPase is related to body weight variation and to peripheral insulin sensitivity. It also indicates that insulin resistent states may be characterised by an alteration of Na, K-ATPase pump activity. 65. Insulin analogues with reduced association tendency: biological activity and absorption rate after subcutaneous administration J. Brange, U. Ribel, A.R. Sorensen, I. Diets, M.T. Hansen, S. Havelund, F. Norris, K. Norris, L. Snel and H. O. Voigt. Novo Research Institute, Bagsva~rd, Denmark Insulin occurs in neutral regular insulin associated into dimers and mainly hexamers which may delay the absorption from subcutis. Recombinant DNA technology and fermentation in yeast were used to produce human insulin analogues in which one amino acid residue in the interface between monomers in the insulin dimer were mutated. The substitutions were: 1) B9Ser to Asp, 2) B12Val to Ile, 3) B12Val to Glu, 4) B26Tyr to Glu, 5) B27Thr to Glu, 6) B28Pro to Asp. Time to 25% disappearance from the injection site after subcutaneous administration of 12SI-labelled insulins (0.8 nmol/kg) to fasted pigs (n=4-8) was (% of human insulin mean+ SEM): 1)69.4+ 7.2,2) 68.5 _+19.0,3) 65.4 + 13.1,4) 52.0 _+11.9,5) 79.2 + 9.7,6) 49.0 + 7.8 respectively. The rapid absorption of the analogues was confirmed by measurements of serum IRI and blood-glucose. Biological activity (% of human insulin) in vitro (free fat cell assay)/in vivo (mouse blood glucose assay) was (95% confidence limits in brackets): 1) 25.5 (25-27)/78.9 (70-89), 2)28.8 (27-30)/86.1 (76-97, 3)0.041 (0.038-0.043)/not done, 4)124.9 (121 129)/103.5 (96-112), 5)107.5 (104-111)/110.0 (100-121), 6) 100.5 (96-105)/104.0 (95-114) respectively. In conclusion: Insulins with substantially faster rate of subcutaneous absorption and with retained biological activity can be obtained by insulin engineering. 66. Puisatility of endogenous insulin secretion in man P. Bratusch-Marrain, S. Gasic, M. Komjati and W. Waldh/iusl. I. Medizinische Universit~itsldinik Vienna, Austria

An oscillatory pattern of hormone secretion has been demonstrated for insulin in animal experiments and isolated pancreas preparations. Since direct measurements of insulin release are not feasible in man, insulin production and portal insulin concentrations were estimated by means of the liver vein catheter technique via quantitation of C-peptide release from the splanchnic area in 6 healthy male volunteers in the fasting state on a minute-to-minute basis. Rapid oscillations of insulin secretion were detected with amplitudes ranging from 50 to 600 pmol/min, and of portal insulin concentration between 5 and 80 mU/1. Intraindividually, no regular rhythmicity was detected by Fourier transformation due to the high frequency noise. Only by averaging data of all subjects Fourier transformation revealed a preponderance of oscillations with a phase time of 13 rain for insulin secretion rates. However, no regular oscillations were detectable for insulin concentrations in peripheral arterial plasma. On the basis of these data it appears questionable to draw conclusions from insulin levels measured in peripheral blood in regard to secretory patterns. 67. A new agent for inhibiting the non enzymatic glycosylation of proteins in diabetes mellitus M. R. Bruno, M. Sensi, E Beales, S. Pagani, L. Valente, M. Giovanelli, G. P. Cioccia and P. Pozzilli. Cattedra Endocrinologia (I), II Clinica Medica, University of Rome, Italy Several studies highlighted the importance of non enzymatic glycosylation in altering the chemical-physical properties of proteins in the diabetic state. We report the in vitro inhibitory effect of a new agent, 1-benzyl-3-Indazol-Oxyacetic acid (Bendazac, sodium salt) on the non enzymatic glycosylation of human serum albumin (HSA), fibrinogen (F) and isolated glomerular basement membrane (GBM). Glycosylation was achieved by protein incubation in increasing concentrations of D-glucose in phosphate buffer (pH 7.4) for 7-10 days at 37 ~ The effect of Bendazac was evaluated at concentrations equal to 1/10 those of glucose. HSA and F glycosylation at 5, 25, 50 and 100 retool/1 glucose concentrations were reduced by 68.3, 69.1, 40.2,

504 A 35.7% and by 9.0, 42.0, 29.9, 33.0% (p<0.01 and <0.05; ANOVA) respectively. No significant inhibition of glycosylation was observed with GBM. However a significant inhibition at 50 and 100 mmol/1 D-glucose (35.4 and 38.3% respectively) was observed when GBM was glycosylated in presence of 5-hydroxy Bendazac, a metabolite of the original compound. We conclude that Bendazac may have a possible role in reducing the extent of non enzymatic glycosylation of proteins, and therefore may help prevent those protein changes which contribute to the development of late diabetic complications. 68. One-year double blind study with combined therapy of glibenclamide and insulin in Type 2 (non-insulin-dependent) diabetic patients with secondary snlfonylnrea failure W. Bruns, M. Scheibe, G.Willkommen and R. Hildebrandt. Sanatorium for Diabetics "Bergfried", Saalfeld, District Hospital, Rudolfstadt, Central Outpatient Clinic for Diabetes and Metabolic Disorders, Berlin, GDR Forty-one mostly overweight Type 2 (non-insulin-dependent)diabetic patients (30female, 11 male; body weight 56.0-90.6kg; age 44-71years; diabetes duration 5-15years) were treated for 12 months with 2 x 5 mg glibenclamide (n=20) and placebo (n=21), respectively, and with individual doses of intermediate insulin, beginning with 14+6 U. After 3 months, tablets were withdrawn for 2 months. Criteria: body weight, MBG of 8 BG-values in 24 h, daily insulin doses and basal C-peptide. Results: SEM in both groups were small and comparable. Placebo versus verum group: body weight-gain + 4.0 and 5.4 kg, respectively; MBG 8.7 and 7.5 mmol/1, respectively; insulin doses 39.0 and 29.7 U, respectively; C-peptide increased from 0.3 to 1.2 and 2.4 ng/ml respectively. Significant differences for MBG and insulin doses (p< 0.05). Withdrawal of tablets showed a significant increase of MBG from 7.5 to 10.5 mmol/1 and insulin requirement from 25.6 to 30.6 U only in the verum group. Conclusions: during combined therapy with glibenclamide and insulin there is an improvement of glibenclamide on metabolic control even after 12 months. The long-term strategy had to consider the danger of stimulated endogenous hyperinsulinism and weight-gain. 69. Hyperaminoacidaemia decreases insulin-mediated glucose metabolism in normal subjects but not in Type i (insulin-dependent) diabetic patients D. Bruttomesso, S. Inchiostro, G. Biolo, E. Duner, M.R. Munari, G. Briani, A. Tiengo and P. Tessari. Malattie del Ricambio and Patologia Medica 1, University of Padova, Italy The metabolic interactions among insulin-mediated glucose disposal, amino acid availability and intermediary metabolites were investigated in Type I (insulin-dependent) diabetic patients and in normal control subjects, using the euglycaemic, hyperinsulinaemic ( - 9 0 mU/1) clamp without or with an amino acid infusion, which increased their concentrations by 50-150% above basal levels. In normal subjects, hyperaminoacidaemia was associated with a reduction in glucose disposal (8.0 _+0.9 vs 10.9 _+0.8 mg/kg, min, p < 0.02), as well as in the increments of lactate (+0.510+0.079 vs +0.962+ 0.138 mmol/1, p < 0.02) and pyruvate concentrations ( + 0.020-+ 0.007 vs + 0.050 + 0.009 mmol/1, p < 0.03). Glucose disposal was directly correlated to increments in lactate concentrations (r= 0.55, p < 0.05). Total ketone body concentrations tended to decrease less in the presence of hyperaminoacidaemia (-0.018+0.010 vs - 0 . 0 5 8 + 0.014 mmol/1, p < 0.1). In the diabetic patients, insulin mediated glucose disposal was decreased as compared to normal subjects (7.0 + 1.1 mg/kg-min, p<0.02) and was not modified by hyperaminoacidaemia (7.3_+0.8mg/kg.min). Also lactate (+0.429+0,024 vs + 0.439_+ 0.075 mmol/l) and pyruvate increments ( + 0.006_+ 0.003 vs 0.017_+0.005 mmol/l), as well as total ketone body decrements ( - 0.037 • 0.028 vs - 0.111 + 0.036 retool/l) were similar without or with accompanying hyperaminoacidaemia respectively. Glucose disposal did not correlate with increments in lactate concentrations. Thus, during euglycaemic, hyperinsulinaemic clamps, hyperaminoacidaemia appears to reduce whole body glucose metabolism in normal subjects but not in Type I diabetic patients. These data suggest that in Type 1 diabetes insulin resistance is not further aggravated by increased amino acid availability. 70. A B islet cell specific BB-rat monoclonal autoantibody (Mab IC-2) detects functional state dependent antigen expression K. Buschard, C.-H. Brogren, C. ROpke and J. Rygaard. Path.-anatomical Inst., Kommunehospitalet; Institute of Med. Anatomy, Dept.A, The Panum Institute; Dept. of Animal Physiology, Royal Vet. Agric. University, Copenhagen, Denmark

Abstracts A monoclonal autoantibody (IC-2) was derived from hybridomas of rat myoloma Y3 cells and splenocytes of a diabetic BB-rat. The selective reactivity with islet cells has earlier been proven. We studied the possible specificity for B islet cells, and the possible variation in autoantigen expression. Islet cells were isolated by cautious collagenase and dispase treatment. The cells were labelled with IC-2 and/ or anti-insulin antibodies. Extensive series of cells were examined by fluorescence microscopy, and for some samples also by flow cytometry. In a double labelling study we found that only anti-insulin positive cells could bind the IC-2 antibody. As insulin treatment has been shown to decrease the incidence of diabetes in the BB-rat, islet cells were examined after reduced B cell strain. Islet cells from Lewis and Wistar Furth rats display 19.3 _+1.1% IC-2 positive cells, while islet cells from 24 h fasting animals showed 7.0 + 1.4% (p< 0.001). Similar results were seen for BALB/c mice (23.7_+2.3% vs t3.1 +4.0, p < 0.05). Also, after a week of insulin treatment autoantigen expression was significantly decreased. Thus, the IC-2 ~mtibody is B cell specific, and expression of the corresponding antigen depends on the functional state of the B cells.

7t. Evidence for a normal inhibitory effect of somatostatin on pancreatic B cells in human obesity V. Cacciatori, P. Moghetti, E. Bonora, M. Querena, G. Zoppini and M. Muggeo. Chair of Metabolic Diseases, University of Verona, Italy Aim of the present study was to evaluate whether the inhibitory effect of somatostatin on pancreatic B cells is normal in nondiabetic obese subjects. Plasma C-peptide concentrations were measured in 8 nondiabetic obese subjects and 10 nonobese healthy control subjects during a 4 h hyperglycaemic (11.05 mmol/1) glucose clamp. Somatostatin was infused (250 meg/h) during the third hour of the study period in order to inhibit glucose-stimulated B cells. Fasting plasma C-peptide averaged 0.39+0.04 pmol/ml (mean _+SEM) in nonobese subjects, and 0.71 +0.05 pmol/ml in obese patients (p< 0.001). In the last 20 rain of the glucose infusion period without somatostatin (100-120 rain), plasma C-peptide averaged 2.70+ 0.30 pmol/ml in control subjects, and 3.00 • 0.17 pmol/ml in obese patients (p= NS). In the last 20 rain of the glucose plus somatostatin infusion period (160-180 min) plasma C-peptide averaged 0.84+ 0.11 pmol/ml in control subjects, and 0.83 • pmol/ml in obese patients, with a percent reduction similar in the two groups (69 + 2% in control subjects, 72_+2% in obese patients). After stopping somatostatin infusion plasma C-peptide increased, and in the period 220-240 rain of the study it averaged 2.90-+ 0.33 pmol/ml in control subjects and 3.11+0.19 pmol/ml in obese patients (p=NS). These results suggest that the inhibitory effect of somatostatin on pancreatic B cells is not impaired in human obesity.

72. Xenotransplantation of microencapsulated canine islets in diabetic NOD mice: prolongation of graft survival with superoxide scavengers R. Calafiore, F. Calcinaro, N. Koh and R. Alejandro. Istituto di Patologia Medica, University of Perugia, Italy and Diabetes Research Institute, University of Miami, Miami, Fla, USA We observed that xenotransplantation of microencapsulated canine islets (MECI) in poly-L-ornithine-Na alginate biomembranes resulted in transient euglycaemia in spontaneously diabetic NOD mice, 8/8 (4.72+0.44 retool/l) at day 3 and 4/8 (4.61+0.83 mmol/1) at day 7 post transplant (p< 0.001 versus untreated controls respectively). We wished to test whether administration of Cyclosporlne A (75 rag-kg -I. day -1, subcutaneously) or free oxygen radical scavengers, polyethylene glycol conjugated superoxide dismutase and catalase (PEG-SOD/CAT, 10.000/50.000UI.kg-~.day -a intraperitoneally) would prolong survival of MECI xenografts in diabetic NOD mice. We found the following results: Cyclosporine A group (n = 9), 9/9 euglycaemic (5.05 + 0.33 mmol/l) at day 3 and 3/9 euglycaemic (5.22+0.16 mmol/1) at day 7 post transplant (p<0.001 versus untreated controls respectively); PEG-SOD/CAT group (n= 13), 13/13 euglycaemic (4.88+0.33 mmol/1) at day 3 and 13/13 euglycaemic (5.22_+0.38 mmol/1) at day 7 post transplant (/7<0.001 versus untreated controls respectively). We conclude that PEG-SOD/CAT but not Cyclosporine A afford complete protection during the first 7 days post transplant suggesting that free oxygen radicals are seemingly generated that adversely affect graft survival in the peritransplant period.

Abstracts 73. Role of free fatty acids in the insulin resistance of non obese Type 2 (non-insulin-dependent) diabetes mellitus patients B. Capaldo, R. Napoli, L. Di Marino, A. Picardi and G. Riccardi. Institute of Internal Medicine and Metabolic Disease 2nd Medical School, University of Naples, Italy FFA abnormalities are regarded as a major factor of insulin resistance in obese Type 2 (non-insulin-dependent)diabetic patients. This does not seem to be the case in lean well controlled insulin-treated Type 2 patients. To assess the role of FFA in the pathogenesis of the insulin resistance of untreated Type 2 diabetes, 6 normal subjects (54 + 4 years) and 5 untreated normal-weight diabetic patients (46 -+ 4 years), made normoglycaemic the day before the study, were studied combining the euglycaemic insulin clamp with the forearm perfusion technique. Insulin-stimulated glucose disposal was markedly lower in diabetic patients (4.3 _+0.7) than in control subjects (7.0 + 0.7 mg-kg 1. rain-1 p < 0.02). Similarly, forearm glucose utilisation was significantly reduced in diabetic patients (4.1 + 1.2) as compared with control subjects (9.1 _+1.4 rag. liter- 1 9r a i n -1, p < 0.05). Basal FFA levels were similar in the two groups and decreased to a similar extent (80%) in response to insulin. Insulin infusion induced a marked increase (p<0.02) in forearm FFA fractional extraction (H3-palmitate) in both groups. Basal FFA forearm utilisation was similar in control subjects and diabetic patients (2.6_+0.5 vs 2.2 + 0.6 umol. liter -1. rain -1, respectively) and decreased to 0.9 + 0.2 and 0.8 '___0.1 ~Lmo1.liter -1. rain-1 after insulin infusion. In conclusion, in non obese untreated Type 2 patients a marked reduction in forearm glucose utilisation occurs in the absence of any abnormality in forearm FFA metabolism. This observation argues against a role of FFA in the insulin resistance of non obese Type 2 patients. 74. Pharmacokinetics of a small subcutaneous insulin bolus in CSIItreated Type I (insulin-dependent) diabetic patients assessed by glucose clamp technique F.Capani, G.Casalini, A.D'Emilio, M.R.Loragno, G.La Nave, E.Vitacolonna, G.Zappone and S.Sensi. Department of Internal Medicine, University of Chieti, Italy The postprandial blood glucose values in CSII-treated diabetic patients depends mainly on the variability of three factors: 1)the amount, 2)the peripheral efficacy of insulin absorbed and 3)the amount of the splanchnic glucose absorption after the meal. In order to explore the variability of the first two factors, five CSII-treated Type 1 (insulin-dependent) diabetic patients without detectable levels of anti-insulin antibodies, were studied by De Fronzo's euglycaemic clamp algorithm after the subcutaneous administration (abdominal region - depth = 10 mm) of 0.1 IU/kg body weight (6-9 1. Units) of semisynthetic human insulin (Actrapid HM Novo). The study was repeated after 3-4 weeks in the same subjects. The total amount of the glucose requested was 50_+5.9 and 61 _+12.1 when the test was repeated (p= NS). The coefficient of variation of all 10 studies was high (37%) in spite of the accuracy of injection techniques. The insulin effect showed a flat curve with a plateau between the 2nd and 4th h and was present until 7th h. These results explain the great variability of post-prandial blood glucose values usually observed after the ingestion of meals and indicate that even small amounts of regular insulin induce an overlapping of insulin effect when three daily injections of regular insulin are administered in both CSII- and conventionally-treated diabetic patients. 75. Beta-blockers in hypertensive obese patients L.Capretti, G. Speroni, A. Pezzarossa and L. D'Amato. Ospedale Civico USSL 54 Lombardia, Codogno (Milano), Cattedra di Endocrinologia, Universitd di Parma, Parma, Italy Controversial metabolic effects often give rise to uncertainties about the use of beta-blockers (BB) in hypertension when associated to obesity, although adrenergic hyperactivity is present in both diseases. Aim of this study was to evaluate the effect of 3 BB with different intrinsic adrenergic activity (IAA) and ill-selectivity (flS) in 16 obese hypertensive patients with normal glucose tolerance (GT) who randomly received Propranolol (PRP; I A A - , f l S - ) (240 rag/day), Acebutolol (ACE; I A A - , f l S + ) (800rag/day) and Pindolol (PIN; I A A + , f l S - ) (37.5 rag/day) for 5 days with a 4 day wash-out between treatments. Oral glucose tolerance test (OGTT) (75 g) was performed in basal conditions (BC) and after each treatment, for glucose (BG), insulin (IRI), cholesterol and triglycerides analysis. Data were evaluated by 2-way analysis of variance (ANOVA). Results: During OGTY BG levels were significantly lower after PRO compared to BC (p<0.005) and PIN (p<0.001) and after ACE corn-

505 A pared to BC (p< 0.01) and PIN (p< 0.001). No statistical difference was found between BC and PIN. The glucose pattern was not modified by BB (peak at 60 rain and nadir at 120 min). All BB significantly reduced IRI response to oral glucose without significant differences between BB. Body weight, HDL-cholesterol, total cholesterol and triglycerides levels were not modified. Conclusions: BB did not worsen GT in obese hypertensive patients, in spite of a reduced insulin secretion, probably through peripheral effects mediated by different degrees of IAA. 76. Beneficial effect of captopril on proteinuria and blood pressure in diabetic patients M.A. Carrasco Bejar, F.J. Arrieta, F.Cordido, S. Casado and J. L. Herrera Pombo. Fundacion Jimenez Diaz, Universidad Autonoma, Madrid, Spain We have assessed change in blood pressure, proteinuria, renal function and glycaemic control in diabetic patients after 20 weeks of treatment with captopril. We studied 21 patients: Group A) 9 with arterial hypertension and proteinuria <200 p~.g/min, Group B)7 with arterial hypertension and protemuna >_200 Ixg/min, GroupC) 5 with normal blood pressure and proteinuria _> 200 ~g/min. The maximum dose used of captopril was 100 rag/day in hypertensive and 50 rag/day in normotensive patients. The blood pressure decreased in Group A (from 180_+ 17/100+6 to 147+ 20/83_+7mmHg, p<0.001), in Group B (from 179+26/97+8 to 151_+20/85+11 mmHg, p
506 A patients and the 7 non-diabetic patients showed no significant differenees in total plasma TG (2.20+0.14 vs 2.30+ 0.25 mmol/1) and in TG-VLDL (1.35 _+0.20 vs 1.46 _+0.23 mmol/1), also when compared with non-uraemic patients. Apo-CII levels were not significantly different between diabetic and non-diabetic patients (3.5 + 0.8 vs 3.1 + 0.9 mg/dl), but significantly different (p < 0.01) when compared with hyper-TG non-uraemic patients (8.3 + 1.7 mg/dl). The differences of Apo-CIII levels were not significantly different in all groups (19.1 _+ 0.6 in diabetic, 17.9 _+1.1 in non-diabetic patients, 19.1 + 2.2 mg/dl in non-uraemic patients). The preliminary result obtained on Apo-CIII isoforms, showed a significant increase in Apo-CIII2 in all uraemic patients compared to hyper-TG non-uraemic patients and a significant increase in Apo-CIIIa in non-diabetic patients and in hyper-TG non-uraemic patients. 79. Crucial role of residual insulin secretion in the efficacy of glipizide added to insulin in the management of Type 2 (non-insulin-dependent) diabetic patients with secondary failure to sulfonylureas M. Castillo, A.J. Scheen, G. Paolisso and P.J. Lefebvre. Division of Diabetes, Institute of Medicine, University of Liege, Liege, Belgium To investigate the effect of a combined insulin plus glipizide treatment on the metabolic control (HbAlc levels) and insulin requirements (Biostator~ assessment) in Type 2 (non-insulin-dependent) diabetes with recent secondary failure to sulfonylureas and to characterise the relative contributions of changes in endogenous insulin secretion (C-peptide response) and insulin sensitivity (glucose clamp) to this effect, ten non-obese patients were treated in a randoraised cross-over order with either insulin alone or insulin plus glipizide (3 x 10 rag/day) during two periods averaging 6 weeks each. During the combined therapy, HbAlc levels decreased in 5 patients (from 8.6+0.7 to 7.1+0.5%; "responders") but not in the 5 others ("non-responders'). The 20-h Biostator~ insulin infusion was significantly decreased in "responders" only ( - 2 9 % , p<0.05). Basal (0.271+ 0.086 versus 0.086 + 0.017 nmol/1) and post-glucagon (0.468 + 0.121 versus 0.180 + 0.060 nmol/1) C-peptide plasma levels were significantly (p< 0.05) higher in "responders" than in "non-responders" and these differences were amplified by glipizide. The insulin-induced glucose disposal was significantly increased by glipizide (+23%, p<0.05) in "responders", an effect observed in the presence of higher free insulin plasma levels ( + 37%, p < 0.02) due to persistent stimulation of endogenous insulin secretion. Thus, combining glipizide with insulin is useful only in Type 2 diabetic patients who still have a significant endogenous insulin secretion capable of being stimulated by this drug. 80. Insulin sensitivity and glucose clearance quantified in humans from labelled intravenous glucose tolerance test and minimal model A. Caumo, A. Giacca, M. Morgese, G. Rivolta, G. Pozza, P. Micossi and C. Cobelli. Istituto S. Raffaele, Univ. of Milan, Milan and Department of Electrical Engineering, Univ. of Padua, Padua, Italy The estimation of insulin sensitivity and fractional glucose clearance has physiopathological and clinical relevance, and several approaches have been proposed in the past to obtain quantitative measures; among these, the intravenous glucose tolerance test (IVGTF). Revised 'minimal model' technique (RMM) uses a simultaneous bolus injection of cold and labelled glucose. The rationale for the hot and cold glucose combined injection is to segregate glucose production and utilisation and the effect of insulin upon them. Therefore, RMM yields tracer estimates of insulin sensitivity (SI*) and fractional glucose clearance (SG*) which, at variance with cold SI and SG, are not affected by changes in hepatic glucose production. Five young normal volunteers underwent the test which was performed by giving a combined bolus injection of 1.2 ~tCi/kg of [3-~H/H glucose and 0.33 g/kg/body weight of cold glucose. Results (values given are means+_SEM): SI*=10.10+1.35 vs SI=7.92+_ 0.6710-4min-1.~U 1.m1-1 (NS) and SG*=0.0078+0.0008 vs SG=0.0212_+0.0018.min -1 (/7<0.01). SI* is markedly greater than SI in all but one subject and SG* results -~ 3-fold smaller than SG in all the subjects. In summary, labelled IVGTT and RMM yield a correct portrait of glucose tolerance. Limitations inherent in cold IVGTT are overcome while test simplicity remains. 81. Electrical anionic charges on erythrocytes are reduced in Type 1 (insulin-dependent) diabetic patients P. Cavallo-Perin, P. Estivi and G. Pagano. Institute of Internal Medicine, University of Turin, Italy

Abstracts The glomerular polyanion represents a charge-selective filter, restricting the passage of negatively charged plasma proteins, such as albumin. A specific loss of charges on the glomerular capillary wall (GCW), associated to an increased clearance of albumin, has been reported in diabetes mellitus and in many nephropathies. We performed a chemical test based on the binding of the cationic dye A1cian Blue 8GX (AB) in 7 Type 1 (insulin-dependent) diabetic patients, aged 25 _+7 years. Albustix-negative, in good metabolic control (HbAI c< 8%) and 10 well-matched healthy control subjects; diabetic patients were normotensive and microalbuminuric (>15 ~tg/min). Each subject repeated the test on two separate occasions. The mean AB binding value was significantly (t-test: p<0.0005) lower in diabetic patients than in control subjects: 84.71+4.71 SEM versus 102.75 _+0.7 ng/106 erythrocytes. These results suggest a possible implication of electrical charges in early stages of diabetic nephropathy. 82. Hyperglycaemia-related a2 macroglobulin and antithrombin III alterations in diabetes A. Ceriello, D. Giugliano, A.Quatraro 1, G.Consoli1, A. Stante2, E Dello Russo and F. D'Onofrio. Istituto di Clinica Medica 1, I Facoltfi di Medicina, Universifft di Napoli, Centro di Diabetologia1, Laboratorio Analisi2, Casa di Cura S. Rita, Taranto, Italy

Reduced Antithrombin III activity (ATIIIa) and increased a2Macroglobulin (aM) participate in the development of complications in diabetes. Ten Type 1 (insulin-dependent) diabetic patients and 10 control subjects were compared. ATIIIa was reduced in diabetic patients (12.5 + 0.74 vs 15.3 + 0.24, p < 0.01 ; mean + SEM), a2Macroglobulin activity (aMa) (17 + 2 + 1.4 vs 13.5 + 0.73 IU/ml, p < 0.01) and a2Macroglobulin concentration (aMc) (91.1+2.7 vs 84.8+ 1.43 mg/dl, p<0.01) were increased, while ATIII concentration (ATIIIc) was unchanged. In diabetic patients a correlation between aMa and aMc existed, while between ATIIIa and ATIIIc failed. An inverse correlation between ATIIIa and aMa, aMc, HbA1 and glycaemia existed, while a direct correlation between aMa, aMc and HbA1 and glycaemia existed. Hyperglycaemia was induced in 3 diabetic patients and 3 control subjects. ATIIIa decreased, simultaneously a M a and aMc increased, while ATIIIc remained unchanged. An inverse correlation between observed glycaemia and ATIIIa, aMa and aMc existed. These data show that hyperglycaemia alters ATIIIa but not ATIIIc and influences an increase of c~M molecule. ATIII and a M both have an anti-thrombin role in the coagulation system; ATIIIa may be reduced by hyperglycaemia, while this effect fails for a M ; thus, there exists an inverse correlation between ATIIIa and a M molecule; a compensative role for the increase of a M may be hypothesised. Moreover, this study shows that hyperglycaemia, by itself, may alter the biological activity of some protein. 83. Excellent metabolic control pays off: a 4-year follow-up of retinal status and glycosylated haemoglobin in 32 patients with Type I (insulin-dependent) diabetes mellitus E. Chantelau, H. Weiss, U. Weber, P. Kornek, G.E. Sonnenberg and M. Berger. Departments of Medicine and Ophthalmology, University, Dtisseldorf, FRG

Retinal status and metabolic control were followed-up in 18 females and 14 males (age at entering the study 26_+ 8 years, duration of diabetes 10_+6 years; mean + SD) during a total of 133 patient-years, i.e. 4.2 years per patient. Annual mean glycosylated haemoglobin (mGHb) was calculated from 7_+ 3 determinations of GHb per patient-year, mGHb is given in % of the mean normal value. Fluorescein-angiography was performed at least once per patient-year, the findings were graded according to a modification of Burger's scale (Diabetologia 1986; 29: 17-22). Results: Upon entering the study, early retinopathy was present in 14 patients, and in 19 patients four years later. During 40/133 patient-years, metabolic control was excellent (mGHb _<125%9 during 70/133 years it was good (mGHb 126-150%), and during 23/133 years it was bad (mGHb > 150%). As compared with the respective previous years, retinal status remained stable during 36 years of excellent control, 56 years of good control, and 16 years of bad control respectively. Worsening of the retinal status occurred during 30% (7/23) of years with bad control, 20% (14/70) of years with good control, and only 10% (4/40) of years with excellent control (p<0.05, chi-square). Conclusion: excellent metabolic control (annual mGHb _<125%) protects against, and bad metabolic control (annual mGHb > 150%) promotes, early diabetic retinopathy.

Abstracts 84. Relationship between blood glucose and lactate concentrations in Type 1 (insulin-dependent) well-controlled diabetic patients V. Cheli, E L. Melga, S. Accoto, P. Buzzo and R. Prando. Department of Internal Medicine, Metabolic Disease Service, University of Genoa, Genoa, Italy

It is not well established whether a good glycaemic control in Type 1 (insulin-dependent) diabetes mellitus is associated with normalisation of intermediary metabolites, other than glucose. Diurnal profiles of intermediary metabolites were performed in 10 normal subjects (N) and 39 Type 1 diabetic patients grouped into well-controlled (Group A) and poorly-controlled patients (Group B), according to a 12 h mean glycaemia lower or higher than 8.3 mmol/1. The 2 h mean post-prandial increase in glucose (AG) and lactate (AL) concentrations was assessed. A significant (p< 0.001) inverse correlation between AG and AL was found in normal subjects ( r = - 0 . 7 0 ) , Group A ( r = - 0 . 7 3 ) and Group B patients (r=-0.79). Variance and covariance analysis of the regression lines, between AG and AL, after each single meal showed that the intercept was significantly lower (p< 0.01) in Group B, compared to that of normal subjects. In Group A the intercept was similar to that of normal subjects only at lunch while it was significantly lower at breakfast (F= 15.5, p < 0.01) and at dinner ( F = 9.3, p < 0.01) reflecting reduced post-prandial lactate increases. In conclusion, in Type 1 diabetes some abnormalities of lactate metabolism were still present, even if good glycaemic control was achieved. Different degrees of hepatic and peripheral insulinisation and time intervening between insulin injection and meal assumption could account for this finding. 85. Effects of sucrose ingestion on glucose turnover in normal and diabetic man and its modulation by a new a glucosidase inhibitor, Bay M 1099 J.-L. Chiasson, F. Ducros, J. Bourque, C. Crdras and A. Chrnier. Clinical Research Institute of Montrral, Montrral, Canada

We have studied the effects of a sucrose meal (68 g) on sucrose absorption, blood glucose, hepatic glucose production and on plasma levels of insulin in normal (n=4) and Type I (insulin-dependent) diabetic (n=4) men. All subjects were studied twice, once under placebo and once under Bay m1099. Diabetic patients were wellcontrolled and were studied while on Biostator. The 2 h post-prandial sucrose absorption was 55.8 g in the control subjects and 60.8 g in the diabetic patients and decreased by 60% and 50%, respectively, after Bay m1099. Basal blood glucose for the 2groups was 4:3+_ 0.2 retool/1 and both increased after sucrose ingestion without and with Bay m1099 to 8.2+0.2 and 7.1 -+0.4 respectively. The mean endogenous glucose production was 12.8+_1.9 and 8.2+2.8 ~xmol. kg -1. min -1 during the basal period for the control subjects and diabetic patients respectively; this was suppressed by 91% and 51% in normal subjects and by 81% and 51% in diabetic patients after sucrose ingestion without and with Bay m1099. Basal insulin was 14-+1.7 and 11+2.2 p,U/ml in normal subjects and diabetic patients; sucrose ingestion resulted in an increase to 71 + 9 and 99.3 -+ 191xU/ml without and to 44_+10 and 48-+8.6 ~tU/ml with Bay m1099 in normal subjects and diabetic patients. In conclusion, the new a-glucosidase inhibitor, Bay m1099, resulted in: 1)decreased sucrose absorption, 2) decreased blood glucose excursion, and 3) decreased plasma insulin. This new drug could be a useful therapeutic drug to prevent post-prandial hyperglycaemia in Type 1 diabetes mellitus. 86. Subcellular localisation of the 64K islet cell antigen in Type 1 (insulin-dependent) diabetes M. R. Christie, A. Lernmark and S. Baekkeskov. Hagedorn Research Laboratory, Gentofte, Denmark

Onset of Type 1 (insulin-dependent) diabetes is associated with the presence of antibodies to islet cell surface and cytoplasmic components. Studies on the nature of islet cell antigens have identified an Mr-64000 human islet protein to which antibodies are found at high incidence preceeding diabetes onset. The aim of this study was to determine the subcellular localisation of the protein using rat islets as antigen source in immunoprecipitation reactions with control and diabetic sera. Detection of the protein in total islet extracts was hampered by a high background of non-specifically bound proteins. Preparation of a particulate fraction of rat islets enabled the identification of an Mr-64000 protein with similar antigenic properties to the human 64K antigen. The protein was extracted into a detergent phase on temperature-induced phase separation of Triton X-114 islet extracts, a characteristic of integral membrane proteins. On fraction-

507 A ation of islet membranes by Percoll density gradient centrifugation, the 64K protein was recovered in a fraction enriched in the plasma membrane marker 5'-nucleotidase. These data are consistent with the 64K antigen being an integral plasma membrane islet protein and support the hypothesis that this protein may be a primary target of autoimmune agression against B celIs. 87. The prevalence of glomerular hyperfiltration in Type I (insulin-dependent) diabetes mellitus A. Ciavarella, P. Vannini, L. Morotti 1, L.C. Borgnino2. Department of Metabolic Diseases, Institute of Clinical Pathology1, Institute of Nephrology and Dialysise, Bologna University, Italy

This research evaluated the prevalence of glomerular hyperfiltration in Type 1 (insulin-dependent) diabetes mellitus and its relationship with metabolic control diabetes duration, albumin excretion rate (AER) and diastolic blood pressure. One-hundred-four Type 1 diabetic patients, 49 male, 55 female, mean age 32_+8 years (16-53), mean duration of diabetes 12 __+8_ years (1-38) were studied. All were on CIT (85%) or CSII (15%) with a mean insulin dose of 41_+ 10 U/day (13-64). 47.7% had background retinopathy, 16.9% pre- or ' proliferative retinopathy, 32% autonomic neuropathy and 20% hypertension. The patients were subdivided into 3 groups: normoalbuminuric (AER <20 txg/min), microalbuminuric (AER between 21-200 ~tg/min) and proteinuric (AER > 200 ~tg/min) group. Fourteen healthy subjects were studied as control subjects. In all were determined: 4-h creatinine clearance, albumin and fl-2-microglobulin excretion rates (RIA), HbAlc (Bio-Rad), serum fruttosamine (Roche), mean blood glucose and glycosuria. Results: Glomerular hyperfiltration (creatinine clearance value > 148 ml/min per 1.73 m ~ as mean + 2 SD of control subjects) was found in 27% of cases and in particular in 35%, 33.3% and 13.5% of normoalbuminuric, microalbuminuric and proteinuric patients respectively. Creatinine clearance (ml/min per 1.73 m e) was significantly higher in normoalbuminuric (142+48) and microalbuminuric (135 +40) than proteinuric (117___ 31) and control (110 + 19) group. A slight but significant correlation was found between creatinine clearance and serum fruttosamine (r=0.24, p<0.01), diabetes duration ( r = - 0 . 3 0 , p<0.01) and AER (r=-0.25, p<0.01). In conclusion high prevalence of glomerular hyperfiltration can be found in Type 1 diabetes mellitus. It correlates with middle-term metabolic control, diabetes duration and AER. 88. Preliminary report on the relationship between in vitro theophylline sensitivity and immunomodulating treatment in recent onset Type 1 (insulin-dependent) diabetic patients G. Ciboddo, F. Crosti, A. Secchi, D. Pavoni, A.E. Pontiroli, E Navone, M.G. Sabbadini, C. Rugarli and G. Pozza. Istituto Scientifico San Raffaele, Cattedra di Patologia Medica V and Clinica Medica VIII, Universitd di Milano, Italy

Sixteen patients with recent-onset Type 1 (insulin-dependent) diabetes entered the trial. Five patients were treated with theophylline (800/day per os) (Group A), 5 patients with prednisone (15 rag/day/ os) (Group B) and 6 patients with placebo (Group C) for 6 months. Patients are currently followed-up for 12-18 month for: fasting blood glucose, HbA1C, insulin requirement, OKT monoclonal antibodies and in vitro theophylline sensitivity of lymphocytes in the ConA system, a marker of suppressive functions. The 3 groups (A, B, C) were equally distributed for the previously reported parameters. At the beginning of the study, 7 patients were theophylline sensitive (th+) (2of G r o u p & 3 of B, 2 of C) and 9 were not ( t h - ) (3 of Group A, 2 of B, 4 of C). Among the 7 th + patients, 4 reached the remission phase: 2 treated with theophylline and 2 with prednisone. Of the 9 t h - patients, only 2 achieved remission from Groups A and B respectively. None of the 6 placebo treated patients achieved remission (2 t h + / 4 t h - ) regardless of theophylline sensitivity. The character t h + / t h - was maintained during the whole follow-up time. No modifications were observed throughout the study in monoclonal antibodies. This preliminary report seems to indicate that theophylline sensitivity in vitro can be considered a precocious marker of the clinical response to immunomodulating drugs in Type 1 diabetic patients. 89. Effects of sulphonylureas on intracellular processing of insulin in monocytes from Type II (non-insulin-dependent) diabetic patients A. M. Ciccarone, L. Benzi, P. Cecchetti, V. Trischitta, A. Masoni, G. Di Cianni and R. Navalesi. Cattedra di Malattie del Ricambio, Ist. Clinica Medica II; Ist. Fisiologia Clinica del CNR, Pisa; Cattedra di Endocrinologia, Catania, Italy

508 A The aim of this study was to elucidate the effect of a sulphonylurea (gliquidone) on the derangements of A14-125 I-insulin (A14-I) processing in monocytes from Type 2 (non-insulin-dependent) diabetic patients (D). Studies were carried out by incubating A14-I with monocytes at 370(2 for 60 min. Six D (age 42_+5 years; 104_+4% of ideal body weight) were studied before (B) and after (A) four weeks treatment with 15 rag/day of gliquidone. Blood glucose decreased in five D (B: 173+13 vs A: 134+5 mg%, p<0.05). In these D, A14-I binding to monocytes increased (B: 2.6 + 0.7% vs A: 4.7 + 1.2%, p < 0.05) but the percentage of insulin that was internalised remained unchanged (B: 31.3 +14% vs A: 32.2+6.5%). Reversed phase HPLC analysis of internalised radioactivity showed three classes of labelled products corresponding to intact A14-I, intermediate degradation derivatives and 125-I tyrosine. Intact A14-I decreased after sulphonylurea therapy (B: 39.6 + 10% vs A: 17.6 + 8%, p<0.05) and conversely 125 I-tyrosine increased (B: 48.6+9% vs A: 74.2_+ 11%, p < 0.05). The D, in which drug had no effect on plasma glucose after four (A I) and eight (AII) weeks of therapy, showed no modification in A14-I binding (B: 4%, A I: 3.4%, A I I : 3.33%), internalisation (B: 18%, A I: 25%, A I I : 18%) and in the percentage of intracellular intact A14-I (B: 25%, A I: 24%, A I I : 39%). We conclude: 1) sulphonylureas enhance intracellular metabolism of A14-I; 2)this effect paralleled the pharmacological action of the drug.

90. The glycaemic index of chickling vetch (lathyrus sativus) tested in normal subjects and in Type 2 (non-insulin-dependent) diabetic patients M. Cignarelli, M. Rosco, M.R. Cospite, G. Garruti, G.M. Nardelli and R.Giorgino. Clinica Medica Generale e Terapia Medica III, Universitg degli Studi di Bari, Bari, Italy The present study was initiated in order to evaluate the glycaemic index (GI) of chickling vetch (CV) a bean widely used until 1950 in southern Italy; carbohydrate 52%, protein 27%, lipid 2% and fibers 5% are the main constituents. Six volunteers with Type 2 (non-insulin-dependent) diabetes (age 57.6+1.9 SEM; BMI 31.4+1.8) and five normal subjects (age 29.3 _+2.2; BMI 21.5 • 0.4) took on separate days 50 g of carbohydrate as white bread or cooked chickling vetch. Diabetic patients did not take their oral therapy on the morning of the test. The blood glucose response area was calculated geometrically and the GI of CV was expressed as a percentage of the mean area of the bread meal (mean_+ SEM). An extremely low mean GI was found in the control group (0.2 + 8.8; range - 2 2 + 24; basal/ peak glycaemic value: bread 4.15 +0.088/5.8+0.094; CV 4.16_+ 0.12/4.78 _+0.14 mm/1). Plasma insulin response to CV (180 rain area) was 21.7 + 4.7% lower than that to bread (p< 0.01) (basal/peak insulin value: bread 11.9 + 0.8/35 + 4.6; CV 12.8 + 1.0/28.6 _+2.5 ~xU/ml). Furthermore, the mean GI of CV was 31.6 + 6.8 in the diabetic group (basal/peak glycaemic value: bread 7.46 + 0.55/12.87 + 1.38; CV 7.29 + 0.408/9.94 + 1.988 mmol/1), whereas insulin response, as area, was 13.5 + 9.4% lower after the legume meal (basal/peak insulin value bread: 18.6 _+1.1/89 + 27; CV 21.8 + 1.4/56 • 23 gU/ml). These findings show that CV has a glycaemic index even lower than lentils, at present considered the legume with the lowest GI (44 + 7 SEM). 91. Effect of glucose and insulin on plasma free fatty acid concentration in human obesity M. Cigolini, C. Zancanaro, P. Moghetti, E. Bonora, V. Cacciatori and M.Muggeo. Institute of Clinical Medicine and Chair of Metabolic Diseases, University of Verona, Italy To evaluate the in vivo effect of glucose and insulin on plasma free fatty acid (FFA) concentrations in simple obesity, plasma FFA were measured during a 4 h hyperglycaemic glucose clamp (11.5 mmol/1) in 10 nonobese and 6 obese nondiabetic subjects. Somatostatin was also infused (250 mcg/h) during the third hour of clamp to inhibit glucose-stimulated insulin secretion. Plasma insulin was similar in the two groups at fasting and throtlghout the study. Plasma FFA were not significantly different in the two groups at fasting, and in both groups decreased during hyperglycaemia plus glucose-induced hyperinsulinaemia (0-120 rnin), rose during hyperglycaemia and inhibition of insulin secretion by somatostatin (120-180 min), decreased again during hyperglycaemia and recovering hyperinsulinaemia (180-240rain). However, plasma FFA were significantly higher in obese than in nonobese subjects both in the B-cell-stimulated period (0-120 min, F=48,90, p<0.001) and in the B-cell-inhibited period (120-180 rain, F = 11.70, p<0.005). The amount of glucose metabolised in the 80-120rain period, which was lower in obese subjects, was negatively correlated with the increase rate of plasma FFA during somatostatin infusion ( r = - 0 . 7 2 , p<0.01). These data indicate that the effect of insulin and glucose on plasma

Abstracts FFA is impaired in simple obesity, and that interactions exist between glucose metabolism and FFA metabolism in vivo.

92. Islet amyloid in Type 2 (non-insulin-dependent) diabetes contains Calcitonin-gene-related-peptide immunoreactivity A.C.Clark, C.E.Lewis, J.F.Morris, G.J.S.Cooper and R.C.Turner. Diabetes Research Laboratories, Radcliffe Infirmary, Oxford. Department of Human Anatomy, University of Oxford, UK Amyloid deposits in the Islets of Langerhans are a common feature of elderly diabetic patients and were found in post-mortem pancreatic tissue of 22 of 24 Type 2 (non-insulin-dependent) diabetic patients (aged 48-86 years), but were not present in that of 10 age-matched control subjects. The amyloid of all 22 diabetic patients showed Calcitonin-gene related peptide (cGRP) immunoreactivity, which was markedly reduced after preabsorption of three different cGRP antisera with either cGRP or with the putative amyloid precursor peptide, Diabetes associated peptide (DAP). Electron microscopical analysis of amyloid containing islets indicated large quantities of fibrillar amyloid between the islet capillaries and endocrine cells, usually penetrating into deep invaginations of islet B cells. Some cellular debris was present in the amyloid material. Both the diabetic patients and non-diabetic subjects had pancreatic islet cells which were variously immunoreactive for insulin, glucagon, pancreatic polypeptide and somatostatin. Scattered islet cells in both the diabetic patients and control subjects showed c G R P / D A P immunoreactivity. These results suggest that a cGRP-like peptide is in certain islet cells and is the subunit peptide of islet amyloid, DAP. 93. Unsuspected risk factors for perforating plantar ulcers in diabetic neuropathy S. Clavel, F.-M. Crausaz, C. Liniger and J. Ph. Assal. Diabetes Treatment and Teaching Unit, Medical Policlinic, Geneva, Switzerland Knowledge about footcare decreases the incidence of perforating plantar ulcers in diabetic patients, but can all patients put this knowledge into practice? Diabetic patients responsible for their own footcare were evaluated to determine their visual acuity (VA) and joint mobility (JM: minimum eyefirst metatarsal head and heel-buttock distances). Three groups were tested: 1) no neuropathy (n= 30); 2) neuropathy but no ulceration (n=21); 3) neuropathy and ulceration (n=38). The groups were comparable for age and diabetes duration. No differences in VA or JM were found between Groups 1 and 2: eye-metatarsum 39 _+16 (mean + SD) vs 41 • 15 cm; heel-buttock 13 • vs 15 + 6 cm. However in Group 3, eye-metatarsum and heel-buttock distances were 67 _+13 and 22+ 7 cm respectively (p< 0.001 for both compared with Groups 1 and 2). VA was normal in Groups 1 and 2 whereas the VA of 27/38 Group 3 patients was insufficient for correct foot examination (VA < 0.2 at 65 cm). Furthermore, all patients of Group 3 had decreased JM or VA or both and were thus physically incapable of adequate footcare. Defective VA and JM are therefore additional risk factors for plantar ulcers in diabetic neuropathy and should be systematically sought when preventive footcare is taught, in order to identify the patients unable to perform it themselves. 94. Sex, diabetes duration and microalbuminuria in Type 1 (insulin-dependent) diabetes mellitus C.F.Close on behalf of Microalbuminuria Collaborative Study Group, London/Poole/Newcastle, UK The clinical features of Type I (insulin-dependent) diabetic patients with microalbuminura (overnight urinary albumin excretion rate (AER) > 30 gg/min) were studied in 1735 (959 M, 776 F) non-proteinuric patients aged 16-60 years, with diabetes onset <39 years, diabetes duration <35years and without arterial hypertension (blood pressure < 160/95). Subjects were screened using first morning urinary albumin concentration (Ualb) and albumin/creatinine ratio. 1394 subjects had Ualb <15 mg/1; in a random sample of 65 of these AER never exceeded 30 ~tg/min. Of 341 subjects with Ualb > 15 gg/ml and/or albumin/creatinine ratio > 3.5 mg/mmol, AER measured in 154 was >30 ~tg/min in 45, yielding a calculated prevalence of 6%. In the first 5 years of diabetes AER ->30 ~tg/min was not observed; diabetes duration was longer in microalbuminuric than normoalbuminuric subjects 19+8 vs 14+9years (p<0.01). Male preponderance was found in microalbuminurics in the ratio 2.5:1 (32M, 13 F) compared to 1.2:1 in normoatbuminurics and 1.2:1 in the total study cohort. Mean age was 36+12 years in microalbuminurics and 32_+10 years in normoalbuminurics. Systolic and diastolic blood pressure was significantly higher in mieroalbu-

Abstracts

509 A

minuric than normoalbuminuric subjects, 132_+17 vs 123+ 14 mmHg (/7< 0.005) and 78_+ 11 vs 73 + 9 mmHg (p< 0.005) respectively. Microalbuminuric subjects are characterised by male preponderance, longer diabetes duration and higher arterial pressure. Microalbuminuria is a sign of early renal disease, not a marker of susceptibility.

I concentration was significantly higher (153 +93 vs 60_+30, A vs B, p < 0.05) and the C / I molar ratio was significantly lower (3.8 _+1.1 vs 8.9+2.9, A vs B, p<0.001) in Group A than in Group B. The low C / I molar ratio together with the high plasma I concentration suggest that a diminution of plasma insulin clearance contributes to the hyperinsulinaemia seen in our NB of Type 1 mothers.

95. Increased neutrophil elastase activity in Type 1 (insulin-dependent) diabetic patients A.Collier1, M.Jackson 2, D.Bell 2, A.W.Patrick 1, D.M.Matthews 1, R. J. Young 1, B.F. Clarke 1 and J. Dawes 3. 1Diabetic Department and 2Department of Medicine, Royal Infirmary, Edinburgh, 3Blood Components Assay Group, Forrest Road, Edinburgh, U K

98. Expression of ductular and exocrine markers on on plnripotent rat islet tumour cells indicate common endodermal origin of endo- and exocrine pancreas G. Contreas, J.L. Jorgensen, T. Beck and O.D. Madsen. Hagedorn Research Laboratory, Gentofte, Denmark

Numerous abnormalities of polymorphonuclear leucocytes (PMN) have been described in diabetes mellitus. The PMN neutral protease, elastase, can cause endothelial damage and could contribute to the development of microvascular complications. The aim of this study was to determine whether PMN elastase, measured by radioimmunoassay, is altered in diabetes. One hundred diabetic patients (92 M, 18 F) aged 29.6_+3.9 years (mean_+SD) with duration of diabetes 12.6_+6.6years and 35 comparable control subjects (25 M, 10 F; 29.8 + 4.45 years) were studied. There was no difference in the total white cell counts (7.3 _+2.1 vs 6.6 _+1.9 x 109/1) but the diabetic group had a higher PMN count (5.0 _+1.7 vs 4.3 _+1.5 x 109/1, p < 0.05). Plasma neutrophil elastase (PNE) (28.7 _+22.2 vs 18.8 _+6.5 Ixg/ml, p < 0.02) and total neutrophil elastase (TNE) (6798_+3061 vs 8735_+ 4352 Ixg/106 PMN, p<0.02) were increased in the diabetic group. PNE was significantly higher in diabetic patients with exudative and proliferative retinopathy than in those with early background or no retinopathy (35.0_+ 32.8 vs 25.5 _+13.8, p < 0.02). There was no correlation with age, duration of diabetes, plasma glucose or HbA. Conclusion: Plasma neutral elastase is increased in diabetes consistent with neutrophil activation; the increase is greater in those with advanced microangiopathy. 96. Glucose synthesis from glycerol in isolated hepatocytes from fasted rats: effects of thyroid status and redox state B.Comte and H.Vidal. Inserm U. 197. Facult~ de M~decine Alexis Carrel, Lyon, France Glucose production from glycerol has been measured in isolated hepatocytes from normal (N), thyroidectomised (Tx) and normal rats treated with various amounts of triiodothyronine (T3) (1, 5, 10, 50 Ixg T3/100 g BW/day, 3 days) with and without ethanol (10 mmol/1) or pyruvate (10 mmol/1). Glucose production (G) (Ixmoles.g-a.h -a, mean_+ SEM) in N (n= 5, 41 _+3) was inhibited by ethanol (25 +_3) and stimulated by pyruvate (77_+6). G in Tx was significantly decreased (n = 6, 15 + 2), completely suppressed by ethanol (0_+ 0.6) and normalised by pyruvate (67_+ 4). G in T3 was significantly increased only in rats receiving the highest T3 treatment but in the other treatment group the suppression by ethanol was suppressed and the stimulation by pyruvate decreased. In no case was lactate production from glycerol affected by the thyroid status. Glycerol-3-P 4was increased in Tx (6.5_0.7 vs 2.7+0.5 in N (#moles.g- I . h - 1) and decreased in T3 (0.26 _+0.2). Gluconeogenesis from lactate, pyruvate, glycerol was increased significantly only in rats treated with pharmacological dose of T3 (50 Ixg T3/100 g BW/day, 14 days). These results suggest that T3 modifies the regulation of glycerol metabolism by increasing the capacity of the liver cells to translocate reducing equivalents. The classical gluconeogenic effect is present only with pharmacological dose of T3. 97. The C-peptide/insulin molar ratio is decreased in the new-born of Type I (insulin-dependent) diabetic mothers T. Constans, Y. Bacq, C. Couet, A. Fignon, M.-A. Garrigue, T. Niyongabo, J. Delarue and F. Lamisse. H6pital Bretonneau, Tours, France To explore the mechanism of the hyperinsulinaemia seen in the newborn (NB) of Type 1 (insulin-dependent) diabetic mothers, we measured the plasma insulin (I, pmol/1) and C-peptide (C, pmol/1) concentrations in the cord's blood from 8 NB of Type 1 mothers (Group A) and 8 NB of non-diabetic mothers (Group B). Type 1 mothers with insulin antibodies > 5% were excluded. Blood glucose control was optimised with a continuous subcutaneous insulin infusion 2 days before and during delivery. The timing of delivery (38.2+0.9 vs 38.2_+0.7 weeks, A vs B; m e a n + S D ; Mann & Whitney; NS), the glycosylated haemoglobin values (7.0_+0.9 vs 6.1_+ 0.9%, A vs B; NS), and the NB blood glucose levels (3.4_+1.0 vs 3.7_+ 1.3 retool/l, A vs B; NS) were similar in both groups. The

A panel of monoclonal (auto)antibodies (Mab) have been generated with strong reactivity to the rat islet tumour cells, MSL These cells possess an endocrine stem celt-like differentiation potential, since monoclonal cultures are highly heterogeneous and express several islet hormones. Some Mab, besides their reactivity to the MSL cells, defined particular cell types on frozen sections of normal pancreas by indirect immunofluorescence. Two Mab stained subpopulations of islet cells, while one Mab reacted to islet B cells and to few scattered exocrine cells. Three Mab stained the ductal tissue, where two of them reacted to the intercalated ducts only, and one Mab bound to the ductal epithelium including the scattered endocrine cells of the ducts. A group of Mab specifically reacted to the acinar cells where either cytoplasmatic or zymogen granule staining was observed. Exocrine and duct-reacting Mab's stained scattered tumour cell populations on sections of monoclonal MSL-tumours. We conclude that the endocrine differentiation potential of MSL cells can be extended to include also ductal and exocrine properties as defined by various Mab. These data strongly support the hypothesis of common endodermal origin of endo- and exocrine pancreas. 99. A double-blind controlled trial of Azathioprine in children with newly-diagnosed Type I (insulin-dependent) diabetes J. J. Cook, I. Hudson 1, L.C. Harrison 2, B. Dean 2, P.J. Colman 2, G. A. Werther, G. L. Warne and J. M. Court. Dept. of Endocrinology & Diabetes and Dept. of Biostatistics 1 Royal Children's Hospital (RCH), and Dept. of Diabetes & Endocrinology, Royal Melbourne Hospital,2 Melbourne, Australia We undertook a double-blind controlled trial of azathioprine (2 mg.kg-a.day -1) in 49 newly diagnosed Type 1 (insulin-dependent) diabetic patients, aged 2-20 years. Subjects were randomised to receive either azathioprine (n=24) or placebo (n=25) for 12 months, beginning within 20 days of diagnosis. Baseline clinical and metabolic characteristics did not differ between the two groups. No subject entered a complete remission, defined as restoration of normal carbohydrate tolerance without other treatment. Partial remission, defined as good metabolic control (haemoglobin Alc < 7.5 g%, pre-prandial blood glucose _<8 mmol/1 on an insulin dose of < 0.5 Ix-kg 1. day-l) occurred in 10 (40%) placebo and 7 (29%) azathioprine subjects at 6 months and in 4 (16%) placebo and 4 (17%) azathioprine subjects at 12 months. (Differences, NS). C-peptide responses to a standard breakfast and the frequency of islet cell and insulin antibodies did not differ overall between the two groups over the 12 month period. Azathioprine caused no significant side effects. We conclude that in the dosage used, this immunosuppressive agent does not influence the remission phase in children with newly diagnosed Type 1 diabetes. 100. Absence of insulin activation of pyruvate dehydrogenase in brown adipose tissue from goldthioglucose obese mice G. J. Cooney, E F. Williams, I.D. Caterson and J.R. Turtle. Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, Australia Lack of diet-induced thermogenesis in brown adipose tissue (BAT) is an important factor in the development of obesity in rodents. Insulin resistance and Type 2 (non-insulin-dependent) diabetes subsequently develop. BAT is insulin resistant in goldthioglucose obese mice as demonstrated by reduced uptake of [1-~4C]-2-deoxyglucose in vivo. The cause of this insulin resistance has not been determined. Since the activity of pyruvate dehydrogenase (PDH) is a major determinant of glucose utilisation in tissues, we have measured the effect of glucose-induced insulin release on the activity of PDH in BAT from control and 4-week goldthioglucose obese mice to determine if insulin resistance in vivo is paralleled by alterations in insulin activation of PDH. PDH was measured spectrophotometrically in BAT from overnight fasted or glucose-injected (1 g/kg) control and

510 A obese mice. Glucose-induced insulin release significantly increased the activity of PDH in BAT from control animals [Fasted= 1.10+ 0.15 units/g, Glucose injected=2.26+0.21 units/g]. However there was no significant difference in PDH activity in BAT from fasted (1.12+0.13 units/g) or glucose-injected (1.26_0A0units/g) obese mice. The absence of insulin activation of PDH in BAT from obese mice may be important in the development of insulin resistance in this tissue. 101. Enalapril reduces albuminuria in hypertensive and normotensive models of diabetic uephropathy M. E. Cooper, T.J. Allen, G. Jerums and A. E. Doyle. Endocrine Unit, Department of Medicine, Austin Hospital, Heidelberg, Victoria, Australia This study has evaluated the effects of angiotensin converting enzyme inhibition with enalapril on the evolution of proteinuria in normotensive and hypertensive diabetic rats. Diabetes was induced by intravenous injection of streptozotocin (STZ) at week 0 in Wistar Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR). Enalapril (35 rag/l) was administered in drinking water from week 0 in WKY and from week 2, 0 or 12 in SHR. Basal albuminuria in all rats was less than 0.5 mg/24 h. Marked increases occurred in 24 h albuminutia after 28 weeks, especially in hypertensive rats (SHR-STZ 59 rag, n= 11 vs WKY-STZ 2.7 mg, n= 8, p < 0.001, medians shown), and also in glomerular basement membrane thickness (SHR-STZ 168 + 8 nm vs 140 _+5 nm, p < 0.01, mean + SEM). Enalapril administered from week 0 in WKY-STZ decreased albuminuria (0.9 mg/day, n= 5, p < 0.01). In SHR-STZ, enalapril administered from week 2, 0 and 12 (coinciding with the increase in proteinuria) reduced 24 h albuminuria at 28 weeks (5.5 mg [n=6, p<0.001], 11 mg [n=8, p< 0.05] and 15.6 mg [n= 8, p < 0.05] respectively). Thus, enalapril may retard diabetic nephropathy in the rat, in the presence or absence of hypertension. In the hypertensive diabetic rat, early commencement of angiotensin converting enzyme inhibition appears to confer additional benefit. 102. Regulation of insulin receptor associated tyrosin kinase by a polyclonal IgG R.Cordera, G. Andraghetti, R.Gherzi, G. Versari, R. De Pirro 1. Dpt. of Internal Medicine, University of Genoa, 1Chair of Endocrinology, University of Ancona, Italy Insulin receptor beta-subunit is an insulin- and ATP-dependent tyrosine ldnage (IRTK), which phosphorylates itself and exogenous substrates. To date the role and regulation(s) of this enzymatic activity are still uncertain. Using a human polyclonal anti-insulin-receptor antibody (pIgG) and partially purified insulin receptors, relationships between insulin binding and tyrosine ldnase activity toward the substrate Glu4-Tyri were investigated. The effect of pIgG on IRTK was studied utilising both insulin or ATP activated insulin receptors. When the reactions were measured at equilibrium, plgG reduced 32P incorporation into Glu4-Tyri by 51%. To better understand the effect of plgG on IRTK the initial rate of the exogenous substrate phosphorylation as function of substrate concentration was studied. When IRTK was activated by insulin, plgG reduced the Vmax (from 61 to 30.8 p m o l . m i n - 1 . m s - 1) without changes of Km; on the contrary, when IRTK was activated by ATP pre-incubation, pIgG was unable to affect the initial rate of the reaction (62 versus 60.8 pmol. min -1. mg-~). These results clearly demonstrate: 1)that pIgG is unable to affect IRTK when insulin receptors are already phosphorylated; 2)that pIgG inhibits IRTK through some conformational change in the ATP binding site of the receptor molecule. Therefore insulin receptor kinase possesses at least two independent regulatory mechanisms: one of which is affected by pIgG. 103. Effects of low protein dietrat weaning on insulin secretion and sensitivity C. J. Crace, I. Swenne, L. Jansson and R. D. G. Milner. Department of Paediatrics, University of Sheffield, U K We have previously shown that feeding a low-protein (5%, LP) diet to young rats caused long-term impairment of glucose-stimulated insulin secretion in vivo and temporary impairment of glucose tolerance which recovered on transfer to normal (18% protein, N) rat chow. A decrease in glucose-sensitivity of pancreatic B cells and/or increased peripheral insulin sensitivity were suggested; and have been investigated in the present study. Rats were weaned at three weeks of age onto LP or N diet and at 6 weeks LP rats were refed N

Abstracts diet. Pancreatic islets were isolated from rats aged 3, 6 and 12 weeks and their insulin secretory response to glucose and arginine tested. Glucose-stimulated insulin release by 6-week LP islets was only 25% of that of 6-week N islets, and the arginine response was absent. Islets from 12-week LP rats showed 40% of the glucose response of 12 week N rats and no arginine-stimulated insulin release. However, parallel studies in vivo demonstrated that LP rats showed a significantly greater fall in plasma glucose than age-matched N rats in response to a bolus of 200 mU/kg insulin. We conclude that, whilst early protein-calorie malnutrition causes long-term impairment of Bcell function, increased insulin sensitivity in vivo allows for adequate glucose regulation in the medium term. 104. Metabolic control and diabetic retinopathy. Italian National Research Council (CNR) study. Two-year report G.Crepaldi, D.Fedele, R.Nosadini, D. Bruttomesso, T. Segato, S.Piermarocchi, Univ. of Padova; P.Brunetti, M.Massi-Benedetti, F. Santeusanio, G. Santoni, G. Lupidi, Univ. of Perugia; G. Pozza, E Micossi, R. Brancato, U. Menchini, F. Bandello, Univ. of Milano; F. Grigoletto, F. Cavarzeran, Fidia Reg. Lab. Abano Terme, Italy A multicenter prospective study was designed by the Italian CNR and carried out by University of Padova, Milano, Perugia and Fidia Research Laboratories, to evaluate the effects of continuous subcutaneous insulin infusion (CSII) or conventional insulin therapy (CIT) on the evolution of background diabetic retinopathy with small and non-confluent ischaemic areas. Thirty-eight Type 1 (insulin-dependent) diabetic patients were blindly assigned by couple randomisation to CSII and CIT after matching for sex, duration of diabetes and ocular status. To assess the evolution of retinopathy, fluoroangiographic changes of ischaemic areas were blindly evaluated and reported as improved, stable, worsened. Degree of ischaemia was also scored in nasal and central regions by means of a quantitative method (Doknmator). Means of 7 daily glucose determinations were: basal CSII 179 _+11, CIT 181 + 9; 24 months CSII 147 + 7, CIT 179 + 9, p < 0.01. Retinal outcome after 24 months was: improved CSII 2, CIT 0; stable CSII 8, CIT 8; worsened CSII 9, CIT 11; p= NS. Systolic blood pressure, higher in patients with retinal worsening, showed correlation with retinal deterioration (logistic regression analysis). No evident correlation between blood glucose levels and retinal worsening was documented. Both CSII and CIT ameliorated blood glucose levels, even if glycaemic control was slightly better in CSII. 105. Anti-immunoglobulin antibodies in diabetic children at diagnosis and follow-up: correlation with islet cell antibodies and other autoantibodies L. Crisa, U. Di Mario, E. Anastasi, D. Andreani, A. Candela, M. P. Raponi, M.Vela and L. Campea. Dept. of Endocrinology and Pediatric Diabetic Unit, University of Rome, Italy The presence of antibodies reacting with immunoglobulins of different species in sera from recent onset Type 1 (insulin-dependent) diabetic patients has been shown by us in pilot experiments. In this study the occurrence of this additional immunopathological phenomenon has been evaluated in sera from Type 1 diabetic patients at diagnosis and at a length of up to three years after, and correlated with that of islet cell antibodies (ICA) and of other organ-specific autoantibodies. Eighteen newly-diagnosed Type I diabetic children and 26 normal subjects were included in the study and followed with time. Serum samples, diluted 1/20, were incubated in goat immunoglobulin-coated wells; after washings, 125-I-anti-human immunoglobulin highly purified antibodies were added. Anti-immunoglobutin antibodies were present at diagnosis in Type 1 diabetic patients (p<0.001 vs normal control subjects) and declined with time after diagnosis (p<0.001). While at diagnosis a significant correlation in positivity, but not in titres, between anti-immunoglobulin antibodies and ICA was found (p< 0.01), in the follow-up study this correlation was lost, the positivity percentage for anti-immunoglobulin antibodies being higher. No correlation was found with other organ-specific autoantibodies. As is also likely in other autoimmune disorders, an immunoglobulin anti-immunoglobulin could play an important part in the complex immunopathogenctic interactions in Type 1 diabetes. 106. Plasma dehydroepiandrosterone sulfate in subjects with various glucose tolerances P.V.M.Cromme, C. Popp-Snijders and E.A.van der Veen. Department of Endocrinology, Free University Hospital, Amsterdam, The Netherlands

Abstracts It has been suggested that the dehydroepiandrosterone sulfate (DHEAS) level in plasma is inversely related to death from cardiovascular disease (CD). The aim of our study was to investigate whether patients with impaired glucose tolerance (IGT) or Type 2 (non-insulindependent) diabetes, being at increased risk of CD, had lower plasma DHEAS levels than subjects with normal glucose tolerance. We determined fasting plasma DHEAS levels in 405 subjects, over 65 years of age. After a standard oral glucose tolerance test all subjects were classified, according to WHO criteria, into non-diabetic subjects (n= 213); IGT (n=98); Type 2 diabetic patients, newly diagnosed (n=62); Type 2 diabetic patients, treated (n=32). DHEAS levels in these groups were: 1.94 (0.1-16.9); 1.82 (0.1-9.2); 2.40 (0.4-14.8); 2.45 (0.1-13.3) ~tmol/1, median (range) respectively. The DHEAS levels in men were significantly higher than those in women: 2.71 (0.1-16.9) vs 1.53 (0.1-14.0) l.tmol/1, p<0.001. Although the DHEAS levels in the diabetic groups tended to be higher than in the non-diabetic and IGT groups, significance was reached only in men (Type 2, treated vs IGT, p < 0.05). A positive correlation between DHEAS and HbAI was seen in the group Type 2, treated (r=0.334, p<0.05), indicating higher DHEAS levels in poorly-controlled patients. We conclude that the increased risk of CD seen in IGT and Type 2 diabetes is not related to decreased plasma DHEAS levels.

511 A 109. Insulin aggregation in solution

M.Dathe, K.Gast, D.Zirwer and B. Mehlis. Institute of Drug Research and Central Institute of Molecular Biology, GDR Academy of Sciences, Berlin, GDR Various conditions influencing the phenomenon of insulin aggregation have been described but the mechanism of particle formation and the role of factors affecting the process are still poorly understood. We report the kinetics of insulin particle growth as revealed by dynamic light scattering. The process of aggregation in insulin solutions of different concentrations was monitored at 37 ~ and continuous rotation of the samples over a period of four weeks. The starting solutions contained hexamers as the largest structures, the hydrodynamic radii of which were determined to be about 2.5 nm. Although the solutions did not evolve perceptible turbidity within the experimental period an increasing scattering intensity was registered. However, this increasing intensity did not result, as expected, from an increasing number of particles with continuously growing dimensions. Instead of this, particles with radii of about 100 nm or larger were exclusively formed, the number of which considerably increased in the course of time. We assume this I00 nm class of insulin aggregates to be the main transient state in the inactivation process of insulin solutions within delivery systems.

107. Improved cardiac performance after a short period of strict metabolic control in diabetic patients

D.Cucinotta, A. Di Benedetto, C. Lamberti, A. Mileto, A. Saitta, M. Cinquegrani and G. Squadrito. Istituto di Medicina Interna, Universitfi di Messina, Italy Non-invasive studies of cardiac performance in diabetic patients without evidence of heart disease often show abnormalities of left ventricular function. To better evaluate the relationship between these abnormalities and metabolic control, we performed an echocardiographic study on 8 poorly-controlled Type 1 (insulin-dependent) diabetic patients (6 males, mean age 38 _+6 years, mean illness duration 17 +__4 years, no evidence of ischaemic heart disease or major diabetic complications) before and after a 24 h period of normoglycaemia, achieved with an artificial pancreas. With respect to a control group, in basal condition (mean blood glucose 15.6+ 1.3 mmol/1) diabetic patients had significant (p< 0.01) alterations of: pre-ejection period (PEP, 119.8 msec in diabetic patients, 94+ 8 in control subjects), left ventricular ejection time (LVET, 270 + 12 and 303 + 15 msec), ejection fraction (EF, 52 + 7 and 70 + 8%), fractional shortening (FS, 2 2 + 4 and 34+5%) and mitral opening delay (MOD, 40 + 5 and 25 _+2 msec). After the 24 h period of normoglycaemia (mean blood glucose 6.8_+ 0.7 mmol/1) there was a significant (p< 0.05) improvement of left ventricular function, both in the systolic (PEP 106+7, LVET 285+18, EF 61 +6, FS 28+3) and in the diastolic (MOD 34+ 3) parameters. These results show that cardiac abnormalities may be observed in poorly-controlled diabetic patients but they seem mainly related to the metabolic disorder, since they significantly improve after normoglycaemia.

110. Glucose tolerance and its evolution in a Caucasian subpopulation at risk

C. H. Daumerie, J.M. Ketelslegers, G. Gerard and A. E. Lambert. Division of Endocrinology and Nutrition, University Hospital St. Luc, Brussels, Belgium Oral glucose tolerance tests (75 g) were performed in 681 male subjects (25-64 years) selected by postprandial glucosuria and/or hypertriglyceridaemia. Some of them were followed during 8 years. Initially, 70% were normoglycaemics, 19% had glucose intolerance (IG) and 11% had diabetes. Body mass indices (BMI) were 26.6+0.2 (mean_+ SEM), 27.8 + 0.3 and 28.3 __+0.4 _ in normoglycaemics, IG and diabetic patients respectively (normoglycaemics < IG and diabetic patients; p<0.01). Peak insulin response was delayed in IG and blunted in diabetic patients (116+7 in IG vs 84+7 btU/ml at 120 rain, p < 0.05). Two h plasma glucose and insulin correlated positively in normoglycaemics (r= 0.38, p < 0.001) but negatively in diabetic patients (r= - 0.29, p = 0.01). After 2 years, 11 of 119 nmxnoglycaemics became diabetic; this was associated with higher initial fasting plasma glucose (multiple logistic model, p < 0.05). 28 of 69 IG and diabetic patients reverted to normoglycaemia (predictive parameter: integrated plasma glucose, p < 0.05). Changes in 2 h glucose and insulin correlated with changes in BMI (r= 0.24 and 0.29, respectively, p < 0.001). After 5-8 years (n = 67), glucose tolerance deteriorated to IG or diabetes in 32% of normoglycaemics and normalised in 48% of IG. In conclusion, 30% of the patients originally studied had IG or diabetes. Initial plasma glucose may be predictive for improvement or deterioration of glucose tolerance.

108. Influence of glycaemic control on insulin absorption rate

P. Damsbo, S. Madsbad, D. Herly, P. Hildebrandt and J. Hilsted. Hvid6re Hospital, Klampenborg, Denmark The rate of subcutaneous absorption (ABS) of 8 IU 125I-labelled soluble insulin (external gamma-counting) and subcutaneous blood flow (SBF) (washout of ~33 Xe) was measured in 8 Type 1 (insulin-dependent) diabetic patients on two randomised days. Day 1: during the last hour of 8 h of hyperglycaemia (14.9_+0.4 moaol/1) and during 3 h succeeding normoglycaemia (6.0+0.6 mmol/1). Day 2: during the last hour of 8 h near-normoglycaemia (7.3 + 0.4 retool/l) and during 3 h of spontaneous recovery after insulin induced hypoglycaemia (nadir: 1.9 + 0.1 retool/l). ABS and SBF were expressed by the rate constant of disappearance (k) calculated by use of the least square method from the respective curves of the logarithmically transformed count rates versus time. On Day 1 ABS (k x 10- 2) was 2.29 _+0.50 during hyperglycaemia and 3.19 + 0.48 during normoglycaemia (/9=0.05). SBF ( k x l 0 -3) was 2.57+0.49 and 2.55+0.45 (NS) respectively. Day2: during near-normoglycaemia ABS was 1.65 _+0.24. The first, second and third hour after hypoglycaemia increased ABS by 20%, 32% (p< 0.05), and 44% (p< 0.01), respectively, in comparison with ABS during near-normoglycaemia. SBF did not change significantly during the study period. In conclusion, the glycaemic control, especially rapid decrements in blood glucose, has influence on subcutaneous insulin absorption. Other factors than subcutaneous blood flow seem also to be of importance for insulin absorption.

111. rDNA human proinsulin produces excellent metabolic control of

Type I (insulin-dependent) diabetes J. K. Davidson, L.D. Satterfield and R.M. Rolfes. Department of Medicine, Emory University Atlanta, Georgia, USA Study objectives: To compare rDNA human proinsulin (PI) and rDNA human NPH insulin (NPHI) in Type 1 (insulin-dependent) diabetes. Design: With Human Investigations Committee approval and signed informed consents, nine were randomly assigned to PI and nine to NPHI. Thirteen males, 5 females mean age 36.3 + 2.6years (SEM). PI group: initial plasma glucose 33.76+ 5.08 mmol/1, haemoglobin Alc 10.5+1%, weight 101+3.6% of ideal. NPHI group: initial plasma glucose 23.92 + 3.31 mmol/1, haemoglobin Alc 10.5+0.3%, weight 102+3.8% of ideal. Results: PI: Initial dose 26.7+3.1 units decreased by 12.9 units in nine months follow-up to dose of 13.8 units+ 1.6 units. Mean follow-up fasting plasma glucose was 124_ 10 mg/dl (range 95-189) and haemoglobin Ale 5.5+0.6%. NPHI: Initial dose 22.1+2.6units increased by 12.3 units in nine months follow-up to dose of 34.4+3.5 units. Mean follow-up fasting plasma glucose was 12.76+2.35 mmol/1 (range 105-460), haemoglobin Alc 5.9+0.5. Conclusions: A mean dose of 13.8 units PI was more effective than a mean dose of 34.4 units NPHI in controlling fasting plasma glucose levels (6.85 vs 12.76 mmol/1, p < 0.05).

512 A 112. Increased responsiveness to insulin-releasing effects of adrenocorticotropin (ACTH) and ACTH fragments in obese hyperglycaemic (ob/ oh) mice C.Day, C.J.Bailey and P.R. Flatt. Department of Molecular Sciences, Aston University, Birmingham and Department of Biochemistry, University of Surrey, Guildford, UK Increased responsiveness to adrenocorticotropic hormone (ACTH) and ACTH fragments might contribute to the hyperinsulinaemia of genetically obese hyperglycaemic (ob/ob) mice. To investigate this possibility, plasma glucose and insulin responses of 11-14 week-old fed lean and ob/ob mice were examined after intraperitoneal administration of ACTH 1-39, ACTH 1-24 and ACTH 18-39, each at a dose of 25 nmol/mouse (50-115 lxg/mouse). ACTH 1-39 produced a marked and rapid increase of plasma insulin in both lean and ob/ ob mice, the effect being much greater in the ob/ob mutant (maximum increases of 5.5+_1.5 and 46./• ng/ml at 10min in lean and ob/ob mice respectively, p<0.000/). In lean mice, plasma glucose concentrations showed a protracted decrease (maximum decrease of 3.7 • 0.5 mmol/1 at 120 min), whereas glucose concentrations were increased (maximum increase of 4.1• mmol/1 at 60 rain) in ob/ob mice. ACTH 1-24 produced qualitatively similar but generally smaller effects than ACTH 1-39, while ACTH 18-39 did not significantly affect glucose and insulin concentrations. In 24 h fasted mice, ACTH 1-39 produced similar but generally smaller effects than in fed mice. The results suggest that (1) effects of ACTH on glucose and insulin homeostasis are conferred by the N-terminal 1-24 sequence, and (2) ACTH can exert acute effects which contribute to the hyperinsulinaemia and hyperglycaemia of ob/ob mice. 113. The influence of pump therapy on the remission period in children with Type I (insulin-dependent) diabetes C. E. de Beaufort I, G.J. Bruining I, C.M.C.J. Houtzagers 2, R.S.R.Aarsen 1, N.C.den Boer3, W.G.F.Grose 3, R.van Strik4 and 1 1 2 J.J. de Visser. Department of Pediatrics, Department of Diabetolo3 gy, Free University of Amsterdam, Holland, Department of Clinical Chemistry, 4Department of Biostatistics, University of Rotterdam, Holland From discovery onwards 30 previously randomised newly diagnosed Type 1 (insulin-dependent) diabetic children were treated either by continuous subcutaneous insulin infusion or by conventional injection therapy. Semisynthetic human insulins (NOVO) were used in both groups. Final follow-up was 2 years. HbA1 values were determined monthly by gelelectrophoresis. Glucagon stimulation tests were performed once every 6 months and urinary C-peptide excretion (24 h urine, collected on ice) was measured monthly to evaluate the endogenous insulin reserve. C-peptide was measured according to the method of Heding. The ages of the 2 groups differed significantly (pump group: 7 girls, 8 boys, 8.9• years; conventional group: 8 girls, 7 boys, 6.5• years). None of the participants dropped out. After 2 years of therapy the pump group showed a significantly better glycaemic control (sHbA1 : pump group: 9.5 • 0.3%, conventional group: 10.9+_0.4%, p<0.05). The insulin doses did not differ after 2 years. Neither urinary C-peptide excretion nor stimulated C-peptide values differed significantly after 2 years (urinary Cpeptide values: pump group - 2.3 • 0.7 nmol/1, conventional group 1.2• nmol/1; stimulated C-peptide values: pump group 0.16 4- 0.03 nmol/l; conventional group - 0.12 • 0.02 nmol/1). Prolonged pump therapy in children results in a better glycaemic control. No difference is found in the endogenous insulin reserve after a therapy by pump or by injections. 114. Effect of anti-insulin serum on pancreatic B-cell proliferation in vitro L. De Clercq, B.R. Reusens-Billen, P. Delvaux, C. Remacle and J. J. Hoet. Department of Cell Biology, Catholic University of Louvain, Louvain-La Neuve, Belgium Type 1 (insulin-dependent) diabetic patients show a progressive destruction of the B cells and the arrest of their regeneration which may lead to total disappearance. Because anti-insulin antibodies are detected in Type 1 (insulin-dependent) diabetic patients after insulin therapy or even before the onset of the disease, the purpose of our study was to analyse the possible influence of the anti-insulin serum (AIS) on fetal rat B-cell proliferation in vitro. Cultured islets were incubated during two days in media comaining various calf serum concentrations without or with AIS in two doses. Incorporation of H3-thymidine in islet cell nuclei was analysed by autnradiography on semi-thin sections. The labelling indexes of the islets cultured in the

Abstracts presence of AIS were significantly lower than in the controls. The low AIS dose reduced the proliferation rate by 27.5% (p< 0.001) and a three-fold AIS dose by 37.2% (p< 0.001) when compared with the controls. Cultures of other cell types (pre-adipocytes, mesenchymal cells) were performed in order to determine if the AIS exerts its inhibitory effect only on the B cell. Our results indicate the inhibitory role of AIS on B-cell proliferation. It may explain the reduced B cell mass observed in Type I diabetic patients. 115. Modest, asymptomatic decreases in arterial plasma glucose already impair brain function and activate hormonal counterregulation in man P.De Feo, V.GaUai, G.Mazzotta, E.Torlone, M.M.Ventura, V.De Angelis and P. Brnnetti. Institutes of Medical Pathology and Clinical Neurology, University of Perugia, Italy To assess the effects of modest decreases in arterial plasma glucose (PG) concentration on brain function and counterregulatory hormones (CR-H), 12 normal subjects were studied during insulin/glucose infusion to maintain either euglycaemic or hypoglycaemic target PG. Cerebral function was evaluated by means of auditory event-related potentials by measuring the latency of the 13300wave, a specific and objective index of cortical function. Cognitive brain function deteriorated as shown by the increased ]?3o0wave latency (from 296 +- 9 to 347 + 26 msec, p < 0.05) when PG was decreased by only 1 mmol/1 over 20 rain (from basal 4.7 + 0.1 to 3.9 + 0.2 mmol/1) and deteriorated further when PG was allowed to decrease at lower levels (PG 3.2• mmol/1, P300 359• PG 2.7• P300 373 _21 msec). After 30 min of restoration of normoglycaemia P30o latency was normalised (317 • 13 msec, p = NS). There was a negative correlation between plateau PG and P300latency (r= -0.61, p < 0.02). Finally, when PG was decreased by only 0.8 mmol/1 (from 4.8• to 4.0+0.3 mmol/1) for 150 min P300 latency remained increased throughout (367+19 vs 301+12 msec, p<0.01) in the absence of symptoms of hypoglycaemia, but all CR-H increased significantly (glucagon, epinephrine, growth hormone and cortisol, p < 0.05). Conclusions: minimal PG decrements (1 mmol/1) are potentially dangerous because they impair brain function in the absence of hypoglycaemic symptoms and activate CR-H. 116. Influence of insulin antibodies on insulin absorption and serumfree insulin levels P. H. E. M. de Meijer, J. A. Lutterman and A. van 't Laar. Department of Medicine (Division of General Internal Medicine) University of Nijmegen, The Netherlands To investigate a possible effect of insulin antibodies (Ab) on the absorption rate of subcutaneously (sc) injected insulin, we compared 2 groups of Type 1 (insulin-dependent) diabetic patients after sc injection of 12 U 125I-Actrapid. Group 1 (n=9) had virtually no Ab (same concentrations as in healthy volunteers), but Group 2 (n = 14) consisted of patients with high concentrations of insulin Ab (more than 25% binding of labelled insulin, added to insulin-free serum in the presence of 1000 pg cold insulin per 25 Ixl serum). Both groups were similar in mean age, sex, weight, duration of diabetes and thickness of sc fat. The day before the study patients used only shortacting insulin, 4 times a day; during the study patients were in a fasting state. Disappearance curves of radioactivity could be described with a 2-compartment model: Y = 100- (K~d.e-(Kd + K12)ti-- K12"e-(K~ + I~d)t)

K~d-K~2

In Group I the rate constants K12, K~a, I~ (mean • SD) were 0.49_ 0.20, 0.40+0.23 and 0.08+0.09% per hour; in Group 2 1.06+2.09, 0.31 + 0.28 and 0.02 + 0.24 (NS). Peak levels of insulin were reached after a mean of 90 min in both groups and were 42.9 + 18.7 and 38.8 + 18.6 ~tU/ml respectively (NS). Mean serum-free insulin curves were not significantly different. In conclusion: insulin antibodies do not influence insulin absorption rate or serum-free insulin levels. 117. The insulin-like effects of human growth hormone in rat adipocytes: a subset of kinase-C mediated pathways of insulin action? P. de Meyts and J. Smal. Department of Diabetes, Endocrinology & Metabolism City of Hope National Medical Center, Duarte, Calif, USA Human growth hormone (hGH) is traditionally diabetogenic, i.e. an antagonist of insulin actions. However, in models devoid of endogenous hGH, it behaves transiently as an insulin-like agent, e.g. stimulates lipogenesis in isolated rat adipocytes preincubated 4 h in medium without GH. This effect is mediated through a receptor different

Abstracts from the somatogenic receptor in that it recognises poorly the 20K variant of hGH. The lipogenic effect of hGH is smaller than that of insulin, and additivity studies suggested that hGH may share a subset of the pathways activated by insulin. In the present work, we compared the lipogenic effects of hGH, insulin, and the phorbol ester 12-myristate 13-acetate (PMA), the maximal effect of which was intermediate between those of hGH and insulin. The responses to maximal doses on hGH and PMA were not additive and the combined response equalled the maximal response to PMA alone, suggesting that PMA and hGH may share the same pathway of action (activation of protein kinase C). The non-additive combined response to insulin and PMA equalled that to insulin alone. These and other results suggest a complex interplay between insulin, hGH and kinase C. 118. L(+)-iactic acid induces ~adrenoceptors redistribution in intact human fat cells

G.De Pergola, S.Di Paolo, G.Garruti, M.Corso, M.Cignarelli, 1M. Pascone and R.Giorgino. Cattedra di Endocrinologia e Medicina Costitutzionale-Clinica Medica III and 1Cattedra di Chirurgia Plastica-Universit~ degli Studi di Bail, Italy In previous studies we showed that 16 mmol/1 lactate significantly inhibits isoproterenol-induced lipolysis in human fat cells. Presently, we inquired whether preincubation of fat cells with 16 mmol/l lactic acid would modify fl-adrenoceptor binding. Isolated human fat cells were pre-incubated for 1 h at 37 ~ in presence or absence of lactate (pH 7.4) then the binding assay was performed. In all experiments, non-specific binding was measured in presence of I m m o l / l ( - ) p r o panolol. Initially, (125I)-cyanopindolol (CYP) was used as radioligand (ranging: 5-500 pmol/1). When the assays were performed at 37 ~ lactate did not influence fl-receptor number (Bmax) and affinity (Kd). In contrast, in assays carried out at 4 ~ in presence of 1actate (125 I)-CYP identified about 35 0V0 less receptors than in control cells (Bmax : control: 38 + 6 fmol/106 cells; lactate: 25 + 4fmol/106cells), while Kd sharply increased (control: 140+ 31 pmol/1; lactate: 202+_53 pmol/1). These results prompted us to hypothesise lactate-induced redistribution of surface fl-adrenoceptors to a sequestered compartment, not accessible to lipophilic (12~I)-CYP at 4~ Therefore, we performed further assays with the idrophilic (3H)-CGP identified 35% fewer receptors than in control (Kd : control: 0.27 + 0.20 nmol/1; lactate: 2.0 • 0.8 nmol/l). 119. The use of Bacitracin in the insulin receptor binding assay of H35 hepatoma cells results in a linearisation of the Scatchard plot

C. P. de Vries, T. W. van Haeften and E. A. van der Veen. Departments of Endocrinology and Internal Medicine, Free University Hospital, Amsterdam, The Netherlands Scatchard plots from insulin receptor binding studies generally show curvilinearity. We assessed the effect of the use of the insulin degradation inhibitor Bacitracin on insulin equilibrium binding kinetics. H35 hepatoma cells were incubated with~ZSJ-insulin, Bacitracin (4 g/ 1) and various insulin concentrations (20 min, 37 ~ Internalised insulin was assessed by an additional incubation at pH 3.5 (20 rain, 4 ~ Control studies still showed a curvilinear Scatchard plot after correction for internalisation. In the presence of Bacitracin total cellbound insulin showed a more linear relationship (r=0.971 + 0.018) in a Scatchard plot. Internalised insulin was unaffected by Bacitracin (52.8+3.9% vs 52.5+6.7%). Receptor-bound insulin (total cellbound corrected for internalised insulin) showed also a more linear Scatchard plot (r= 0.958 + 0.029). Addition of Bacitracin resulted in a 35 0'/0 decrease of insulin binding sites without affecting the affinity as reflected by a comparable insulin concentration reducing 125j-insulin binding with 50% (ca. 20 ng/ml). In conclusion: the presence of Bacitracin in the rat hepatoma cell insulin-binding assay results in a more linear Scatchard plot suggesting one class of insulin receptors without negative cooperativity. 120. Glomernlar size and charge selectivity in Type I (insulin-dependent) diabetes mellitus

T.Deckert, B. Feldt-Rasmussen, R. Djurup ~, M. Deckert. Steno Memorial Hospital, DK-2820 Gentofte, Denmark, 1University Hospital of Copenhagen, Dept. of Clinical Chemistry, Copenhagen, Denmark Renal fractional clearances of albumin, total IgG, IgG4 and betaz-microglobulin were studied in 70 patients and 11 control subjects. In diabetic patients with normal urinary albumin excretion ( < 30 rag/24 h), fractional IgG clearance was 2-3-fold higher than in control subjects, whereas fractional clearance of the anionic plasma

513 A proteins IgG4 and albumin was similar to that of control subjects. These alterations indicate an increase in anionic pore charge within the glomerular basement membrane concomitant with an increase in either pore size or impairment of tubular reabsorption. Diabetic patients whose urinary albumin excretion has started to rise (30-100 rag/24 h) are characterised by unchanged glomerular pore size, but decreased anionic pore charge, since fractional IgG was unchanged while fractional IgG4 and albumin clearance were 3 to 4-fold increased. In patients demonstrating urinary albumin excretion of >100 rag/24 h a further decrease in size selectivity was found. Fractional beta2-microglobulin clearances were similar to that of control subjects in all groups. Thus, the increase in large pore area seen in patients with clinical nephropathy is preceded by loss of anionic charge in the glomerular basement membrane, probably due to loss of heparan sulphate. 121. Effect of the loss of the first phase of insulin secretion on glucose production and disposal in man

R. A. De Fronzo and L. Luzi. Department of Medicine, Yale University, New Haven, Connecticut, USA and Department of Medicine, Istituto Scientifico San Raffaele, Milan, Italy To examine the effect of the loss of first phase (FP) insulin secretion on total body glucose homeostasis, 6 normal subjects ( a g e = 2 4 + 1 years; IBW=100+_I%) were studied with the hyperglycaemic ( + 75 mg%) clamp technique combined with 3H-3-glucose. Subjects participated in three studies: Study I - 150min hyperglycaemic clamp; Study II - hyperglycaemic clamp plus somatostatin (6 g g / min), plus basal glucagon (0.4 ng/kg, min) replacement, plus insulin infusion designed to mimic the second phase (SP) insulin secretion; Study III - hyperglycaemic clamp plus somatostatin, basal glucagon, and an insulin infusion designet to mimic both FP and SP. Fasting plasma insulin was similar in all studies (7+1 [tU/ml); FP (peak, 0-10 min) was 57 _+7, 18 + 4, 65 _+10 in Studies I, II, III, respectively; FP (mean, 0-10 rain) was 31 + 3, 15 • 1, 45 +3 (Studies I, II, III respectively). Basal hepatic glucose production, 2.3 _+0.2 mg/kg per min, was suppressed by 90% at 20 min and remained suppressed thereafter in Studies I and III. In contrast, in Study II hepatic glucose production was inhibited by only 50% (1.1 +0.2 mg/kg per rain) after 60 min (p<0.01 vs Studies I and III), but eventually decreased by 90% at 75 min. Total body glucose disposal averaged 5.3• 5.9+0.5, and 6.5+0.5 mg/kg per min in Studies I, II, III. Conclusion: loss of FP insulin secretion (an early finding in both Type 1 (insulin-dependent) and Type 2 (non-insulin-dependent) diabetes) causes a markedly delayed inhibition of hepatic glucose production but does not impair peripheral glucose disposal. 122. Mexilitine - a new treatment of chronic painful diabetic neuropathy

A. Dejg~rd, J. Kastrup and P. Pctersen. Hvidore Hospital, Klampenborg, Denmark In a randomised double-blind cross-over study we evaluated the effect of Mexilitine - per oral given close structural analogue of lidocaine - on the symptoms and signs of chronic painful diabetic neuropathy. Sixteen patients participated (10 M, 6 F; 14 Type 1 (insulindependent), 2 Type 2 (non-insulin-dependent) diabetic patients, age median 50, range 30-64 years). Mexilitine was given at a dose of 10 mg/kg body weight per day. There was a significant reduction in pain assessed with a visual analogue rating scale during treatment with Mexilitine (p< 0.02). Likewise, assessment of a five-item clinical symptom score (pain, dysesthesia, paresthesia, nightly exacerbation and sleep disturbances) showed significant improvement during treatment with Mexilitine compared to placebo (p< 0.01). Mexilitine had no effect on tendon reflexes, vibration threshold levels, beat-tobeat variation in heart rate during deep breathing, and postural blood pressure response. Mild side effects due to Mexilitine were seen in three patients. This study demonstrates a beneficial clinical effect of Mexilitine on the symptoms but not the signs of chronic painful diabetic neuropathy. 123. Arginine-stimulated insulin response in trained and untrained man

F. Dela, K.J. Mikines and H. Galbo. Dept. of Medical Physiology B, Panum Institute and Dept. Med. University of Copenhagen, Denmark Glucose-stimulated insulin response is lower in trained than in untrained man. We investigated whether this training-induced B cell adaptation also includes the arginine-stimulated insulin response. Five sedentary (~r02m~= 44.8 • 1.1 ml. rain- 1 9kg- 1 (mean .+. _ SE)) and

514 A 7 physically trained C~r02max = 66.5 _+2.4 ml. min -1. kg -1) men had Larginine infused (10 mg-min -1 .kg -~) for 90 min. Glucose metabolism was measured by primed, constant 3-3H-glucose infusion. Plasma insulin, glucose and 3H-glucose were measured in arterialised blood. We found that glucose concentration increased similarly from 5.3 + 0.1 to 6.9 + 0.2 mmol/1 and from 5.6 + 0.1 to 7.0_+ 0.2 mmol/1 in trained and untrained subjects, respectively. Insulin showed a biphasic response peaking at 8 and 30 min after start of arginine infusion, and was lower (25.4 + 3.0 and 42.9 + 5.5 lxU/ml) in trained than in untrained (43.2_+ 8.1 and 74.9 + 10.4 txU/ml) subjects. Glucose turnover was similar in trained and untrained subjects throughout the experiment: R~= 1t4 = 2.1 mg. min -1. kg-1 at basal (n = 12). R a peaked at t = 5 m i n (6.5mg.min-l-kg -1) and Ra peaked at t = 7 0 m i n (5.6 rag.rain -1 .kg-l). Conclusion: The insulin response to arginine infusion is lower in trained than in untrained men, despite similar glucose kinetics. Thus it seems that training induces an adaptation in the arginine stimulated B cell secretion.

124. A study of 424 extended haplotypes of the major histocompatibility complex in Type 1 (insulin-dependent) diabetes: evidence for both cis and trans interactions I. Deschamps, N.Lallemand, M.Busson, P. Prevosi, A. Marcelli, M. Schmid, J.C. Poirier, H. Lestradei and J. Hors. Inserm U. 290, Hopital Herold, and Inserm U.93, Hopital St-Louis, Paris, France The susceptibility to Type 1 (insulin-dependent) diabetes in HLAnon DIL3/DR4 heterozygous patients (66% of patients) is still poorly characterised. In order to analyse non D1L3, non DR4 (DRX) haplotypes, we obtained from 212 Type 1 diabetic and 108 normal families extended patient and control haplotypes for HLA-A, B, C, DR and the Bf and C4A, B complement components. Gene and haplotype frequencies and linkage disequilibria (delta) were compared. Cis and trans haplotype combinations were analysed in DR3/X, D R 4 / X and D R X / X subgroups. HLA DtL3, DR4, BfF1 and C4BQ0 were increased and DR2 and DR5 decreased among patients (p< 10-3). In cis situation, the DR3 haplotypes differed significantly between patients (B18, C4BQ0 associated) and controls (p< 10-3). In trans situation, the dramatic excess of DR3/4 was not the only heterozygous effect observed. DRX haplotypes were found heterogeneous among subgroups. In DR3/X patients, DRX facing B18-DR3 was different from random (p< 0.01). Among D R X / X patients, DR1 and C4BQ0 were increased, DR2 was never found homozygous or combined with DR5, and DR homozygotes were decreased. These results suggest that diabetes susceptibility may be heterogeneous and generated by different cis and trans interactions between genes or gene products of the HLA region.

125. In search of antMdiotypic antibodies in recent onset Type I (insulin-dependent) diabetic patients U. Di Mario, L. Crisd, F. Dotta, M. MercaUi, S. Dib and G.S. Eisenbarth. Dept. of Endocrinology, University of Rome, Italy, Joslin Diabetes Center, Boston, Mass, USA The possible presence of anti-idiotypic antibodies in the early stages of Type 1 (insulin-dependent) diabetes has been investigated by the use of anti-human pancreatic islet monoclonal antibodies (MAb.ISL). The following monoclonals have been used: HISL1, HISL4, HISL5, HISL8, HISL14, HISL19, HISL22 raised against an homogeneate of viable pancreatic islets and 41=2, A2B5, 3G5 raised against other antigens but reacting with human islets. Non-islet related monoclonals and other species immunoglobulins have been used as controls. To highly adsorbent polypropylene tubes, precoated with goat anti-mouse immunoglobulins, MAb.ISL have been added as a pool. Sera from 51 recent onset Type I diabetic patients and 47 normal subjects have been put to incubate in duplicates. 125I-anti-human immunoglobulins have been then added and radioactivity was counted after washings. Type i diabetic sera showed a significantly increased binding (p< 0.001 vs normal subjects). When tubes were coated by either individual MAb.ISL or control antibodies, diabetics showed a higher binding vs controls (p< 0.001) but there was no substantial difference in binding between the different monoclonals and, surprisingly, with the control antibodies. Thus circulating immunoglobulins reacting with anti-islet monocional antibodies are here proved to be present in newly-diagnosed diabetic patients, but they appear to be anti-immunoglobulin antibodies instead of antiidiotypic antibodies.

Abstracts 126. Insulin specific IgE in diabetic patients treated with human insulin Th. Discher, H. G. Velcovsky, D. Sch~ifer, W. H6sl and K. Federlin. IIIrd Medical Clinic and Policlinic, Center of Internal Medicine University of Giessen, Farbwerke Hoechst AG, Frankfurt/Main, FRG The use of human insulin is increasing in many countries. It is suggested that immunological side effects of insulin treatment will be rare or even disappear. In order to prove this hypothesis, in a multicenter study sera of 276 patients were examined at various intervals during 2 years after the beginning of therapy. IgE was measured using a solid-phase technique and human-bovine-porcine insulin as antigen. In 45 patients (16.36%) insulin specific IgE in concentrations above 0.5 U / m l was detected, maximal values reaching 5 U / ml. In 12 patients (4.34%) IgE was elevated continuously, in 33 patients (11.9%) elevated IgE was only found on one occasion. In 69 patients (26%) insulin-specific IgE ranged in a borderline between 0.29 and 0.5 U / m l and was detectable only by bovine insulin being used as antigen. These antibodies were mainly found in diabetic patients treated for the first time with insulin, the distribution between Type I (insulin-dependent) and Type 2 (non-insulin-dependent) diabetic patients was equal. Interestingly, elevated IgE levels were frequently observed during the first month of therapy and even after 2 weeks. Patients switching from other insulins to the human hormone rarely developed IgE. In spite of high IgE values clinical insulin allergy was not observed in this study. However, a few single cases with Type i allergy to human insulin outside the study will be reported. 127. Retinal photography in diabetic patients J. Dolben, J. Vora, J. Atiea and D. R. Owens. Diabetes Research Unit, University of Wales College of Medicine, Cardiff, UK The efficacy of photocoagulation in the treatment of diabetic retinopathy has been well proven. Detection of patients at risk is therefore important. Previous investigations with the polaroid non-mydriatic fundal camera (Canon CR3 NM) suggest that this might be a solution for screening in busy diabetic clinics. We have therefore compared retinal images of 127 eyes of diabetic patients obtained on fluorescein angiograms, 35 mm transparencies and polaroid prints. When the photographs were graded for quality, 22.5% of polaroid prints were unusable. In comparison, assessable retinal images were obtained in 93.5% of eyes with 35 mm transparencies and 98.5% of fluorescein angiograms. Microaneurysms, cotton wool spots and macular oedema were seen more frequently on fluorescein angiograms but exudative lesions and haemorrhages were more difficult to diagnose. The pick-up rate for the latter lesions was equivalent for polaroid prints and 35 mm transparencies. Of the two eyes with new vessels, these were not seen on polaroid prints and one was also missed on the 35 mm transparency, It is concluded that good quality polaroid prints could be of value in the context of a large population of Type 2 (non-insulin-dependent) diabetic patients but this method of screening in Type 1 (insulin-dependent) patients is of questionable value. 128. Does a decreased ratio of C-peptide UlI-secretion contribute to the byperinsulinism of rats with insulinoma? M. Dolderer, R.D. Fufig~inger, W. Beischer, M. Maas, V. Maier and E. F. Pfeiffer. Department of Internal Medicine University of Ulm, FRG The secretion of islet cell hormones under hyperinsulinism was studied with the isolated perfused insulinoma rat pancreas (IP). Pancreata were perfused with Krebs-Ringer buffer under successive additions of arginine, arginine + glucose, glucose alone and somatostatin. Pancreatic hormones were measured in the perfusate radioimmunologically. Insulin secretion showed the expected hypersecretory response in IP versus controls. C-peptide secretion was comparable to insulin secretion in controls. In IP C-peptide II responded significantly to all stimuli, whereas C-peptide I secretion was completely blunted. Secretion of glucagon, somatostatin and pancreatic polypeptide was not significantly different in controls and IR This unexpected phenomenon required further investigations: in extracts of pancreata C-peptide I levels were twice as high in IP as in controls. Column chromatography of IP extracts showed a peak in higher molecular region after determination of C-peptide II, but not for C-peptide I. C-peptide II may be stored in a higher molecular weight form (e. g. proinsulin-C-peptide II) in granulas, C-peptide I with a lower molecular weight form may escape from secretory granulas into the cytosol by diffusion and thus no more is available for secretion. Former investigations showed predominant feedback inhibition of insu-

Abstracts lin secretion by C-peptide I as compared to C-peptide II. Hence decreased ratios of C-peptide I/II-secretion may contribute to the hyperinsulinism in rats with insulinoma. 129. Exercise and posture-related changes of atrial natriuretic peptide in diabetic patients with and without cardiac autonomic neuropathy J.E.Donckier, M. Buysschaert, P.De Coster, A.C. Berbinschi, J.M. Ketelslegers and A.E.Lambert. Cliniques Universitaires de Mont-Godinne, 5180 Yvoir and Unit6 de Diab6tologie et Nutrition, Brussels, Belgium To assess the effect of exercise and posture on the release of atrial natriuretic peptide (ANP) in cardiac neuropathy, we studied plasma ANP concentrations during 3 exercise levels, followed by 5 min recovery, 30 min lying and 30 rain standing in 3 groups: normal subjects (Group I, n=13), diabetic patients without (Group II, n=7) and with (Group III, n=6) cardiac neuropathy. ANP (pg/ml) increased in the 3 groups at the second and third exercise levels and after recovery: in Group I, from 20.9_+2.5 to 44.2_+7.8 at level 3 (mean+SEM, p<0.001); Group II, from 20.1_+1.7 to 45.3_+6.2 (p<0.005); Group III, from 28.2_+3.8 to 60.1 _+14.8 (p<0.05). There was no significant difference of ANP between groups at each exercise level. The increase of ANP correlated with the rate-pressure product (RPP) in the 3 groups (I: p<0.001; II: p<0.01; III: p < 0.05). Group III patients had a lower increase of RPP at exercise level 3 than Groups I and II (p< 0.05). A postural drop of ANP was only observed in Group III (p< 0.02). In conclusion, these data suggest that RPP-ANP relationship during exercise and ANP postural drop are altered in patients with cardiac neuropathy. This could result from autonomic dysfunction of heart and blood vessels. 130. Myocardial metabolism in Type I (insulin-dependent) diabetic patients. Evidence of an inhibiting effect of ketones on heart lactate utilisation A. Dora, R. Nosadini, A. Avogaro, C. Vigorito, L. Sacc~, A. Valerio, E. Duner, R. Rengo and G. Crepaldi. Patologia Medica I, Policlinico Universitario, Padova-Clinica Medica I, Naples, Italy The Framingham study showed an increased risk of congestive heart failure in Type 1 (insulin-dependent) diabetes, without coronary heart disease presence. Recently, Taegtmeyer hypothesised that Krebs cycle impaired activity causes a lower ATP availability in Type 1 diabetic heart. We sampled artery and coronary sinus at rest and during atrial pacing, infusing a4C-lactate and 3H-hydroxybutyrate in 7 Type 1 diabetic patients with 10_+2 mmol/1 mean blood glucose value and 8 normal subjects (N) who underwent diagnostic cardiac catheterisation and show patent coronary arteries a posteriori. Blood flow was measured by thermodilution. Ketone body uptake was higher (39+14 in Type 1 diabetic patients, 11 +3 in N, ~tmol/ min, mean +_SD) at rest. Lactate was simultaneously produced and taken up in N and Type 1 patients. In N lactate uptake (22+ 7 gmol/min) exceeded production (8-+ 5), in Type 1 patients lactate production was the predominant process (22-+ 11 vs 13 -+ 8). Lactate oxidation from 14CO2 production was 10_+2 Ixmol/min in Type 1 patients and 20+ 2 in N (p<0.01). Lactate uptake was inversely related to that of ketone bodies. Atrial pacing stimulated glucose and lactate uptake abolishing lactate release in Type 1 patients. Conclusions: lactate utilisation is impaired in Type 1 diabetic heart by the excess of lipid metabolites. Our results support the hypothesis of an altered myocardial fuel metabolism in Type 1 diabetic patients. t31. Ganglioside expression in whole pancreas and isolated islets F. Dotta, U. Di Mario, C. Tiberti, R. Nasak, P. G. Colman, G. S. Eisenbarth, L. Lenti, D. Lombardi and G.M. Pontieri. Dept. of Endocrinology and Dept. of Experimental Medicine, University of Rome, Italy. Joslin Diabetes Center, Boston, Mass, USA Sialoglycoconiugates, especially gangliosides, have been suggested as targets of anti-islet-cell autoanodies in Type 1 (insulin-dependent) diabetes. We have studied the pattern of ganglioside expression in pancreas and isolated islets, and the ganglioside antigen reacting with anti-islet-cell monoclonal antibody 3G5, using high performance liquid chromatography (HPLC) and high performance thin layer chromatography (HPTLC). The major gangliosides detected in glycolipid extracts of both human and bovine pancreas were found to be GM3 and GD3. However, isolated human and bovine islets were found to express GM3 and a doublet at GM2 by both HPLC and HPTLC, indicating the islet-specific expression of gangliosides comigrating with GM2. The glycolipid extracts were also separated by HPTLC and directly immunostained with 3G5. The migration po-

515 A sition of the 3G5-antigen was above GM1 and a little below GM2 and did not correspond to any major ganglioside of whole pancreas but did comigrate with the lower band of the major islet ganglioside doublet migrating at the position of GM2. In conclusion, major ganglioside(s) expressed in islets comigrate with GM2 and are not major components of whole pancreas; GD3, shown to be a major band of whole pancreas, is not detectably expressed in islets; the islet-cell-antigen recognised by 3G5 comigrates with a major islet ganglioside at or near the GM2 marker. 132. Are insulin autoantibodies different from exogenous insulin induced antibodies? N. Dozio, F. Sodoyez-Goffaux, M. Koch and J.-C. Sodoyez. Laboratory of Experimental Nuclear Medicine, University of Liege, Liege, Belgium Anti-insulin antibodies from four patients with insulin autoimmune hypoglycaemic syndrome were characterised and compared to those found in Type 1 (insulin-dependent) diabetic patients. Specificity was studied by competition experiments using 125-I-labeled human, bovine and porcine insulins and different species of insulin and derivatives as competitors. Species specificity was variable in both types of patients but the results suggested the presence of monoidiotypic antibodies in autoimmune patients whereas in Type 1 patients, antibodies recognising different epitopes were identifiable. The size of 125-I-insulin labelled immune complexes was evaluated by FPLC. A single species of immune complex coeluting with the monomeric IgG fraction was found in autoimmune patients whereas aggregates of 2 or more IgG were found in patients receiving exogenous insulin. No characteristic pattern of anti-insulin IgG subclasses was found in autoimmune patients. By contrast, in Type 1 diabetic patients, antiinsulin IgG1 were predominant over IgG3 and/or IgG4. In conclusion, differences can be found between anti-insulin autoantibodies and exogenous insulin induced antibodies. The former are monoidiotypic although not necesessarily monoclonal whereas the latter are neither monoclonal nor monoidiotypic. 133. Glucose tolerance and insulin efficiency in aging J. Duhault, D. Ravel, M. Boulanger and O. Della-Zuana. Institut de Recherches Servier, France The hyperglycaemia seen in an aging population may be closely related to the development of Type 2 (non-insulin-dependent)diabetes mellitus. We have explored the probable multifactorial etiology of this age-related defect by studying glucose tolerance and the glucosestimulated insulin response in an animal model of aging, the older male Sprague-Dawley rat (52 weeks). The rats were fasted 24 h prior to the glucose tolerance test (2 g/kg). The older rats showed mild glucose intolerance relative to the young animals (23 weeks). Basal insulinaemia ( + 67%) and insulin secretion ( + 84%) by older rats in response to hyperglycaemic stimulus were exaggerated, therefore insulin efficiency was decreased in these animals. Perifusion studies seeking to determine the cellular mechanisms responsible for the insulin/glucose abnormalities have demonstrated that there is an agerelated diminution in glucose-stimulated (16.7 mmol/1) insulin release ( - 7 3 % ) by pancreatic islets isolated from older rats. Chan.ges in the function of the tissular insulin receptors were examined usmg solubilised, lectin-purified insulin receptors prepared from the liver of young and old rats. Although there was no age-related change in insulin binding to receptors of liver membranes, a decrease in receptor kinase activity ( - 6 0 % ) indicates a defect in transmembrane signaling through the insulin-receptor. These changes may be important in the altered metabolic state that is observed in older patients and in obese or diabetic patients. 134. Glomerular and albuminuric response to moderate hyperglycaemia in Type 1 (insulin-dependent) diabetic patients with incipient nephropathy R. P. F. Dullaart, S. Meijer and H. Doorenbos. Departments of Endocrinology and Nephrology, University Hospital Groningen, The Netherlands To evaluate kidney response to moderate hyperglycaemia, glomerular filtration rate (GFR), renal plasma flow (RPF) and urinary albumin excretion were measured in two 2-h periods after 4 h stabilisation, using l:sI-iothalamate, 131I-hippurate and radioimmunoassay, respectively, in Type I (insulin-dependent) diabetic patients with microalbuminuria. Blood glucose was maintained at 6.1 + 1.3 mmol/1, then raised to 10.7 + 2.3 retool/1 by intravenous glucose in 12 patients (Group I) (nocturnal albumin excretion median 22.63,

516 A range 12.63-192.01 p~g/min) and was kept constant (6.3_+1.4 vs 6.1 ___1.3 retool/l) in 19 control subjects (Group II) (nocturnal albumin excretion 19.96; 10.01-185.23gg/min (NS vs Group I)). Results: Group I: GFR remained constant 118_+25 vs 119+ 18ml/min/1.73 m 2 (mean_+SD) but increased in the 11 patients without elevated GFR (GFR <146 ml/min/1.73 m 2) (113+18 vs 118 _+18 ml/min, p < 0.05). RPF did not change (508 + 140 vs 499 _+ 99 ml/min/1.73 m 2 (NS)). Albumin excretion decreased from 20.92 (2.25-182.32) to 15.70 (3.00-147.37) gg/min, p=0.02, while tubular glucose reabsorption increased (6.3_+ 1.6 vs 10.1_+2.0 mmol/1 glomerular filtrate/1.73 m 2, p<0.01). Group II: GFR and RPF remained constant (118_+32 m l . m i n - l . 1.73 m 2 vs 118 + 36 ml. rain -~. 1.73 m 2 (NS) and 478 _+156 vs 471 + 153 ml. min -1.1.73 m2). Albumin excretion and tubular glucose reabsorption did not change [18.50 (2.19-320.85) vs 15.37 (2.64-293.88) p~g/min (NS) and 6.2 + 1.7 vs 6.1 + 1.4 mmol/1 glomerular filtrate/1.73 m2]. Conclusion: In incipient nephropathy moderate hyperglycaemia induces an increase in GFR only in patients without elevated GFR. We hypothesise that the decrease in albumin excretion during hyperglycaemia results from enhanced tubular reabsorption induced by increased tubular glucose reabsorption.

135. The thymidine incorporation of pancreatic islets is diminished in normoglycaemic diabetes prone BB rats A. Dunger, I. Kl6ting, W. Besch and H.-J. Hahn. Central Institute of Diabetes "Gerhardt Katsch", Karlsburg, G D R The number of pancreatic B cells still detectable in BB rats during the development of diabetes is the result of immune destruction and renewal rate. The BB rats, which are characterised by an immune dysregulation immunogenetically belong to the RTlu haplotype. Therefore, we investigated the influence of the RTlu haplotype with varying genetic background on D N A synthesis of isolated islets. Islets of similar size obtained from newborn Lewis (RTla, RTll, RTln) and Wistar (RTlu, RTla) strains were cultured in one and the same experiment for 16 h in the presence of H3-thymidine. While the replicatory activity was not related to MHC, pancreatic islets obtained from the two Wistar rat strains are characterised by a higher incorporation rate compared to Lewis rats. However, islets from normoglycaemic BB rats which dispose of a variant MHC of the RTlu haplotype had a significant reduced incorporation rate. Consequently, the results demonstrating the importance of genetic background for regulation of replication do not support the hypothesis that the RTlu haplotype per se is connected with a lower islet replication. Nevertheless, they underline that a lower islet replication possibly as a result of the variant MHC haplotype RTlu may contribute to the B cell deficiency observed in diabetic animals.

136. GH response to 1-29 GRF and basal somatomedin-C levels in children and adolescents with Type I (insulin-dependent) diabetes S. Duran Garcia, C.Velez, J.E. Hurtado and C. Ceballos. Departamento de Medicina, Catedra de Endocrinologia, Hospital Universitafio, Sevilla, Spain We have studied 35 Type 1 (insulin-dependent) diabetic patients between 8 and 17 years of age (12.5+2.5), 19 females and 16 males, with an evolution time between 1 and 10.6 years (3.4+2.4). Basal and post-stimulus (100 ug of 1-29 GFR, i.v. bolus, SERONO) growth hormone (GH) determinations were performed by RIA. Basal Somatomedin-C (SmC) was analysed using the RIA method of the Immunonuclear Corporation. Glycaemic control was evaluated during at least the year prior to the hormonal studies using monthly HbAlc (TBA-test) determinations. Basal and post-stimulus GH levels were not significantly different in diabetic patients from those found in control subjects. SmC levels were decreased in 36-100% of female and in 75-85% of male diabetic patients as compared with age- and sex-matched control subjects. No relationship was detected between SmC levels and GH, chronological age, growth velocity, time of diabetes, pancreatic reserve or HbAlc levels. A linear relationship was found between SmC levels and skeletal maturity (r: 0.3820, n~35, p<0.05) and in female diabetic patients in the pubertal stage (Tanner criteria, r: 0.5307, n= 19, p < 0.05). GH response to 1-29 GRF was similar in hyperglyeaemic (HbAlc > 9%) or normoglycaemic (HbAlc _<9%) patients. In conclusion: Abnormal regulation of GH response to 1-29 GRF and of basal SmC levels was detected in children and adolescents with Type 1 diabetes.

Abstracts 137. Glucose tolerance in adult offspring of diabetic mothers L. Dvo~fikovfi and H. Pfibylovfi. Dept. Int. Med. Faculty of Paediatrics Charles University and Institute for the Care of Mother and Child, Prague, Czechoslovakia The generation of offspring of diabetic mothers who were successfully treated during pregnancy has grown up only recently. We were interested in the state of these offspring in early adulthood. We evaluated a group of 131 offspring aged 20.5_ 3.6 years. Seven children developed diabetes; in 124 we performed oral glucose tolerance test. One-hundred-seven had normal results, 10 offspring had an impaired glucose tolerance, and 2 had diabetes. In 5 others, significant increase of the blood glucose values in the first and second hour after glucose load occurred when compared with a control group (p< 0.05 and p < 0.001 respectively). Glucose load led to essentially higher elevation of insulin levels not only in offspring with impaired glucose tolerance but in the whole examined group. A prospective follow-up of children of diabetic mothers has shown that they represent a high risk group which needs a long-term dispensarisation. 138. Diurnal profiles of insulin, C-peptide, proinsulin and gastric inhibitory polypeptide (GIP) in control subjects and Type 2 (non-insulindependent) diabetes mellitus R. Ebert, M. Mann and W. Creutzfeldt. Dept. of Medicine, University of G6ttingen, FRG In the present study diurnal profiles for insulin, C-peptide, proinsulin and the insulinotropic gut peptide GIP were measured in 22 control subjects and 31 Type 2 (non-insulin-dependent) diabetic patients following ingestion of a 1600 kcal diet. The diabetic patients were either placed on diet alone (n= 13) or diet plus glibenclamide (n= 18). In control subjects the greatest insulin response occurred after breakfast, in diabetic patients in the evening with a blunted initial insulin release in the morning. The insulin/C-peptide ratio was highest at 09.00 hours in controls and at 19.00 hours in both diabetic groups. The proinsulin responses were augmented in diabetic patients reaching a maximum at 14.00 hours. In both diabetic patients and control subjects only marginal increments of proinsulin were seen in the evening. The GIP responses were attenuated in diabetic patients (integr. responses: 2.5+0.16 in control subjects, 1.8+0.1 in diabetic patients on diet, 1.3 + 0.06 nmol/1 x 24 h in diabetic patients on diet and glibenelamide). These data show that Type 2 diabetic patients, in contrast to control subjects, exhibit the maximal insulin response in the evening, probably due to an impaired hepatic insulin extraction. Proinsulin secretion occurs till 17.00 hours without relevant release in the evening, and the secretion of GIP, lasting till 03.00 hours, is impaired in diabetes meUitus under a strict dietary regimen. 139. Long-term survival of mouse pancreatic islets in vitro following acute exposure to streptozotocin: altered insulin response to secretagogues D. L. Eizirik, E. Strandell and S. Sandler. Dept. of Medical Cell Biology, Uppsala University, Uppsala, Sweden Recent studies suggest that during the course of diabetes there may be alterations in the pancreatic B-cell response to various stimuli. In the present investigation, isolated mouse pancreatic islets were exposed to 2.2 mmol/1 streptozotocin (SZ) and then cultured for six days. There was no difference in the islet DNA content between the two groups of islets, but the insulin content was decreased by 50% after SZ. The islet ATP content was unaffected in the SZ group, whereas glucose oxidation rates at 16.7 mmol/1 glucose were reduced by 45%. In comparison to the insulin release at 1.67 mmol/1 glucose, the insulin release increased 4.1 _+0.6 (16.7 mmol/l glucose), 5.1 _+1.0 (5.6 mmol/1 glucose + 10 mmol/1 arginine), 4.9_+1.0 (10 mmol/1 leucine + 2 mmol/1 glutamine) and 49.0_+8.7 (16.7 mmol/1 glucose + 5 retool/1 theophylline) times in the SZ-treated islets. When compared to the insulin secretion of the control islets, the combination of glucose plus theophylline could partially counteract the SZ-induced inhibition of insulin release. The present study suggests that acute exposure of islets to SZ induces a long-term suppression of insulin release, probably due to impaired islet metabolism. This suppression can partly be overcome by phosphodiesterase inhibitors. 140. Does glycaemic control affect high density lipoprotein cholesterol in Type 2 (non-insulin-dependent) diabetes? A prospective study R. S. Eleles, S. G. H. Rains, G. A. Wilson and W. Richmond. St. Mary's Hospital, London, UK The conflicting evidence on whether high density lipoprotein choles-

Abstracts terol (HDL-C) in Type 2 (non-insulin-dependent) diabetes is raised by improvement in diabetic control, may be due to different modes of treatment. Insulin may raise HDL-C by stimulation of lipoprotein lipase (LPL), which catabolises triglyceride rich lipoproteins with concomitant production of HDL and HDL-2. The effect of oral hypoglycaemic drugs is unclear. We have compared the effects of Glibenclamide with Metformin in a three months cross-over study, in 35 Type 2 diabetic patients uncontrolled on diet on serum lipids and post-heparin LPL. Both drugs reduced fasting blood glucose (Glibenclamide 13.7+4.3 to 10.5+_4.1mmol/1, p<0.001:Metformin 13.8+_4.1 to 10.6+_3.4, p<0.001). Neither drug altered mean HDL, HDL 2C, or serum triglycerlde. Metformin, but not Glibenclamide, reduced serum and low density lipoprotein cholesterol. There was no significant change in LPL on Glibenclamide, but on Metformin LPL fell from 7.6+2.1 to 6.7+_2.3 `amol fatty acid.ml-~.h -1 (p< 0.05). Analysis of covariance showed the individual changes in HDL-C and HDL-2C to be unrelated to changes in glycaemic control but positively related to changes in LPL (p<0.05) and inversely related to changes in serum triglyceride (p< 0.0001). HDL-C is influenced by alterations in LPL and serum triglyceride rather than by glycaemic control. 141. Diabetic nephropathy: acute effects of captopril on renal haemodynamics and albuminuria L. D. Elving, J.F.M.Wetzels, E.de Nobel, A.J. Hoitsma and J. H. M. Berden. Department of Medicine, Divisions of General Internal Medicine and Nephrology, University Hospital Nijmegen, The Netherlands The acute effects of captopril on systemic and renal haemodynamics and albuminuria were studied in 9 proteinuric Type I (insulin-dependent) diabetic patients with normal blood pressure ( < 140/90 mmHg). Mean age ( _+SD) was 35 + 10 years, mean duration of diabetes 24+ 10 years. Blood pressure, glomerular filtration rate (GFR: insulin clearance), effective renal plasma flow (ERPF: p-aminohippurate clearance) and albumin excretion rate (AER) were measured from 1.5 h before until 3 h after a single dose of 25 mg captopril. Blood pressure decreased slightly from 130/79+ 4/3 to 124/74+4/3 mmHg (mean+ SEM:NS). AER decreased from 677_+ 221 to 561 _+184 `ag/min (p< 0.01), maximal percentual decrease ranging from - 3 % to - 3 3 % (median -21%). GFR decreased from 123+13 to l 1 7 + 1 4 m l / m i n (p<0.05:median A% -8%). ERPF increased from 609 + 56 to 714+ 55 ml/min (p< 0.01: median A% +19%). As a result filtration fraction (FF, i.e. G F R / ERPF), which reflects intraglomerular pressure, decreased significantly (0.204+_0.014 vs 0A70+_0.014:p<0.01). AER corrected for GFR also decreased significantly (maximal A% median - 1 7 % ; range - 4 % to - 2 5 % ; p<0.01), indicating that the decrease of GFR hardly contributed to the decrease of AER. The decrease of AER correlated significantly with the decrease of FF (R~ = 0.75; p < 0.02). In conclusion: in normotensive diab~etic patients captopril causes a prompt reduction of albuminuria, which is not mediated by lowering of blood pressure or GFR, but by lowering of the intraglomerular capillary pressure. 142. Altered deformability, shape and size of red blood cells in ob/ob mice K. G. Engstr6m. Dept. of Histology and Cell Biology, University of Ume~, S-90187 Ume~, Sweden The deformability of red blood cells (RBCs) is essential to normal microcirculation. Reduced deformability may contribute to circulatory dysfunctions in diabetes mellitus. Red blood cells in adult obese hyperglycaemic mice (ob/ob) are considerably less deformable than those of normal mice. To search for a morphological explanation, RBCs from ob/ob and normal mice were suspended in salt balanced buffer (300 mosm/1), sucked into the tip of a 1.82 am pipette, photographed and measured in a computerized image analysis device. The ob/ob mouse RBCs exhibited significantly increased membrane area, cell volume and minimum cylindrical diameter ('MCD' = diameter of the narrowest cylinder capable of encompassing a red blood cell with fixed area and volume). Twenty percent of the ob/ob mouse RBCs, and only 2% of control RBCs, had MCD > 3 gm, i.e. the pore diameter of Nuclepore filter previously used to analyse RBC deformability. Cross-sectional profiles with minimum corpuscle bending resistance were computed for RBCs of mean area and volume. The profiles of ob/ob-mouse RBCs predicted an enhanced resistance to bending. It is concluded that changes in shape and size explain in the decreased deformability of diabetic ob/ob-mouse RBCs.

517A 143. Maternal environment and fetal genome determine the outcome of diabetic pregnancy U.J.Eriksson. Department of Medical Cell Biology, University of Uppsala, Biomedicum, Uppsala, Sweden Previous experimental studies have implicated a genetic component in the induction of malformations in the offspring. We have now compared the outcome of diabetic pregnancy in two outbred (sub)strains of Sprague-Dawley rats (with Low-incidence (L) and High-incidence (H) of skeletal malformations in the offspring) and hybrids between them. The fetuses of diabetic L mothers had no skeletal malformations and the lowest frequency of resorptions (8-9%), regardless of embryo type (L/L or L/H). If the diabetic mother was H or from the hybrid strain (L/H), and the offspring were of the mixed L / H type, then we found increased resorption (16-21%) and skeletal malformation (3-5%) rates. If instead the embryos contained a major H genome (either H / H or H / ( L / H ) ) further increased resorptions (23-30%) and skeletal malformations (17-19%) resulted. The L / L and H / H embryonic susceptibility to defined teratogens (3-6 mg/ml D-glucose, 4-8 mmol/l B-hydroxybutyrate) were compared in whole embryo culture and found to be similar. In our model, diabetes seems to cause a disturbance in early stages of chondrogenesis. This developmental disturbance is not directly associated with increased levels of D-glucose and B-hydroxybutyrate. These findings are in concert with the notion of a mixed genetic-environmental etiology of malformations in (diabetic) pregnancy.

144. Phorbol esters inhibit the insulin stimulated tyrosine phosphorylation of fat cell proteins B. Ermel, B. Obermaier, E. Schleicher and H.U. H~tring. Institut for Diabetesforschung, KNner Platz 1, FRG The first steps of cellular insulin action include receptor binding, activation of the tyrosine kinase,, the receptor fl subunit and subsequent phosphorylation of so far not defined cellular substrates. Using phosphotyrosine antibodies we were recently able to identify possible substrate proteins in rat fat cells. Phorbol esters are known to inhibit the insulin receptor kinase and to induce insulin resistance of fat cells. We studied the effect of TPA on the insulin stimulated tyrosine phosphorylation of the putative substrate protein in 32p labelled rat fat cells. Pretreatment of 32p labelled fat cells with TPA (10 9 tool/l) inhibits the insulin stimulated phosphorylation of bands of approx. 40-50 kDa, 60 kDa, and 116 kDa. The data support the idea that protein kinase C is an inhibitory regulator of the signal function of the insulin receptor kinase. Furthermore, the coincidence of TPA-induced insulin resistance with decreased phosphorylation of these proteins gives additional evidence for a role of these phosphoproteins in insulin signal transmission.

145. Effect of acarbose | on colonic function and metabolism C.Fabian 1, W.Scheppach 1, M.Spengler 2 and H. Kasper 1. ~Dept. of Medicine, Wurzburg University, FRG, 2Bayer Pharma, Wuppertal, FRG The glucosidase inhibitor acarbose | (Bay g 5421) is used to delay intestinal glucose absorption, but may also affect bacterial carbohydrate fermentation in the colon by glucose malabsorption. Twelve healthy volunteers were put on a controlled diet for 2 four-week periods during which they received either acarbose or placebo (doubleblind/cross-over design). Acarbose was given in 3 daily doses of 50 mg (lst week), 100 mg (2nd week), 200 mg (3rd, 4th week). In the 4th week fecal wet weight was measured and the mean transit time assessed with radio-opaque pellets. Breath hydrogen and plasma acetate (parameters of bacterial fermentation) were measured for 12 h following ingestion of a starchy test meal; the area under curve was calculated for hydrogen and acetate. Student's t-test was used throughout for comparisons. Fecal wet weight (g/day) was higher (p< 0.005) in the acarbose period (207.5 + 3.5 SEM) than in the control period (123.7 _+3.2). The mean transit time (h) was 55.9 _+3.2 in the acarbose trial compared with 42.9 _+3.2 in the control trial (p< 0.025). The areas under curve for breath hydrogen (ppm. rain. 103) were 52.6 + 3.5 (acarbose) and 13.8 + 3.5 (control) (p< 0.005). For the plasma acetate areas (gmol/1. min. 103) no significant difference between acarbose (89.6+2.8) and control (63.1+2.8) periods was found. The results suggest that acarbose may influence colonic function and metabolism the consequences of which are largely unknown.

518 A 146. Intracellular metabolic effects of fast-acting monomeric insulins K. Falholt, J. Brange, Aa. Volund and L.G. Heding. Novo Research Institute, Bagsvaerd, Denmark Using recombinant D N A technology it is possible to make human insulin analogues (INA). Change of B9--+Asp plus B27-~Glu resulted in a monomer INA with fast absorption from subcutis. Three groups of 5 pigs were given, respectively, 40 U INA, 40 U human Actrapid and the corresponding Actrapid medium subcutaneously daily for 8 days. Plasma INA peaked at 15 min and human Actrapid at 45 rain. After 8 days of treatment an intravenous glucose tolerance test revealed decreased k-values of the Actrapid and INA groups. Samples of aorta, muscle, and liver were collected for enzyme and substrate analyses. Glucose-6-phosphate-dehydrogenase (G-6-PDH) from muscle was significantly reduced in the Actrapid and INA treated pigs compared with medium, mean +_SEM: 0.09 + 0.01 and 0.10 +_0.01 vs 0.16_+ 0.02 p-mol/g, p < 0.05. No significant change in the activity of this enzyme was found in aorta and liver, the same holds true for the activity in all three tissues of phosphofructokinase, hexokinase, pyruvate kinase, a-glycerophosphate DH and malic enzyme. The triglyceride content was decreased ten-fold in aorta in the INA-treated pigs compared with those treated with Actrapid and medium (4.8 +_2.4 vs 39.2 + 18.4 and 41.1 + '19.4 p-mol/g). In conclusion, these data demonstrate that after 8 days of treatment with a quick absorbed INA the cellular metabolic profile was close to normal. Long term studies are required for evaluation of the effect of insulin treatment on triglyceride in the arterial wall. 147. Diabetogenic effects of Diltiazem in rats H. C. Fehmann, R. Haverich, F. Strckmann and W. Creutzfeldt. Med. Univ. Klinik Grttingen, FRG Calcium antagonists are widely used clinical drugs. A diabetogenic potency, i.e. inhibition of insulin' release, is well known for Nifedipine and Verapamil. Up to now no experimental data on Diltiazem and its effect on insulin secretion have been available. Male Wistar rats (180-220 g) were injected with Dfltiazem 6.25 mg/kg bw intraperitonally once (D; n=6) or twice (DD; n=5) daily for 7 days, controls (C; n=8) received saline. After an overnight fast in one part of the animals in each group the pancreas was removed and extracted for insulin, in the other part the pancreas was isolated and perfused with KRH-buffer (37 ~ pH 7.4) containing 0.2% BSA. Insulin secretion was stimulated after equilibration (10 rain, 2.8 mmol/1 glucose) with 20 retool/1 glucose for 50 rain. Insulin after extraction, in serum and peffusate was measured with a specific RIA, glucose with the hexokinase method. Results are given as mean_+ SEM. D and DD had no effect on pancreatic insulin content (g/g wet weight, C: 93+3; D: 97+5; DD: 72+'10.5) and plasma glucose (mmol/1 C: 5.85 + 0.165 ; D: 6.46 _+0.165; DD: 5.47 +- 0.331). Plasma insulin levels were slightly diminished. After stimulation of insulin secretion from the perfused pancreas with 20 mmol/l glucose the first (ng/ 10 min, C: 120+22; D: 85+-6; DD: 4'1+7) and the second peak (ng/40 min, C: 485 _+62; D: 136 +_14; DD: 72 +_9) of insulin release was markedly decreased in the D- and DD-treated animals. Conclusion: pretreatment of rats with Diltiazem in the upper therapeutical range induces an impaired insulin secretion from the in vitro perfused pancreas. The question of a clinical significance of these results has to be answered by further investigations. 148. Comparison of thermogenic activity induced by the new sympathomimetic Ro 16-8714 between lean and obese subjects J. P. Felber1, C. Henny 1, A. Biickert2, Y. Schutz3, E. J~quier3, J.P. Felbe?. 1Division of Endocrinology and Clinical Biochemistry, C.H.U.V., Lausanne, 2Hoffmann-La Roche, Basel, 3Institute of Physiology, University of Lausanne, Switzerland The aim of the present study was to assess the thermogenic response of lean and obese individuals to the new ~adrenoceptor agonist Ro 16-8714. Following an overnight fast, a placebo or Ro 16-8714 (0.17 m s / k s fat free mass) was given per os to 6 lean subjects and to 6 obese subjects. The rate of energy expenditure (EE) and the substrate utilisation were determined by indirect calorimetry (hood system) before and for 6 h following the drug administration. Heart rate and blood pressure as well as plasma glucose, insulin and free fatty acid (FFA) concentrations were also measured at regular intervals throughout the study. The increment relative to base-line (mean +_SEM) in EE was similar in the two groups and averaged 4.0 + 1.4% and 12.2+1.4% with placebo and with Ro 16-8714 respectively in lean subjects, whereas the values reached 3.5 +-'1.2% and 14.4+2.0% in obese subjects. Heart rate, systolic blood pressure, insulin and

Abstracts FFA were increased without any significative difference between the two groups. Plasma glucose was not modified. This study shows that Ro 16-8714 is a potent thermogenic agent both in lean and obese subjects. 149. Growth-hormone and diabetes mellitus: role of the metabolic control. A multiple nenropituitary study M. Ferdeghini, O.Giampietro, R.Miccoli, M.Cerri, G. Penno, E. E. Mtiller and R. Navalesi. Cattedra di Malattie del Ricambio, Istituto di Clinica Medica II, CNR Clinical Physiology Institute, University of Pisa; Dept. of Pharmacology, Univ. of Milan, Italy Diabetic patients are considered to have high basal GH and to be abnormally responsive to GH provocative stimuli, including neuropeptides. We evaluated: in 11 Type 1 (insulin-dependent) diabetic male patients (1DM) (26+2 years; HbA1 9.5+0.2%) the GH after intravenous G H R H (1 p-g/ks b.w.) or clonidine (0.15 ms); in 9 1DM (35 _+4 years; HbA1 10.5 _+1%) and 7 Type 2 (non-insulin-dependent) diabetic male patients (2DM) (50_+4years; HbA1 8+0.3%) the GH after i.v. LHRH (25 p.g); in 23 I D M (31+2years; HbA1 10.1 +0.3%) and in 7 2DM (50+_4 years; HbA1 8+0.3%) the GH after i.v. TRH (200 p-g). In every protocol, diabetic patients had nearnormal fasting and average plasma glucose levels and control males were also studied. GH after GHRH and clonidine was prompt, significant and superimposable in normal subjects and diabetic patients. Only three 1DM and one 2DM showed a non-specific GH response to TRH, but five 1DM and two 2DM had an "inappropriate" GH release after LHRH. They, in whom saline studies and cortisol behaviour excluded a "stress-related" GH response, showed the highest HbA1 (11%). In well-controlled Type 1 diabetes the hypothalamic-pituitary neurotransmission is correct, and the occurrence of "inappropriate" GH responses of somatotropes to non-specific stimuli (such as TRH or LHRH), which may be found even in Type 2 patients, seems dependent on the quality of the long-term glycometabolic control. 150. Heart-rate variability during sleep in diabetic patients with and without autonomic neuropathy L. Ferini-Strambi, M.C. Librenti, G. Galimberti, P. Pinto, S. Smirne and G. Pozza. Sleep Disorders Center and Dept. of Medicine, University of Milan and Istituto Scientifico S. Raffaele, Milan, Italy Since direct electrophysiological methods for the investigation of the autonomic nervous system (ANS) functioning are not currently available in clinical practice, several more or less simple tests have been proposed for assessing different autonomic functions. The most widely used tests are non-invasive and investigate cardiovascular reflexes by measuring the heart rate (HR) variation during wakefulness both at rest and after various stimuli. A normal HR variability is currently regarded as a reliable index of ANS integrity. However, most tests have some limitations: they require collaboration from the patient and can be modified by his emotional state. Moreover, some tests may be dangerous for some patients, such as handgrip test for patients with cerebrovascular or cardiac disorders, and Valsalva manoeuvre for the subjects with diabetic neuropathy. During sleep HR shows tonic changes along with the deepening of NREM stages. Moreover, phasic modifications of HR occur during REM periods and in relation to body movements all during sleep. In this way, sleep supplies natural and repetitive changes of ANS activity. We studied 26 diabetic patients with signs of cardiac autonomic function on the traditional tests (DAN; mean age=39.2 years), 10 diabetic patients without cardiac autonomic neuropathy (D; mean age=36.1 years) and 30age-matched control subjects (C; mean age = 38.5 years). In each subject we measured the maximal HR variation related to body movements (Rmax) during sleep. Rbm during NREM sleep was significantly lower in both groups of diabetic patients in comparison to controls (DAN = 1.24 + 0.10; D = 1.42 _+0.07; C=1.58+0A5). Similar results were obtained during REM sleep ( D A N = I . 2 5 _ 0 . 0 9 ; D=1.42_+0.07; C=1.65_+0.21). We can conclude that the study of HR variability during sleep may be an useful tool for the assessment of autonomic dysfunction. 151. In vivo hormonal regulation of insulin receptors in isolated rat adipocytes M. Fickovfi, L. Macho and S. Z6rad. Institute of Experimental Endocrinology, CPS SAS Bratislava, Czechoslovakia In our previous experiments 24 h hypokinesia (motion restriction) was observed to cause a significant fall in insulin receptors number in rat adipocytes and elevated concentrations of corticosterone and

Abstracts catecholamines. In the present study the role of the above hormones in the regulation of insulin receptors was investigated under in vivo conditions. Endogenous catecholamines were eliminated by surgical removal of the adrenal medulla and by treating the rats with guanethidin ( M E D E X + G U A N ) . Corticosterone was eliminated by adrenalectomy (ADREX). The effectiveness of the surgery was checked by serum analysis of corticosterone, epinephrine and norepinephrine. Insulin binding capacities -R0 (fmol/106 adipocytes) under basal conditions vs hypokinesia were: control animals - 322.3 + 78.2 vs 105.7+26.2 (p<0.025), ADREX - 216.8+52.8 vs 236.2+ 50.2, M E D E X + G U A N - 204.0+24.8 vs 111.7+13.2 (p<0.01). The results suggest that 24 h permanent elevation of corticosterone levels (in absence of catecholamines) caused down regulation of insulin receptors in rat adipocytes. 152. Delayed axonal transport of cytoskeletal proteins in streptozotocin diabetic rats B. Figliomeni, P.Macioce, G.Filliatreau and L.Di Giamberardino. Fidia Research Laboratories, Abano Terme, Italy; D~pt. de Biologic, C. E. N. de Saclay, Gif-sur-Yvette, France Sprague Dawley rats, 200-250 g, were made diabetic with a single injection of streptozotocin (STZ, 70 rag/ks). Two weeks later a group of animals was injected in the fifth dorsal root ganglion with 50 ~Ci 35S-methionine. Another group received after three weeks of diabetes an intraspinal injection of 130 gCi 35S-methionine. Then, after 2 and 3 weeks, respectively, the sciatic nerve was dissected out, cut in consecutive 6 mm segments, homogenised in a 0.5% Triton-containing buffer, separated into a supernatant (enriched in soluble and membrane-bound proteins) and a pellet (enriched in polymerised cytoskeletal fibrils), and analysed by gel electrophoresis and autoradiography. In sensory fibers the transport rate of neurofilament proteins was retarded by 70%; the other proteins, particularly those carried by slow component a (SCa), were also delayed but to variable extents. In the motor fibers, a much less prominent modification of the axonal transport was observed. These results indicate that the proteins carried with the slow axonal transport, predominantly the cytoskeletal polymers carried with SCa, are delayed in STZ diabetic rats. Also, the sensory axons appear to be more severely affected than motor axons. 153. Transfer to recombinant human proinsulin; resultant clinical and immunological effects S. E. Fineberg, N. S. Fineberg, J. H. Anderson and J. A. Galloway. Department of Medicine, Indiana University and the Lilly Clinic for Clinical Research, Indianapolis, Ind, USA Transfer to human proinsulin (HPI) from human insulin (HI) could result in clinical and immunological benefits. 66 Type 1 (insulin-dependent) and 63 Type 2 (non-insulin-dependent) diabetic patients were maintained on HI for 7 months, then randomised to treatment with HPI (Group I) or continued on HI (Group II) for an additional 6 months. Significant anti-proinsulin and anti-insulin antibody values were > 1.2 and 2.4%. Beef, pork, and human 12sI and unlabelled antigens were used. Groups were closely comparable at baseline including: clinical characteristics, insulin dose in units/kg, C-peptide levels, glycaemia, and previous insulin exposure. Anti-proinsulin antibodies were more likely in Type 1, p<0.002 and in patients exposed to beef insulin, p<0.001. Anti-proinsulin and anti-insulin cross-reacted among species. Treatment with HPI resulted in disappearance of anti-HPI over 6 months, 11.6 to 1%, p<0.001, but not anti-HI. Both groups decreased HgbAlc, i. e. Group I: 6.64 to 6.01%, p < 0.0001. Units/kg decreased in Group I at 3 months, 0.68 to 0.58, versus no change in Group II, p < 0.001. Further, in Group I individuals lacking anti-HPI at baseline, new antibodies did not develop. Transfer to HPI controls glycaemia at a lower dose/kg and is associated with reduction of pre-existent antibodies without production of new anti-HPI. 154. Relationship between ~-endorphin levels and glomerular filtration rate in short-term Type 2 (non-insulin-dependent) diabetic patients with good metabolic control M. Fioravanti, S.B. Solerte, F. Petraglia 1, A.L. Patti, C.Viola, A. R. Genazzani 1 and E. Ferrari. Department of Internal Medicine, Medical Clinic II, University of Pavia, Italy, 1Department of Obstetrics and Gynecology, University of Modena, Italy fl-endorphins (/3-EP) have been recently involved in the regulation of glomerular filtration rate (GFR) in Type 1 (insulin-dependent) diabetic patients with glomerular hyperfiltration and overt nephropathy.

519 A This study concerns the evaluation of plasma fl-EP, together with renal haemodynamics, in short-term Type 2 (non-insulin-dependent) diabetic patients without obesity and in good metabolic control. In 14 Type 2 diabetic patients with glomerular hyperfiltration (mean G F R = 1 5 6 + 1 7 ml/min/1.73 m 2) and 16 matched Type 2 patients with normal GFR (102+7 ml/min/1.73 m 2) plasma fl-EP, fl-lipotropin (fl-LPH), plasma renin activity (PRA), ACTH, cortisol, GH and aldosterone levels were studied together with GFR and renal plasma flow (RPF). Increased plasma fl-EP and PRA levels were found in hyperfiltering diabetic patients (13 _+2 fmol/ml and 4.3 _+ 1 n g / m l / h respectively) compared to patients with normal GFR (8.4_+ 1.5 fmol/ml, p < 0.001 and 2.1 + 0.5 ng/ml/h, p < 0.001 respectively). Moreover a positive correlation between fl-EP and PRA was observed in the whole group of diabetic patients (r=0.61, p<0.001). No changes in ACTH, cortisol, GH and aldosterone levels were demonstrated, whereas lower plasma /?-LPH concentrations were found in hyperfiltering (6.0 + 2 fmol/ml) than in normofiltering diabetic patients (8.6 + 2 fmol/ml, p < 0.01). Plasma fl-EP levels significantly correlate with both GFR (r=0.70, p<0.001) and RPF (r=0.67, p<0.001) in our patients. Circulating //EP levels might therefore be responsible for the occurrence of early glomerular alterations in diabetes.

155. Superoxide radical and the fructosamine reaction C. M. Florkowski, A. F. Jones, C. Le Guen, J. Winkles, P.E. Jennings and A. H. Barnett. Departments of Medicine, East Birmingham Hospital; Clinical Chemistry, Selly Oak Hospital and University of Birmingham, U K Non-enzymatically glycated proteins, specifically ketoamines, are reductants at alkaline pH, a property used in the fructosamine assay of serum glycation. We report the superoxide radical dependency of this reaction. Assays were performed in a 100 retool/1 sodium carbonate buffer, pH 10.35, measuring the reduction of nitroblue tetrazolium or cytochrome C by diabetic sera or albumin glycated in vitro. Superoxide dismutase (SOD) in concentrations up to 100 rag/1 markedly inhibited the reaction, with half-maximal inhibition achieved at concentration of 0.5 rag/1 SOD. At maximal inhibition (100 ms/1 SOD), the reducing activity of diabetic sera and glycated albumin were inhibited by 50% and 70% respectively. Dialysis of serum increased the maximal level of inhibition to 70%. The superoxide radical is a reductant at alkaline pH, and reducing activity that can be eliminated by SOD is classically attributed to this radical. We conclude that the fructosamine reaction depends upon the generation of the superoxide radical by protein ketoamines in a one electron reduction of oxygen. A single electron oxidation of the ketoamine would necessarily be involved. The free radical intermediates of the glycated protein may be of pathological significance in protein browning, known to be accelerated at alkaline pH, by inducing protein oxidation and cross linking.

156. Influence of the protein concentration on fructosamine values R. FlOcklger, T. Woodtli and W. Berger. Departments of Research and Internal Medicine, University Clinics, Basel, Switzerland The fructosamine test appears to be a convenient, inexpensive, and precise method for the quantitation of serum or plasma protein glycation which is amenable to automation. We evaluated the biological variability of results from this assay. Plasma was collected from 4 Type 1 (insulin-dependent) diabetic patients at hourly intervals over 48 h while maintaining normal physical, eating, and sleep-time habits. The fructosamine assay was performed with the CobasBio centrifugal analyser utilising commercial reagents and a glycated albumin standard. Total protein was also determined. Fructosamine values fluctuated by up to 1 mmol/1 in parallel to the protein concentration (range: 5-8 g/dl), causing overlap between results of patients in poor and satisfactory glycaemic control. Fructosamine values and protein concentration were linearly related. Correcting fructosamine values and protein concentration were linearly related. Correcting fructosamine values for protein concentration reduced one third of the variability between samples (coefficient of variation 7.1% vs 4.5%). The variability, which is only in part due to fluctuations in protein concentration, introduces uncertainty in assessing averaged glycaemia of up to 7.73 mmol/L It is not clear how this drawback may be overcome. Unless the factors causing the variability are fully established and correction is possible, results from the frnctosamine assay have to be interpreted with caution.

520 A 157. Improved skin perfusion in the diabetic neuropathic foot with euglycaemia induced by short-term insulin infusion M. D. Flynn, M. Boolel, H.M. Flynn, G.C. Carter, J.E. Tooke and RJ.Watkins. Diabetic Dept., King's College Hospital, London and Clinical Microvascular Lab., Dept. of Physiology, Chafing Cross and Westminster Hospital Medical School, U K Insulin is a hypoglycaemic and vasoactive agent. We have demonstrated that short-term insulin infusion causes major changes in foot skin perfusion, increasing the nutritional capillary flow more than the shunt flow. Five male diabetic patients aged 48-68 years were studied after omission of the previous evening's therapy. They acclimatised lying supine for 30 min in an environmentally controlled room at 22 + 0.5 ~ Insulin was infused at 8.5 _+3.4 (mean + SD) units over 15 rain and then at a constant rate of 3.6_+1.1 units/h. Mean blood glucose fell from 15.4_+ 1.9 to 6.8 + 1.9 (p= 0.05 Wilcoxon rank sum test) without hypoglycaemia. Mid-foot arterial inflow increased from a mean of 1.37 to 1.46 ml.min -~.100 ml -a tissue (NS) but great toe pulp laser Doppler flow (predominantly arteriovenous shunt flow increased by 106.7 _+59.9% (.p= 0.05) above rest flow at intermediate glucose levels but was not significantly increased at euglycaemia. In contrast, nutritional capillary blood flow measured by television microscopy increased progressively to a peak of 136.1_+95.2% above restflow (p=0.05). The preferential partitioning of blood flow between shunt and nutritive circulation achieved by insulin infusion and euglycaemia demonstrated here may have important therapeutic implications in the management of the ulcerated diabetic neuropathic foot. 158. Stiff man syndrome - Type I (insulin-dependent) diabetes mellitus: a common primary autoimmune pathogenesis? F. Folli, M. Solimeja, G. C. Coral, E. Bosi, G. Po~a, P. Decamilli and A. H. Vicari. Clinica Medica VII, C N R Center for Cytopharmacology and Clinica Neurologica, Milan, Italy Sitff man syndrome (SMS) is a rare CNS disorder, characterised by spontaneous muscle rigidity and painful spasms, due to a simultaneous activation of agonist and antagonist muscles. We describe the first case of SMS associated With Type 1 (insulin-dependent) diabetes. Autoimmune screenings showed: positive complement-fixing islet cell antibodies, thyroglobulin and thyroid microsomal antibodies, gastric parietal cell antibodies and smooth muscle antibodies. HLA phenotype was: A 1/28, B 8/44, Cw 5/x and D R 3 / 4 . The patient was euthyroid with no goiter. Blood counts were normal. We searched for signs of autoimmunity against the CNS. An impressive pattern of immunoreactivity in the gray matter, and particularly in the GABAergic system was found when serum or cerebrospinal fluid were used to immunostain rat brain sections. By double immunofluorescence and by immunoblot, we identified glutamic acid decarboxilase (GAD) (an intracellular enzyme involved in the biosynthesis of GABA) as the major antigen. Recent studies support the concept that SMS is probably due to a selective damage of the GABAergic system. GAD and GABA are present also in the B cells of pancreatic islets. Our findings suggest a possible common primary autoimmune pathogenesis in a syndrome involving two GABA producing cellular types. 159. A moderate switch from high- to low-glycaemic index foods for 3 weeks improves the metabolic control of Type I (insulin-dependent) diabetic patients A.M. Fontvieille, M.Acosta, S.W. Riskalla, F.Bornet, P. David, M. Letanoux, G. Tchobroutsky and G. Slama. Department of Diabetes, Hotel-Dieu, Paris, France Glycaemic indexes are calculated using test meals in acute conditions. Their use has not yet proved to be effective in routine diabetic diet counselling. We studied 8 Type 1 (insulin-dependent) diabetic patients (4 men, 4 women); mean age: 43.5 + 3.5 years; BMI: 24.1 + 1.9; diabetes duration: 14.6 + 2.4 years (mean-+ SEM). Each patient received in random order and with a cross-over design two diet prescriptions lasting 3 weeks each equivalent in term of calories, CHO, fat, protein and fiber intake. Thus, diet H enriched in bread and potato bearing a high mean glycaemic index (mGI) was compared to diet L enriched in pasta and rice bearing a low mGI. Diet L contained 2152+79kcal/day; prot.=17.4-+0.5%; fat=35.0+1.0%; CHO = 46.1 -+ 1.6%; mGI = 46.5 -4-0.9% ; Diet H: 2118 + 96 kcal/day; prot.=16.9+0.6%; fat=36.0+1.0%; CHO=45.4+1.6%; m G I = 60.1 _+1.8% (mGI L vs H: p < 0.001). At the end of the L period the following parameters were improved: frnctosamine (% of decrease: - 18 -+ 7% from 2.77 + 0.21 to 2.17 + 0.24 mmol; p < 0.05), triglycer-

Abstracts ides (-16_+7% from 1.40+0.21 to 1.18+0.20 mmol; p < 0.05), phospholipids ( - 8 + 1% from 3.36 + 0.16 to 3.07 + 0.13 mmol; p < 0.01), daily insulin needs ( - 6 + 2 % from 50.2-+5.45 to 47.4+5.9 U.I.; p < 0.05). Mean fasting plasma glucose levels were lower at the end of the L period but not significantly (p<0.1). No significant variations were observed concerning body weight and other circulatory lipid levels. Increasing the proportion of low glycaemic index foods ingested is thus recommendable in Type 1 diabetic patients on a chronic basis. A similar study in 12 Type 2 (non-insulin-dependent) diabetic patients and in 12 Type 1 diabetic patients over a 10-week period is underway. 160. Recombinant human growth hormone regulation of insulin gene transcription in human fetal pancreatic islets B. Formby, A. Ullrich1, L. Coussens 1 and C.M. Peterson. Sansum Medical Research Foundation, Santa Barbara, USA, ~Department of Developmental Biology, Genentech, San Francisco, Calif, USA Immunoreactive human growth hormone (hGH) reaches a maximal concentration in human fetal (HF) serum at 20 weeks of gestational age. The total body mass of the human fetus increases about 100-fold from 10 to 20 weeks of gestation. Insulin has an essential growth-promoting influence in the fetus, which prompts the question as to whether hGH can function as a growth peptide with insulin-releasing activity by stimulating the transcriptional rate of the insulin gene. Pancreatic islets were isolated from human fetuses (n=28) of 18 to 22 weeks of gestational age. Insulin gene transcription rate was quantified by insulin mRNA and assayed by blot hybridisation analysis using 32p-cDNA probe encoding human insulin. By 48 h of culture insulin gene transcription was stimulated by 1.25 gg recombinant h G H / m l to 214% of control value. Insulin secretory capacity was expressed as a fractional stimulatory ratio (FSR = Fz/F 0 of fractional secretion rates during two successive 1 h static incubations. A maximal significant increase in FSR-value to 273% of control was found with 1.25 ~g recombinant h G H / m l after 48 h of culture. We conclude that insulin gene transcription rate is enhanced by recombinant hGH concurrent with an increase in insulin secretory capacity, hence providing evidence for a regulatory function of hGH during HF development. 161. Activation of the sympathetic nervous system during insulin-induced hypoglyeaemia is not prevented by plasma volume substitution H. A. Frandsen, J. Hilsted, C. Berne, J. Fagius and N.J. Christensen. Department of Internal Medicine and Endocrinology F. Herlev Hospital University of Copenhagen, Denmark and Dept. of Int. Med. and Neurology, University Hosp., Sweden Activation of the sympatho-adrenal system during insulin-induced hypoglycaemia could represent baroreceptor-modulated muscle nerve sympathetic activity (MSA) due to plasma volume reduction. We investigated whether the increase in norepinephrine and MSA could be preventend by plasma volume substitution. Methods: In two experiments hypoglycaemia was induced in healthy volunteers by an intravenous bolus of regular insulin, 0.15 IU/kg body weight. In a controlled experiment 7 subjects were once given only insulin and in another experiment in addition a constant intravenous infusion of 250 ml liquid (215 ml NaC1, 9 mg/ml and 35 ml albumin, 200 mg/ml) between 20-40 min after insulin. Plasma catecholamines were measured at 10 min intervals. In 7 other subjects given 350 ml liquid 5-35 rain after insulin, MSA was recorded by microneurography in the fight peroneal nerve. Results: In the controlled experiment with and without volume substitution the nadirs of blood glucose at 30 min (1.40+0.06 mmol/1 and 1.34+0.25 mmol/l respectively) coincided with the peak values of plasma norepinephrine (0.42+ 0:04 ng/ml and 0.45 +0.10 ng/ml). The latter and the integrated responses were identical. MSA peaked at 30 rain with 42 + 4 bursts/min from 18 + 2 bursts/min. Conclusions: Sympathetic nerve activation is not influenced by plasma volume substitution, but could be caused by central nerve activation. 162. Somatostatin as a possible mediator of insulin and glucagon pulsatile secretion M. Frank, D.R. Matthews, R. Spivey, M.A. Burnett and R.C. Turner. Diabetes Research Laboratories, Radcliffe Infirmary, Woodstock Road, Oxford, U K To determine whether, in man, somatostatin (SS) would have the correct characteristics for a paracrine controlling hormone for insulin and glucagon secretory pulses, we have studied 6 normal subjects and 12 maturity-onset diabetic (DM) patients in the basal state, tak-

Abstracts ing 1 rain integrated blood samples for 2.5 h. After 1 h basal we have given 10, 20 or 40 gg pulses SS over 10 s, leaving 16 min intervals for 4 pulses. Insulin suppression was detectable with 10 p~gSS and maximal at 20 p~g SS (T 1/2=3.8 min) while 40 p.g caused no further suppression. Serial array averaging allowed the mean suppression profiles to be examined, and these were identical to the waveform of insulin in normal basal subjects with maximal suppression after 7 min (p<0.001) recovering to baseline at 16 rain. Glucagon suppression was faster in normal subjects (6 min) than in DM (11 rain) which may be a defect in DM allowing hyperglucagonaemia. In keeping with this, glucose concentrations fell cyclically after each SS pulse by mean 0.2 retool/1 (p<0.01) recovering back to baseline, while in DM glucose rose 0.1 mmol/1 (p<0.05). Glucose homeostasis in N was unaffected despite halving insulin concentration for a period. In conclusion: somatostatin fulfills the criteria for a paracine control mechanism within the pancreas to co-ordinate and synchronise pulsations. 163. Early signs of neuropathy and microangiopathy in young Type 1 (insulin-dependent) diabetic patients: correlation with long-term metabolic control V. Frighi, J.W. Loughnane, P. Pozzilli, A.C.Tam, J.M.Thomas, J. E. Taylor, D. Andreani and E.A.M. Gale. Dept. of Diabetes and Immunogenetics and Computer Services Dept., St. Bartholomew's Hospital, London. Endocrinologia I, Universit/l di Roma "La Sapienza', Italy

The relation of metabolic control, duration of diabetes and HLA phenotype to neurological and microvascular complications was investigated in 80 Type 1 (insulin-dependent) diabetic patients (age: 20 +_5 years; duration of diabetes: 10 + 5 years). At investigation 35% (28/80) showed early signs of somatic (11/80), autonomic (8/80) or somatic and autonomic neuropathy (9/80). All were asymptomatic. Ten patients showed background retinopathy, 12 had an albumin excretion rate > 15 lxg/min. Metabolic control was assessed retrospectively (random plasma glucose values since diagnosis and HbA1 values in the last 5 years). Statistics: Mann-Whitney and chi square tests. Patients with neuropathy had higher median plasma glucose and HbAI values than patients without complications (11.4 vs 9.7 retool/l, p < 0.005; 12.3% vs 11.3%,p< 0.005). Significantly higher median plasma glucose levels were found in both patients with AER >15 gg/min (11.7mmol/1) and patients with retinopathy (11.3 mmol/l) than in uncomplicated patients (9.7 mmol/1, p < 0.005 and p < 0.001 respectively). Only patients with retinopathy had longer duration of diabetes than patients without complications (13.5 vs 10 years, p<0.05). No differences were found in HLA (A, B, C, DR) antigens distribution. Conclusion: poor metabolic control over the years is associated with early signs of neurological and microvascular complications in young Type 1 diabetic patients. 164. Galanin-induced inhibition of the insulin release from cultured islet cells in rats Xiao W. Fu and A.M. Sun. Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, Canada

It has been reported that galanin elicits a significant hyperglycaemia in conscious dogs and a fall in plasma insulin. The present study aimed at investigating the effect of galanin on in vitro insulin and glucagon secretion from rat pancreatic islet cells. Rat islets were isolated from pancreas by collagenase digestion method cultured for 24 h at 37 ~ in CMRL-1969 supplemented with 10% bovine fetal serum and various concentrations of glucose. After preincubation, the islets were incubated with medium containing galanin for 1 h at 37 ~ with continuous shaking. Insulin and glucagon concentrations were measured by radioimmunoassay. Insulin release from pancreatic islets was inhibited dose-dependently by galanin (3 x 10- 8 retool/ 1, 6 x 10 -7 mmol/l, 3 • 10 -7 mmol/1, 3 x 10 -6 mmol/1). The most prominent inhibitory effect of galanin (3 • 1 0 - 6 mmol/1) on insulin release was found with medium containing normal levels of glucose (5.5 retool/l); insulin release was reduced to 27.8% (SE + 3.1%, p < 0.01) as compared with the control. In contrast, glucagon release was not significantly changed by adding galanin to the medium. Thus, galanin seems to act as a modulator to control insulin release from the pancreatic B cells in normal condition. 165. Kinetics of human and porcine insulins in normal subjects and Type t (insulin-dependent) diabetic patients S. Fugleberg, B. Thorsteinsson and C. Binder. Steno Memorial Hospital and Hagedom Research Laboratory, Gentofte, Denmark

521 A The kinetics of insulin display saturability at supraphysiological plasma insulin concentrations in normal subjects and in Type 1 (insulin-dependent) diabetic patients. Existence of saturability invalidates studies based on assumed first order kinetics. We therefore studied whether saturation kinetics are apparent at physiological plasma insulin concentrations. Human and porcine insulin was infused intravenously at various rates into 4 normal subjects (age 21-24 years) and 6 Type 1 diabetic patients (age 26-39 years, diabetes duration 3-25 years, insulin antibodies, 0-2.3%), using a doubleblind cross-over design and euglycaemic glucose clamp (3.9+ 0.1 mmol/1). The relationship between steady state plasma free insulin concentration and plasma disappearance rate was studied by mathematical model validation procedures. Both human and porcine insulin obeyed saturation kinetics in normal subjects and first order kinetics in diabetic patients at the insulin concentration range studied (0-2 nmol/1). No differences in parameter estimates existed between the two species of insulin within the study groups. The median clearance rate of insulin in normal subjects was 31 ml. kg -a. rain- 1 (range 22-63) at infinitesimal plasma insulin concentrations versus 21 ml-kg -a-min -1 (14-26) in the diabetic patients (p<0.01). Thus, at physiological plasma insulin concentrations both human and porcine insulin disappear faster via the saturable mechanism(s) found in normal subjects than via the apparently linear mechanism(s) found in diabetic patients. 166. Circulatory disease mortality in the WHO Multinational Study J. H. Fuller, J.A. Head and the WHO Multinational Study Group. University College and Middlesex Hospital Medical School, UK Cause-specific mortality rates over a follow-up of 6-7 years have been obtained for 4740 middle-aged patients originally attending ten centres participating in the WHO Multinational Study of Vascular Disease in Diabetics. Causes of death were assigned using standardised procedures based on all available information including death certificates, autopsy reports and clinical records. Circulatory disease (ICD 390-459) mortality rates were highest in Berlin for males (19.5/1000person years) and highest in Warsaw for females (9.7/1000 person years). Mortality rates were lowest in Tokyo for both sexes. For all centres except Zagreb, circulatory disease mortality rates were substantially higher in male diabetic patients compared to females. The proportion of deaths ascribed to ischaemic heart disease (ICD 410-414) varied considerably between centres, being highest for London (M: 63%; F: 43%) and lowest in Hong Kong for males (33%) and Warsaw for females (27%). Of the several base-line factors examined, mean centre systolic blood pressure had one of the strongest relationships to circulatory disease mortality rates (M: r=0.54, p<0.05; F: r=0.63, p<0.05) and made a significant contribution to overall centre differences in mortality. 167. High release of soluble interleukin-2 receptors by peripheral mononuelear cells in Type 1 (insulin-dependent) diabetes A. Galluzzo, C. Caruso 1, C. Giordano, M.A. Modica 1, A. Marco and M. EZambito a. Clinica Medica and aPatologia Generale, University of Palermo, Italy Peripheral blood mononuclear cells (PBMC) from 10 Type 1 (insulin-dependent) diabetic patients (5 newly-diagnosed and 5 longstanding), mean age 17 + 9.6 years, and 5 control subjects were investigated for their capacity to generate soluble interleukin-2 receptors (IL-2R) following cellular activation in vitro after stimulation with PHA (50 mcg/ml). Soluble IL-2R were measured in cell-free supernatants and sera with a "sandwich" enzyme-linked immunoabsorbent assay. Serum soluble IL-2R in the Type 1 diabetic patients were higher (405+47.3 U/ml) than in control subjects (250+78 U/ml) (p< 0.001). As regards supernatant IL-2R production after 24 h incubation, the Type 1 diabetic PBMC produced larger quantities of soluble IL-2R with medium alone (63.3+25.8 U/2.5xl06cells) than the controls (20 + 7.05 U/2.5 • 106 cells). Comparable amounts of released IL-2R in normal subjects and diabetic patients were found in supernatants after 24 h PHA activation, although the newly-diagnosed patients seemed to release more soluble IL-2R. The high levels of released IL-2R in Type 1 diabetes may be related to activated T lymphocyte clone(s). Analysing the T cell phenotypes with monoclonal antibodies by immunofluorescence after 24 h culture period, this hypothesis was confirmed by a higher percentage of activated T cells (TAC +) in Type 1 diabetic patients than in control subjects. The elevated production of secreted IL-2R in Type i diabetes, not correlated with glycaemic and glycosylated haemoglobin levels, may reflect the excessive lymphocyte activation in the disease.

522 A 168. The insulinotropic effect of various proteins given with glucose in Type 2 (non-insulin-dependent) diabetic patients M. C. Gannon and F.Q. Nuttall. Metabolic-Endocrine Section, V.A. Medical Center and University of Minnesota, USA We previously reported that 50 g protein as lean beef, given with 50 g glucose, results in a greater insulin response in Type 2 (non-insulindependent) diabetic patients than the sum of the two ingested individually. Fifty g protein was just as potent as 50 g glucose in stimulating insulin secretion. Thus we were interested in determining whether other proteins would be as effective. Serum glucose and insulin responses were determined in 6-16mate, untreated, mild Type 2 diabetic patients after ingestion of meals consisting of 50 g glucose+25 g protein from 7 sources (beef, turkey, gelatin, egg white, cottage cheese, fish, or soy), given in random order as a breakfast meal. Insulin and glucose were determined at 0, 0.5, 1, 2, 3, 4, and 5 h. The net areas under the glucose (GA) and insulin (IA) curves also were determined. The GA was smaller following all glucose+protein meals ( 8 - 1 2 m m o l . l - l . h -1) compared to glucose alone (13 mmol.l-l.h='f). The IA was larger following all glucose+protein meals (99-173 lxU.h-l.m1-1) compared to glucose 0 alone (75 g U . h - 1 .ml- 1). The 0V0 I A : G A ranged from 152)/0 (egg white) to 355% (soy) when glucose alone was set at 100%. It is clear that the insulinotropic effect of protein was synergistic with that of glucose for all proteins studied, as previously reported for beef. 169. Prevalence of microalbuminuria in a multicenter study of diabetic patients in Northern Italy P. Garancini, G. Calori, P. Valsania, P. Micossi and G. Gallus. Unit of Epidemiology and Department of Medicine, S. Raffaele Institute, Milan, Italy Data from the literature show that a raised albumin excretion rate (AER) is a predictor of clinical proteinufia and renal failure in Type 1 (insulin-dependent) diabetes and that microalbuminutia is a predictor of early mortality in matutity-onset diabetes. No studies investigating the prevalence of diabetic microalbuminuria involve large samples of diabetic patients and use standardised methods in urine collection. In order to evaluate the prevalence of diabetic nephropathy a multicentre population-based study was undertaken in 1983-84 in 12 antidiabetic centers in the Lombardia region. Out of a total population of 17,704 diabetic patients 1160 were randomly selected within strata of duration of disease ( < 5 ; 6-10; 11-20; > 20 years) and AER assessment was available in 542 patients. Albuminutia was analysed in a central laboratory using a radioimmunologic method from an overnight urine collection. The standardised prevalence rates were 25.8% and 3.0% for microalbuminufia (AER > 30 ~g/min) and clinical proteinuria respectively. The specific rates were higher in the insulin-treated than in the non-insulin-treated patients who otherwise account for more than 60% of the total prevalence of microalbuminuria and clinical albuminuria. The lower rate of clinical proteinuria compared with data from other countries may suggest that Italian diabetic patients develop less severe complications. Results on analysis of risk factors associated to microalbuminufia will be presented.

Abstracts ients versus 75.8+9.3 in the others (NS); in the second, 85.4_+ 9.7 mmHg versus 78.7 + 7.5 (p< 0.001); in the third, 84.6 +- 5.3 mmHg versus 79.3 ___6.4 (p< 0.05). To detect cardiovascular autonomic neuropathy two tests (deep breathing and orthostatism) were performed in 133 (45.7%) patients. In 26 high microalbuminuric Type I patients, 19 (73%) had cardiovascular neuropathy, in the other 107 only 45 (42%), p < 0.01. 171. Cyclosporin A treatment does not influence natural killer cell activity and antibody-dependent cellular cytotoxicity in Type I (insulindependent) diabetes mellitus S. Gaube, C.C. MUller, Ch.Zielinski and G. Schernthaner. Department of Medicine II, University of Vienna, Austria Cydospotin A is currently used as an immunosuppressive agent in newly-diagnosed Type 1 (insulin-dependent) diabetes. Since a possible effect of Cydosporin A on natural killer cell activity (NK) and antibody dependent cellular cytotoxicity (ADCC) is unknown, we studied 37 patients with recent onset Type 1 diabetes mellitus before and shortly after (one week) the initiation of insulin treatment and subsequently in monthly intervals for up to 12 months. Nineteen of these patients included in a multicentre controlled study underwent parallel treatment with Cyclosporin A. N K activity in Type 1 diabetic patients was decreased in comparison to that in 61 control subjects tested simultaneously. No difference in the N K activity between patients treated with Cyclosporin A and insulin and those treated with insulin alone was found (p> 0.1). A decrease of ADCC was seen in the diabetic patients before insulin treatment as compared to control subjects (p< 0.0025). During the follow-up ADCC of Type i diabetic patients reached normal levels. Neither in the initial decrease nor in the subsequent normalisation of ADCC was a difference between Cyclosporin A treated and only insulin treated patients seen. In conclusion, both N K activity and ADCC seem to be impaired in diabetic patients before initiation of insulin therapy. Cyclospofin A therapy did not interfere with the normaiisation of N K and ADCC under insulin treatment.

170. Blood pressure and autonomic neuropathy in a microalbuminuric population L. Gardete-Correia, J. Boavida, E. Pereira, L. da Silva, J. Nunes-Corraa and A.Silva Graga. Associag~o Protectora dos Diab6ticos de Portugal, Lisbon, Portugal

172. Distal revascularisation in diabetic ischaemic foot. Results of a one year follow-up G.Ghirlanda, S.Agnes, G.Mingrone, C.Vulpio, M.Carnassale, M. Bianchi, A. Borzone, C. Ianna, C. E. M. Catagneto and A.V. Greco, Rome, Italy Up to now the supposed presence of peripheral microangiopathy has hindered the application of the newer techniques of distal revascularisation in diabetic patients with ischaemic foot, leading to major amputations. Recently the existence of a typical microangiopathy has been questioned, whereas a peculiar pattern of arterial obstruction at infrapopliteal level has been found. In 23 out of 34 Type 2 (non-insulin-dependent) diabetic patients with an ischaemic foot and an arteriographic obstruction of infrapopliteal arteries coupled with patency of dorsal and/or plantar arteries, we performed a distal revascularisation with autologous inverted saphenous vein. We obtained at one year a 70% by-pass patency rate, with limb salvage. One patient died in the post-operative period from myocardial infarction. Mortality rate (4%) was smaller than mortality for major amputations (10%), and similar to that observed in non-diabetic patients submitted to the same operation. We concluded that in diabetic patients an ischaemic foot is usually the consequence of stenosis and/or occlusions of the infrapopliteal arteries, leading to a large indication to distal revascularisation for limb salvage. One year bypass patency rate is similar to that observed in non-diabetic patients.

We studied blood pressure and presence of autonomic neuropathy in 291 Type 1 (insulin-dependent) diabetic patients. Albustix negative (onset -<35, age >15-<50years, evolution >2years, blood pressure < 160/95 mmHg) investigated for microalbuminuria in the last 18 months. In 47 Type 1 patients (16%) with elevated microalbuminutia (> 30 p~g/min) systolic pressure was 128.5 _+13.0 mmHg versus 120.2+11.6 in 244Type 1 patients with microalbuminuria < 30 gg/min (p< 0.001); diastolic pressure was 83.9 _+8.7 mmHg in the high microalbuminuric Type 1 patients versus 77.4 + 8.3 in the others (p<0.001). We divided the 291 Type 1 patients into 3 groups: evolution -< 10 years (130 Type 1 patients, age 25.3 +8.1 years); 11 to 20 (108 Type 1 patients, age 29.4+_9.2 years); >_21 years (53 Type 1 patients, age 41.3 + 7.2 years). Systolic pressure in the first group was 125.5 +_11.4 mmHg in high microalbuminuric Type I patients versus 116.8+-14.5 in the others (/7<0.05); in the second, 128.2+12.9mmHg versus 120.6+12.3 (p<0.02); in the third, 133.1+15.1 mmHg versus 125.6 +_10.4 (NS). Diastolic pressure in the first group was 80.4 + 8.4 in high microalbuminutic Type 1 diabetic pat-

173. Plasma rates of insulin appearance after intraperitoneal or subcutaneous administration: a dose-response study A.Giacca, A.Caumo, G.Galimberti, G.Petrella, M.C. Librenti, P. Micossi and G. Pozza. Istituto S. Raffaele, University of Milano Medical School, Italy A dose-response study was designed to compare insulin rates of appearance (Ra) during sequential administration of A: basal rate (mean 0.8+-0.3U/h); B: 2 . 5 U / h ; C: 7 U / h of human insulin (H0echst HOE21GH U-100) through intraperitoneal (IP) or subcutaneous (SC) route. Five C-peptide negative Type 1 (insulin-dependent) diabetic patients were studied; insulin was continuously infused IP through an implanted programmable device, SC through an external device. The sequence of the studies in the same patient was random. Before starting the study 1 U of insulin was injected intravenously to evaluate the plasma clearance rate (PCR). A steady-state of plasma free insulin was reached and maintained for at least

Abstracts 20min. Steady-state plasma free insulin were: IP: A5.6+2.7; B 8.6_+1.9; C 25.3+8.2 I.tU/ml+SD. SC: A6.5_+3.4; B 15.8_+8.7; C: 37.2_+ 10.7 ~tU/ml_+ SD, p < 0.025 IP vs SC. PCR were 17.7 + 5.4 during IP and 15.9+3.5 ml.kg -1 -min -a during SC infusion, NS. Ra were: IP: A5.9+_4.5; B 7 . 7 + 2 ; C 2 5 . 3 - + 8 . 2 m U / m i n + S D . SC: A6.8-+6; B 12.9+7.3; C 35.5+16.4 mU/min-+SD, p<0.025 IP vs SC. During IP infusion about 20% of the infused insulin reached the blood stream; during SC, about 30%. It is concluded that: I) IP infusion resulted in lower circulating free insulin at the three flow-rates. 2) The difference was attributable to a significantly lower rate of appearance. The observation that a lower percentage of insulin reached the blood stream during IP than during SC infusion is in favour of a higher hepatic extraction and degradation of insulin following IP administration. 174. Urinary albumin excretion rate (UAER) after short-term low-dose dopamine infusion in Type I (insulin-dependent) diabetes mellitus O.Giampietro, R.Miccoli, R.Anichini, A.Bertolotto, L.Di Palma, G. Penno, A.Clerico and R. Navalesi. Cattedra di Malattie del Ricambio, Istituto di Clinica Medica II, C.N.R. Clinical Physiology Institute, University of Pisa, Italy We studied the effects on albuminuria of a 30-min low-dose ( < 3 ~tg.kg -~. min -a) intravenous dopamine infusion in 30 Type 1 (insulin-dependent) normoalbuminuric ( < 15 gg/min) diabetic patients (14 M, 16 F; aged 29 -+ 11 years) and in 10 age-matched control subjects (7 M, 3 F; aged 27 _+2 years). In diabetic patients, basal- and after-dopamine urinary albumin excretion rate (UAER) were higher (p<0.05) than in control subjects (6.9_+2.6 vs 4.2_+0.8 p~g/min; 29.9 _+34.2 vs 10.9 _+11 l.tg/min respectively). After dopamine, UAER increase was significant (p< 0.01) in diabetic patients but not in control subjects. In 6 diabetic patients and 6 control subjects who had repeated dopamine infusion at the same doses and experimental conditions, results achieved were superimposable to the first test. In 7 diabetic patients and 6 control subjects, control saline infusion did not affect UAER. Among diabetic patients the rate of "responders" to dopamine (UAER >than 2 SD above the after-dopamine mean value of control subjects) increased (p< 0.05) with the rise of diabetes duration. Responders and non-responders did not differ as to prevalence of retinopathy. A short-term low-dose dopamine infusion is a simple, repeatable procedure to test glomerular membrane permeability in normoalbuminuric Type I diabetic patients. It could be usefully employed as a provocative test for microalbuminuria. 175. Salivary insulin is related to plasma-free insulin basally and after subcutaneous injection of insulin in insulin-treated diabetic patients R.Giannarelli, P. Marchetti, A.Zappella, P.Cecchetti, A.Masoni, M. Giannecchini, C. Grossi, k Cruschelli, F. Caricato and R. Navalesi. Cattedra Malattie del Ricambio, Istituto Clinica Medica II, Istituto Fisiologia Clinica CNR, University of Pisa, Italy In insulin-treated diabetic patients plasma insulin is largely bound to its antibodies, with the unbound hormone (plasma free-insulin, PFIRI) representing the metabolically active fraction. Measuring PFIRI is troublesome, especially for the dynamic equilibrium of insulin with its antibodies; therefore, the measured PF-IRI levels may not represent the actual in vivo values. We have previously shown that salivary insulin (S-IRI) is related to plasma IRI in normal subjects and Type 2 (non-insulin-dependent) diabetic patients after oral glucose. Presently we evaluated the relationships between PF-IRI (measured after polyethylene-glycol precipitation within 30 s of drawing blood samples) and S-IRI (measured by a sensitive, precise and accurate radioimmunoassay) in 46 diabetic patients (age 3 8 + 2 years, mean + SEM; duration of diabetes 11 _+1 years; duration of insulin therapy 9 ___1 years). The daily insulin dose was 53 + 4 U. Fasting PFIRI and S-IRI resulted 13.5 + 1.8 ~tU/ml and 4.1 _+0.6 ~tU/min respectively. S-IRI was significantly correlated (r=0.42, p<0.01) to F-IRI. Both fasting PF-IRI and S-IRI were correlated positively (p< 0.01 and p < 0.05 respectively) with the intermediate-acting insulin evening dose. Five patients were additionally studied over a 6 h period after subcutaneous injection of insulin. Again, a significant correlation emerged between PF-IRI and S-IRI (r=0.59, p<0.01). Thus, S-IRI may represent an alternative and simpler way to evaluate the plasma values of free-insulin. 176. Histamine content of plasma and tissues in diabetic rats D. S. Gill, C. S. Thompson and P. Dandona. Metabolic Unit, Department of Chemical Pathology & Human Metabolism, Royal Free Hospital and School of Medicine, London, U K

523 A In view of the previous observation that the histamine content of the aorta in rats with streptozotocin-induced diabetes mellitus is increased significantly over that in normal rats, we investigated the effect of experimental diabetes on the histamine content of the plasma, the aorta, the kidney, the heart, the lung and the brain. Our data show that the median (range) histamine content of the plasma and the other organs mentioned above is significantly greater in diabetic rats (n=13) than that found in controls (n=14) plasma: 28.6 (14.9-35.3) ng/ml in controls vs 71.3 (44.6-96.5) in diabetic rats, p < 0.01; aorta: 348 (326-384)ng/mg protein vs 671 (621-695), p < 0.001 ; heart: 3.83 (3.39-4.09) ng/mg protein vs 4.83 (3.96-5.33), p < 0.01; kidney: 0.46 (0.37-0.53)ng/mg protein vs 0.84 (0.73-0.96), p<0.001; lung: 5.57 (3.01-7.19)ng/mg protein vs 7.52 (4.73-10.13), p<0.01; brain: 0.56 (0.37-0.79)ng/mg protein vs 0.76 (0.49-1.0), p<0.02. This increase in plasma and tissue content may contribute to the increase in capillary and endothelial permeability known to occur in diabetes, and may have a role to play in the pathogenesis of diabetic micro- and macroangiopathy.

177. Increased soluble IL-2 receptor levels in the sera of Type I (insulin-dependent) diabetic patients C. Giordano, A. Marco, C. Caruso 1, F. Pant6 and A. Galluzzo. Clinica Medica and apatologia Generale, University of Palermo, Italy Recently, the presence of a soluble form of IL-2 receptor in human sera and in supernatants of PHA-stimulated lymphocytes has been demonstrated. It has been suggested that autoimmune diseases could be characterised by a defect in production of released IL-2R, unlike immunopriliferative disorders which are characterised by overproduction. Our aim was to investigate the levels of circulating soluble IL-2R in 28 newly diagnosed Type 1 (insulin-dependent) diabetic patients (mean age 16.4_+ 6.8; time from diagnosis 45 + 37 days), in 24 age-matched healthy blood donors and in 5 patients with Hodgkin's disease, as a group with high serum IL-2R levels. Using a "sandwich" enzyme immunoassay, we dosed released IL-2R, utilising two monoclonal antibodies, anti-TAC and 7G7/B6, that recognise distinct epitopes on the human IL-2R molecule. We found that newly diagnosed diabetic patients have higher released IL-2R levels (413 +_197 U/ml) than control subjects (250 + 78 U/ml) (t7< 0.005). The mean levels of released IL-2R in patients with Hodgkin's disease were confirmed higher (705+407 U/ml) than controls (p< 0.001). The persistence of circulating soluble IL-2R high levels was observed in 18/28 diabetic patients after 6 months from diagnosis (470 _+196 U/ml). The increased levels were not correlated with glycaemic and glycosylated haemoglobin levels and patients' age. The increase in released IL-2R may mean that more IL-2 is available for an exchange with surface IL-2R to maintain B-cell pancreatic autoimmunity. Our findings suggest a potentially significant role for the released IL-2R in the regulation of IL-2 dependent lymphocyte functions in Type 1 diabetes. We believe that soluble IL-2R may provide a new tool for the study of immunological dysregulation in diabetes. 178. Increased B-cell responsiveness to ~-endorphin is present in human obesity D.Giugliano, D.Cozzolino, T.Salvatore, A.Ceriello, R.Torella and F. D'Onofrio. Istituto Medicina Generale, Terapia Medica e Malattie del Metabolismo, First Faculty of Medicine, University of Naples, Italy The finding that naloxone, an opiate antagonist, may inhibit feeding in a variety of situations indicates a role for endogenous opioid peptides in the regulation of food intake. Naloxone (5 mg i.v.) was given in both lean (n= 7) and obese (n= 7) healthy subjects. In lean subjects, there was a slight trend for insulin and C-peptide levels to decrease below basal values, with no glucose change. Obese subjects showed an exaggerated suppression of insulin and C-peptide and a slight decrease of glucose. Glucagon was suppressed in both groups. An infusion of human fl-endorphin (0.05 mg/h) produced only minor changes in plasma glucose, insulin and C-peptide concentrations in lean subjects (n=6), but caused marked increments in obese (n= 6); glucagon rose in both groups, but its response was greater in obese subjects. A ten-day treatment with naloxone (1.2 mg twice a day) did not change the metabolic and hormonal responses to an oral glucose load (75 g) in lean (n=6) but significantly inhibited the insulin and C-peptide responses to glucose in obese (n= 6) people. These results suggest that an increased oioid drive to the pancreatic B cell and an increased responsiveness of insulin to fl-endorphin are present in human obesity.

524 A 179. Fasting plasma C-peptide, glucagon stimulated plasma C-peptide, and urinary C-peptide in relation to type of diabetes H. J. Gjessing, L.E. Matzen and A. Froland. Medical Department, Fredericia Hospital, Denmark Recent epidemiological data indicate rarity of Type 2 (non-insulindependent) diabetes mellitus before the age of 40 years and insulin treatment of many Type 2 diabetic patients in order to control hyperglycaemia. In order to relate B-cell function to clinical type of diabetes fasting plasma C-peptide, glucagon stimulated plasma C-peptide, and 24 h urinary C-peptide were determined in 132 insulin-treated diabetic patients. Almost the same number of patients were classified as C-peptide non-secretors whether using fasting (n = 53), stimulated (n= 51), or urinary C-peptide (n= 55). Among patients with fasting C-peptide ranging from 0.20-0.30 nmol/l (n = 14), stimulated C-peptide from 0.30-0.60nmol/1 (n=17), and urinary C-peptide from 3.5-7.0 nmol/1 (n=20) or 0.45-1.20 nmol/~mol creatinine/24 h (n = 25) about 70% were diagnosed after the age of 40 years. Between 90-100% of patients with C-peptide levels above these ranges were diagnosed after the age of 40 years. Between 40-50% of patients with preserved B-cell function and C-peptide levels below these ranges were diagnosed after this age. Among patients with C-peptide levels below the ranges, a large proportion were ketose-prone and treated with insulin within first year of diagnosis. Patients with C-peptide levels above had the highest values of BMI and fasting plasma free insulin. These data suggest that the described ranges distinguish between Type 1 and Type 2 diabetes. 180. Production of human monoclonal autoantibodies to insulin R. M. Gledhill, I.H. Mirza 1, U. Keller, R.D.G. Leslie, M.R. Norman and T.J.Wilkin. King's College School of Medicine, London SE5, 1Dept. Medicine II, Southampton General Hospital, UK, and 2Institute of Microbiology & Hygiene, Basel, Switzerland Hybridomas producing human antibodies against insulin have been produced from the peripheral blood of a patient with hypoglycaemia caused by an IgG autoantibody to human insulin. Mononuclear leucocytes (1.2 • 108) were cultured with pokeweed mitogen (1%) for 4 or 7 days, after which they were fused with the mouse x human heteromyeloma cell line SHM-D33, using PEG 4000 (50%) as fusogen. Fused cells were plated in 96 well plates containing human skin fibroblast feeder cells (Detroit 532). Hybridomas were selected by culture in HAT medium containing ouabain (5 • 10 -7 mol/1). Colonies grew in about 70% of the wells and about 50% of these colonies (approximately 500) produced human antibody. ELISA assay detected 3 colonies which were producing antibodies to insulin. Cloning by limiting dilution produced stable clones. All the clones produced IgM antibodies which bound to porcine as well as human insulin. These hybridomas are probably the first cell lines derived from a patient with autoantibodies to insulin and should contribute to the analysis of binding specificity and affinity of insulin autoantibodies.

181. Stimulation of calcium net uptake is not functioning in pancreatic islets of fetal rats C. Glocker and H. P. T. Ammon. Department of Pharmacology, Institute of Pharmaceutical Sciences, University of Ttibingen, FRG Calcium uptake into islet cells is closely related to glucose-induced insulin release9 Fetal islets exhibit little or no insulin secretion when being challenged by glucose. Calcium net uptake into collagenaseisolated pancreatic islets of adult rats and of 21.5 day-old fetuses was determined. Islets were incubated for 30 min with 45 Ca 2 + (26 ~tCi/ ml). Insulin secretion (90 rain) was measured by RIA. Glucose (0, 3, 16.7 retool/l), glyceraldehyde (10 retool/l), leucine (20 retool/l), bBCH (20 mmol/1), tolbutamide (100 l.tg/ml), glibenclamide (0.5 lxg/ ml, 5.0 lzg/ml), potassium (20 mmol/1, 40 retool/l) and arginine (20 mmol/1) significantly stimulated Ca 2+ net uptake into islets of adult rats. None of the above substances affected net uptake of calcium into fetal islets. Glucose, glyceraldehyde and potassium (both in the absence of medium glucose), leucine, b-BCH, tolbutamide, glibenclamide and arginine (together with 3 mmol/1 glucose) stimulated the secretion of insulin from islets of adult rats. However, using fetal islets glucose and glyceraldehyde had no effect, leucine, bBCH, tolbutamide, glibenclamide, potassium and arginine exhibited only a poor insulin secretory response. It is concluded that failure of stimulation of insulin secretory response by fetal islets may at least in part be due to the inability of the above-mentioned substances to increase calcium net uptake.

Abstracts 182. Insulin/C-peptide secretion in a North American population: age and Type 2 (non-insulin-dependent) diabetes in the Wadena City Health Study F. C. Goetz, A. Bender, S. Bushhouse, K. Davis, R. French, M. Geisser, J.Mandel, K.Muckala and P.Oestreich. University of Minnesota and Minnesota State Department of Health, Minneapolis, Minnesota, and Tri-county Hospital, Wadena, Minn, USA The impaired glucose tolerance of old age may be due primarily to impaired tissue responsiveness to a normal insulin supply. To test this hypothesis, we are measuring 4-h urine C-peptide (an index of integrated insulin secretion) and plasma C-peptide in an age-stratified, randomly-selected sample of 600 non-diabetic adults, and in 120 known diabetic patients, based on the population of Wadena, Minnesota (4,700 inhabitants), a rural community in central North America. Two stimulants to insulin secretion are used: a 75-g oral glucose load (as part of the National Diabetes Data Group tolerance test) and a 700-calorie liquid mixed meal, Ensure-Plus (Ross Laboratories). Novo antibody M1221 is used for C-peptide immunoassay. Results in the first 217 subjects in the random sample, expressed as nmol urine C-peptide/4 h (mean + SD) are: After glucose challenge, age 20-39, 8.4+3.6 (n=40); age 40-59, 8.6+6.0 (n=62); age 60+, 9.1 _+6.0 (n= 72). After meal challenge, age 20-39 10.8 _+6.5 (n= 41); age 40-59 10.1+4.9 (n=61); age 60+, 10.4___5.6 (n=72). These results support the conclusion that there is no significant decline in insulin secretion in surviving older humans. In fact, plasma C-peptide was significantly higher (p<0.001) in subjects age 60+. The results also provide comparative data for interpretation of C-peptide results in Type 2 (non-insulin-dependent) diabetic patients from the same population. 183. Endogenous protein substrate for transglutaminase in pancreatic islets R. Gomis, A. Casanovas and W.J. Malaisse. Endocrinology and Diabetes Unit, Hormone Laboratory, Hospital Clinic, Barcelona University, Barcelona, Spain and Laboratory of Experimental Medicine, Brussels Free University, Brussels, Belgium Transglutaminase may participate in those motile events associated with proinsulin conversion and insulin release in the pancreatic B cell, but the endogenous substrates for the enzyme were not characterised. The labelling by [2,5-3H]histamine of endogenous proteins was examined in both intact and homogenised islets. In homoge9 nates, protein labelhng represented a Ca 2 + -dependent process inhibited by glycine methylester (but not sarcosine methylester) and tolbutamide. After electrophoresis, some radioactivity was recovered in the stacking gel, whereas autoradiographs of the resolving gel revealed the labelling of a Mr 84000 protein. The labelling of both the material located in the stacking gel and the Mr 84000 protein was suppressed in the absence of Ca 2+ or presence of glycine methylester. Although transglutaminase activity was found in both particulate and soluble subcellular fractions, the endogenous substrates were located mainly in the particulate matter. In intact islets, the incorporation of [2,5-3H]histamine represented a temperature-sensitive, timerelated and concentration-dependent process stimulated by D-glucose in the presence, but not absence, of extracellular Ca2+. Preincubation of the islets at a high D-glucose concentration prior to homogenisation also increased Ca2+-dependent transglutaminase activity as judged from the labelling of material either precipitated by TCA, recovered in the stacking gel or migrating at the Mr 84000 level. 184. Complementarity of antibodies against islet cell antigens in Type 1 (insulin-dependent) diabetes F. Gorus, M. Vercammen, P.in't Veld, W. Gepts, O. Segers, G. Somers, M.Van De Winkel and D.Pipeleers. Dept. of Metabolism & Endocrinology Vrije Universiteit Brussel, Belgium The clinical onset of Type 1 (insulin-dependent) diabetes is often associated with circulating antibodies which bind to islet cell antigens. The antibodies are identified through their binding to cytoplasmic (ICA), surface (ICSA) or hormonal (insulin autoantibodies= IAA) islet cell antigens. We have investigated whether a correlation exists in the occurrence of ICPh ICSA and IAA in patients younger than 20 years at diagnosis. The antibody screening was positive for 2/30 IgM and 0/30 IgG fractions from age-matched control subjects. In recently diagnosed Type 1 diabetic patients, 19/30 scored positive in at least one of the tests. In 18/19 positive samples, binding occurred with the IgG class; it was quantified by FACS analysis for ICSA, by ELISA for IAA and by dilution for ICA. Positivity for

Abstracts ICA was noted in 12/27 patients, for ICSA in 7/30 and for insulin in 4/30. No correlation was detected in the tested islet-cell markers, not even when the comparison was only conducted on strongly positive samples (above mean control + 6 SD). These results indicate that the present methods for detection of ICA, ICSA and IAA assess binding to different target molecules. Their information concerning autoimmune reactions against islet cell antigens is thus complementary rather than convergent. 185. Effects of dietary fish oil supplement, MaxEPA in Type 2 (non-insulin-dependent) diabetic patients A.A.Grace, L.D.Curtis, M.A.Vella, K.L.E.Ling, A.C.Dickson, A. R Wilson and D.J. Betteridge. Department of Medicine, University College London, U K Diabetic patients have an excess morbidity and mortality from large and small vessel disease; lipid and platelet abnormalities may contribute to this increased risk. We have studied the effects of MaxEPA, a dietary fish oil supplement rich in the co3 fatty acids eicosapentaenoic acid and docosahexaenoic acid on lipoprotein and platelet metabolism in Type 2 (non-insulin-dependent) diabetic patients. Twenty treated Type 2 patients were randomly allocated on a double-blind basis to eight weeks treatment with either MaxEPA or olive oil capsules as placebo (9 capsules; 9.9 g oil/day). Nine patients (8 M, 1 F; aged 56+14years) received MaxEPA and 11 patients (7 M, 4 F; aged 57 + 10 years) received placebo. Significant increases were observed in the platelet content of eicosapentaenoic (p= 0.02) and docosahexaenoic acid (p= 0.02) in the MaxEPA group and there was a significant fall in platelet factor 4 concentrations in platelet poor plasma (p<0.002). In addition a significant fall in very low density lipoprotein triglyceride (p<0.05) was observed in the treated group. We conclude that dietary fish oil supplementation with MaxEPA in Type 2 diabetic patients may have advantageous effects on lipoprotein and platelet metabolism which may be of importance in the prevention of vascular disease. 186. Abnormal osmoregulated vasopressin responses in a Type 2 (noninsulin-dependent) diabetic patient with a past history of hyperosmolar coma P. J. Grant, K. K. Hampton and R. G. Jones. Department of Medicine, The General Infirmary, Leeds, UK Elderly non-diabetic subjects have decreased thirst, increased osmoregulated vasopressin (aVP) secretion and reduced urinary concentrating ability. Type 2 (non-insulin-dependent) diabetic patients occasionally present with hyperosmolar non-ketotic coma (HONK) with severe fluid loss, the aetiology of which is unknown. To investigate the role of aVP in H O N K we investigated the thirst and osmoregulated aVP responses in a 73 year-old Type 2 diabetic patient with a history of HONK. Blood glucose was maintained at 5 mmol/1 with an insulin infusion. Overnight water deprivation of 16 h was followed b~ 500 ml water loading and 6N saline infusion (0.06 mtkg- 9min- ) over I h. Samples were taken for plasma aVP, osmolality, glucose and electrolytes and urinary osmolality. Thirst was estimated using a 100 mm visual analogue scale and urinary output measured hourly. Urinary osmolality, output, thirst and plasma osmolality responded appropriately. Plasma aVP however, did not rise above the lower limit of detection during water deprivation and rose to 1.5 and 1.3 pg/ml during 6N saline infusion. In six non-diabetic subjects receiving 6N saline, plasma aVP rose to median values of 4.4 pg/ml. The results show blunted aVP responses to osmotic stimuli which together with impaired renal concentrating ability may provide the mechanism whereby severe hypovolaemia occurs in the presence of intercurrent illness, in Type 2 diabetic patients with hyperosmolar coma. 187. T cell reactivity in newly diagnosed diabetic patients and the effect of treatment with isophane insulins R. Gregory 1 and W.G. Reeves 2. Departments of Medicine ~ and Immunology 2, Queens's Medical Centre, Nottingham, U K We have conducted a prospective study of T cell responses in diabetic patients during the first 6 months of treatment with human or bovine isophane insulin. T cell proliferation was measured in vitro at 0, 0.5, 1, 3 and 6 months to insulin, protamine and Candida albicans antigens. We report the results for 12 patients. None showed reactivity to human insulin at time zero: Stimulation Index (SI) = 1.4 + 0.4 vs matched controls 1.8 + 1.1 (mean_+ SD). Their responses to Candida albicans antigen were not impaired (SI=22.4+14.4 vs 28.4+28.2) despite hyperglycaemia (blood glucose = 19.6 + 8.3 mmol/1; HbAa =

525 A 13.5 + 2.6% : normal range < 8.5%). Four patients developed protamine specific responses after I month of treatment (SI = 6.5 _+1.8 vs 1.3 + 0.5). By contrast only one patient had responded to insulin (bovine) by 3 months (SI=4.5 vs 1.8 +0.8). We find no evidence of either impaired antigen stimulated T cell responsiveness or of spontaneous autoimmunity to insulin in newly-diagnosed Type 1 (insulindependent) diabetes. Contrary to popular belief, protamine is considerably more immunogenic than either bovine or human insulin. 188. Screening for at risk microalbuminura in Type t (insulin-dependent) diabetes mellitus A.Grenfell on behalf of Microalbuminuria Collaborative Study Group, London/Poole/Newcastle, U K Overnight urinary albumin excretion rate (ONAER) >30 ~g/min strongly predicts the later development of clinical nephropathy in Type i (insulin-dependent) diabetes mellitus. One thousand seven hundred and thirty-five non-proteinuric Type 1 diabetic patients aged 16-60 years <39 years at diabetes onset and with diabetes duration < 35 years were screened using first morning urinary albumin concentration and albumin/creatinine ratio (Ua/c). Urinary albumin concentration was >15 mg/ml and/or U a / c >3.5 mg/mmol in 341 subjects. O N A E R measured subsequently in 173 of these was > 30 ~g/min in 28. ONAER in a random sample of 65 subjects with urinary albumin concentration <15 rag/1 was <30 ~g/min in all cases. Initial U a / c >2.0 rag/retool identified (predictive value 28.8%) subsequent AER >30 l~g/min with 98.7% sensitivity and 92.9% specificity. U a / c > 2.5, > 3.0 and > 3.5% rag/retool identified AER > 30 ~g/min with predictive values of 32.8%, 28.8% and 32.6% respectively. However both sensitivity and specificity are lowered. Within sample (n=203) U a / c > 2.0 rag/retool identified ONAER > 30 ~g/min (predictive value 57%) with 97.8% sensitivity and 89% specificity. First morning U a / c > 2.0 rag/retool seems to be a useful initial screening test for identifying at risk microalbuminuria in Type I (insulin-dependent) diabetes mellitus. Repeated quantitative measurements may be needed for the precise characterisation of an individual's albumin excretion status due to inherent variability. 189. Insulin receptor tyrosine-kinase phosphorylates in vitro a 67 kDa protein of a novel class of calciproteins bound to cytoskeletal actin F. Grigorescu, F. Martin, J. R Capony, J. C. Cavadore, J. Demaille and J. Mirouze. Department of Endocrine and Metabolic Diseases, Lapeyronie Hospital and INSERM U.249, University of Montpellier I, France Activation of the insulin receptor tyrosine-kinase is an early biochemical event in insulin action at the cellular level. In an attempt to identify intracellular substrates of the kinase, we have studied the ability of the insulin receptor to phosphorylate Ca +2 and actin-binding proteins, which along with lipocortins and calpactins have been recognised as a novel class of regulatory substrates for epidermal growth factor (EGF) receptor kinase and oncogene product pp60 ~c. A 67 kDa protein has been purified to homogeneity from bovine aorta by F-actin affinity and ion-exchange chromatographies. When lectin purified insulin receptor from human or rat was incubated with this protein, in the presence of Mn +2 (4 mmol/1) (g-32p)ATP (50 lxmol/1) and Ca +2 (2 mmol/1), insulin increased the phosphate incorporation into the 67 kDa protein by 3 to 4-fold. Insulin-like growth factor I (IGF-I), but not EGF was equally potent. A calcium chelator, EGTA (3 mmol/l) abolished the reaction. The phosphoaminoacid analysis revealed exclusively phosphorylated tyrosine. These data indicate that the 67 kDa protein may represent a new cellular substrate of the insulin receptor. Further studies on the interaction of insulin receptor with these regulatory calciproteins may give new insight into the molecular mechanism of insulin action. 190. In vivo thromboxane and prostacyclin formation in diabetes meilitus V. Grill, O.Vesterqvist and K. Green. Dept. Clinical Chemistry and Blood Coagulation and Dept. Endocrinology Karolinska Hospital, Sweden Studies on in vivo formation of thromboxanes and prostacyclins in man have been hampered by lack of adequate methodology. We have assessed synthesis by measuring the urinary metabolites 2,3-dinor-thromboxane B2 (2,3-dn-T• and 2,3-dinor-6-keto PG F1~ by gas chromatographic mass spectrometric methods. Mean 24 h excretion of 2,3-dn-TxBz in 20 Type 1 (insulin-dependent) diabetic patients was similar to non-diabetic subjects (398 + 68 and 344+_29 pg/mg

526 A creatinine); however excretion in diabetic patients was more variable. Normalisation of blood glucose (BG) by Biostator (3 patients) failed to affect excretion of 2,3-dinor-TxB2. Excretion was also similar before and after 7-12 days of initial insulin therapy despite reduction of mean BG. (2,3-dinor-TxB2 337 _+78 before, 425 + 136 pg/ mg creatinine after insulin, NS, BG 12.7+0.9 before, 7.4_+ 0.7 mmol/1 after insulin, p<0.01). A longer period (1-4months) lowered levels of HbAlc (from 13.5_+0.6 to 8.9_+0.8% p<0.01), yet excretion of 2,3-dn-TxBz was unaltered (337+62 before vs 328+ 69 pg/mg creatinine after insulin, n = 8). Excretion of 2,3-dinor-6-keto PG F~a was similar in diabetic patients and non-diabetic subjects (177 + 29 vs 205 -+ 30 pg/mg creatinine) and was unaltered by shortterm or long-term metabolic control. We conclude that control of BG does not play a major role in thromboxane or prostacyclin synthesis. Variability of excretion of 2,3-dinor-TxB2 may indicate existence of some diabetic patients with enhanced synthesis and possibly increased risk for vascular disease. 191. Urinary excretion of kappa light chains in Type I (insulin-dependent) diabetic patients L. C. Groop, A. M~ikipemaa, S. Stenman and A.-M. Teppo. Fourth Department of Medicine, Department of PediatricS, Helsinki University Central Hospital, Helsinki, Finland

In order to study factors which influence the urinary excretion of kappa light chains (KLC), we measured its excretion in urine together with that of albumin and beta-2-microglobulin (BM) in patients with newly-diagnosed and long-standing Type I (insulin-dependen0 diabetes and their age-matched control subjects. Newly-diagnosed patients excreted more KLC than control subjects (8.2_+ 1.5 vs 1.5 _+ 0.4 g/24 h, p<0.001). Starting insulin therapy did not normalise the excretion of KLC. Moderate exercise did not influence the urinary protein excretion in these patients. In patients with long-standing diabetes KLC (49.4+ 13.0 vs 3.0-+ 1.0 g/24 h, p<0.001) and albumin excretions (41.9_+ 13.0 vs 11.3 _+2.9 g/24 h, p<0.01) were increased compared with control subjects and they were further enhanced by exercise (albumin: 7.3_+3.0 to 44.3_+32.8 l.tg/min, p <0.01; KLC: 11.3 +_2.9 to 41.0-+13.0 ~tg/min, p<0.05). To examine the effect of hyperglycaemia, we measured the excretion of KLC, BM and albumin in 10 normal subjects before and after each two h of hyperglycaemic clamp (+3, +7, +17 mmol/1). The excretion of albumin and BM rose in a dose-dependent manner during hyperglycaemia, whereas that of KLC was unchanged. Conclusions: (1)urinary excretion of KLC (and other proteins) is increased in patients with newly-diagnosed Type 1 diabetes; (2) acute hyperglycaemia does not influence the KLC excretion; (3) the excretion of KLC in patients with long-standing diabetes is increased during exercise. 192. Risk factors and markers for coronary heart disease in patients with Type 2 (non-insulin-dependen0 diabetes mellitus P.-H. Groop, K.J. T6tterman, C. Saloranta and k Groop. Fourth Department of Medicine, Helsiniki University Hospital, Helsinki, Finland

One hundred and twenty Type 2 (non-insulin-dependen0 diabetic patients (54 F, 66 M) were examined for risk factors associated with coronary heart disease (CHD). Thirty-two patients (26.7%, 14 F, 18 M) had a positive history of CHD, whereas no CHD was found in 88 patients (40 F, 48 M). Patients with C H D differed from those without C H D with regard to age (mean+SEM) (60+1 vs 56+ 1 years, p < 0.001) and prevalence of hypertension (71.8% vs 37.5%, p < 0.001), but not with regard to duration of diabetes, smoking habits, sex and HLA-antigens. Glyeaemic control (FPG, HbA1) was similar in both groups. Fasting C-peptide (0.58+_0.05 vs 0.38+_ 0.02nmol/l) and insulin (IRI) conc. (0.17+0.01 vs 0.12+ 0.01 nmol/1) were higher in CHD-positive patients than in CHDnegative patients (o<0.01). Patients with CHD had higher serum VLDL-triglyceride (Tg) (2.1 _+0.4 vs 1.1 + 0.1 mmol/l, p < 0.02) and LDL-Tg (0.72_+0.1 vs 0.46_+0.01 mmol/1, p<0.02), but lower total HDL-cholesterol (1.2_+0.05 vs 1.4_+0.03mmol/1, p<0.01), HDlm-cholesterol (0.59 + 0.04 vs 0.77-+ 0.03 mmol/1, p < 0.01) and apoprotein A1 conc. (110-+4 vs 121_+2 mg/dl, p<0.05), than patients without CHD. No difference in LDL-cholesterol was observed between the two groups. A positive correlation was observed between IRI and Tg (r=0.32, p<0.001), whereas IRI and total HDLcholesterol ( r = - 0 . 3 3 , p<0.001) and HDI_2-cholesterol ( r = - 0 . 3 8 , p<0.001) were inversely correlated. No correlation was seen between IRI and HDL3-cholesterol (r= 0.03, NS). Conclusion: age, hypertension, increased insulin, VLDL-Tg and LDL-Tg and decreased HDL-cholesterol and HDLz-cholesterol concentrations were asso-

Abstracts ciated with CHD, indicating that known cardiovascular risk factors are operative in patients with Type 2 diabetes. 193. Unemployment in Type I (iasulin-dependeut) diabetic patients without severe complications of diabetes - employment related data of 645 patients in 10 diabetes centers M. Grtil3er, V. Scholz, V. JOrgens and M. Berger. University of Diisseldoff, Department of Metabolism and Nutrition WHO Collaborating Center for Diabetes, Dtisseldorf, FRG

There is still reluctance on the part of some employers to hire diabetic patients. In a government sponsored intervention study, a diabetes teaching and treatment programme for Type 1 (insulin-dependen 0 diabetic patients has been instituted and evaluated in 10 city hospitals. Consecutive Type 1 diabetic patients from the 10 centers are recluited into the study for a three-year longitudinal follow up. Patients with blindness or renal insufficiency are excluded, aged between 15 and 40 years. The initial diabetes-related data including social parameters of 645 patients are available. Results: 351 of 645 patients were working, 51 patients were registered as unemployed, 243 housewives, pupils and students. No correlation was observed between the duration of diabetes and unemployment. Unemployed diabetic patients had been hospitalised more frequently in diabetes hospitals, they achieved fewer correct answers in a diabetesrelated knowledge test after the teaching programme. The unemployment rate in the Type 1 diabetic patients was 12.7% (unemployed+employed=100%). The unemployment rate of the general population was 10.7% in 1985 (official FRG statistics). In unemployed diabetic patients the professional qualification seemed to be higher compared to the unemployed general population. Conclusion: independent of diabetes duration, those Type 1 diabetic patients without severe complications showed similar employment characteristics to the general population. This should be indicated to employers' organisations. 194. Fruetosamine:iuterest in diabetic control nosis of glucose intolerance disorders P. J. Guillausseau, M.A. Charles, V. Codard, J. Timsit, Ph. Chanson, F. Delie, E. Eschwege, betzki. Departments of Diabetology and Lariboisirre, Paris, and Statistical Research Villejuif, France

evaluation and in diag-

F. Paolaggi, J. Peynet, F. Rousselet and J. LuBiochemistry, H6pital Unit (INSERM U21)

Fructosamine (F) and HbA1 were assessed in 53 control subjects and 322 unselected diabetic patients. Intra- and inter-assay variation coefficients were 1.6 and 5.8% for F and 4.3 and 5.3% for HbA1. F and HbA1 were increased in Type1 (insulin-dependent) (2.67+ 0.8 mmol/1, 9.1 + 1.9%; n= 107) and in Type 2 (non-insulin-dependent) diabetic patients (2.07_+0.65, 8.3 _+1.8%, n=215) compared to control subjects (1.34 + 0.24, 6.1 + 1.2%) (p< 0.001). In Type 2, F and HbA1 were greater in patients on sulfonylureas plus metformin than on diet, sulfonylureas or metformin alone. A significant correlation was found between F or HbA1, and fasting (FBS) (r=0.52-0.54) or postprandial blood sugar (PPBS) (r= 0.60-0.61), and between F and HbA1 (r= 0.71) (p< 0.001). We found stronger correlations between FBS or PPBS and F or HbA1 in Type 2 (0.62-0.67) than in Type 1 diabetic patients (0.29-0.38) while correlations between F and HbA1 were similar in both groups (0.67-0.68). In normal, glucose intolerant, or diabetic subjects according to OMS criteria but with FBS < 7.8 mmol/1, F and HbA1 sensitivity was 40 and 10% for diabetes, 5 and 0% for IGT while specificity was 95% and 100% when normal upper limit was m + 2 SD; when upper limit was m + l SD, F and HbA1 sensitivity was 40 and 30% for diabetes, 40 and 35% for IGT, specificity being respectively 91 and 90%. These preliminary data indicate that F and HbA1 are of similar poor value in mild glucose intolerance disorders diagnosis. 195. The initial reduction and later increase of cytoplasmic Ca 2+ is a physiological response to glucose in normal B cells E. Gylfe. Department of Medical Cell Biology, University of Uppsala, Sweden The cytoplasmic calcium concentration (Ca 2+i) was measured in suspensions of fura-2 loaded pancreatic B cells from ob/ob-mice by continuously recording the 340/380 nm fluorescence excitation ratio. This technique resulted in a several-fold increase of the signal-tonoise ratio. It was possible for the first time in single experiments to demonstrate both the initial reduction and later increase of Ca 2 + i after glucose exposure. These actions were apparent after preincubation for 40-50 min in medium containing 1.28 mmol/1 Ca 2+ and

Abstracts 3 mmol/1 glucose. However, as in the case for 45Ca effiux, the bimodal nature of the changes became more distinct following prolonged exposure to 0.5 mmol/l Ca 2+. After initial omission of sugar, the rise of the glucose concentration to 20 mmol/l resulted in a longer reduction of Ca2+i succeeded by a more sluggish increase. " As the Ca 2+ concentration was raised from 0.5 to 5 or 10 mmol/1, Ca2+ i increased in B cells incubated in 3 mmol/1 glucose. When elevating the Ca 2+ concentration only 5 min prior to raising glucose to 20 mmol/1, the sugar-induced reduction of Ca2+i became much more pronounced hke" the subsequent increase, which was also more rapid.z+ 9 The results emphasise the role of a saturable B cell pool of Ca in glucose reduction of Ca2+ i. 196. Monitoring of subcutaneous adipose tissue glucose in vivo in man with use of novel microdialysis technique E. Hagstrrm, P. Arner and J. Bolinder. Department of Medicine, Karolinska Institute, Huddinge Hospital, Huddinge, Sweden

Subcutaneous adipose tissue glucose was measured with a microdialysis technique. A semipermeable microdialysis probe (8 x 0.5 mm) was implanted subcutaneously and continuously perfused (5 g l / rain). Glucose was determined in 15-min fractions by a glucose-oxidase method. The in vitro recovery was about 10% in glucose solutions ranging from 1 to 20 mmol/l. Intraprobe variation was small (2.4___0.9%, mean_+ SEM). Gluteal subcutaneous and blood glucose was monitored in nine normal-weight volunteers, before and after a 75 g oral glucose load. After glucose ingestion blood glucose peaked at 35 min (60 • 8% above baseline). The corresponding maximum increase in subcutaneous glucose (64_+ 15%) occurred 45-60 min after glucose. Thereafter blood and subcutaneous glucose decreased to baseline levels almost identically. Calculated at a 10% recovery rate the subcutaneous glucose concentration was 12.0+3.3% of blood glucose in the fasted state, and the ratio between subcutaneous and blood glucose didn't change after glucose ingestion. When two probes were implanted simultaneously in the same individual, identical kinetics were recorded during the oral glucose load. Similar decreases in blood and subcutaneous glucose levels were observed after insulin administration to hyperglycaemic diabetic patients. It is concluded that microdialysis of subcutaneous adipose tissue may provide a feasible technology for continuous glucose monitoring in non-diabetic and diabetic man. 197. Presence of IL-2 receptor expressing cells within pancreatic islets of diabetic BB rats: treatment of diabetic rats with a monoclonal antiinterleukin-2 receptor antibody and cyclosporin H. J. Hahn, I. K16ting, S. Lucke, B. Kuttler, H.D.Volk, R.v. Baehr, H. Stein, H. Gerdes and T. Diamantstein. Central Institute of Diabetes Karlsburg and Institute of Clinical Immunology, Humboldt-University Berlin (GDR); Dept. of Pathology and Dept. of Immunology, Free University Berlin (West), G D R

In diabetic BB rats the pancreatic islets are characterised by intense lymphatic infiltrations. We investigated the nature of cells infiltrating the islets using monoclonal antibodies (mab) directed against defined surface epitopes (OX-19, OX-8, IL-2R (ART-18)). At the time of diabetes diagnosis within the pancreas, around and within pancreatic islets phenotypically OX-19 +, OX-8 + and ART-18 + cells were observed. To clarify the role of ART-18 + cells, newly-diagnosed diabetic BB rats remained either untreated (n= 8; diabetic control) or were treated with cyclosporine (1.5 mg/kg bw) and IL-2R mab (1 mg/kg) (n= 17) for 10 days, a scheme which prolonged heart and islet allografts. Seventy percent of the treated BB rats with a plasma glucose between 9.5 and 13.0 mmol/l at diabetes diagnosis maintained normoglycaemia (<8.2 mmol/1) up to 120 days. The pancreatic B cells are well granulated having an insulin content of 60% when compared with nondiabetic age-matched BB rats. The study supports the idea that ART-18 + cells play a pivotal role in pancreatic B-cell destruction. The application of an anti-TL-2R mab offers a new strategy of a more selected immunotherapy directed against activated mononuclear cells. 198. Parameters of infection in Type 2 (non-insulin-dependent) diabetic patients G.Halaby, A.Khoury, H.Mourad-Khoury, S.Jambart and E.Baz. Service d'Endocrinologie and Laboratoire d'Immunologie HotelDieu de France Hospital, Beirut, Lebanon

The authors have studied the following parameters in 40 patients: T4/T8, CRP, C3, C4 and HbAIC, in 10 infected diabetic patients (ID), in 10 non-infected diabetic patients (NID), in 10 infected non-

527 A diabetic patients (IND) and in 10 non-infected non-diabetic patients (NIND). The ratio of T4/T8 was lower in ID (0.95) and NID (1.13) vs IND (1.65) and N I N D (1.75, p<0.05). The CRP (rag/l) was lower in ID (4.5) vs IND (25.2, p < 0.01). No difference was found between NID and NIND. Infection in diabetic patients decreases C3 (g/l) compared to NID (0.87 vs 1.07, p<0.05) and compared to IND (1.03, p < 0.05). There is no difference for C4 (g/l) between ID and NID (0.48 vs 0.52), nor compared to IND (0.49). But all were increased compared to N I N D (0.35, p<0.05). The HbA1C was lower in ID compared to NID (6.9% vs 10.6%, p<0.05); this correlates with decreased hematocrit (32% vs 43%, p<0.05). These results suggest that the diabetic patient has an altered T4/T8 and that infection decreased C3 and CRP. The HbA1C is low due to the decrease in hematocrit in infected diabetic patients.

199. Twelve-hour metabolic profiles in patients with insulinoma P.J.Hale and M. Nattrass. The General Hospital, Steelhouse Lane, Birmingham, U K The hyperinsulinaemia of patients with insulinoma has important implications for energy substrate supply yet there are few reports on circulating substrates other than glucose. Twelve-h metabolic profiles were compared in six patients (4 men) with histologically proven benign insulinoma studied pre-operatively, mean age 41 years (range 26-73 years) and mean % IBW 131% (105-158%) with 6 normal subjects (2 men) mean age 40 years (24-60 years) and mean % IBW 122% (113-137%). Fasting blood glucose in insulinoma patients was lower 2.9+0.3 (mean+SEM) vs 5.0• (p<0.001) and plasma insulin higher 20.0 • 3.9 vs 7.2 • 1.6 mU/1 (p< 0.01). Significant differences over the 12 h sampling period were sought using 2-way analysis of variance. Glucose (12 h mean in insulinoma patients 4.1 vs 5.7 mmol/1 in normal subjects F = 129.8 p < 0.001) pyruvate (0.061 vs 0.079 mmol/1 F = 50.2 p < 0.001) and glycerol (0.065 vs 0.080mmol/1 F=8.5 p<0.005) were lower, insulin (30.9 vs 19.9 m U / l F=99.6 p<0.001), lactate (0.95 vs 0.82 mmol/1 F=17.7 p<0.001), alanine (0.322 vs 0.296 mrnol/1 F=6.0 p<0.05) and NEFA (0.55 vs 0.46 mmol/1 F=20.9 p<0.001) were higher. Blood total ketone bodies (0.089 vs 0.081 mmol/1 F = 8.8 p < 0.005), while higher throughout most of the day, were lower in the later afternoon. The failure to suppress N E F A and total ketone bodies concentrations is of particular interest due to their potential roles as alternative energy sources.

200. Glycosylated fibrinogen: a new index in the management of diabetes M.R. Hammer, P.N.John, M.D. Flynn, A.J. Bellingham and R. D. G. Leslie. King's College Hospital, London, U K A protein with a shorter half-life than haemoglobin A1 might be more valuable in the management of diabetes. We have developed a simple, reliable method of measuring glycosylated fibrinogen (halflife 3-4 days) and compared values to Hb-A1 in 25 normal subjects and 22 selected diabetic patients. Fibrinogen was extracted from plasma and applied to an m-aminophenylbaronate column. The glycosylated fraction was eluted with sorbitol and quantitated using Coomassie blue.

(25) Normal subjects (11) Well-controlled diabetic patients (11) Poorly-controlled diabetic patients

% glycosylated % glycosylated haemoglobin fibfinogen mean + SD 6.7 + 0.9 4.8 + 0.9 8.7 • 0.4 6.8 • 0.6 16.8•

13.8•

The levels of both proteins were significantly different (p<0.001) among the three groups. In twelve patients with improving control, glycosylated fibrinogen fell more rapidly than glycosylated haemoglobin (by 15% versus 3.5% of the initial value respectively by 3 days, p < 0.01). In nine patients with deteriorating control the levels of glycosylated fibrinogen rose more rapidly than glycosylated haemoglobin (by 38% versus 8% of the initial value respectively by four weeks, p < 0.02). We suggest that glycosylated fibrinogen is a useful adjunct to blood glucose and haemoglobin Aa in the management of diabetes.

528 A 20t. Non-enzymatic glycation of human circulating lymphocytes and metabolic control in diabetes mellitus H. P. Hammes, B. Ernst, H. Laube and K. Federlin. IlL Medizinische Klinik und Poliklinik, Justus-Liebig-Universit~it Giel3en, FRG Many tissue and circulating proteins undergo non-enzymatic glycation in diabetes, followed by functional changing. Since life-time of circulating human lymphocytes extends from days to even years, i.e. long enough to be excessively glycated, we intended to study whether or not glycation of lymphocytes occurs in diabetes and the possible correlation to metabolic control. Four Type 1 (insulin-dependent) diabetic patients and 5 Type 2 (non-insulin-dependent) diabetic patients, aged 24-62 years, diabetes duration 1-20 years, were selected. Seven subjects aged 20-81 served as controls. After isolation from 20 ml venous blood samples (Lymphoprep) and homogenisation, glycated lymphocytes (GL) were measured by affinity chromatography. HbA1 was determined by column chromatography and blood glucose by Beckman analyser. GL was significantly higher (p< 0.005) in diabetic patients than in control subjects: 14.2%+3.8% (mean+SD) vs 8.6% + 2.8%, the highest values being 1.35 times higher than the normal range. GL showed a positive but insignificant trend (p> 0.05) vs blood glucose (r=0.54) and HbA1 (r=0.52). Patients with sepsis and acute viral infections had the highest values (15.5-19.2%). Conclusion: circulating lymphocytes are subject to excessive glycation in diabetes with a trend to metabolic control. It is conceivable that this may even contribute partly to the increase of infection in diabetes. 202. Stimulatory effects of a diglyceride lipase inhibitor, RHC 80267, on glucose metabolism in isolated islets of Langerhans S. E. Hansen, K. Capito, C.J. Hedeskov and P. Thams. Department of Biochemistry A, University of Copenhagen, Denmark It is supposed that a cleavage of phosphatidylinositoldiphosphate to diglyceride and inositoltriphosphate is involved in regulation of insulin secretion. To further elucidate the role of diglyceride in secretion, we have examined the effect of a known diglyceride lipase inhibitor, RHC 80267, on islet metabolism. RHC 80267 inhibited islet diglyceride lipase, but had little or no effect on phospholipase A2, phospholipase C, or adenylate cyclase. Surprisingly, formation of tritiated water from 5-tritiated glucose was significantly enhanced at 10 and 16.7 mmol/1 of glucose. The activation was dose-dependant in the range from 10 to 140 gmol/l of RHC 80267, the latter resulting in a 5-fold increase in glucose utilisation. The effect seems to be islet-specific, since, in experiments with hepatocytes and leucocytes, no stimulation was seen. There was no effect of RHC 80267 on 14CO2-formation from a4C-labelled glucose and no effect on ATP-concentration or insulin secretion. Both phorbol ester, which in some respects mimics diglyceride, and indomethacin stimulated glucose utilisation but to a lesser extent than RHC 80267. These observations may suggest that the effect of RHC 80267 is on the triosephosphate-level and may be due to a pulling, somehow, of triosephosphates into lipid metabolism. 203. Glycolipids are able to mimic the insulin effect on glucose transport in rat fat cells H. U. H~iring, Ch. Mtihlbacher and F. Machicao. Institut f~ir Diabetesforschung Miinchen und Hormon Chemic Mtinchen, FRG Recent study comparing the effects of insulin and phorbol esters (TPA) on translocation of glucose carriers (cytochalasin B binding sites) and 3-O-methylglucose transport (GT) in fat cells suggested that a second step in addition to carrier translocation is involved in insulin action on glucose transport. In an attempt to find substances which selectively stimulate this second step we tested among others glycolipids, as a role of these substances in insulin action was recently proposed. We found that a glycolipid fraction designated P1 isolated from the drug Actovegin has a partial insulin-like effect on glucose transport (basal 9.8%, P1 24%, insulin 40%) without inducing cartier translocation. In combination with TPA, which has alone also only a partial insulin-like effect on glucose transport, but which induced carrier translocation just as insulin does, P1 can almost mimic the full insulin effect on glucose transport (basal 9.8%, P1 24% and TPA 34%, insulin 40%). The data confirm the hypothesis of a two-step model for glucose transport activation and suggest that the insulin effect on the second step of this process might involve glycolipid release. 204. Eight hours of moderate hyperglycaemia do not cause preferential secretion of proinsulin in normal subjects S. G. Hartling and C. Binder. Steno Memorial Hospital, 2, Niels Steensens Vej, Gentofte, Denmark Relatively elevated proinsulin (PI) concentrations is a characteristic

Abstracts of cystic fibrosis patients with impaired glucose tolerance, of healthy siblings of Type 1 (insulin-dependent) diabetic patients and of dysregulated Type 2 (non-insulin-dependent) diabetic patients. The aim of this study was to test if 8 h hyperglycaemia would induce a similar alteration in B-cell function in normal subjects. Plasmaconcentrations of insulin, C-peptide, and PI were measured in 9 (4 m/5 f) healthy normal-weight subjects during 8 h of hyperglycaemia (6.5-9 mmol/1). None had any first-degree relatives with diabetes. Glucose (20%) was infused continuously after a bolus injection. The infusion rate was adjusted, aiming at a blood glucose of 7-9 mmol/1. There was a gradual rise both in insulin and C-peptide during all 8 h. Proinsulin increased quickly during the first 2 h, thereafter PI rose slowly or remained constant at a median value of 25 pmol/1. The fasting PI/C-peptide ratio was 0.5%, from 2 to 8 h it was about 3%. The fasting PI/insulin ratio was 2% increasing to 7% after 2 h. Conclusion: the B-cell secretion pattern was not altered after 8 h of moderate hyperglycaemia in normal subjects. 205. Comparative biopotency studies of des-[(B26-30)pentapeptide] insulins in rat hepatocyte cultures H. Hartmann 1, S. Zachmann 1, M. Casaretto 2, I. Probst 3, W. Creutzfeldt 1 and D. Brandenburg 2. 1Department of Medicine, GSttingen, 2German Wool Research Institute, Aachen, 3Institute of Biochemistry, University of G6ttingen, FRG An enhanced biological activity has been suggested for the shortened insulin analogues des-[(B26-30)pentapeptide]-insulinamide (I) and [TyrB25]des-[(B26-30)pentapeptide]-insulinamide (II) for the stimulation of glucose metabolism in isolated rat adipocytes when compared to the effects of native insulin. The present work evaluates the biological activity of the analogues in primary cultures of adult rat hepatocytes where insulin-mediated short- and long-term regulatory effects have recently been characterised. Dose response curves for 1) the inhibitory action on hepatic glycogenolysis, 2) the antagonistic action towards glucagon-stimulated glucose output, 3) the induction of regulatory enzymes, e.g. glucoldnase and pyruvatkinase, and 4) the glucagon-antagonistic action towards the induction of phosphoenolpyruvate-carboxykinase revealed an identical responsiveness and similar potency (half-maximal effective concentrations about 0.2 mmol/1) for both analogues compared to native insulin. It is concluded that I and II have full biological activity on cultured hepatocytes. The enhanced effects on adipocytes reported especially for II might suggest a preferential action on adipose tissue. 206. Serum concentration of basement membrane antigens in diabetic children and adult Type t (insulin-dependent) diabetic patients Ch. Hasslacher and H. Schmidt. Medical and Pediatric Clinic, University of Heidelberg, FRG As shown by studies in animals, induction of diabetes leads to an alteration of the metabolism of capillary basement membrane (BM), which seems to be modulated by sex steroids. Therefore, we measured radioimmunologically the serum concentration of laminin (Lain), a noncollagenous BM protein, in 35 diabetic and 30 non-diabetic children before puberty as well as in 65 adult Type 1 (insulin-dependent) diabetic patients and 50 non-diabetic control subjects. In control subjects, Lam levels of children were 18% higher than in adults, but did not show a relation to sex. Lam levels of diabetic children were not different to those of non-diabetic control subjects, irrespective of the metabolic control (HbA1). However, in adult diabetic patients Lain levels showed a positive correlation to HbA1 (r= 0.67, p < 0.01) and to the progression of microangiopathy. The different results found in children before puberty and adults suggest that sex steroids may play an important role in the regulation of BM metabolism in diabetes. 207. The effect of the octapeptide somatostatin analogue Sandostatin in Type 2 (non-insulin-dependent) diabetes E. Haupt, E. Oerter, Ch. Rosak and H.G. Harris. Klinikum Bad Kissingen der BfA, Saale-Klinik, Bad Kissingen, FRG Type 2 (non-insulin-dependent) diabetes is characterised to a larger extent by peripheral insulin resistance than by B-cell deficiency. Moreover high levels of insulin have been implicated in the development of macroangiopathy in diabetic patients. Substances like long acting somatostatin analogues have been shown to inhibit the secretion of insulin, GH and glucagon which are all involved in the pathophysiology of the diabetic syndromes. Therefore we report the effect of a long acting somatostatin octapeptide Sandostatin in Type 2 diabetes. Sixteen Type 2 diabetic patients (7 F, 9 M, aged 54.9+_

Abstracts 1.73 years, diabetes duration 7.8 + 1.47 years, BMI 27.06__ 0.69) well controlled by diet (HbAI 7.81_+0.18%) were included in a prospective randomised placebo-controlled cross-over study where 25 l-tg Sandostatin or placebo were administered three times subcutaneously immediately before meals, during 7 days. On the last day (7th) of each study period a 17 point blood glucose profile was performed, at hourly or half-hourly intervals. In 6 patients C-peptide and GH were also measured during the profile. Blood glucose control was evaluated by computing mean values, mol/1 value and AUC. No significant differences between study periods was found under Sandostatin and placebo. Patients who received Sandostatin in the first study period showed lower glycaemic excursions after breakfast. C-peptide and GH levels were significantly reduced during Sandostatin in all patients. During Sandostatin fat and starch stool content slightly increased as well as stool weight. Stool chymotrypsin was reduced. No serious gastrointestinal side effects were noted. Sandostatin inhibits peripheral hyperinsulinism in insulin-resistant Type 2 diabetes without concomitant worsening of blood glucose homeostasis. This can be accounted for by inhibition of increased GH levels. Hyperinsulinism is considered an additional risk factor for macroangiopathy in Type 2 diabetes and may be controlled by medication.

208. Phosphorylation-dephosphorylation of phosphatidylinositol-specific phospholipase C in mouse pancreatic islets C. J. Hedeskov, K. Capito, S. E. Hansen and P. Thams. Department of Biochemistry A, University of Copenhagen, Denmark During insulin secretion elicited by glucose and muscarinic receptor occupancy phosphatidylinositol-specific phospholipase C catalyses a continuous formation of the second messenger diglyceride. We have therefore examined some regulatory properties of this enzyme. Phosphatidylinositol-specific phospholipase C activity in mouse islet homogenates decreased 60-95% when the homogenates were incubated at 37 ~ for 1 h in the presence of down to micromolar concentrations of Ca + +. Ca + +-induced inactivation was augmented by Mg + + and abolished when pH was lowered to 4.9 or Tp lowered to 4 ~ Inactivation was not caused by Ca + +-activated proteases. Inactivation was, however, potentiated by calmodulin (1 lxmol/1) and the Ca++-cal modulin stimulated inactivation was completely abolished by trifluoperazine (50 p~mol/1). This observation is strong evidence that dephosphorylation by protein phosphatase 2B had caused the inactivation. Inactivation of phosphatidylinositol-specific phospholipase C was completely reversible at 37 ~ in the presence of ATP and an ATP-regenerating system, suggesting that phosphorylation reverses inactivation. It is concluded that the phosphatidylinositol-specific phospholipase C system in pancreatic islets is subject to regulation by phosphorylation/dephosphorylation. It remains to be clarified whether it is the phospholipase C itself or a modulatory protein such as lipomodulin that is phosphorylated/dephosphorylated.

209. Follow-up study of the endocrine pancreas in normoglycaemic BB/ DK rats (diabetes-prone) B. Hehmke, S. Lucke, I. K16ting, W. Besch and H.-J. Hahn. Central Institute of Diabetes "Gerhardt Katsch" Karlsburg, G D R Partially inbred BB rats of our colony develop an insulin-dependent diabetic syndrome (incidence 50-70%) resembling in many features the human disease. However, normoglycaemic BB rats are genetically affected as well and exhibit a diminished immune responsiveness. Therefore, 55 normoglycaemic BB rats were included in a follow-up study and investigated at an age of 70, 90, 120 and 200 days by determination of body weight, plasma glucose, glucose tolerance, complement-dependent antibody mediated cytotoxicity (C'AMC) and in pancreatic biopsies of insulin content, relative islet and B-cell volume density and the degree of insulitis. After the 70th day of age, normoglycaemic BB rats are characterised by decreased pancreatic insulin content (p<0.01), reduced relative islet and B-cell volume density (p< 0.01), glucose intolerance, a temporarily enhanced C'AMC and a marked degree of pancreatic islet inflammation compared to 50 dayold rats. From these results it is concluded that 1) all animals of our BB colony respond to hitherto unknown factor(s) in an age-dependent manner and 2) B-cell destruction also occurs in long-term normoglycaemic BB rats, but despite signs of functional, morphological and immunological alterations similar to those in diabetic BB rats, these animals overcome or recover from this process.

529 A 210. Absorption kinetics of human proinsulin and human NPH insulin from the deltoid, abdominal and femoral regions R.J.Heine, M.Oolbekkink and E.A.van der Veen. Departments of Internal Medicine and Endocrinology. Free University Hospital, Amsterdam, The Netherlands In this study we compared plasma insulin levels and insulin action profiles of human proinsulin (P) and NPH insulin (NPH) after subcutaneous administration in three commonly used injection sites in eight healthy male volunteers. The glucose clamp technique was used for 12 h after the injections of P and NPH (0.50 IU/kg) in a random order in the deltoid, abdominal and femoral regions. Areas under the curve from 0-360 min (AUC 360) of the glucose requirements were significantly affected by the site of injection for P, being greatest for the abdominal site and the smallest for the arm: 3101.6+433.1 (SEM) vs 1356.5 + 234.9 mg/kg (p= 0.02, analysis of variance). Injection sites did not influence the action profiles of NPH. AUC 360 for P and NPH were similar for the deltoid region and significantly greater for P than NPH after administration in femoral and abdominal region: 2517.5 _+288.8 vs 1989.9_+ 323.9 mg/kg and 3101.6_+ 433.1 vs 2389.5_+334.9mg/kg (p<0.05). Similar differences existed for AUC 720. Plasma insulin levels reflected the insulin action profiles. In conclusion, the absorption kinetics of NPH insulin are not affected by the site of injection whereas the rate of absorption of proinsulin is faster from the abdominal than from the deltoid and femoral regions. 211. Glucose tolerance and insulin sensitivity are not enhanced through pulsatile insulin infusion in Type 1 (insulin-dependent) diabetic patients L. Heinemann, G.E. Sonnenberg, J.Gerich, J.J. Benn, D. Kelly, P. Soenksen and M. Berger. Dept. of Metabolic Diseases & Nutrition, University of Dtisseldorf, West Germany; Mayo Clinic, Rochester, Minn, USA; St.Thomas' Hospital, London, U K It has been postulated that through repeated intravenous insulin infusion (insulin pulsing) the (hepatic) insulin-sensitivity can be enhanced resulting in an improvement of glucose-tolerance in Type 1 (insulindependent) diabetes. In a multi-center study, we have investigated the effects of insulin pulsing (10 pulses of human insulin 0.035 U/kg IBW intravenous each of 20 s duration with intervals of 6 min-three times per day with adequate oral glucose supply) on two successive days on glucose-tolerance in 19 Type 1 diabetic patients (mean age 28 years; duration of diabetes 12 years; BMI 24.4 kg/m2). Before and after the insulin protocol the patients, which were adjusted through an intravenous insulin infusion to a glycaemia of 5.5 mmol/1 overnight, were subjected to an oral glucose tolerance test (1 g glucose/kg, OGTF), after receiving a subcutaneous injection of 0.15 U / k g human-insulin 30 min in advance. Four h later the patients underwent an exercise challenge on a bicycle-ergometer. We measured: glycaemia, serum free insulin and respiration quotient (RQ). Free insulinconcentrations (maximal to 40 ttU/ml) and the RQ (rising during the OGTT and exercise) remained unaffected by the insulin pulsing. Glucose-tolerance was significantly deteriorated (maximal before insulin pulsing 11 mmol/1, after 14 mmol/1). Conclusion: insulin pulsing did not enhance oral glucose tolerance in Type I diabetes. 212. Glibenclamide directly stimulates clonal growth of isolated chondrocytes in vitro E. Heinze, U. Vetter, R.D. Fussg~inger and W. Pirsig. Universit/itskliniken, University of Ulm, FRG When hypophysectomised rats were treated with glibenclamide for 10 days serum insulin, serum somatomedin and skeletal growth increased. It is unknown if glibendamide can directly enhance skeletal growth. Therefore chondrocytes from 60 g male normal or hypophysectomised rats were isolated with collagenase-trypsin and the viability determined with trypan blue. Then 1000 cells were incubated in 1 ml of a semi-solid medium (= BM Wissler, 5% heat inactivated FCS, 0.8% methylcellulose, gcntamycin, vitamins, glibenclamide, human growth hormone in an atmosphere of 5% CO2, 5% 02) for 14 days at 37 ~ Colony formation was determined. Results: Viability of chondrocytes was 75-85%. Colony formation without glibenclamide (20-50 colonies) was taken as 100% (mean_+ SEM). In 12 normal rats 25 (90_+4%) inhibited colony formation. Cells from hypox, rats formed less colonies, suggesting growth hormone dependence. Therefore the response of hGH plus glibenclamide was tested in 8 normal rats. Colony formation was enhanced with 25 ng/ml: 108 _+3%, glib. 25 ng/ml plus 12.5 ng hGH: 120 _+5%) in an additive fashion suggesting similar mechanisms for glibenclamide and hGH on clonal growth of chondrocytes.

530 A 213. Effects of platelet-derived growth factor and somatomedin C on the DNA replication of fetal rat islet cells in tissue culture C. Hellerstr6m, C.-H. Heldin, D.J. Hill and I. Swenne. Department of Medical Cell Biology and The Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden and Department of Paediatrics, Children's Hospital, University of Sheffield, Sheffield, UK Previous studies indicated that DNA replication of the fetal pancreatic B cell is stimulated by growth hormone, an effect partly mediated by the autocrine release of somatomedin-C (SM-C). Serum potentiated the effects of growth hormone and the present study evaluates to what extent this was related to the presence in serum of platelet-derived growth factor (PDGF). Fetal rat islets were cultured in the presence of PDGF and different concentrations of glucose, serum and SM-C. The effects on DNA replication and release of insulin and SM-C were studied. PDGF stimulated DNA replication in the presence of 1% fetal calf serum but not in medium without serum. SM-C stimulated DNA replication both in the presence and absence of serum. The stimulatory effects of PDGF and SM-C were additive and the effects of PDGF could not be blocked by an antibody to SM-C. PDGF furthermore failed to stimulated release of either insulin or SM-C into the culture medium. The results suggest that the effects of PDGF and SM-C on islet DNA replication are not synergistic and could be independent. In addition, hitherto undefined serum factors may be needed for the stimulatory action of PDGF on the DNA replication of pancreatic islet cells. 214. In vitro metabolism of low density lipoprotein isolated from hyperglycaemic Type 2 (non-insulin-dependent) diabetic patients E.Helve, P.T.Kovanen and M.-R.Taskinen. Third and Second Departments of Medicine, University of Helsinki, and Vihuri Research Institute, Helsinki, Finland Glycosylation of low density lipoprotein (LDL) in vitro impairs the receptor-mediated catabolism of LDL by inhibiting its binding to LDL receptors. To examine whether receptor-mediated catabolism of in vivo modified LDL of hyperglycaemic Type 2 (non-insulin-dependent) diabetic patients is impaired, we determined the ability of normal cultured skin fibroblasts to bind and degrade LDL obtained from patients with poor control and again after correction of hyperglycaemia by insulin therapy. Six Type 2 diabetic patients (aged 55 to 64 years) with poor glycaemic control despite maximal oral therapy were treated with insulin over 4 weeks. Glycaemic control improved markedly (mean+_SEM for blood glucose 12.6_+2.0 vs 7.0+_ 2.1 mmol/1, p<0.01, and for HbA1 11.0+0.8 vs 8.9_+1.1%, p<0.01 before and after insulin respectively). In spite of improvement of glycaemic control the lipids and protein contents of LDL were similar before and after insulin therapy. Furthermore, receptor-mediated binding and degradation of both pre- and postinsulin LDL were identical, and they did not differ from those obtained with LDL from nondiabetic control subjects. The results demonstrate that in Type 2 diabetic patients hyperglycaemia of the observed range (HbA1 9.6-11.7%, nondiabetics 6-9%) was not able to modify LDL in vivo to such extent that it would have impaired its receptor-mediated catabolism as measured in vitro. 215. The ionic dependency of acetylcholine effects in B cells J.C. Henquin, M.C.Garcia, M. Bozem and M. Nenquin. Unit6 de Diab6tologie et Nutrition, University of Louvain, Brussels, Belgium, and I Physiologisches Institut, University of Saarland, Homburg/ Saar, FRG Acetylcholine (ACh) amplifies insulin release whenever the B-cell membrane is already depolarised by a primary stimulus. Under these conditions, the small depolarisation by ACh itself may become sufficient to activate Ca channels. However, it is not known how ACh causes this depolarisation and whether its suppression abolishes the releasing action of ACh. In this study, the Na- and Ca-dependency of ACh effects was investigated in mouse islets. In a control medium containing 3 mmol/1 glucose, ACh accelerated Rb effiux from islet cells, slightly depolarised the B-cell membrane but neither induced electrical activity nor stimulated insulin release. At 10 mmol/1 glucose, ACh accelerated Rb effiux, increased electrical activity, stimulated Ca influx and augmented insulin release. All these effects of ACh were suppressed by omission of extracellular Na, except the increase in release that was inhibited by 75% only. In contrast, omission of Ca abolished insulin release but did not prevent acceleration of Rb effiux. Activation of phospholipase C and early mobilisation of cellular Ca by ACh were not impaired in Na-free solutions. It is concluded that ACh depolarises the B-cell membrane by increasing its

Abstracts Na + permeability and that the resulting augmentation of Ca influx largely (not totally) accounts for the amplification of insulin release. 216. Mechanisms of the permissive action of glucose on arginine-indnced insulin release M. P. Hermans, W. Schmeer and J. C. Henquin. Unit6 de Diabrtologie et Nutrition, University of Louvain, Brussels, Belgium, and I Physiologisches Institut, University of Saarland, Homburg/Saar, FRG Mouse islets were used to study how glucose accelerates and amplifies (permissive action) arginine-induced insulin release. At 3 retool/1 glucose, arginine decreased the resting membrane potential of B cells (10 mV), did not evoke electrical activity, slightly accelerated Ca influx and efflux, and increased release only slowly and weakly. When the membrane was depolarised by threshold (7 mmol/1) or stimulatory (10-15 mmol/1) concentrations of glucose, arginine rapidly induced or augmented electrical activity, markedly accelerated Ca fluxes, and triggered a strong and fast increase in release. When glucose-induced depolarisation was prevented by diazoxide, the effects of arginine were similar tO those observed at 3 mmol/1 glucose, except that the delayed increase in insulin release still exhibited some glucose-dependency. Depolarisation, at low glucose, by tolbutamide or high K largely or partially mimicked the permissive effects of high glucose. The releasing effect of arginine was Ca-dependent under all experimental conditions. In conclusion, depolarisation of the B-cell membrane by glucose is essential for its permissive action. It enables the small depolarisation by arginine itself to activate Ca channels more rapidly and efficiently. Intracellular changes induced by glucose may also play a secondary role. 217. Differential sensitivity to galanin of pancreatic insulin, somatostafin and glucagon secretion K. Hermansen. Second University Clinic of Internal Medicine, Aarhus Kommunehospital, Aarhus, Denmark Galanin, a 29 amino acid peptide, has most recently been found in nerves of the pancreas. To determine how this pancreatic neuropeptide influences the endocrine pancreas we examined the effects of galanin on the hormone release from the isolated perfused dog pancreas. We found that 1)galanin (1 pmol/1-10 nmol/1) dose-dependently inhibited insulin and somatostatin secretion at 5.5 mmol/1 glucose. The islet cell threshold for galanin was between 1 and 10 pmol/1 for the B cell, between 10 and 100 pmol/1 for the D cell, while the high galanin concentration of 10 nmol/1 was needed to inhibit the glucagon secretion. 2)Galanin (100pmol/1) caused a more pronounced inhibition of B- and D-cell secretion at high (11 mmol/1) rather than at low glucose (1.3 mmol/1). In contrast, suppression of the A-cell secretion was only seen at low glucose (1.3 mmol/1). 3) Perfusion of 1 ~mol/1 atropine, 1 p~mol/1 phentolamine and 1 l.tmol/1 propranolol had no effect on the galanin-mediated (100 pmol/1) inhibition of B- and D-cell secretion. 4)The potency of galanin (1-100 pmol/1) to inhibit insulin secretion was similar to that of somatostatin (1-100 pmol/1). The potent inhibition of insulin and somatostatin by galanin and the presence of this peptide in pancreatic nerves point to a physiologically important role of galanin for the secretion of insulin and somatostatin, but probably not for glucagon. 218. DQ region polymorphisms predict susceptibility to Type I (insulindependent) diabetes mellitus in South-Indian subjects G. A. Hitman,1 P. K. Karir,1 J.A. Sachs,2 A. Ramachandran,3 C. Snehalatha,3 M.Viswanathan3 and V. Mohan.3 1Medical Unit and 2Bone and Joint Research Unit, The London Hospital, Whitechapel, London, UK and 3Diabetes Research Centre, Madras, India Recently close markers for Type I (insulin-dependent) diabetes mellitus in Western 'Caucasoid' subjects have been defined based on DQ region (both a and flgenes) restriction fragment length polymorphisms. In order to define the genetic contribution of Type I diabetes in an Indian population we have analysed unrelated Dravidian (South-Indian) subjects (58 with Type 1 diabetes and 43 control subjects) using Southern blot hybridisation methods and HLA-DR~z, -DQa, -DQfl, and -DPaprobes. In Type I diabetic patients an increased frequency of the Taq 1 DQfl restriction fragment length polymorphisms designated T2omega/T6 (relative risk= 10.6) and homozygotes for Taq 1 DQa 4.6 (relative risk = 11) was found in the patient groups. The highest relative risk for diabetes was obtained by comparing patients and control subjects who either co-inherited DYT2omega/T6 with certain DQa restriction fragment length polymorphisms or with DQ~z 4.6 homozygotes, the combination accounting for 55.5% of diabetic patients and none of the controls (relative

Abstracts

531 A

risk= 101; 95% confidence limits 93-109). Furthermore these combinations are not seen in Caucasoid subjects. Such a tight association of DQ related genetic markers and Type 1 diabetes mellitus has not been previously observed in studies of Caucasoid subjects. 219. Marked difference in the effects of therapy with isoenergie diet plus glibenclamide versus hypoenergic diet on adipocyte insulin action in obese Type 2 (non-insulin-dependent) diabetic patients E. Hjallund, B. Richelsen, O. Hother Nielsen, N.S. Norensen and O.Pedersen. Medical Department III, Aarhus Amtssygehus, Denmark Adipocytes from untreated Type 2 (non-insulin-dependent) diabetic patients express severe impairements in basal and insulin-mediated glucose transport and metabolism. To assess if these defects can be reversed we compared the effects of isoenergic (8000 + 1000 k J) dieting plus glibenclamide (daily dosage 4.0_+_0.7 rag) therapy (study A) versus hypoenergic (5500 + 800 kJ) dieting (study B) in 2 groups of moderately obese, newly-diagnosed Type 2 diabetic patients. Each group comprised 8 diabetic patients who were treated for a period of 3 months. In study A body weight remained constant, fasting p-glucose decreased from 10.1 + 0.6 to 7.4+ 0.2 mmol/l (p< 0.01), fasting Pullns~ ~nd~~ t fr ~ 4t~ + 5 _gDUg~umlo s(PetO~5p) o~her~tasl211a~n_.

-

--

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.

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-

lism remained impaired. In study B average body weight decreased by 7kg, fasting p-glucose decreased from 11.2+1.2 to 6.8+ 0.8 mmol/l (p< 0.01), fasting p-insulin and adipocyte insulin binding were both unchanged whereas basal, halfmaximal and maximal insulin-stimulated glucose transport and metabolism all were normalised compared with the findings in a matched group of nondiabetic control subjects. In conclusion: In obese Type 2 diabetic patients lowenergy-dieting normalises the insulin resistance of adipose tissue, whereas weight-maintaining dieting plus glibenclamide giving the same degree of glycaemic control fail to improve insulin action. 220. Detection of antibodies to protamine-insulin binding region after NPH-insulin treatment H. Hobler1, M.Zoltobrocki2, G. Schemthaner3 and K. Federlin4. 1Bayer AG, Wuppertal; 2Hoechst AG, Frankfurt a.M.; 3II.Med. Univ. Klinik, Wien; 4II1. Med Klinik d. Univ. Giessen, Giessen, FRG Detection of insulin antibodies alone reveals only some of the antibodies evoked by NPH-insulin treatment. We developed enzymelinked immunosorbent assays (ELISA) which can be used to detect antibodies against both protamine, the sustained release additive in NPH-insulins, and "junction" antibodies directed to protamine-insulin binding region of NPH-insulin. This is made possible by introducing the protamine or intact NPH-insulin antigen indirectly in a sterically controlled manner, following adsorption of a poly-(L-glutamate, L-tyrosine) n 4:1 mixture at the ELISA plate. In a follow-up study on 20 diabetic patients treated solely with NPH-human insulin, high titre "junction" antibodies (ELISA) but no antibodies against protamine (ELISA) and/or insulin (ELISA, RIA) were detectable in one patient (1/20) in checkups 6 and 12 months after commencing treatment with NPH-human insulin. In another group of 20 diabetic patients undergoing long-term treatment with NPH bovine/porcine insulins, "junction" antibodies were detected in 2 patients (2/20). Conclusion: the formation of high titre specific "junction" antibodies can be evoked by NPH-insulin. These antibodies remain undetected using insulin or protamine antibody assay alone. 221. The scapula :triceps skin-fold thickness ratio in Type 2 (non-insulin-dependent) diabetic patients T. D. R. Hockaday, B. Pim, S. Humphreys and V.Thursfield. Sheikh Rashid Diabetes Unit, Radcliffe Infirmary, Oxford, UK The mid-upper-arm posterior skin-fold thickness (T) and that at the inferior scapula angle (S) were measured in 207 Type 2 (non-insulindependent) diabetic patients when diagnosed. As expected, the scapula:triceps (S:T) ratio was higher in 120 males (1.95 + SD 0.71) than in 87 females (1.14+0.33), p<0.0001. Both S and T correlated positively with fasting plasma insulin concentration, but the correlation coefficient was stronger with S (0.40-0.49) than T (0.25-0.37) initially and 1, 3 and 5 years post-diagnosis. These correlation coefficients were little affected by allowing for the positive association between fasting plasma insulin and fasting plasma glucose, but those 2 With with T disappeared on allowing for Body Mass Index (kg/m). S, however, the partial correlations, though lessened, remained highly significant 1, 12 and 60 months after diagnosis (p< 0.02). As among non-diabetic subjects the S :T ratio was related to development of

electrocardiographic abnormality (Minnesota coding), but only among the 47 females analysed was there a repeated trend to higher values among those whose electrocardiogram changed over 5 years from diagnosis than among those in whom it remained normal, e.g. mean of 0, 1 and 3-year S:T ratios was 1.34+0.35 in the 12 former and 1.10+0.24 in the 35 latter (p<0.05), with p<0.01 if only 1 and 3 years were considered. 222. Glucose inhibits the high affinity (Ca++Mg++)ATPase in the plasma membrane of a glucose responsive insulinoma M. Hoenig, R.J. Lee and D.C. Ferguson. Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia, USA A rise in cytosolic calcium has been implicated as a trigger of insulin release and D-glucose inhibits the efllux of calcium from islets. We examined the effect of glucose on the (Ca + +Mg + +)ATPase of a purified plasma membrane preparation from a glucose-responsive insulinoma. Enzyme activity was quantitated by the release of 32p from [gamma-32p]-ATP. A high affinity enzyme with a Km (ATP) ranging from 20-30 lxmol/1 was identified. This enzyme was assessed as a total ATP concentration of 30 .amol/l, while lower affinity activity was studied at 250 ~tmol/1 ATP, both in 2 mmol/l CaC12 and 2 mmol/1 EGTA at p H 7.5. Glucose inhibited (Ca ++ Mg + +)ATPase in a dosedependent manner, with no significant inhibition between 0 and 5 mmol/1, 50% inhibition at 11 mmol/l and almost complete inhibition (6.2+1.1% of zero glucose control, mean+SEM, n=10) with 30 mmol/1 glucose. The inhibition reached maximum rapidly ( < 1 min) and was rapidly reversible ( < 1 rain). This effect was stereospecific for the alpha-anomer of D-glucose. No effect of glucose was observed on the Km enzyme. These findings support the concept that glucose acts directly at the B-cell plasma membrane to inhibit the efflux of calcium from the cell, thereby raising intracellular calcium and triggering the release of insulin. 223. Receptor affinity of human growth hormone variants on insulin producing cells J. Hoiriis Nielsen, A. Moldrup and N. Billestrup. Hagedorn Research Laboratory, Gentofte, Denmark The pituitary gland secretes both authentic 22,000-dalton growth hormone (22KhGH) and a 20,000-dalton variant lacking amino acid residues 32-46 (20KhGH). In spite of the same growth-promoting activity in vivo, 20K has lower affinity for GH-receptors on hepatocytes and adipocytes in vitro, and reduced diabetogenic action has been suggested. This could be due to a relatively higher affinity for the B cells, and the aim of the present study was to compare the affinity of GH-variants for GH-receptors on rat insulinoma cells. The receptor assay was performed by incubating 12SI-hGH and unlabelled compounds with RIN-5AH cells and separating cells and medium by centrifugation through dibutyl phtalate. Compared with PHLC-purified 22K the receptor potency was 92% for recombinant hGH (Nordisk Gentofte, DK), 55% for deasamido-hGH and 2.3% for 20K. It is concluded that 1)recombinant hGH has the same affinity as pituitary 22K, 2) the lower affinity of clinical grade hGH is due to the content of 20K and desamido-hGH and 3) the affinity of 20K is reduced to the same extent as reported on hepatocytes and adipocytes. Thus the discrepancy between the in vivo and the in vitro action of 20K seems not to be explained by a higher affinity for insulin-producing cells. 224. Optimal therapy for improved outcome in gestational diabetes P. A. Hollander, J. Davidson, D. Reader and R. Bergenstal. International Diabetes Center, Minneapolis, Minn, USA The increased incidence of maternal and fetal complications seen in gestational diabetes can be related to hyperglycaemia; however, glucose levels that predict increased risk continue to be defined and tightened. We have developed a two-level treatment program for gestational diabetes with goals of fasting blood glucose (FBG) < 5 mmol/l and 1 h postprandial BG < 6.6 mmol/1. Placement is based on the 3 h oral glucose tolerance test. For FBG < 5.5 mmol/l and abnormal post glucose challenge values, entrance is at level one, a program of diet therapy and home blood glucose monitoring (HBGM). If BG goals are not met at level one or initial FBG is > 5.5 mmol/1, placement is at level two where insulin therapy is initiated through an ambulatory insulin program. Fourteen patients with initial FBG > 5.5 mmol/1 (range 5.6-16.6) entered at level two. Forty-eight patients entered at level one with nine patients moving to level two. Birth weight averaged 3170 + 272 grams. Macrosomia was seen in 7.4% of infants. Thirty percent of mothers had caesarean sections. Neo-natal

532 A

Abstracts

complication rate was 5.2%. In summary, HBGM, a structured program of diet and insulin therapy, and stringent BG goals resulted in maternal and fetal complication rates in gestational diabetes comparable to the general population.

ter CSII, and in normal subjects, expressed enzyme activity was highly correlated with clamp glucose requirement (r=0.86 and 0.83 respectively, p < 0.05). Thus clamp insulin insensitivity in Type 2 diabetes is strongly related to abnormalities of glycogen deposition in skeletal muscle.

225. Twice-daily subcutaneous human proinsulin provides a satisfactory basal insulin supplement in Type 2 (non-insulin-dependent) diabetic patients R. R. Holman, J. Steemson and R. C. Turner. Diabetes Research Laboratories, Radcliffe Infirmary, Oxford, UK The greater half life and hepatic effect of proinsulin, as compared with insulin, suggests that it might provide a suitable basal insulin supplement in diabetic subjects. Ten Type 2 (non-insulin-dependent) diabetic patients on stable long term therapy with human ultralente insulin and with fasting plasma glucose values <9 mmol/1 underwent a four week randomised cross-over study of twice daily subcutaneous biosynthetic human proinsutin and once daily semisynthetic human ultralente insulin given in the evening. At the end of each twoweek study period patients were admitted for a standard 24 h metabolic profile. Very similar plasma glucose profiles were seen in all patients with no suggestion of a "proinsulin run-out" prior to the next injection. Mean basal plasma glucose levels were significantly lower on proinsulin (4.8 + 0.7 vs 5.8 _+0.6 mmol/1, p = 0.008), probably because of inexact dose equivalence. Three patients experienced a total of 4 hypoglycaemic episodes, 2 on proinsulin and 2 on ultralente insulin. No significant differences were seen in the glycaemic response to meals, beta-hydroxybutyrate or lipid levels. Conclusion: Biosynthetic human proinsulin, given as a twice daily subcutaneous injection, appears to provide a satisfactory basal insulin supplement in Type 2 diabetic patients.

228. Body fluid volumes and transcapillary colloid osmotic gradient in diabetic nephropathy E. Hommel, K.Auldand, E. R. Mathiesen and H.-H. Parring. Hvid6re Hospital, Klampenborg, Denmark The transcapillary fluid balance in Type 1 (insulin-dependent) diabetic patients with nephropathy was investigated. Subcutaneous interstitial fluid (suction blister technique), colloid osmotic pressure, albumin concentration, plasma volume (125I-albumin space), glomerular filtration rate and extracellular fluid volume (51Cr-EDTA) were measured in 9 normal subjects (I), 9 normoalbuminuric diabetic patients (II), 16 diabetic patients with nephropathy (III), and 14 diabetic patients with peripheral oedema due to nephropathy (IV). Glomerular filtration rate (ml/min/1.73 m 2) in the different groups was 103 + 13 (I), 120___19 (II), 111+_19 (III) and 94+_25 (IV). The ratio between plasma volume and interstitial fluid volume was reduced in nephropathic patients 0.23 + 0.04 (IV) compared to 0.30 + 0.03 (I), 0.27 +_0.04 (II) and 0.25 + 0.03 (III) (p< 0.01). The ratio between interstitial fluid and plasma albumin concentration was reduced in group IV, 0.30, compared to the other groups: 0.43 (I), 0.44 (II), and 0.42 (III) (p< 0.01). The transcapillary colloid osmotic gradient was slightly reduced in the oedematous patients compared to the remaining subjects. The decrease in interstitial fluid albumin concentration and colloid osmotic pressure compensates the tendency to formation of oedema in diabetic nephropathy.

226. Are the enteroglucagons encoded by the glucagon gene? J. J. Holst, J. Olsen and T. Lund. Institute of Medical Physiology C, the Panum Institute, and Lab. Molecular Endocrinology, Rigshospitalet, University of Copenhagen, Denmark The large fragments of pancreatic proglucagon (PG) produced in the intestine differ markedly from the pancreatic products of PG. Possibilities: t)two genes; 2)differential proRNA-splicing; 3)differential processing of PG. We studied this in 9 hamsters using eDNA encoding for hamster pancreatic PG, cloned into pGEM-plasmids. Pancreas, ileum and colon were extracted for peptide studies (gel filtration profiles by assays for all regions of PG or RNA studies, cRNAs, complementary to two fragments of the eDNA (nucleotides 1-259 and 259-1199) were syntracted RNA and digested with RNAse a and T1, followed by PAGE and autoradiography. Pancreas contained "major proglucagon fragment" (PG 71-160) and glucagon (PG 33-61), 132 pmol/g. Ileum contained 41 pmol immunoreactive glucagon/g: 41% PG 1-69, 59% PG 33-69; other major products were PG 78-107 and PG 121-160 ("GLP-1 and GLP-2"). Colon was similar (16 pmol/g) but also contained glucagon. Both cRNAs were completely protected against RNAse by RNA from all tissues. A mismatch of > 1 nucleotide abolishes protection. The hybridising RNAs were therefore identical. Thus, 3) is correct. 227. Skeletal muscle glycogen synthase activity and insulin insensitivity in Type 2 (non-insulin-dependent) diabetes E D. Home and Y. T. Kruszynska. University of Newcastle upon Tyne, UK The relationship between skeletal muscle glycogen synthase activity, glycogen deposition, and insulin sensitivity has been investigated in Type 2 (non-insulin-dependent)diabetes. Six patients were studied after a period on diet (HbA1 10.6_+1.1 (SD) %) and after continuous subcutaneous insulin infusion (CSII; HbAa 7.5+_1.0%). Six matched control subjects were also studied. Quadriceps muscle biopsies were taken before and after a 4-h euglycaemic clamp. Diabetic patients were insulin insensitive (glucose requirement: diet 3.5 +-0.4 (SE), CSII 4.0 _+0.5, control 7.5 + 0.5 rag- kg -1- rain -1, p < 0.05) with lower skeletal muscle glycogen deposition on both study days (diet 0.8+_1.4, CSII 3.3 +_1.5, control 9.9+ 1.2 mg/g protein, p < 0.01). Despite similar basal concentrations muscle glycogen concentrations at the end of the clamp were lower after CSII than in control subjects (61 +_2 vs 69+_2 mg/g protein, p<0.05). Total glycogen synthase activity did not vary between patients or control subjects, or between fasting and clamp states. The expressed activity of glycogen synthase increased significantly between basal and end of clamp (F = 8.50, p < 0.05), and was then significantly lower in patients than in control subjects (diet 15.8 +_3.2, CSII 14.3 + 1.7, control 29.6 ___1.8 U/g wet wt, p < 0.01). Af-

229. The effect of antologons serum and some drugs on in vitro lymphocyte-mediated eytotoxicity in Type I (insnlin-dependen0 diabetic patients M. Horvfith, M. Varsfinyi, I. Balfizsi and L. Romics. 3rd Department of Medicine, Semmelweis University of Medicine, Budapest, Hungary The aim of work was to study the modulating effect of serum, immunosuppressive drugs and insulin on specific lymphocyte-mediated cytotoxicity against pancreatic tissue. Twenty patients with Type 1 (insulin-dependent) diabetes mellitus and 20 sex- and age-matched healthy controls were investigated. As targets 300 ~g/ml human pancreas extract ("0" Rh pos)-coated Sacr-labelled chicken erythrocytes were applied. As effector cells 1 x 106/ml separated T-lymphocytes were used. Cytotoxic capacity of T-cells was measured without and with inactivated autolgous serum (50 ~tt), Oradexon (Organon, 50 Izg/ 50 Ixl), azathioprone (Wellcome, 50 p.g/50 fxl), Cyclosporin A (Sandoz, 5 ng/50 p~l),and MC Actrapid insulin (Novo, 0.1 IU/50 gl). The results were expressed by maximal number of killed target cells. A significant lymphocyte-mediated cytotoxicity was observed in 18 of 20 Type 1 diabetic patients. The cytotoxicity was decreased by autologous serum in 17 cases (mainly in ICA positive 14 patients). Oradexon, azathioprine, insulin also diminished the cytotoxicity in 16 patients independently of their ICA or HLA-DR positivity. The most decrease of cytotoxicity (approaching to the control level) was observed in presence of Cyclosporin A, which proved to be a very potent in vitro immunosuppressive drug in all 18 Type 1 diabetic patients. 230. Type 2 (non-insulin-dependent) diabetic patients show reduced sensitivity to a fall in plasma glucose J. P. Hosker, M.A. Burnett, E. G. Davies and R. C. Turner. Diabetes Research Laboratories, Radcliffe Infirmary, Woodstock Road, Oxford, UK To study the ability of the B cell to "turn-off" in response to falling plasma glucose, 2 experimental protocols were used. Five diet-treated diabetic patients and 6 normal subjects were clamped at a glucose level of 15 mmol/1 for 2.5 h and then reclamped at 10 mmol/1. Fall of plasma insulin levels after change of clamp level was compared between the 2groups. For time periods - 1 0 - 0 r a i n , 1-10 min and 11-20 rain after change of clamp level, mean_+ SD plasma insulin levels for normal subjects were 178 + 141, 183 _+136 and 148 + 97 mU/1 respectively (/7<0.02 between - 1 0 0min and 11-20min values), and for diabetic patients were 61+41, 58+38 and 56+35mU/1 (NS). In a second protocol 12 diet-treated diabetic patients and 11 normal subjects were given a glucose infusion at 5 mg/kg !BW/ min for 1 h, after which the infusion was switched off and plasma insulin levels followed for 30 min. For periods - 10-0 min, 1-10 min,

Abstracts 11-20 min and 21-30 rain after infusion was switched off, mean+_ SD plasma insulin levels for normal subjects were 19-+ 8, 17 +__7, 13 +_4 and 10-+3mU/l respectively (p<0.01 between - 1 0 - 0 m i n and 10-20 rain, and p<0.005 between - 10-0 min and 21-30 min values) and for diabetic patients were 18___7, 19+_6 and 17+_ mU/1 (NS). Conclusion: "Turn-off" of B-cell secretion in response to falling plasma glucose levels is impaired in diet-treated Type 2 diabetes. 231. Mechanisms of action of metformin in patients with Type 2 (noninsulin-dependent) diabetes mellitns O. Hother Nielsen, O. Pedersen, O. Schmitz, H. Beck-Nielsen, J. Bak, P.H.Andersen and N.Schwartz Sorensen. Medicinsk afdeling III, Aarhus Amtssygehus, Aarhus, Denmark Ten obese patients with Type 2 (non-insulin-dependent)diabetes mellitus were examined in a double-blind cross-over study. Metformin (MF) 0.5 g thrice daily or placebo were given for 4 weeks. At the end of each period fasting and day-long postprandial values of plasma glucose, insulin, c-peptide and lactate were determined. Whole-body insulin action was assessed using the euglycaemic insulin clamp in combination with 3-3H-glucose tracer technique, and insulin action at the cellular level was studied in isolated adipocytes. Metformin therapy reduced fasting and postprandial p-glucose (p<0.05) without changes in p-C-peptide or insulin. P-lactate levels were increased (p< 0.05). Insulin-stimulated glucose uptake in peripheral tissue rose (p<0.05) whereas basal as well as insulin inhibited hepatic glucose output was unaffected. Insulin receptor binding and the effects of insulin on glucose transport, glucose oxidation, glycolysis, lipogenesis and lipolysis in fat cells were unchanged. Conclusions: (1) The major blood glucose lowering effect of MF is due to an increased glucose utilisation (glycolysis) in skeletal muscles. (2) No effect of MF could be shown on hepatic glucose production, glucose utilisation in adipose tissue or pancreatic B-cell secretion. 232. A long-term experimental and clinical study of an aldose reduetase inhibitor, ONO-2235, on diabetic neuropathy N. Hotta, H. Kakuta, N. Koh, F. Sakakibara, J. Nakamura, R. Kitoh, H.Matsumae and N.Sakamoto. The 3rd Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan The experimental study of an aldose reductase inhibitor (ARI), ONO2235 (50 mg.kg -1. day-l), was done in streptozotocin diabetic rats maintained on a fructose-rich diet for 1-8 months. On the other hand, a long-term clinical trial of ONO-2235 (300 mg/day) for 24 months was made in twelve diabetic patients who suffered from either spontaneous pain or paresthesia. From the observations of MNCV in tail nerve and the morphometric analysis of the myelinated fibers in sural nerve by using a computer, the development of neuropathy in diabetic rats maintained on a fructose-rich diet was completely reversed by ARI. These changes were well correlated with the levels of sorbitol and myo-inositol in nerves. In a clinical trial of ONO-2235, the improvement in either subjective or objective symptoms was observed in a time-dependent manner and ONO-2235 efficacy on diabetic neuropathy was about 75%. All of these symptoms were in good correlation with the sorbitol content and glyceraldehyde reductase activity in red cells. These results indicate that increased polyol pathway activity is related to the pathogenesis of diabetic neuropathy and an ARI, ONO-2235, has an effective and safety drug in a long-term treatment of diabetic neuropathy. 233. Modulation of insulin release and islet protein phosphorylation by a phorbol ester (TPA) and clomiphene citrate (CC) S. J. Hughes and S.J.H. Ashcroft. Department of Clinical Biochemistry, University of Oxford, UK Activation of protein kinase C in the B cell may be involved in the insulin secretory mechanism although the nature and role of protein kinase C substrates are unknown. We have studied the effects of TPA and CC, an activator and inhibitor of protein kinase C respectively on glucose-stimulated insulin release, protein kinase C activity and phosphorylation of proteins in rat islets. Glucose-stimulated insulin release, potentiated by TPA (100nmol/1) was inhibited by CC (50 gmol/1). 50 gmol/1 CC had a modest inhibitory effect (21%) on glucose-stimulated insulin release. In islet homogenates, TPA stimulated protein kinase C activity was inhibited by CC. Stimulation of intact islets by TPA after preincubation with 32p-phosphate resulted primarily in enhanced phosphorylation of a protein Mr 36,700-+ 600 (mean +_SEM, n = 7). This was blocked by CC. Subcellular fractionation revealed the presence of the phosphoprotein in the post-nuclear membrane fraction of islet homogenates. These results show that the

533 A phosphorylation state of membrane protein Mr 36,700 parallels the modulation of insulin release induced by TPA and CC and supports a role for protein kinase C in insulin secretion. 234. Exogenous insulin does not influence T-lymphocytes activation in Type I (insulin-dependent) diabetes M. J. Hussain, B. A. Millward, R. D. G. Leslie, D. A. Pyke and D. Vergani. Dept. of Immunology and Diabetes, King's College HospitaL, London, U K Increased levels of activated T-lymphocytes expressing HLA DR antigens are present in newly-diagnosed Type 1 (insulin-dependent) diabetic patients. Activated T-lymphocytes carry insulin receptors, but the role of exogenous insulin in modulating this activation in Type 1 diabetes is not known. To define whether insulin administration influences T-cell activation in Type 1 diabetes we studied 12 newly-diagnosed Type 1 diabetic patients before insulin treatment and at 1, 7 and 60 days after the commencement of insulin replacement. T-lymphocytes were purified by a rosetting technique and the presence of the HLA DR marker of activation was assessed using fluorescein labelled anti-HLA DR monoclonal antibody (Becton & Dickinson). We found that the levels of activated T-lymphocytes were increased above the normal levels (5%) before administration of insulin (9.68+0.45) and 1 (9.22_+0.60), 7 (7.51+0.86), and 60 (8.98+0.61) days thereafter. These results demonstrate that the activation of Tlymphocytes in recently-diagnosed Type I diabetic patients is not due to administration of insulin and persists after treatment. 235. Microalbuminuria, retinopathy and hypertension in an unselected diabetic out-patient population A. S. Hutchison1' 2, D. O'Reilly1, J. Gray 2 and A. MacCuish 2. 1Dept. of Biochemistry and 2Diabetic Unit, Glasgow Royal Infirmary, Glasgow, Scotland Overnight albumin excretion rate (AER) was measured, using a sensitive and precise radioimmunoassay, at least once in 415 unselected diabetic outpatients, with the aim of documenting the prevalence of, and associations between, microalbuminuria (an overnight AER of > 30 gg/min), retinopathy, and hypertension in the clinic population. One hundred ninety (45.8%) of the patients were insulin-treated, and 225 (54.2%) were managed with diet and/or oral hypoglycaemics. The overall prevalences were: 12% with microalbuminuria; 17.8% with retinopathy; 21.9% with hypertension. Insulin-treated patients were more likely to have retinopathy (22.6% vs 13.8%; p<0.05) but less likely to have hypertension (12.1% vs 30.2%, p<0.01) than noninsulin-treated patients. Microalbuminuria was more likely to occur in retinopaths (17 of 74; p<0.01 compared with non-retinopaths), but was not significantly associated with hypertension. No differences between the treatment groups were demonstrated. In this study we have shown a significant association between retinopathy and an increased AER. It is worth noting, however, that the majority of patients with retinopathy (77%) did not have microalbuminuria, and the majority of patients with microalbuminuria (66%) did not have retinopathy. This would suggest that different mechanisms are involved in the pathogenesis of diabetic retinopathy and nephropathy. 236. Acceptability and feasibility of continuous subcutaneous insulin infusion - preliminary report on a multicentre WHO-study I.Hiittl, H. Bibergeil, U. Fischer, G.Albrecht, D.Gottschling, R. Bohnefeld and Ch. Ltider. Central Institute of Diabetes "Gerhardt Katsch" Karlsburg, GDR In order to study acceptability, psychosocial implications, glycaemic control and economic consequences of optimised conventional insulin therapy (CIT) versus continuous subcutaneous insulin infusion (CSII), a WHO multicentre study was initiated, which involves approximately 400 patients in Europe. In the course of this study we randomly allocated 40 Type 1 (insulin-dependent) diabetic patients who were crossed over after 6 months. There were the following findings in 30 patients who have completed the study so far: on CSII glycaemia was more easily controllable by the patients themselves, blood glucose criteria under ambulatory conditions were significantly better and the insulin doses were significantly lower (p< 0.01). Running the pump treatment was more expensive by 14% in terms of equipment, but equally expensive with regard to the overall time spent by patients and staff. The number of days off work did not differ significantly (10.8_+2.1 versus 14.3+_2.3/6 months). Unexpectedly, CSII acceptance was 100%. The main reasons for this were greater flexibility in life-style and enhanced well-being as stated by the patients spontaneously and as analysed from questionnaires. These psychosocial impli-

534 A cations have to be considered in estimating the cost/effect ratio of a regime. 237. An abberrant secretory process in islet cell tumours J. C. Hutton, M. Peshavaria, C. A. Hynds and H.W. Davidson. Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, UK Insulinomas in man are commonly associated with high circulating levels of proinsulin and processing intermediates. We have investigated the basis of this abnormality using rat transplantable insulinoma as a model, studying the biosynthesis and secretion of a series of granule matrix proteins, notably proinsulin and carboxypeptidase H. Insulinoma cells released 30-40% of 3SS-methionine-labelled insulin and carboxypeptidase H in a secretagogue-insensitive process within the first hour post synthesis. By contrast islets released less than 5% of their newly synthesized proteins over this period. Pulse-chase studies showed that predominantly proinsulin and a lesser amount of desdibasic proinsulin were released. Carboxypeptidase H release followed an identical time course to proinsulin and was present in the media initially as a 56,000 Da precursor and finally as the 54,000 Da mature form after 30 rain. Maturation of this enzyme is attributable to changes in glycosylation rather than prote.olysis. These results together with a study of the effects of chloroqume suggested that the unregulated release process occurred from elements of the Golgi prior to the transfer of secreted proteins to secretory granules. There is thus a dysfunction in the normal mechanism of protein se.gregation in these cells. This would account for the abnormality seen m man. 238. Growth hormone suppression in diabetic retinopathy S.L.Hyer, P.S.Sharp, R.A.Brooks, J.M.Burrin and E.M.Kohner. Department of Medicine, Hammersmith Hospital, London, UK Growth hormone (GH) suppression may be beneficial in the treatment of diabetic retinopathy. Five patients (range 20-45 years) with active proliferative retinopathy despite repeated laser photocoagulation and four non-diabetic matched control subjects received, in randomised order, oral atropine (anticholinergic drug with good CNS penetration), oral propanthelene (anticholinergic drug with poor CNS penetration), and a long acting somatostatin analogue SMS 201-995 (Sandoz) subcutaneously, in an effort to suppress GH secretion. Atropine 1.2 mg 4-hourly markedly inhibited GH secretion in the control subjects with GH peaks of less than 10 mU/1 throughout 24 h monitoring. Patients showed GH suppression except at times of hypoglycaemia when GH peaks, range 12-26 mU/1, occurred. Propanthelene (30 mg tds and 30 mg qid) failed to suppress GH peaks in controls (range 12-129 mU/1) or in patients (12-80 mU/1). Twentyfour h GH secretion was unchanged. SMS 201-995 completely suppressed GH secretion in the controls at a dose of 50 ~g tds but even at doses of 200 lxg tds (n=5) or 500 lxg tds (n=2) failed to suppress GH secretion in the patients. This group of diabetic patients appear to be resistant to GH suppression with SMS 201-995. Atropine is of limited usefulness because of uninhibited GH release at times of hypoglycaemia and because of unpleasant side-effects. 239. Glucose recruits pancreatic B cells to biosynthetic activity P. A. In't Veld, F. Schuit, W. Gepts and D.G. Pipeleers. Dept. of Pathology and Dept. of Metabolism & Endocrinology, Vrije Universiteit Brussel, Belgium The rate of protein synthesis by pancreatic B cells increases more than 10-fold when extracellular glucose rises from 1 to 10 mmol/1; no further stimulation is measured for higher nutrient levels. The doseresponse curves are similar in single and in structurally coupled B cells. When purified single B cells are incubated with [3H]-leucine before their analysis in autoradiographs, marked differences are noted in the glucose responsiveness of the biosynthetic activities of individual cells. At I mmol/1 glucose, only 15 percent of the cells are densely labelled and clearly distinguishable from the remaining cells with background levels of silvergrains. Raising the glucose concentration to 5 mmol/1 increases linearly the number of positive cells up to 65 percent. At 10 and 20 mmol/l glucose, an intense biosynthetic activity is discerned in 85% of the cells. This dose-dependent recruitment of B cells to a biosynthetic activity parallels the dose-response curve of glucose-induced protein synthesis as measured by the trichloroacetic acid precipitable radioactivity. When an intact pancreas is perfused with [3H]-leucine at different glucose concentrations, its islet B cells display a similar heterogeneity in their biosynthetic response to glucose. It is concluded that the pancreatic B-cell population is composed of cells with a different glucose sensitivity of their biosynthetic activity.

Abstracts 240. Intermediary metabolism in Cushing's Syndrome during euglycaemic-hyperinsnlinemic clamps S. Inchiostro, G. Biolo, G. Fantin, M.C. Marescotti, G. Merola, R. Nosadini, F. Mantero, A.Tiengo and E Tessari. Malattie del Ricambio, Patologia Medica 1 and Semeiotica Medica, University of Padova, Italy The concentrations of intermediary metabolites and amino acids were determined in six patients with Cushing's Syndrome (CS) and in six control subjects (C) in postabsorptive conditions and during sequential euglycaemic-hyperinsulinaemic clamps (plasma insulin= N50, -100 and ~1500 mU/1). Basal insulin was greater (p<0.05) in CS than in C (15+2 vs 10+1 mU/1), while plasma glucose concentrations were comparable. In CS, basal concentrations of lactate (1.150_+0.14mmol/1), pyruvate (0.08+0.01 mmol/1) and alanine (0.498 + 0.029 mmol/1) were greater (p< 0.01) than in C (0.58 _+0.09, 0.03 _+0.002 and 0.295 + 0.026 mmol/1, respectively). Insulin-mediated glucose disposal was lower (p<0.05 or less) in CS than in C at each clamp step. During the clamps, lactate and pyruvate did not change from basal in CS, while in C they increased significantly in a stepwise fashion. Alanine decreased significantly in CS but not in C. Among the other amino acids, only lysine and isoleucine were higher in CS than in C. A significant positive correlation (r=0.50, p<0.05), was found between glucose disposal and the increments in lactate concentrations in C but not in CS. In conclusion, our study underscores the presence of alterations in the concentrations of 3-carbon compounds in CS, both in the postabsorptive state and during euglycaemic hyperinsulinemia, indicating abnormalities in the glycolitic and/or gluconeogenic pathways, possibly linked to insulin resistance. 241. Comparative study of insulin antibodies in diabetic patients with primary and secondary Insulin-dependence C. Ionescu-Tirgovi~te, Z. Mirodon, D. Chela, E. Sfintu, M. Simionescu, A. Nicolau and I. Mincu. Cfinic of Diabetes, Nutrition and Metabolic Diseases, Bucharest, Romania Insulin antibodies (% binding) were determined by RIA method in 404insulin-treated diabetic patients divided in two groups: (a)primary insulin-dependent patients: 300cases, 170M, 130F, mean age_+ SD 29.2 + 7.5 years, disease and insulin treatment duration of 7.7 _+6 years; (b) diabetic patients needing insulin (secondary insulin-dependence): 104cases, 47M, 57 F, aged 53.4_+2.1years, duration of diabetes 13.1 _+8.3 years, and of insulin treatment 3.1 + 2.1 years. Both groups of patients had treatment with the same types of insulin preparations. In 297 cases, all belonging to group (a), we also determined the fasting C-peptide. Results: the titre of insulin antibodies was significantly (p< 0.001) higher in patients with secondary insulin-dependence than in those with primary insulin-dependence (22.96_+ 15.1% vs 10.25 + 9.89) in spite of the longer duration of the insulin treatment in the latter group; the mean C-peptide value found in primary insulin-dependent diabetic patients with a binding capacity < 10% was significantly (p< 0.001) lower than that found in 11 primary insulin-dependent diabetic cases with a binding capacity > 20% (0.091 +0.57 vs 0.273 +0.376 pmol/1); no correlation was found between insulin antibodies titre and both insulin dose used as treatment and degree of metabolic control as assessed by HbA1. 242. Non-enzymatic glycosylation of low-density lipoprotein in diabetic patients. Results of an affinity chromatography method C.M.Jack, B.Sheridan, L.Kennedy and R.W.Stout. Department of Geriatric Medicine, The Queen's University of Belfast, and Sir George E Clarke Metabolic Unit, Royal Victoria Hospital, Belfast N. Ireland Low density lipoprotein (LDL) glycosylation has generated interest because molecular modification of LDL alters its cellular uptake and metabolism and may contribute to the increased atherosclerosis found in diabetes mellitus. In vivo LDL glycosylation was quantified using aminophenylboronate affinity chromatography. Mean LDL glycosylation in 55 diabetic patients was 10.1 +3.3%, which is significantly greater (p<0.001) than the mean value of 4.6+_ 1.0% of 38 nondiabetic subjects. In diabetic patients, LDL glycosylation was not significantly different in males and females, nor between different age groups. No significant difference was found between Type 1 (insulindependent) and Type 2 (non-insulin-dependent) patients. No sex or age difference was found in non-diabetic subjects. Glycosylated LDL correlated with HbA1 (r=0.65) and with glycosylated plasma proteins (r= 0.68), but the strongest positive correlation was between glycosylated LDL and plasma glucose at the time of sampling (r= 0.72). Longitudinal studies of hyperglycaemic diabetic patients showed eie-

Abstracts vated glycosylated LDL levels which did not fall appreciably till after 5 days of intensive therapy. Conclusion: Diabetic patients have increased LDL glycosylation, which is not influenced by age or sex and is dependent on ambient plasma glucose levels. Glycosylated LDL appears to be metabolised at a slower rate than native LDL in vivo. 243. Influence of glucose ingestion on lactate turnover in man R.A.Jackson, M.I. Hawa and B. M. Sim. Cobbold Laboratories, The Middlesex Hospital, Mortimer Street, London, U K The rise in plasma lactate concentrations after glucose ingestion is well recognised and is thought to reflect increased splanchnic lactate production. However, the changes in lactate kinetics underlying this response have not been studied. Therefore, using a primed constant infusion of L( + )-U-14-C-lactate, we investigated systemic lactate turnover before and after a 75 g oral glucose load in 10 normal men aged 21-23 years. Concurrently, forearm lactate balance and glucose uptake and systemic glucose turnover were studied. Lactate specifc activity was determined on arterialised venous plasma. After glucose loading, plasma lactate levels rose from 0.99 + 0.06 mmol/1 basally to peak concentrations (1.71 _+0.12 retool/l) at 60 min before declining. As lactate levels increased, forearm muscle took up lactate. Lactate appearance rate (Ra; ~mol-kg -1- min -1) was 9.9 + 1.1 basally and increased after glucose ingestion to a peak of 15.9 _+1.8 at 60 rain, declining thereafter, while glucose Ra peaked at 30 min. Lactate disappearance rate peaked at 90 min (15.7 _+1.5 ~mol. kg -1. min-l), falling thereafter. The increments in lactate Ra from 0-60 and 0-180 rain were 14.6 and 42.1 mmol respectively, reflecting the metabolism of 1.3 and 3.8 g glucose respectively. Conclusions: 1) Lactate turnover is increased by 61% from 0-180rain after glucose ingestion. 2)The 1.7-fold rise in lactate concentrations reflects the splanchnic metabolism of only small amounts of glucose. 244. Reduced perception of early hypoglycaemic symptoms in Type 1 (insulin-dependent) diabetic patients after changing from porcine to human insulin E.J~iggi, A.Teuscher, K. Btitikofer and W. Berger. Department of Internal Medicine, University Hospitals Basel and Berne, Switzerland Mitigation of hypoglycaemic symptoms has been reported in patients treated with human insulin preparations (HM). In the present study 54 out of 70 patients (mean age 42.1 years, diabetes duration 16.7 years), who were transfered from porcine (MC) to human insulin, were analysed with respect to hypoglycaemic symptoms. Ninetythree % reported differences in the awareness of hypoglycaemia when changed to HM (p < 0.001). Typical early symptoms, such as perspiration, tremor and hunger were two times more frequent in patients on MC regimen (p< 0.001), whereas symptoms of neuroglycopenia - inability to concentrate, visual disturbances and odd behavior - were significantly more often reported by patients on HM (p< 0.01). Severe hypoglycaemic events occurred more frequently during HM treatment periods (p<0.01). Fifty-four % of the patients felt safer when using MC compared to only 20% who considered HM to more secure. In 16 out of 56 patients MC had to be reinstated because of insecurity due to impaired awareness of hypoglycaemia. In 9 patients, experimental hypoglycaemia was provoked by HM and MC administration. No differences were found in cortisol-, norepinephrine-, epinephrine-, glucagon-, and growth hormone-responses. Conclusions: 1) Transfer from MC to HM in Type 1 (insulin-dependent) diabetic patients can mitigate early hypoglycaemic symptoms. 2)This phenomenon is not due to alterations in the counterregulatory hormone responses. 245. Tight metabolic control improves cerebral functions in older Type 2 (non-insulin-dependent) diabetic patients W.Jagusch, D.v.Cramon 1, R. Renner and K.D. Hepp. Diabetes Center and Dept. of Neuropsychology 1, Bogenhausen City Hospital Munich, FRG The extent of therapeutic efforts and the target values of metabolic control in the elderly Type 2 (non-insulin-dependent) diabetic patients are currently a matter of controversy. It seemed therefore of interest to assess the possible influence of the disease and the effect of improved metabolic control on mental functions in these patients. In 32 Type 2 diabetic patients (age 65.9 + 8.8 years; mean + SD) with an average HbAac of 10.4+ 1.5% and a mean capillary blood glucose of 10.82+2.66 mmol/1, memory, learning and speed of central information processing were tested with sensitive neuropsychofogical methods. Twenty patients were tested after improved metabolic control in intervals between 4 and 21 days, with a mean blood glucose at

535 A 7.35 + 1.87 mmol/1. Thirteen nondiabetic persons with a mean age of 71.1 _+5.5 years were used as control subjects. Verbal learning ability improved by 29.7% (p<0.01), and decision as a measure for central information processing by 5.6% (p<0.05), in contrast to the control group which showed no change after repeated testing. We conclude that marked improvement of diabetic control leads to enhanced mental efficiency and hence to a better quality of life in the elderly diabetic patient. 246. Effect of osmolality on the metabolic availability of glucose given orally during prolonged muscular exercise B. Jandrain, F. Pirnay, M. Lacroix, F. Mosora, A. Scheen and P.J. Lefebvre. Division of Diabetes, Institute of Medicine, University of Li6ge, Belgium Increasing osmolality of carbohydrate-containing solutions has been reported to diminish gastric emptying rate, thus limiting substrate delivery during exercise, but running at a moderate intensity has been shown to significantly enhance gastric emptying rate. The aim of this study was to investigate the metabolic availability of a 50 g glucose load diluted in water - either 200 ml (A: 1204 mmol/1), 400 ml (B: 644 mmol/1) or 600 ml (C: 439 mmol/1) - ingested by 5 healthy male volunteers after 15 min adaptation to a moderate-intensity (45% ~'O2m~,) long-duration (4 h) exercise. Indirect calorimetry was used to evaluate carbohydrate (CHO) and lipid (LIP) oxidation and "naturally-labelled 13C-glucose" to follow the oxidation of the exogenous glucose (ExoG). Total energy expenditure (about 9730+290 kJ/4 h), protein (about 14.5+1.3 g/4 h) and LIP (A: 128+4, B: 132+15, C: 124+ 12 g/4 h; NS) oxidation were similar in the 3 tests. No statistically significant difference was observed in either total (A: 237 +20, B: 258 + 17, C: 276 + 20 g/4 h; NS) or endogenous carbohydrate oxidation (A: 195 + 21, B: 214 + 18, C: 227 + 27 g/4 h; NS). ExoG oxidation was slightly but not significantly reduced with the more concentrated solution (A: 43+4, B: 43+4, C: 4 9 + 7 g / 4 h ; NS). In conclusion: within a large range of osmolalities, a 50 g glucose load ingested soon after initiation of moderate-intensity long-duration exercise is a perfectly available metabolic substrate. 247. Reduced erythrocyte (Na+-K +) ATPase activity and red cell deformability in Type I (insulin-dependent) diabetic patients with retinopathy: normalisation under calcium entry blocker therapy H. U. Janka, J. Mayer, A.G. Ziegler, A. Nuber, H. Walter, A. Gtinther and H.Mehnert. Schwabing City Hospital and Diabetes Research Unit, Munich, FRG Hypertension and reduced cell deformability (RCD) have been documented repeatedly in diabetic patients with microangiopathy. In addition, an association of impaired cation transport systems, including blood cell (Na + -K + ) and (Ca 2 + -Mg 2 + ) ATPase, with these abnormalities have been reported. The aim of this study was to examine the influence of calcium entry blocker therapy with nitrendipine on erythrocyte cation ATPase activity and RCD. Erythrocyte membranes were prepared by iso-osmotic freeze-haemolysis and ATPase activity was measured by liberation of inorganic phosphate (Pi)- RCD was determined by red cell micropore filtrability. In comparison to 18 healthy control subjects, (Na+-K +) ATPase activity in 31 diabetic patients with retinopathy was significantly reduced (5.6 _+2.3 nmol Pi/ mg per min vs 8.4+4.7, p<0.05). After one-week treatment with nitrendipine (10 mg bid) normalisation of (Na+-K +) ATPase took place (8.9 + 4.1 nmol Pi/mg per min). This effect was paralleled by a significant improvement of RCD, but there was no correlation with blood glucose or hemoglobin Ale. (Ca2+-Mg 2+) ATPase activity, however, did not change. Thus, depressed erythrocyte (Na+-K +) ATPase activity in diabetic patients with retinopathy can be increased by calcium entry blocker therapy, indicating the molecular mechanism by which these drugs affect blood pressure and red cell deformability. 248. Non-esterified fatty acids augment insulin-stimulated muscle glucose utilisation in vivo: a route to insulin resistance? A.B.Jenkins, L.H.Storlien and E.W. Kraegen. Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, Australia Increased availability of non-esterified fatty acids (NEFA) is associated with reduced glucose disposal in Type 2 (non-insulin-dependent) diabetes and obesity. The acute effects of elevated N E F A (triglyceride+heparin infusion giving NEFA elevations of 1-4 mmol/1) on glucose utilisation (Rg') and storage in peripheral tissues in vivo were assessed using tissue accumulation of intravenous bolus 3H-2-deoxyglucose and ~4C-glucose. Fasted conscious rats were studied in the basal state and during a hyperinsulinaemic (150 mU/1) euglycaemic

536 A clamp. NEFA suppressed basal ( - 6 8 % , p<0.001) and clamp (-49%, p<0.001) Rg' in heart and basal ( - 3 9 % , p<0.001) Rg' in skeletal muscle. NEFA stimulated clamp Rg' (+72%, p<0.001) and .glucose storage (glycogen: + 170%, p<0.01; lipid: + 100%, p<0.01) m muscle, and basal Rg' in brown adipose tissue (BAT) (+200%, p<0.01). Clamp BAT Rg' was suppressed by N E F A ( - 4 2 % , p < 0.001). Thus there are heterogeneous and unexpected tissue responses to NEFA. Non-esterified fatty acid-induced suppression of insulin stimulated Rg' in BAT may reflect reduced thermogenesis and/or lipogenesis. Acute elevations of N E F A augment insulin-stimulated glucose utilisation and storage product accumulation in muscle. This may lead to insulin resistance via end-product inhibition of glucose storage pathways and competetion between mobilised intramuscular substrates and circulating glucose for oxidative pathways. 249. Abnormalities in plasma lipoproteins and fibrinogen concentrations in Type I (insulin-dependent) diabetic patients with increased urinary albumin excretion T. Jensen, S. Stender I and T. Deckert. Steno Memorial Hospital, Gentofte, Denmark, 1KAS Gentofte, Dept. of Clin. Chemistry, Hellerup, Denmark Type 1 (insulin-dependent) diabetic patients with diabetic neprhopathy have a huge increase in relative mortality of cardiovascular diseases compared with patients without nephropathy. Lipoproteins and fibrinogen were investigated in 74 long-term diabetic patients subdivided into three sex-, age- and diabetic duration-matched groups. I: normal urinary albumin excretion ( < 3 0 m g / 2 4 h; n=37), II: microalbuminuria (30-300 mg/24 h; n = 20), III: overt clinical nephropathy ( > 300 rag/24 h; n=17). Plasma cholesterol in the fasting state was group I: 4.82_ 0.77 mmol/1, group II: 5.23 + 1.03 mmol/1, group III: 6.08 +_1.20 mmol/1; p < 0.01. After ultracentrifugal separation VLDL- and LDL-cholesterol were higher in groups II and III compared with group I, while HDL-cholesterol was indistinguishable in the three groups. Plasma fibrinogen was group I: 7.68 (5.16-11.52) gmol/1; group II: 8.68 (5.24-12.85) ~xmol/1; group III: 9.62 (6.04-13.73) ~tmol/1; p < 0.01. The differences were not explained by differences in metabolic control (HbAlc), blood glucose, insulin dose, free plasma insulin, body mass index or blood pressure. In conclusion: abnormalities in plasma lipoproteins and fibrinogen concentrations are an early event in the development of diabetic nephropathy. 250. Platelet thromboxane A 2 synthesising capacity is enhanced by fasting but diminished by diabetes mellitas in the rat J.Y.Jeremy, C.S.Thompson, D.P. Mikhailidis, O. Epemolu and P.Dandona. Metabolic Unit, Department of Chemical Pathology & Human Metabolism, Royal Free Hospital & School of Medicine, London, U K Diabetes mellitus and anorexia nervosa are known to be associated with platelet hyperaggregability and increased thromboxaneA2 (TXA2) release. The effect of long-term streptozotocin-induced diabetes mellitus, fasting and semistarvation on platelet TXA2 synthesising capacity was therefore investigated. Platelet rich plasma was centrifuged in an Eppendorf microfuge and pellets washed with isotonic saline. Pellets were frozen to - 7 0 ~ thawed, 1 ml Krebs Ringer bicarbonate buffer added, sonicated and incubated at 37 ~ for 3 h. Aliquots of supernatants were taken for radioimmunoassay of TXB2 (total TXA2 synthesising capacity; TSC). TSC was significantly (t9< 0.01) reduced in platelets from diabetic rats of 62 days duration, an effect prevented by insulin administration (controls, 330; diabetes, 230; insulin-treated diabetes, 320 pg TXB2/106platelets/3 h). In contrast, 48 h and 72 h fasting and 9 days semi-starvation significantly (p< 0.01) elevated TSC (controls, 340; 48 h, 390; 72 h, 540; 9 days, 520). These data indicate that (1) despite the metabolic and endocrine similarities of diabetes and fasting, these conditions elicit diametrically opposite effects on platelet TXA2 synthesising capacity; (2) TXB2, as a novel means of assessing platelet TXA2 synthesis, may reveal changes not apparent with other methods, e.g. TXA2 release consequent to platelet aggregation. 251. Do Guar granules improve obese Type 2 (non-insulin-dependent) diabetic patients poorly controlled on both sulphonylureas and biguanides? C. Johnston, N. Sailer, J. L. Barton and G. S. Spathis. St. Helier Hospital, Carshalton, Surrey, U K Improving obese Type 2 (non-insulin-dependent) diabetic patients poorly controlled on both sulphonylureas and biguanides is difficult.

Abstracts We assessed the benefit of adding Guar granules (Guarem or Guarina) in 21 patients, 13 female, aged 60 + 2 years (mean + SEM). After a month's run-in with the addition of only dietary advice their mean body-weight was 88.2 + 4 kg, fasting glucose 11.4 _+0.7 mmol/1, HbA1 10.3 + 0.5%, fructosamine 3.0_+ 0.1% and cholesterol 6.8 + 0.3 mmol/l. None of these values differed significantly from entry. Patients were then randomised to either preparation for three months administered to a maximal tolerated dose not exceeding 15 grams a day. After Guar mean body-weight was 88.0+4kg, fasting glucose 12.0+ 0.8 mmol/1, HbA1 9.7 _+0.5%, fructosamine 3.0 + 0.1% and cholesterol 6.3 + 0.2 mmol/1. None of these differences approached statistical significance (paired t test). Ten patients were unable to tolerate the maximal dose. In all patients gastro-intestinal side-effects measured using a symptom scale rose from a mean of 1.6 + 0.5 (maximum 15) to 4.7 + 0.2 after Guar (p< 0.001). There was a difference in the metabolic effect of symptoms with either product. We conclude that in this group of diabetics the addition of Guar granules confers no metabolic benefit and is associated with a significantly increased frequency of side-effects.

252. Effects of noradrenaline on protein phosphorylation in electrically permeabilised islets of Langerhans P. M. Jones, D.M.W. Salmon and S.L. Howell. Department of Physiology, King's College London and Department of Pharmacology, St. George's Hospital Medical School, London, U K Noradrenaline causes a marked inhibition of glucose-induced insulin secretion from intact islets of Langerhans and of Ca2+-induced insulin secretion from electrically permeabilised islets. We have now investigated whether the noradrenaline inhibition of secretion involves the activation of protein kinases by studying the effects of noradrenaline on patterns of Ca2+-dependent protein phosphorylation in electrically permeabilised islets. Collagenase isolated rat islets were permeabilised by 5 exposures to an electric field of 3.4 kV/cm and incubated for 1 rain at 37 oC in glutamate-based Ca 2 + / E G T A buffers containing 0.3 retool/1 [7/32 P]-ATP (1.2 C1/mmol) in the presence or absence of noradrenaline (10 ~tmol/l). Proteins were separated by SDS-polyacrylamide gel electrophoresis and 32p incorporation assessed by autoradiography. Increasing the Ca 2+ concentration from 50 nmol/1 to 10 ~tmol/1 caused increased 32p incorporation into a number of proteins, particularly a protein of 80 kDa molecular weight, and the dephosphorylation of a 60 kDa protein. Noradrenaline had no detectable effects on protein phosphorylation/dephosphorylation at either substimulatory (50nmol/1) or stimulatory (10 ~xmol/1) concentrations of Ca 2+. These results suggest that the inhibition of insulin secretion by noradrenaline reflects an action at a late stage of the exocytotic pathway, beyond the activation of protein kinases and the phosphorylation of their specific substrates.

253. Glomerular hyperfiltration and albuminuria - a 5 year prospective study in Type I (insulin-dependent) diabetes mellitus S. L. Jones, M.J. Wiseman, G.C. Viberti and H. Keen. Unit for Metabolic Medicine, Division of Medicine, United Medical and Dental Schools, Guy's Hospital, U K Glomerular fltration rate (GFR), albumin excretion rate (AER) and blood pressure were measured in 30 Type I (insulin-dependent) diabetic patients at entry into, and after 5 years of a prospective study. Thirteen hyperfilterers had initial GFR >135 (mean 155; range 137-196) ml/min per 1.73 m 2 and 17 normofilterers had GFR < 135 (mean 117; range 93-132) ml/min per 1.73 m 2. Two hyperfilterers and three normofilterers had AER > 20 I.tg/min at entry. Mean blood pressure was 9 0 + 7 mmHg in the hyperfilterers and 93 +11 in the normofilterers. Over 5 years, on average, GFR fell by 0.45 ml. rain -a. month -1 in the hyperfilterers and by 0.22 ml. min -1. month -1 in the normofilterers (p<0.05). GFR fall rate was related to initial GFR in the hyperfilterers only (r=0.6, p<0.05). After 5 years no hyperfilterer had AER > 20 ]xg/min. Albuminuria had increased in one normofilterer with initial AER <20 l-tg/min to 167 and in another to 28 pg/min. After 5 years mean blood pressure in the hyperfilterers was 88 + 9 and in normofilterers 94+10 mmHg; one normofilterer and no hyperfilterer received antihypertensive therapy. Initial GFR and GFR fall rate do not predict increase in albuminuria or blood pressure over 5 years. This study does not support a relationship between high GFR and microalbuminuria. The relationship of hyperfiltration to kidney damage in human Type 1 diabetic patients remains to be established.

Abstracts 254. Aldose reductase and iris changes in galactose-fed rats P. F. Kador, H. Terubayashi, Y. Akagi and J.H. Kinoshita. National Eye Institute, National Institutes of Health, USA Rats fed 50% galactose enriched diets are commonly employed in investigations into the role of aldose reductase in ocular diabetic complications. In the absence of synechiae, the irises of these rats display both a delay in dilation and a failure to fully dilate in response to treatment with mydriatics. This delay in dilation initially occurs 8 weeks after galactose feeding and is accompanied by a hypersensitivity to phenylephrine. This is followed at week 12 by the formation of fibrous tissue containing collagen, fibroblasts and macrophages which progresses beneath the posterior surface of the iris from the pupillary margin toward the limbal area. At the same time increased iridal blood vessel permeability to horseradish peroxidase and 125I-albumin can also be detected. The delay in dilation, fibrous tissue formation and altered vessel permeability are absent in galactose-fed rats concomitantly administered the aldose reductase inhibitors tolrestat and sorbinil. These studies provide the first experimental evidence that aldose reductase is associated with diabetes-linked iris changes and provide a new potential model for the evaluation of aldose reductase inhibitors. 255. Effect of culturing at physiologic low glucose concentration on glucose-mediated insulin secretion coupling in rat pancreatic islets P. Kaiser, E.J. Verspohl and H.P.T. Ammon. Department of Pharmacology, Institute of Pharmaceutical Sciences, University of Tuebingen, FRG In contrast to supraphysiologic glucose concentrations (16.7 mmol/1), long-term culturing of rat pancreatic islets in the presence of lower (5.6 mmol/1) glucose has been shown to diminish their secretory ca~6acity. The effect of glucose on NADPH, reduced glutathione (GSH) Rb + efflux, and 45Ca+ + net uptake were investigated in freshly prepared islets and in islets maintained for 1 or 5 days in culture (presence of 5.6 mmol/l glucose). The insulinotropic effect of 16.7 mmol/1 glucose decreased with the period of culturing, though both protein content and ATP levels per islet were not affected. Both NADPH and GSH decreased with time of culture. 86Rb+ efflux, which is decreased by enhancing the glucose concentration from 3 to 5.6 mmol/1 in freshly isolated islets, was not affected by culturing whatsoever, not even after 14 days of culture (no insulin responsiveness). The 45Ca+ + net uptake was decreased during culturing. The data indicate (1) that the diminished glucose-stimulated release of insulin during culturing is not due to cell loss or simple energy disturbances, (2)that more likely it is the result of a diminished 45,Ca + + net uptake as a consequence of the inability of islet cells to maintain proper NADPH arts GSH levels, and (3) that potassium (86Rb + ) efflux may not be related to changes of NADPH and GSH. 256. Single channel recordings from human isolated pancreatic B cells M. Kakei, F.M.Ashcroft, R.P. Kelly and R. Sutton. University Laboratory of Physiology, Oxford, UK Single channel recordings from rodent B cells and B-cell lines have indicated that an ATP-sensitive K+-channel plays an important role in glucose stimulated insulin release. We have recorded single channel currents with similar properties to those found in rodent cells in membrane patches from human B cells isolated from cadaver organ donors. In cell-attached patches the channel has a conductance of 56 pS and a reversal potential between - 45 and - 80 mV (pipette potential) when the pipette contains 140 mmol/1 K +. In inside-out patches exposed to symmetrical 140 mmol/l K +, the reversal potential was 0 mV suggesting the channel is K+-permeable; and the conductance was 64 pS. The channel was inhibited by adding 1 mmol/1 ATP to the intracellular solution and activated by 0.5 mmol/l ADP (5 mmol/1 Mg + +). It was also blocked in a dose-dependent manner by the sulphonylurea tolbtuamide; and by 1 mmol/1 sparteine. These results are consistent with the idea that the ATP-sensitive K+-channel may play a role in mediating insulin release in response to glucose and sulpbonylureas in man. A channel of 100 pS with a reversal potential of - 7 0 mV and activated by depolarisation was also recorded in cell-attached patches. A similar channel has been reported in rat B cells. 257. Glomerular hyperfiltration, albuminuria and the duration of Type I (insulin-dependent) diabetes W.J. Kalk and A. Feigenbaum. Department of Medicine, University of the Witwatersrand Medical School, Johannesburg, South Africa Glomerular hyperfiltration may occur early after the onset of diabetes

537 A and is considered a risk factor for diabetic nephropathy. We therefore examined the duration of diabetes in relation to glomerular filtration rate (GFR; 51Cr-EDTA) and urinary albumin excretion rate (AER; radioimmunoassay) in 58 Type 1 (insulin-dependent) diabetic patients (aged 14-30 years, onset <20 years, duration 1-10 years). The duration of diabetes at the onset of intermittent and persistent proteinuria was studied in additional subjects. An elevated GFR (> 140 ml/min per 1.73 m 2) was found in 24 (41%) of patients. There was a progressive rise in the observed median duration of diabetes as "risk factors" for nephropathy increased: elevated GFR (166.5+5.1 ml/min per 1.73 m z) and normal AER (18.2+3.2 ixg/min) - 4.0 years; elevated GFR (165.7 _+7.72) plus microalbuminuria (AER 80.9 _+11.3 lxg/min) - 8.0years; intermittent proteinuria (GFR 141.3_+11.7; AER 156.9_+ 66.9) - 9.5 years; persistent proteinuria (GFR 100.1-+ 12.7; AER 1072.6_+438.4) - 14.9 years. However, 13 of 34 with normal GFR (107.7-+3.6) had an increased AER (59.2+8.2 gg/min) also with median duration of diabetes of 8.0 years. The mean blood pressures of the microalbuminuric groups were similar. The apparent sequence of changes in renal function supports a role for hyperfiltration as a probable risk factor, in some subjects, for future microalbuminuria and overt nephropathy. Other risk factors may exist in patients without hyperfiltration.

258. Fluoroscein secretion in diabetic and non-diabetic saliva. Relation to retinopathy B. Karamanos, K. Petrou, G. Digenis, Th. Kardoyiannis and J. J. Roussos. Diabetic Center, Hippokration Hospital, Athens, Greece To compare the handling of Fluorescein by salivary glands of diabetic patients with that of non-diabetic subjects, we studied 17 diabetic patients and 7 non-diabetic subjects. During the Fluorescein angiogram of the retina, Fluoroscein was measured in the serum and saliva (after parotid duct canulation) 30 rain and 120 min after the intravenous bolus Fluorescein injection. Fluoroscein levels were measured with a spectrofluorometer in arbitrary units. Fluoroscein level in saliva at 30 min was not different between diabetic patients and non-diabetic subjects (4.7 + 0.6 vs 4.0_+ 0.8, p > 0.05), but at 120 rain it was higher in diabetic patients (1,2 + 0.2 vs 0.6 +_0A, p < 0.05). The Fluoroscein ratio serum/saliva was lower in diabetic patients both at 30 min and 120 min (0.6+0.1 vs 1.2+0.3, p<0.05; and 0.5+0.1 vs 1.1 _+0.3, p < 0.02, respectively), indicating that for a given serum level more Fluoroscein is secreted by the parotid in diabetic patients than in non-diabetic subjects. Dividing diabetic patients in two groups according to the presence of retinopathy, as judged by the Fluoroscein leakage in the retina, it was found that Fluoroscein level in saliva at 30 rain was higher in the group with retinopathy, 6.1 _+1.4 vs 3.8 _+0.5, p < 0.05. Conclusions: diabetic patients have a distinctly different pattern of Fluoroscein secretion in saliva compared to non-diabetic subjects. Since in diabetic patients Fluoroscein secretion in saliva parallels Fluoroscein leakage in the retina, Fluoroscein in saliva could be used as index of early diabetic retinopathy.

259. A repetitive DNA sequence used to detect linkage of polymorphic DNA fragments with diabetes in the BB rat W. Kastern and I. Kryspin-Sorensen. Hagedorn Research Laboratory, Gentofte, Denmark Type 1 (insulin-dependent) diabetes in the BB rat is thought to be attributable to at least two genes, one lying within the major histocompatibility complex (MHC) and the other(s) elsewhere in the genome. We have begun a search for the extra-MHC diabetes genes using minisatellite D N A probes. These probes are D N A sequences that are moderately repetitive in the rat genome and which are both interspersed and tandemly repeated. These qualities make them hypervartable, resulting in a greater degree of heterozygosity or polymorphism between individuals. Their repetitive nature and high degree of polymorphism allow a greater portion of the genome to be screened for linkage of polymorphic sequences to diabetes than with single copy gene probes. We have isolated such sequences from the rat genome and have used them to screen the I=2 progeny of crosses between our high incidence of diabetes-inbred BB rat line and other control lines and strains. In a cross between diabetic BB and Long Evans Hooded rats, we detected a polymorphism which is inherited in 5/6 diabetic F2 animals. This association indicated that this probe detected a D N A sequence which lies on the same chromosome in the vicinity of one of the diabetes genes in BB rats.

538 A

Abs~acts

260. Serum laminin P1 concentration in diabetic patients with and without microangiopatbies S. Kawazu, T.Takahashi, T.Watanabe, K. Negishi, M. Suzuki, S. Katayama and J.Ishii. The 4th Department of Internal Medicine, Saitama Medical School, Japan

immunity arises against both soluble and surface antigens of islet cells. Autoimmunity against the soluble antigens tested is rather directed against those from B than from alpha-cells. Neither antibodies to soluble nor to surface antigens of islet cells are associated with deterioration of glucose tolerance in these subjects.

It is well known that thickness of capillary basement membranes or basement membrane-like materials increase in diabetic patients with vascular complications depending on the severity and/or duration of diabetes. Therefore, it seems reasonable to attempt to estimate whether laminin P1 concentration in sera from diabetic patients increase or not, in relation to diabetic microangiopathies, in which basement membranes are known to be accumulated and thickened. RIA-Laminin P1 kit (Hoechst Co.) was used for the determination of serum laminin P1, a non-collagenous glycoprotein of lamina lucida of basement membranes or basement membrane-like materials. Serum laminin P1 concentrations in patients with diabetic nephropathy and/or retinopathy, when diagnosed by the persistent proteinuria using test paper (Albustix R; Ames Co.) or fundoscopy, are significantly higher than those in age-matched patients without diabetic microangiopathies. These findings were independent of the levels of the blood glucose or HbA1 determined at the same time. Moreover, significant relationships are found between serum laminin P1 concentrations and daily excretory rates of urinary protein. Thus, serum laminin P1 could be used as a useful indicator to predict the progression of diabetic microangiopathies.

263. Acid-base effects on ketone body production and lipolysis in man: a mechanism of feedback regulation of ketogenesis U. Keller, V. L. Hood, J. Miles and W. Stauffacher. Division of Endocrinology and Metabolism, University Hospital, Basel, Switzerland To assess whether acute acid-base changes affect ketone body kinetics or plasma nonesterified fatty acids (NEFA), normal subjects were studied during administration of 3 mmol/kg per 2 h NaC1 (n=6); NaHCO3 ( n = 6 ; acidosis I); or NH4C1 ( n = 6 ; acidosis II). Pancreatic hormone concentrations were maintained constant by infusion of somatostatin with insulin and glucagon replacements. Total ketone body production (determined by combined 3-14Cacetoacetate and 1,3-13C-B-hydroxybutyrate infusions) was 4.0_+0.9 lxmol/kg per min in NaC1 controls; it increased after 3 h during alkalosis (pH 7.44+ 0.01) to 6.1 + 1.7 lzmol/kg per min (p < 0.025); it decreased during arginine-HC1 (pH 7.28_+0.01) to 2.8_+0.6 ~mol/kg per min (p<0.02), and during NH4C1 to 3.3+0.8 gmol/kg per min (p<0.02). These changes preceded parallel changes in ketone body utilisation and resulted in altered total ketone body concentrations (205 _+43 during acidosis, 426_+ 82 ~mol/1 during alkalosis, p=0.01). Plasma NEFA were 556_+40 and 899 + 100 limol/1 during acidosis and alkalosis respectively (p < 0.001). Glycerol concentrations paralleled NEFA, indicating acid-base effects on lipolysis. The data demonstrate that modest physiological pH changes affect NEFA availability and ketone body production, suggesting that acid production exerts a feedback inhibitory effect on ketogenesis.

26t. Metformin increases response to insulin in Type I (insulin-dependen0 diabetes H. Keen, A. C. G. Collins and J.J. Bending. Unit for Metabolic Medicine, UMDS (Guy's Campus), London, UK

Biguanides are said to reduce post-prandial insulin requirements in Type 1 (insulin-dependent) diabetes by increasing binding to receptors. We studied 8 C-peptide negative Type 1 diabetic patients receiving constant insulin dosage via continuous subcutaneous insulin infusion. In a randomised double-blind cross-over design, patients received metformin 500 mg or placebo (PB) thrice daily for 3 weeks with 1 week's washout between. Mean 7-point diurnal capillary plasma glucose (heparin fluoride tubes collected at home) was significantly (p<0.05) lower (6.1 _+1.6 mmol/1) during metformin therapy than at baseline (BL) and following PB (7.7 _+1.5 retool/1 and 7.8 + 2.0 retool/1 respectively). Body weight (PB 84_+12 kg; metformin 83 _+12 kg) and insulin dosage (PB 52 + 18 U/24 h; metformin 52 _+ 18 U/24 h) were unchanged. A trend towards more frequent hypoglycaemic episodes was noted (PB 0 episodes; metformin 7 episodes). Fasting venous plasma glucose (metformin 6.1 + 3.0 mmol/1) was not different (BL 7.8 _+2.9 mmol/1; PB 6.9 + 3.1 mmol/1, NS) at hospital assessment but significantly (p< 0.02) lower (9.3 + 3.8 retool/l) 90 min following a standardised mixed breakfast (BL 13.4_+3.6 mmol/1; PB 13.2_+2.4 mmol/1). Mean fasting plasma free insulin concentration (mefformin 26.8+18.4 mU/1) was unchanged (BL 18.6_+8.4 mU/1; PB 22.2+ 15.4 mU/1) but significantly (p<0.01) higher 90 rain after breakfast (metformin 39.8 _+18.0 mU/l vs PB 28.4_+9.3 mU/1). Thus, metformin addition to insulin therapy reduces post-prandial blood glucose rise and the effect appears to be associated with increased insulin concentrations. 262. Autoantibodies against insulin, C-peptide, glucagon and islet cell surface antigens are not related to impaired glucose tolerance in firstdegree relatives of diabetic patients H. Keilacker, I. Rjasanowski, B. Ziegler, K. P. Woltanski, K. D. Kohnert and M.Ziegler. Central Institute of Diabetes "Gerhardt Katsch", Karlsburg, GDR Autoantibodies against soluble and surface antigens of islet cells were determined in 305 first-degree relatives of diabetic patients as well as in 70 control subjects. Insulin (AIA), C-peptide, and glucagon (AGA) autoantibodies were detected by "125I-peptide binding assay and islet cell surface antibodies by immunofluorescence. Excepting AGA, all autoantibodies were significantly more frequent in relatives of diabetic patients than in control subjects. Prevalence of AIA was significantly higher in relatives of Type 2 (non-insulin-dependent) diabetic patients than in those of Type I (insulin-dependent) diabetic patients. Dividing first-degree relatives into subgroups with Type 1 diabetes, impaired, borderline, previously abnormal (but normal at the time of investigation) or normal glucose tolerance, generally there were no significant differences of prevalences of either antibodies among these groups with different severity of disturbance of glucose tolerance. Conclusions: in first-degree relatives of diabetic patients auto-

264. Cardiovascular autonomic neuropathy in diabetes mellitus, in chronic alcoholism and in patients suffering from diabetes and alcoholism simultaneously P. Kempler, A. Vfiradi, I~.Reg6s, B. Veszter, L. Oravecz and I~.Kiss. Ist Clinic of Medicine, Semmelweis University Medical School, Budapest, Hungary

Cardiovascular autonomic neuropathy is a well known complication of diabetes mellitus and of chronic alcoholism (CA). It has not yet been investigated, however, in patients suffering from diabetes and CA simultaneously (DAS). We studied cardiovascular reflexes in 126 Type 1 (insulin-dependent) diabetic patients, in 99 alcoholics, in 17 DAS-patients and in 40 control subjects. Parasympathetic integrity was evaluated by beat-to-beat variation, Valsalva-ratio and standingratio, sympathetic function by blood pressure response to standing and to sustained handgrip test. A significant reduction of all parasympathetic parameters and of sustained handgrip test was found in all patient groups compared to control subjects (p<0.001). In the DASgroup, a significant decrease in beat-to-beat variation (8.41 _+3.59 vs 15.19_+9.89, p<0.01), in standing-ratio (1.0_+0.02 vs 1.05 +0.09, p < 0.05), in Valsalva-ratio (1.33 -+ 0.2 vs 1.49 + 0.32, p < 0.05) and in duration of diabetes (3.7-+3.3 vs 11.5-+9.2, p<0.001) was found compared to nonalcoholic diabetic patients. DAS-patients showed a significant reduction of beat-to-beat variation (8.41 +3.59 vs 11.85+ 6.99, p < 0.05) and of standing-ratio (1.0_+0.02 vs 1.03 + 0.06, p < 0.05) also in comparison with nondiabetic alcoholics. Thus, in patients with both etiological factors of autonomic neuropathy cardiovascular damage is more profound and occurs earlier. 265. Comparison of variability, potency, and kinetics of subcutaneous human proinsulin and human NPH insulin F. Kennedy, M. Mitrakou and J. Gerich. Division of Endocrinology, University of Pittsburgh, Pittsburgh, Penn, USA; Mayo Clinic, Rochester, Minn, USA Human proinsulin (HPI) may be useful in the treatment of diabetes as an intermediate-acting insulin-like preparation. Because intermediate-acting insulins are notoriously variable, we undertook the present study to determine whether HPI was also very variable. The variability of responses to HPI and human NPH insulin were, therefore, compared in 9 normal volunteers. Subjects were injected subcutaneously 3 times with each agent in the periumbical abdominal areas (NPH 0.3 ~/kg, n=9; HPI 0.3 ~/kg, n=5; HPI 0.15 p/gk, n=4) in random order and euglycaemia was maintained for 16 h using the biostator. Variability was assessed as coefficients of variation (CV) for peak glucose infusion rates (PGIF), total glucose infused and time of PGIF. Total glucose infused was 71 g for NPH, 172 g for 0.30 ~/kg HPI and

Abstracts 59 g for HPI 0.15 pJkg. Responses to NPH were biphasic, whereas responses to HPI were monophasic. The CVs were 36 + 2% (NPH) vs 19+5% (HPI) for total glucose infused; 24+5% (NPH) vs 9 + 1 % (HPI) for time of peak; and 33+4% (NPH) vs 14+3% (HPI) for PGIF; all p<0.02. Conclusions: after subcutaneous injection, responses to HPI are much less variable than responses to NPH; a unit of subcutaneous HPI, as currently defined, equals about two units of subcutaneous NPH. 266. Effect of hypoglycaemia on erythrocyte 2,3-diphosphoglycerate in vitro and in insulin-treated diabetics S. Kerenyi and P.Vargha. 1st Medical Department of Semmelweis University Medical School, Budapest, Hungary 2,3-diphosphoglycerate (DPG) is an erythrocyte glycolytic intermediary playing an important role in the regulation of tissue oxygen delivery. We investigated the hypothesis that hypoglycaemia may be associated with hypoxia by its effect on DPG. Blood glucose (BG) and DPG were investigated hourly in nine series on heparinised venous blood samples incubated in parallel at 4 and 37 ~ respectively. At 4 ~ no change in BG or DPG occurred for 8 h whereas at 37 ~ BG decreased during the first hour, and hypoglycaemia was achieved 1 h later. In contrast a significant fall in the DPG occurred only after 6 h of incubation, and the fall in DPG was delayed by 5 h compared to BG. Further on a highly significant positive correlation was found in between the BG decrease and the delayed DPG fall (r=0.424, p < 0.001 ; n = 63). The effect of in vivo hypoglycaemic episodes on DPG levels was investigated in ten insulin-treated diabetic females (age 23-63 years). Mean DPG levels following a hypoglycaemic episode [BG 2.4+0.5 mmol/1 (mean_+SD)] were significantly lower than those found 48-72 h later when no hypoglycaemia was present (DPG 4.85 +0.68 vs 6.27 +0.81 mmol/1 RBC respectively, p<0.001). Thus DPG could be considered as a new parameter signifying transient tissue hypoxia after hypoglycaemic episodes in insulin-treated diabetic patients. 267. Fluorescein leakage through the blood retinal barrier during hyperand normoglycaemia in the lower range in juvenile Type I (insulin-dependent) diabetic patients A. Kernell, L.Jorfelt and J. Ludvigsson. Depts. of Pediatrics and Clinical Physiology, Faculty of Health Sciences, University Hospital, Link6ping, Sweden Is it the hyperglycaemia or the hypoinsulinaemia that is detrimental for the blood retinal barrier (BRB) Type 1 (insulin-dependent) diabetic patients? Aims: To study the function of the BRB during constant hyperglycaemia (220 mmol/1) and normoglycaemia in the lower range (3 mmol/1). Material and Methods. Ten Type 1 diabetic patients aged 20.5 (18.0-22.0) years (median, 95% conf interval) and 12.5 (6.0-15.0) years disease duration and with fasting C-peptide levels 0.02 (0.0-0.13) pmol/1 were examined. They were examined both during hyper- and low normal glycaemia employing clamp technique for 1-5 hours. Leakage through the BRB was demonstrated by vitreous fluorophotometry. Results: the fluorescein leakage through the BRB was significantly higher during hyperglycaemia than during low normoglycaemia (11.0 (3.0-17.0) ng/ml and 5.0 (2.0-8.0) ng/ml; p < 0.05). There was also a significantly lower level of IRI (immunoreactive insulin) in hyperglycaemic compared to low normoglycaemic clamp (31 (18-40) ~xU/ml and 103 (82-120) ~xU/ml; p<0.01) but not in C-peptide level or in HbAac. There was no correlation between fluorescein leakage and IRI at blood glucose 20 nmol/1 or at 3 nmol/1. Conclusion: a near normal blood glucose level seems to be important for an intact BRB. 268. Measurement of subcutaneous glucose concentration in sheep with an amperometric enzyme electrode W. Kerner, H. Zier, G. Steinbach, J. Haas, R. Warhadpande, J. Briickel and E. F. Pfeiffer. Department of Internal Medicine I, University of Ulm, FRG A membrane limited amperometric enzyme electrode was developed. The sensor consists of a central platinum wire (0.03 mm) surrounded by stainless steel tubing (1.0 mm outer diameter). By successive dipcoating procedures, layers from cellulose acetate, glucoseoxidase (crosslinked with glutardialdehyde) and polyurethane are placed on its surface. The platinum wire represents the anode and is polarised at 700 mV against steel. In vitro results: Electrodes are stable for at least 10 days. They exhibit a linear range extending to 25 mmol/1 glucose. Response times (t9e/0) are less than 100 s. The sensors are not dependent on stirring. Dependency of glucose measurement upon

539 A dissolved oxygen is negligible above oxygen concentrations of 0.015 retool/1, in vivo results: in six experiments, sensors were placed into subcutaneous tissue of a sheep. After intravenous injection of 1 mg glucagon, mean (_+ SEM) sensor output increased from 3.4_+ 0.5 to 5.5 +0.9 hA, while mean plasma glucose rose from 3.1 +_0.3 to 6.5 _+0.8 mmol/1. The time delay between both curves was 10-20 min. It is concluded that measurement of glucose concentration in subcutaneous tissue is possible with this sensor. Plasma and tissue glucose curves are nearly parallel. 269. Effects of 1"3 and dexamethasone on the activity of hepatic glucose6-phosphatase in normal, mildly diabetic and severely diabetic rats A.Khan, H.L6w and S.Efendi6. Department of Endocrinology, Karolinska Hospital, Stockholm, Sweden Glucose-6-phosphatase is a microsomal enzyme mainly found in the liver and kidneys. It seems to play an important role in the regulation of glycogen synthesis. In the present study we have determined the activities of the two components of hepatic glucose-6-phosphatase system, i.e. glucose-6-phosphate translocase and glucose-6-phosphate hydrolase in normal, mildly diabetic and severely diabetic rats. Furthermore the effects of 1"3 and dexamethasone were evaluated in these groups of animals. Mild and severe diabetes per se increased the activities of the translocase and hydrolase components of the enzyme. Treatment with T3 or dexamethasone to all these groups of animals increased the activities of both the components of the enzyme as well. In control and mildly diabetic rats blood glucose and plasma insulin levels were uneffected with I"3 treatment, while dexamethasone increased blood glucose and insulin concentrations. In severe diabetic animals none of the hormones altered blood glucose level. Dexamethasone treatment increased insulin concentrations above the low levels noticed in severely diabetic animals but 1"3 was without effect. The latency of the enzyme system was increased in normal and mildly diabetic rats treated with T3. Dexamethasone treatment diminished the latency of the enzyme system in normal and mildly diabetic rats and significantly decreased in severely diabetic rats. We conclude that the action of I"3 and dexamethasone on the activity of glucose-6-phosphatase can explain an increased glucose effiux from the liver. The hormones effectively increased glucose concentration to a level at which glucose phosphorylation can exceed glucose-6-P dephosphorylation. 270. The effect of insulin and IGFt on human osteoblasts in tissue culture M.A.Khokher and P.Dandona. Metabolic Unit, Department of Chemical Pathology & Human Metabolism, Royal Free Hospital and School of Medicine, London, U K The effect of insulin and insulin-like growth factor (IGF0 on alkaline phosphatase production and [3H] thymidine uptake by human osteoblasts was investigated. Human osteoblasts were cultured by the method of Beresford et al. (1983). The cells were plated at 5 x 104cells per well, alone and in various combinations of insulin (1-1000 m U / 1), IGFa (10-80 mg/1); H7 (1-5-isoqunolinylsulfonyl-2-methylpiperazinc, 1-1000 rag/l), a specific protein kinase C inhibitor; verapamil (1-1000 mg/1), a calcium blocker; and amiloride (10-100 mg/1), a N a + / H + exchange inhibitor. Both insulin and IGF1 stimulated alkaline phosphatase production (45-155% over basal) and thymidine incorporation (7-73% over basal). H7, verapamil and amiloride were inhibitory to basal and insulin- or IGFl-stimulated alkaline phosphatase production and [3H] thymidine incorporation. We conclude that (a) insulin and IGF1 stimulate human osteoblasts; (b) this stimulation is mediated by the activation of protein kinase C and calcium influx; and (c) N a + / H + exchange mechanism may also have a role in mediating this stimulatory effect. 27L Macrophage directed immune intervention suppresses the development of experimental diabetes U. Kiesel, S. Epstein and V. Kolb-Bachofen. Diabetes Research Institue, Institute for Biophysics and Electron Microscopy, University of DUsseldorf, FRG Multiple treatment of male C 57 BL/6J mice with low doses of streptozotocin (5 x 40 mg/kg body weight on consecutive days) leads to hyperglycaemia and hypoinsulinaemia. Diabetes development occurs 1- 2 weeks after administration of the B-cell toxin and can be prevented by conventional immunosuppression. Electron microscopy of pancreatic islets was performed shortly before diabetes onset. Lysed B cells were observed in all islets investigated. However, only a few infiltrating immune cells were observed, most of which could be iden-

540 A tiffed as macrophages. Some were found engaged in phagocytosis of B-cell debris. The role of macrophages was studied in immune intervention trials. The daily administration of prostaglandin synthesis inhibitors acetyl salicilic acid (2 x 25 mg/kg) or of BW755C inhibited the development of hyperglycaemia as did the injection of macrophage toxic silica particles (p< 0.05-0.001). In conclusion, functional and morphological data support an essential role of macrophages in islet inflammation and diabetes development.

272. Regulation of LDL receptor activity and cholesterol synthesis by prostaglandins in freshly isolated human mononuclear lenkocytes A.Klass, W.Krone, H.N~igele, B. Behnke and H.Greten. Medizinische Kernklinik und Poliklinik, Universit~its-Krankenhaus Eppendoff, Hamburg, FRG Diabetes mellitus is associated with abnormalities in cholesterol and arachidonic acid metabolism. Furthermore, lipoproteins and prostaglandins play an important role in atherogenesis. To investigate a possible connection between prostaglandins and cholesterol metabolism the effects of PG El, E2. F2a and the stable prostacyclin analogue iloprost on LDL receptor activity and cholesterol biosynthesis were examined in freshly isolated human mononuclear leukocytes. PG Ea (1 gmol/1), PG E2 (1 I.tmol/1) and iloprost (1 lxmol/1) inhibited accumulation of 125I-LDL by 57, 32 and 54%, degradation by 55, 47 and 60% and cholesterol synthesis from 14C-acetate by 70, 62 and 47%, respectively, in cells incubated in lipid-free medium for 20 h. PG F2, had no effect. PG E~ decreased the LDL receptor number without changing its affinity. The PG action was mimicked by db-cAMR The results demonstrate that PG Ea, E2 and iloprost affect LDL receptor activity and cholesterol synthesis. Decreased vascular prostaglandin formation may play a role in impaired cholesterol homeostasis and premature development of atherosclerosis in diabetic patients. 273. Postprandial hyperglycaemia in obesity is an important compensatory mechanism to achieve normal glucose disposal rates R. Klauser, R. Prager, G. Schernthaner and J.M. Olefsky1. Medical Department II, University of Vienna, Austria; Weterans Administr. Hospital, La Jolla, Calif, USA We recently found that postprandial hyperinsulinaemia is not able to compensate the insulin resistance of obese subjects and proposed that postprandial hyperglycaemia might be more important in promoting glucose disposal (Rd) via the mass action of glucose. We performed oral glucose tolerance tests (OGTY) in 6 normal and 6 obese subjects measuring glucose and insulin levels. Afterwards two glucose clamp studies were done. In Study 1 insulin was infused in a dynamic square wave fashion to mimic the individual post OGTT insulin levels at euglycaemic glucose levels. In Study 2 glucose and insulin infusions were varied to mimic post OGTI" levels in each subject. During all studies overall glucose turnover was measured by infusion of 3H3-glucose. During the OGTI" the obese subjects exhibited significantly higher insulin (p< 0.005) and glucose levels (p< 0.03). Rd during the euglycaemic clamp (Study 1) was significantly lower in obese compared to normal subjects (time 50:2.8 vs 4.5, time 110:3.1 vs 5.7, time 170:3.0 vs 5.1 mg/kg, min, p < 0.01) despite higher post OGTF insulin levels in obese subjects. In Study 2 Rd was not significantly different between obese and normal subjects (time 50:5.6 vs 6.2, time 110:4.5 vs 5.4, time 170:3.5 vs 4.8 mg/kg.min; NS). We conclude that postprandial hyperglycaemia in obese subjects is more important to achieve normal Rd via the mass action of glucose than postprandial hyperinsulinaemia. 274. Kinase activity of internalised insulin receptors H. H. Klein 1, S. Matthaei 2, G. Freidenberg and J. M. Olefsky. Veterans Administration Medical Center, San Diego, Calif, USA and 1Department of Medicine, University of Luebeck and 2Department of Medicine, University of Hamburg, FRG Exposure of intact adipocytes to insulin activates the insulin receptor kinase. The kinase activity state of the receptors can be preserved during receptor isolation and subsequently be determined in vitro. We have now applied this technique to investigate whether insulin receptors remain activated kinases following insulin-induced internalisation. Rat adipocytes were incubated with or without insulin and subsequently disrupted to prepare subcellular fractions (PM=plasma membranes, LDM =low density microsomal membranes and HDM = high density microsomal membranes). From these fractions insulin receptors were isolated and insulin receptor number and insulin receptor kinase activity determined. Exposure of the cells to insulin resulted in a loss of approximately 30% of receptors from PM

Abstracts which was completely accounted for by the appearance of receptors in LDM and HDM, indicating that insulin had induced endocytosis of receptors. Measurement of histone kinase activity revealed that exposure of the cells to insulin had resulted in an 8- to 15-fold increase in the kinase activity per receptor in all fractions. Although the absolute degree of activation was slightly lower in LDM and HDM compared to PM, the results demonstrate that the insulin receptor kinase remains (at least partially) active while receptors are translocated to organelles found in LDM and HDM.

275. Insulin degradation by red cells is not directly related to changes in membrane fluidity I.Klimeg, S.Z6rad, E.Svfibovfi and L. Macho. Institute of Experimental Endocrinology, Center of Physiological Sciences, Slovak Academy of Sciences, Bratislava, CSSR Mono-125I-(tyr A14)-insulin degradation (ID) in relation to changes of membrane fluidity (induced by temperature elevation or n-hexane addition) was studied in human erythrocytes using TCA precipitation, gel filtration (GF) and EPR spectroscopy. At 15~ ID in the medium was immeasurable, but it grew during 3 h of incubation at 37 ~ from 0 to 0.41 + 0.02 (expressed as ratio of degraded to non-degraded insulin). Following the addition of n-hexane (76.6 ~tmol per 2 x 109 cells in 500 lxl) at 15 ~ ID increased to 2.5 + 0.1. GF of the medium revealed increased amounts of both low-molecular degradation and high-molecular aggregation products of insulin metabolism at 37~ (70.2%; 8.7%) or at 15 ~ plus n-hexane (89.5%: 3.4% of the recovered radioactivity) as compared to data obtained at 15 ~ (9.2%; 0.9%). GF of acidic extracts of erythrocytes gave similar results. EPR spectroscopy showed a 37 ~ drop of the ordering parameter (0.930+0.004 vs 0.640+ 0.005). At 15 ~ n-hexane induced small decrease of the ordering parameter (0.930+0.004 vs 0.895_+ 0.010, p < 0.01). These data provide new evidence on stimulatory effect of nhexane on ID by erythrocytes. Fluidity changes are probably not essential for the n-hexane action on ID. However, both raised temperature and n-hexane stimulate formation of high-molecular insulin aggregates. 276. Application of immunogens delays or prevents the development of Type I diabetes in BB rats I. K16ting, S. Sadewasser, S. Lucke, L. Vogt, O. Stark I and H.J. Hahn. Central Institute of Diabetes "Gerhardt Katsch" Karlsburg, GDP,, 1Institute of Biology, Department of General Medicine, Charles University, Prague, CSSR Infections in BB rats as macroscopically demonstrated by lung abcesses led to a strong reduction of incidence of Type 1 (insulin-dependent) diabetes (70.6 v 14.9%) suggesting environmental factors influencing the expression of Type 1 diabetes. This prompted us to study the action of exogenous immune stimuli on the development of Type 1 diabetes in inbred BB rats (Fll). 100 ~g crystallised bovine insulin and 0.1 ml tuberculin were peritonially applied two times a week from the 7th until the 20th week of life in three first- and three second-litters of same parents as experimental and control litters, respectively. Body weight, plasma glucose, lymphocytes and glucose tolerance were monitored weekly and monthly, respectively between the 3rd and the 30th week of life. Also, the immune status (graft-versus-host reaction) and the occurrence of insulitis (pancreatic biopsy) were studied after 20 and 30 weeks of age. The insulin application had no effect on the incidence (7/28 vs 7/30) but delayed significantly the age at onset (135 +_6 vs 89 _+5 days). After tuberculin the incidence (2/15 vs 7/22) was reduced but no effect on the age at onset (83 + 25 vs 107 + 10 d) was observed. Only in the tuberculin group the immune status was drastically altered. All others measured exhibited no difference. The findings suggest the influence of environmental factors on the expression of Type 1 diabetes in BB rats. 277. Glucagon is effective on glucose homoeostasis in pancreatectoraised diabetic patients J. Klujko, J. Reiter 1, G. Schwall I and F. H. Schmidt. Research Laboratories, Boehringer Mannheim GmbH, W.Germany, achirurg. Klinik, Klinikum Mannheim, FRG Hard facts about the effect of pancreatic glucagon on hepatic glucose production or its glucose output in healthy humans or diabetic patients are few and far between. Pancreatectomised diabetic patients should provide ideal conditions for the investigation of the glucagon effects on glucose homoeostasis. Six totally pancreatectomised patients (4 F, 2 M) were given in a cross-over test on two consecutive days for 3 h either insulin (I), 50 mU/kg per h or insulin + glucagon,

Abstracts 50 mU/kg per h (I) 1 ttg/kg per h (G) by infusion. I + G was thus administered in equimolar amounts (approximately 0.3 mmol). Blood glucose was monitored by means of a Biostator. In addition to the concentration-time curves of glucose in the blood (HK method), the hepatic glucose production (HGP) and peripheral glucose utilisation (PGU) between min 120-150 were measured by means of a tracer-kinetics analysis. Results: Under I there was a continuous fall in blood glucose from a mean of 14.20 mmol/l to 7.62 mmol/1 (min 180). Under I + G there was a fall from a mean of 14.64mmol/1 to 12.65 mmol/l (rain 180). The values between min 90 180 were significantly different (p< 0.05). The AUC (0-180') for I was 34.430 and for I + G 2.8"lmmol/1.min -1. The HGP values following I + G were 2.87 mg/kg per min, which was about 100% higher than following I alone (1.44 mg/kg per min). In contrast, no significant differences were found for the PGU. Accordingly, glucagon has a clear antagonistic action on insulin-inhibited glucose production or glucose output of the liver. 278. A five year feasibility study of infusion pump treatment: glycaemic control G. Knight, A. M. Jennings, A. J. M. Boulton and J. D. Ward. Royal Hallamshire Hospital, Sheffield, U K In 1982, 382 insulin-treated diabetic patients were offered the use of CSII, intensified conventional treatment (ICT) or a non-intensified regimen. With subsequent follow up in an ordinary clinic 45 (45.5%) of 99 patients continued CSII for 5 years. Glycosylated haemoglobin (HbA1) was measured to compare glycaemic control in the SCIItreated patients with those using injection therapy. Fifty percent of the 382 patients have been reviewed so far. In the CSII group, mean HbA1 (SD) (normal range: 5.8-7.8%) was 9.9% (1.4) at 5 years, compared with 10.8 (1.6) pre-study (p< 0.05). The lowest result obtained was at I year: 9.34 (1.1) (p: NS: year 5 vs year 1; p<0.00"1, year 1 vs pre-study). HbA1 of patients who had used ICT pre-study: 11.0 (1.8); year 1:10.4 (1.6); year 5:11.0 (2.1) (compared with 5 year CSII result, p<0.01). For all injection-treated patients 5 year HbA"1 was 11.1 (2.1) (p< 0.001 compared with CSII). We conclude that approximately 50% of CSII-treated patients chose to continue for 5 years and that the CSII-treated group maintained a significant reduction in mean HbA"1 during this period compared with injection-treated groups. For suitable patients CSII appears to be an acceptable method of obtaining long term improved glycaemic control. 279. Anti-islet activity of mononuclear blood cells from newly-diagnosed Type I (insulin-dependent) diabetic patients S.Knospe, E. Krhler, I. Rjasanowski and D. Michaelis. Central Institute of Diabetes "Gerhardt Katsch", Karlsburg, G D R In a previous study we have found that lymphocytes from newlydiagnosed Type I (insulin-dependent) diabetic patients affect rat islets treated with the patient's own complement-inactivated serum. The islet destruction is caused by ADCC (antibody-dependent cell-mediated cytotoxicity) and was measured by the 51Cr-release assay. Non-T lymphocytes are responsible for the anti-islet cytotoxicity. Other authors have observed an increased CMC (cell-mediated cytotoxicity) with lymphocytes from Type 1 diabetic patients measured by insulin release of incubated rat islets. In this study, ADCC and CMC were compared in the same patients. Also it was investigated whether antiislet ADCC is influenced by lymphocates from healthy probands and whether CMC may be the result of a direct islet cell-lymphocyte interaction or be caused by soluble factors as produced by lymphocates. Lymphocyte-mediated CMC and ADCC are not correlated to each other. Anti-islet ADCC as elicited by lymphocytes and serum from diabetic patients are not influenced by the addition of lymphocytes from healthy probands. Lymphocytes or their culture supernatants provoked comparable insulin release which is not necessarily dependent upon lymphocyte contact to islets. It is concluded that the lymphocyte-mediated insulin release does not properly reflect an anti-islet CMC process. 280. Effects of glucose tolerance and treatment of abnormal tolerance on mortality in MalmShus County, Sweden W.C.Knowler, G.Sartor and B.Scherst~n. Lund University, Dalby and Lund, Sweden; and National Institutes of Health, Phoenix, Ariz, USA Mortality according to glucose tolerance was studied to determine the prognosis of impaired glucose tolerance (IGT). In 1962-65, 228,833 subjects were screened for glycosuria. Of 2477 with glycosuria, 2180 were given 3 0 g / m 2 oral glucose tolerance tests and

541 A grouped according to normal tolerance (NORM, mean 2-h capillary blood glucose=5.0mmol/1), IGT (9.0 mmol/1), or diabetes (D, 16.0 mmol/1) by WHO criteria. Among subjects >_25 years old with NORM, IGT, or D, age-sex-adjusted mortality through 1983 was 39+2, 49+4, and 71+4deaths/1000person-years (_+SD) for all causes (p<0.001 for difference in 3 groups), and 24_+ 2, 25 _+3, and 40_+ 3 for vascular causes (cardiovascular, cerebrovascular, or renal disease) (p< 0.001). Two hundred-six men with abnormal tolerance by local, but not WHO, criteria (mean 2-h glucose=6.7 mmol/1) were randomly assigned to diet with tolbutamide, diet only, or no treatment, which was continued through 1975. Age-adjusted all-cause mortality through 1983 did not differ significantly among treatment groups (34+_9, 52+10, 45_+19), but vascular mortality was 10_+5, 31 _+8, 38 + 19 in those assigned to tolbutamide, diet only, or no treatment (p<0.05). Thus compared with NORM, D had higher all-cause and vascular mortality, and IGT had higher all-cause but similar vascular mortality. Treatment of abnormal glucose tolerance apparently reduced vascular but not total mortality. 281. Restoration of vascular reactivity during the first month of Type 1 (insulin-dependent) diabetes treatment is followed by a gradual deterioration M.Kobbah, T.Tuvemo and U.Ewald. Department of Pediatrics, Uppsala University Hospital, Sweden Disturbances of vascular function precede structural changes in Type I (insulin-dependent) diabetes. We studied prospectively vascular reactivity in newly-diagnosed diabetic children to detect the early functional changes in diabetic angiopathy. Methods: Skin vasodilation in response to hypoxia was evaluated using a transcutaneous Po2 electrode at 37 ~ Trace elements, lipids, lipoprotein and carbohydrate control indices were analysed. Subjects: All children in our county acquiring Type 1 diabetes 1983-84 (n= 38) were studied. Fiftyeight healthy children served as control subjects. Vascular reactivity was examined at diagnosis before insulin treatment and repeated 1, 6, 12 and 24 months later. Results: At admission vascular reactivity was reduced compared to controls (3.0_+0.2 vs 3.9_+0.1 kPa, mean_+ SEM, p<0.001). No correlation to age, sex, puberty, HbAlc, plasma glucose or pH was found. Insulin therapy rapidly restored the metabolic derangements. At one month also vascular reactivity was normalised (3.9 _+0.2). When examined at 6, 12, and 24 months a gradual deterioration of vascular function was noted (3.7_+ 0.1, 3.5 _+_0.2 and 3.0_+ 0.2 respectively. At 6 months a significant reduction of serum magnesium was noted correlating to the decrease in vascular reactivity. Conclusion: Vascular function was impaired at admission. Its normalisation at one month and deterioration in the following years could not be explained by any single metabolic factor. 282. Urine excretion of some prostaglandins and its relationship to the renin-angiotensin-aldosterone system in diabetes mellitus D. Koev, S. Zaharieva and K. Andonova. Institute of Endocrinology and Gerontology, Sofia, Bulgaria Urine excretion of prostaglandins (PG) E2, F2~, 6-keto-PGFl~ and tromboxan B2 (TxB2) in 21 diabetic patients and 25 control subjects was investigated before and after 5 days sodium-free diet and diuretic. Urine excretion of PGE2 was significantly lower in diabetic patients in comparison with control subjects (277 +_65 ng/24 h for Type 1 (insulin-dependent) and 267_+72 ng/24 h for Type 2 (non-insulindependent), versus 562 + 42 ng/24 h and 638-+ 69 ng/24 h for the matched control subjects. After sodium depletion an increase of PGE2 excretion in diabetic patients was established, but it was again lower in comparison with control subjects. PGF2~ and 6-keto-PGFla excretion were similar in both groups. TxB2 excretion was higher in Type 2 diabetic patients. There were no changes of PGF2a, 6-ketoPGF la and TxB2 excretion after sodium depletion. Plasma renin activity was lower in diabetic patients both before and after sodium depletion compared with that of control subjects. Plasma aldosterone was similar in diabetic patients and control subjects. It is concluded that the lower urine excretion of PGE2 in diabetic patients is related to the decreased activity of renin-angiotensin system. One may speculate that the increased plasma renin activity+plasma aldosterone:urine PGE2 ratio is responsible for the higher prevalence of arterial hypertension in the diabetic population. 283. Electrocardiographic abnormalities and retinopathy in relation to serum Ca, Mg, Cu and Zn in diabetic patients A. Kofinis, N. Kalliakmanis, A. Tsomi, N. Zachariou, Ch. Tountas and B.Karamanos. Diabetic Center, Hippokration Hospital, Athens, Greece

542 A

Abstracts

To investigate the possible relation of diabetic complications with the level of certain cations in serum we studied 100 diabetic patients. ECG abnormalities were evaluated by the WHO criteria of the Minnesota Code while retinopathy was assessed by fundoscopy and fundus color photographs. The mean of 5 post-breakfast plasma glucose (PG) and HbA1 measurements over a year was considered as index of the quality of diabetic control. Serum Ca, Mg, Cu and Zn were measured by atomic absorption. Abnormal ECG was found in 20% of the patients who compared to the others were older (68.3 ___1.7 vs 59.4_+ 1.5, p < 0.01), had longer diabetes duration (16.2_+ 3.0 vs 9.8 _+ 0.8, p<0.01), had no different diabetic control (PG: 10.6+0.7 vs 9.7+0.3 mmol/1, p < 0.05; HbAI: 6.9+0.2 vs 6.7_+ 0.2%, p < 0.05), but they had higher Mg (1.11+0.05 vs 1.01 + 0.01 mmol/1, p < 0.05) and Cu (15.1+1.4 vs 12.8+0.2 mmol/1, p<0.01). Retinopathy was present in 44.4% who had longer diabetes duration (14.5___1.2 vs 8.1 + 0.9, p<0.001), worse control (PG: 10.4+0.5 vs 9.2+0.3 mmol/1, p < 0.05), and higher Ca (2.31 + 0.02 vs 2.24 + 0.01 mmol/1, p < 0.05). Conclusions: 1) An association has been found between ECG abnormalities and Mg and Cu serum levels, as well as retinopathy and Ca levels. 2 ) E C G abnormalities are related to age and diabetes duration but not to diabetes control, while retinopathy is related to diabetes duration and control but not to age.

286. Role of vascular permeability in the pathogenesis of Type I (insulin-dependent) diabetes in mice H. Kolb, S. Martin, K. Maruta and V. Burkart. Diabetes-Forschungsinstitut, Diisseldorf, FRG We analysed the role of enhanced capillar permeability in the pathogenesis of Type 1 (insulin-dependent) diabetes in two experimental systems. Multiple low doses of streptozotocin cause the development of insulitis and diabetes in male C57BL/6 mice 7-14 days later. Daily treatment with methysergide (30 mg/kg) or disodiumcromoglycate (DSCG, 20 mg/kg) suppressed diabetes development (blood glucose 8.90 + 0.39 mmol/1 and 9.45 + 0.61 mmol/1, respectively, compared to 15.41 +0.94 mmol/1 in diabetic controls). In a second experimental system we observed that 10-20 l.tU of cytokines interleukin I beta and interferon gamma caused an increase in vascular permeability when injected intracutaneously (p< 0.005). Prior treatment of animals with methysergide (50 mg/kg) or DSCG (20 mg/ml) led to suppression of cytokine-induced permeability increase. The protective effect of antagonists of vasoactive amines in low dose streptozotocin diabetes may be due to counteracting cytokine-enhanced permeability of capillaries. The inhibition of edema formation in pancreatic islets represents a new type of intervention which is effective in at least one animal model of Type 1 diabetes.

284. A monoclonal islet cell antibody competing with autoantibodies in Type 1 (insulin-dependent) diabetic patients for binding sites on B cells K. D. Kohnert, G. Contreas, H. Keilacker, K. F/ilt, B. Ziegler, B. Hehmke and M. Ziegler. Central Institute of Diabetes, Karlsburg, GDR, Hagedom Research Laboratory, Gentofte, Denmark, Department of Pathology, University of Lund, Malmr, Sweden

287. Increased sensitivity, but normal responsiveness of the antilipolytie action of insulin in adipocytes from diabetic rats S.J.Koopmans, J.Bosman, J.K.Radder and H.M.J.Krans. Department of Endocrinology & Metab. Dis., University Hospital Leiden, The Netherlands Isolated epididymal adipocytes from 3-day streptozotocin (65 mg/ kg)-diabetic rats showed increased sensitivity, but decreased responsiveness of insulin-stimulated glucose transport and increased insulin (125I) binding. The aim of this study was to elucidate whether the antilipolytic effect of insulin on isoproterenol (iso)-stimulated lipolysis parallels insulin's action on glucose transport. Isolated (0.5 mg/ml collagenase) epididymal adipocytes from 7 control (body weight+ 300 g) and 5 diabetic Wistar rats (plasma glucose > 20 mmol/1, plasma insulin <3 9U/ml) were incubated in Tris-HC1, Krebs Ringer buffer (pH 7.4, 37 ~ 2% BSA, 5 mmol/1 glucose). Glycerol release by adipocytes was measured 30 min after simultaneous addition of 10 -6 mol/1 iso and increasing amounts of insulin. The effect of insulin on lipolysis was calculated as the difference between iso-stimulated lipolysis in the absence and presence of insulin. Results: 10 -6 mol/1 iso-stimulated lipolysis was similar in adipocytes from control and diabetic rats. In diabetic adipocytes the dose-response curve of insulin's antilipolytic action was shifted to the left (EDs0 controis: 2.1x 10- 10 mol/1, EDs0 diabetics: 10- 11 mol/1; p<0.05). Maximal inhibition of lipolysis, however, was identical. This indicates an increase in insulin sensitivity but no change in insulin responsiveness. We conclude: in contrast to insulin-stimulated glucose transport, there is no postbinding defect in insulin's antilipolytic action in adipocytes from diabetic rats.

Islet cell autoantibodies in Type 1 (insulin-dependent) diabetes may detect different islet autoantigens. The aim was to produce monoclonal antibodies able to block the binding of autoantibodies to islet cells. Monoclonal antibodies (mcAb) were generated by fusing mouse myeloma cells with spleen lymphocytes of Balb/c mice immunised with human or rat islets. Numerous mcAb reactive with the surface of rat islet and islet tumor RIN 5AH-cells were identified by cell-ELISA and immunoperoxidase (IP) staining. In a RIN 5AH-cell surface antibody radioimmunoassay only one mcAb, 29aC6, was specifically displaced by autoantibodies in sera from 10 Type I diabetic patients of recent onset. IP-staining on sections of human, rat, and mouse pancreas revealed reactivity with islet cells, but not with rat liver, kidney, and thyroid. The 29aC6 mcAb mediated complement-dependent cytotoxicity in a 51Cr,release test with neonatal rat islet cells. SDS polyacrylamide gel electrophoresis of purified RIN 5AH-cell membranes followed by immunohlotting suggested that 29aC6 mcAb recognised a Mr39K cell component. These results demonstrate that 1) it is feasible to produce mcAb which block islet autoantibody binding, and 2) that such mcAb should be useful in defining different islet cell autoantigens in Type I diabetes. 285. There is no isotope effect in the disposal of 3-H3-glueose V. A. Koivisto, H. Yki-J~trvinen, J. Kolaczynski and R.A. DeFronzo. Helsinki University Hospital, III Department of Medicine, Helsinki, Finland; Clinical Diabetes and Nutrition Section, NIH, Phoenix, Arizona, and Yale University, School of Medicine, New Haven, Conn, USA Glucose labelled with tritium is commonly used in the clinical studies of glucose metabolism. It has been recently suggested that the use of 3-H3-glucose underestimates the rate of glucose turnover due to an "isotope effect" induced by the label. To examine this possibility, we compared the disposal of unlabelled and 3-H 3-labelled glucose during an euglycaemic hyperinsulinaemia induced by insulin clamp technique in 10 healthy males, aged 32_+ 2 years (mean + SEM). The glucose pool was initially labelled with primed (40 ~Ci) continuous (0.5 IxCi/min) infusion of 3-H3-1abelled glucose for 120 min. Thereafter, plasma insulin was acutely raised and maintained at 79 + 3 mU/1 (CV+1%). During hyperinsulinaemia, basal 3-H3-glucose infusion was discontinued and the steady state level of plasma unlabelled glucose (5.0+0.2 mmol/1, CV 5+2%) and glucose specific activity (116 ___3 dpm/mmol, CV 3 + 1%) Were maintained with a variable infusion of 20% glucose labelled with tritium (303__+15 ~tCi/500 ml). The rate of glucose disposal was virtually identical, when measured with unlabeUed (7.66+0.48mg/kg/min) and labelled (7.58___ 0.44 mg/kg per min) glucose. The correlation coefficient between the two measurements was 0.85, p < 0.001. Thus, during euglycaemic hyperinsulinaemia, 3-H3-tracer has no isotope effect on the disposal of glucose.

288. C-peptide secretion during initial remission in diabetic children K.St.Koprivarova, M.I.Atanasova and O.Koparanova. Clinic of Diabetes and Metabolic Disorders, Research Institute of Pediatrics, Medical Academy, Sofia, Bulgaria In order to investigate C-peptide secretion during initial remission, glucose-glucagon tolerance test (GGTI') has been performed in 15 children with Type 1 (insulin-dependent) diabetes mellitus. The results have been compared with a control group of 34 healthy children and with 12 Type i diabetic children with one year duration of the disease. The data show normal basal C-peptide levels in children with remission phase (1.24_+ 1.4 ng/ml), while those from the 12 diabetic children out of remission are significantly lower (0.6 _+0.5 ng/ml, p < 0.05). Compared with the control group, the glucose stimulated Cpeptide secretion in initial remission is significantly lower (5.58 + 2.75 ng/ml vs 1.945 + 1.75 ng/ml respectively). The other diabetic patients showed similar results. The maximal glucagon stimulated Cpeptide levels in initial remission were significantly lower compared with the control group also (3.3 + 2.6 ng/ml vs 10.93 + 4.66 ng/ml respectively). The glucagon stimulated C-peptide secretion was significantly higher than the glucose stimulated (3.3 __2.6 ng/ml vs 1.945 _+ 1.75 ng/ml). The glucose levels were not influenced from the higher C-peptide levels during the G G T r in the initial remission, which is probably due to serious disturbances in the glucose metabolism. There is no correlation between the initial remission and the family history of diabetes, HLA antigens and titres of Coksackie viruses neutralising antibodies.

Abstracts

289. Left ventricular performance in well-controlled short-term Type 1 (insulin-dependent) diabetes A. Koprowski, A. Rynkiewicz, T.Zdrojewski, S. Horoszek-Maziarz and B.Krupa-Wojciechowska. IIKlinika Chor6b Wewn~trznych Akademia Medyczna, Gdafisk, Poland Simultaneous two-dimensional and targeted m-mode echocardiogram was performed on 19 males with short-term well-controlled Type 1 (insulin-dependent) diabetes (age 26+7 years) and in 20 healthy males (age 25___3 years). Resting heart rate in diabetic patients was 2 =59 min -1 and in normal subjects 63 min -1 (NS). Systolic and diastolic left ventricular (LV) dimensions did not differ between the two groups. Cardiac index was higher in diabetic patients ( 2 =3004 m l / m 2 rain) than in normal subjects (.~ =2575 m l / m 2 min, p<0.05). Mean rate of circumferential fiber shortening was significantly higher in diabetic patients ( 2 = 1.13 circ/sek) in comparison to normal subjects ("~ =1.01 circ/sek, p<0.03). LV mass was increased in diabetic patients to 2 =223 g (in normal subjects .~ =198 g, p < 0.04). Total peripheral resistance was significantly lower in diabetic patients ( 7 = 2009 dynsec cm 5) than in normal subjects ( =2387 dyn sekcm 2, p<0.05). End-systolic wall stress was significantly lower in diabetic patients (~ =65 gcm 2) than in normal subjects ( 2 =77gcm2). In short-term well-controlled Type 1 diabetes there are changes in left ventricular structure and function which are probably due to adaptation to increased peripheral blood flow. 290. Genetic variants of complement factor 3 in Type I (insulin-dependent) and Type 2 (non-insulin-dependent) diabetes mellitus S.Krantz, M. Lober, I. Rjasanowski, D. Michaelis, R. Menzel and F. Stelter. Institute of Biochemistry, E.M.A. University, Greifswald, Central. Institute of Diabetes, Karlsburg, G D R There is a well documented linkage between Type 1 (insulin-dependent) diabetes and the genes within the major histocompatibility complex. However, it is unlikely that genetic predisposition to Type 1 diabetes can be completely accounted for by genes linked to chromosome 6. Previous studies have suggested an association between complement factor 3 (C3) coded for by genes on chromosome 19 and autoimmunological disorders and atherosclerosis. In this study associations of C3 variants with Type I (n = 286), Type 2 (non-insulin-dependent) diabetic patients (n= 255), first grade relatives of Type 1 diabetic patients (n = 95), non-diabetic persons suffering from myocardial infarction (n= 177) compared to a control group (blood donors, n= 512) were investigated. We have also looked for evidence of an association between C3 and HLA DR3 and/or 4 genes in Type 1 diabetic patients and their relatives. Type 1 diabetic patients did not show any differences in the distribution of C3 phenotypes in comparison to the control subjects. Associations of distinct C3 variants with HLA DR3 and/or 4 could not be observed in Type 1 diabetic patients and their relatives. In Type 2 diabetic patients there was a significantly (X2-test) diminished C3 F allelic frequency without an association to atherosclerosis, because patients with coronary heart disease failed to give indications of an association with C3 polymorphism. 29L Effect of porcine 8000 dalton immunoreactive gastric inhibitory polypeptide on the isolated perfused pig pancreas T. Krarup, J. J. Holst, S. Knuhtsen, A.J. Moody and O.V. Nielsen. Hvidore Hospital, Institute of Medical Physiology C, Panum Institute, Novo Research Institute, Department of Surgical Gastroenterology C, Rigshospitalet, Denmark Most GIP antisera measure two IR-GIP forms; one somewhat larger (8 kDa) than the insulinotropic 5 kDA IR-GIE Nothing is known about the biological effect of 8 kDa IR-GIR This form was isolated from an extract of porcine upper small intestine by gel filtration (Sephadex G-50, fine grade) and repeated HPLC, using two antisera which detect the 8 kDa form. In order to determine a physiological concentration of the 8 kDa form, plasma from six normal subjects taken before and 1 h after a 490 calorie test meal was gel filtered and the amount of 8 kDa immunoreactivity calculated. Plasma 8 kDa IRGIP increased from 5 + 1 before to 20 _+3 pmol/1 after the meal. Natural porcine and human 5 kDa IR-GIP and porcine 8 kDa IR-GIP were then perfused, in random order, through the isolated porcine pancreas (n= 3 for each) at a glucose concentration of 7 mmol/l. During the 3 perfusing experiments porcine 5 kDa IR-GIP was 268+3, human 5 kDa IR-GIP 118+4 and porcine 8 kDa IR-GIP 35 + 2 pmol/1 above the basal level. IRI in the effluents was 263 _+9, 127 _ 3 and 91 _+3, respectively, percent of the basal level. No change in glucagon or somatostatin secretion during the perfusions occurred. In conclusion: both porcine and human 5 kDa IR-GIP stimulated in-

543 A sulin secretion, whereas porcine 8 kDa IR-GIP had no such effect. 8 kDa IR-GIP seems not to be an insulinotropic hormone.

292. Ineffectiveness of bromocriptine in suppressing growth hormone secretion in Type I (insulin-dependent) diabetes mellitus J.Krassowski, E.Ros{onowska, J.Kurta, P.Szulc, W.Jeske and S.Zgliczyflski. Department of Endocrinology, Medical Center of Postgraduate Education, Warsaw, Poland Growth hormone (GH) hypersecretion despite hyperglycaemia is a frequent finding in Type I (insulin-dependent) diabetic patients. We have studied the effect of bromocriptine, which suppresses GH in states of GH hypersecretion (acromegaly or excessive growth), on GH profile in Type I diabetic patients. Sixteen normal subjects (mean age 27.1 years) and five nonobese Type 1 diabetic patients (mean age 28.6 years) were studied. Diabetic patients were studied on two occasions: before and on the 4th day of bromocriptine treatment (15 mg daily). Growth hormone was determined at hourly intervals with all the subjects maintaining their usual activity. The data were analysed with Halberg cosinor method. The control group exhibited normal rhythm of GH secretion with mesor value of 2.5 ng/ml and acrophase at 02.13 hours. In diabetic patients no rhythm of GH secretion was observed with mesor of 5.9 ng/ml and acrophase at 00.28 hours. Bromocriptine treatment did not restore GH rhythmicity while mesor was increased to 6.9 ng/ml and acrophase shifted to late afternoon hours (17.45). Blood glucose levels were unaffected by bromocriptine. The results of this study show that Type 1 diabetic patients have no diurnal rhythm of GH secretion and that elevated GH levels are not suppressed during bromocriptine administration. 293. Effect of Miglitol on blood glucose excursions following test meals with slowly- and with rapidly-absorbable carbohydrates J.Krebs, G.Schauberger, L. Niklas and H.Otto. Zentralkrankenhaus Bremen-Nord, Klinikum flir Innere Medizin, Bremen, FRG Miglitol (Bay m 1099) reduces postprandial blood glucose (BG) excursions by delaying absorption of carbohydrates (CH) subsequent to inhibition of intestinal disaccharases. Aim of study was to examine whether this effect is likewise demonstrable after ingestion of rapidlyabsorbable CH (white bread, test meal A) and slowly-absorbable CH (rye bread with high portion of unground or broken grain, test meal B) as well. Ten persons with impaired glucose tolerance had 4 breakfasts each: test meals A and B, both without and with 50 mg Miglitol, respectively. Mean maximal BG rise after test meal A was 92 mg/ml without, 52 mg/ml with Miglitol, after test meal B: 3.48 mmol/l without, 1.66 mmol/1 with Miglitol (p<0.01%). Mean areas under BG increments after test meals with Miglitol were 65.7% in A, 41.9% in B, when areas after meals without Miglitol were taken as 100%. The same test design was applied to 10 Type 2 (non-insulindependent) diabetic patients well controlled with glibenclamide. Again mean maximal BG rises and mean areas under BG increments were reduced by Miglitol, irrespective of whether slowly- or rapidlyabsorbable CH were given (p<0.01%). Conclusion: Miglitol is effective in lowering postprandial BG rises after test meals with rapidlyand with slowly-absorbable CH as well. 294. Increased ketone body clearance during treatment of diabetic ketoacidosis A. J. Krentz, P.J. Hale, B. M. Singh and M. Nattrass. The General Hospital, Steelhouse Lane, Birmingham, U K During the treatment of diabetic ketoacidosis glucose infusion is commenced when blood glucose has fallen to 14 mmol/1. It is unclear whether hypertonic glucose and insulin enhances clearance of ketone bodies. Of 17 patients with diabetic ketoacidosis 9 were infused with 5% dextrose ( + 10 U/1 insulin) and 8 with 10% dextrose ( + 40 U/I insulin) at a rate of 250 ml/h for 6 hours. Blood glucose (mean + SEM) at the start of the infusion was 12.8_+1A (5% group) and 13.7+ 0.9 mmol/1 (10% group) and did not change significantly in either group during the infusion. Total ketone bodies fell from 7.72 _+0.58 to 2.55+0.34 (5% group) and from 8.69_+0.81 to 1.35_+0.54mmol/1 (10% group). The fall was significantly greater in the 10% group (p< 0.05). Blood glycerol was elevated in both groups and rose in the 10% group (0.19 _+0.04 to 0.28 _+0.04 mmol/1, p < 0.05) but was unchanged in the 5% group. Blood alanine levels were low and fell in the 5% group (0.20 + 0.01 to 0.10 + 0.01 mmol/l, p < 0.01). Blood lactate and pyruvate fell in the 5% group and there was no difference in lactate/ pyruvate ratios between groups. The use of 10% dextrose and increased insulin at this stage of treatment significantly enhances the fall in ketone bodies.

544 A 295. Mechanism of regulation of LDL receptor activity by insulin and catecholamines in human mononuclear leukocytes W.Krone, H. N~igele, B. Behnke and H.Greten. Medizinische Kernklinik und Polikiinik, Universit~its-Krankenhaus Eppendorf, Hamburg, FRG The LDL receptor is one determinant of plasma LDL cholesterol levels and is important in atherogenesis. Poorly controlled Type I (insulin-dependent) diabetic patients are characterised by insulin deficiency and catecholamine excess. These hormonal changes may increase L D L levels by affecting the LDL receptor pathway. We studied the mechanisms by which insulin and catecholamines act on LDL receptor activity in human mononuclear leukocytes. Incubation of cells in a lipid-free medium increased LDL binding, accumulation and degradation. Insulin (10 mU/ml) stimulated this induction by 40%, whereas epinephrine (0.1 mmol/1) showed a 60% inhibition. Linearization of LDL-concentration curves revealed that insulin raised and epinephrine lowered the number of LDL receptors without altering binding affinity. The catecholamine effect was mediated by beta2-adrenoceptors since the unspecific beta-agonist isoproterenol, the beta2-specific agonist terbutaline and db-cAMP also suppressed LDL receptor activity and the unspecific beta-antagonist propranolol reversed the epinephrine action. These results demonstrate that insulin stimulates and catecholamines suppress LDL receptor activity. Insulin deficiency and elevated catecholamines may cause increased LDL levels and premature atherosclerosis in Type 1 diabetic patients. 296. Hyperperfusion of the leg in newly diagnosed Type I (insulin-dependent) diabetes mellitus K. Kr6nert, W. Grauer, M. Giinderoth-Palmowski, A. Schuler, C. Zimmermann, D.Luft and M. Eggstein. Med. Univ. Klinik, Abt. Innere Med. IV, Neurol. Univ. Klinik, Tiibingen, FRG Hyperperfusion of retina and kidney have been documented in shortterm diabetes. Differences in tissue oxygen tension may derive from alterations in microcirculation. In order to evaluate whether newly diagnosed Type 1 (insulin-dependent) diabetic patients exhibit abnormal tissue oxygen tensions, 7 newly diagnosed Type 1 diabetic patients and 6 control subjects matched for age and sex were studied: clinical neurological status, motor and sensory nerve conduction velocities, EMG, cardiovascular reflex tests, oxygen tension (pO2) of the anterior tibial muscle of the right leg at rest determined by a needle probe while breathing 1) ambient air, and 2) an oxygen enriched gas (FiO2 = 0.4) for 30 min, capillary pO2 and pCO2. The parameters reflecting peripheral and autonomic nerve integrity did not differ between both groups. While breathing ambient air, the muscle tissue pOe was significantly higher (p= 0.025) in the diabetic patients than in the control subjects. In contrast to the controls, the diabetic patients did not clearly augment muscle tissue pO2 while breathing the oxygen enriched gas, despite of an equal increase in capillary pO2 (2 p < 0.05). The increased oxygen tension of the anterior tibial muscle may indicate an abnormal microcirculation of muscle tissue in newly diagnosed Type 1 diabetes mellitns. 297. Improvement of patient care and reduction of excessive sulphonylurea use in general practice by a structured diabetes teaching and treatment programme in elderly Type 2 (non-insulin-dependent) diabetes mellitus P. Kronsbein, u Joergens, I. Muehlhauser, V. Scholz, A. Venhaus and M. Berger. Med. Dep. for Metabolic Diseases and Nutrition, University of Duesseldorf, FRG In many countries, an excessive use of sulphonylurea drugs is documented, whereas structured diabetes education is widely neglected. The aim of this study was to evaluate a programme for improvement of patient care in Type 2 (non-insulin-dependent) diabetes at the level of the general medical practice. We have developed a diabetes teaching and treatment programme (DTTP) consisting of 4 lessons (90 min each with 1 week intervals, group teaching delivered by paramedical personal); the DTTP was based upon individualised treatment goals, urine testing and primarily non-drug therapy. The effects of the DTTP were prospectively followed over 1 year in 48 Type 2 diabetic patients (mean age 65 years, initially 70% on sulphonylurea drugs) in 5 general practices (intervention group) and compared to 49 comparable patients treated in 3 general practices without the DTI'P (control group). After 1 year, in the intervention group mean body weight had fallen from 76 to 73 kg, non-fasting triglycerides from 3.5 to 2.8 mmol/l; only 35% of patients were on sulphonylurea drugs (each p<0.05). H b A l c was unchanged (7.1 vs 7.0). In contrast, no changes in any of these parameters were observed in the control group during

Abstracts the study period. This DTFP appears of significant value in limiting the excessive use of oral antidiabetic agents in elderly patients with Type 2 diabetes mellitus while various aspects of diabetes care are substantially improved. 298. Morphological changes of the secretion granules in the pancreatic endocrine system in short-term streptozotucin diabetes J. P. Kroustrup and K. Hermansen. University Inst. of Pathology and Sec. University Clinic of Internal Medicine, Arhus Kommunehospital and University of Arhus, Arhus, Denmark Short-term streptozotocin diabetes is associated with abnormal pancreatic A-, B-, and D-cell function. Whether the derangement of hormone secretion is reflected in changes in the morphology of the secretion granules of the hormone producing cells is unknown. To study this, the relative and total volume of secretion granules of the endocrine cells from pancreas in the basal state of short-term streptozotocin diabetic dogs were investigated by means of electron microscopic, stereological methods. We found that the secretion granules of the remaining B cells in streptozotocin diabetic dogs were smaller than normal, constituting a reduced relative volume of the cells. The total volume of the A cells was unchanged while the relative volume and the size of secretion granules were reduced. The total D-cell volume and the relative as well as the total volume of the secretion granules of the somatostatin cells were normal; however, the D-cell granules were found to be slightly enlarged. The morphological changes of the Aand B-cell secretion granules were correlated with the simultaneous levels of plasma glucagon and insulin. Based upon these results, the hypothesis is proposed that an increased function of the pancreatic A- and B-cells has a morphological counterpart in smaller secretion granules. 299. Suppressive effect of T lymphocyte subsets from healthy subjects on B-cell cytotoxicity in Type i (insulin-dependent) diabetes mellitus J. K_rug, E. Lampeter, B. Bierwolf and D. Lohmann. City Hospital of Leipzig, G D R The aim was to investigate which subpopulation of the peripheral blood lymphocytes (PBL) from healthy subjects is responsible for the suppressive effect on B-cell cytotoxicity of PBL from patients with newly diagnosed Type I (insulin-dependent) diabetes. This could be demonstrated previously in an in vitro assay with isolated rat islets using the nonstimulated insulin release as a marker of cytotoxicity. The PBL from healthy subjects were incubated with the monoclonal antibodies BMA030 (CD3), 040 (CD4) and 081 (CD8) (Behring, FRG). After the incubation the lymphocytes were put on goat-anti-mouseIgG coated plastic dishes and incubated for 15 min on ice. The nonadherent cells (negative selection) were harvested and added to PBL from patients with Type 1 diabetes in a ratio of 1 : 4 for evaluation of the suppressive effect of the different preparations. The depletion was controlled by flowcytometric analysis (FACS440). We could demonstrate that the cell preparation, depleted of CD8 + PBL, no longer showed a suppressive activity. The cell preparations, depleted of CD4 __.PBL, were able to suppress B-cell cytotoxicity (n= 5, p < 0.02). Our results indicate that T lymphocytes within the CD8 _+PBL population from healthy subjects are responsible for the suppressive effect on cytotoxicity of PBL from patients with Type 1 diabetes mellitus. 300. B-cell malfunction and the time course of recovery following chronic overinsulinisation of normal rats Y.T.Kruszynska, L.Villa-Komaroff and P.Halban. Joslin Diabetes Center, Boston, Mass, USA Appropriate insulin therapy may preserve or improve B-cell function whereas overinsulinisation may be detrimental. To study this and the time course of recovery, normal rats were overinsulinised for 4 weeks using Alzet mini-pumps (fed blood glucose 2.5-4.5 mmol/1; controls 4.1-7.0 mmol/1). B-cell function was assessed by a 3 h glucose clamp (10.0 mmol/1) performed 1, 48 and 120 h after insulin withdrawal [n=6 in each group; (p<0.001 vs controls)]. One hour after withdrawal, 3 h clamp insulin was 1.6+0.1 vs 9.3+__1.0 lxg/l in controls; area under insulin curve (AUC~) 4.8 + 0.4 vs 20.3 + 2.2 Ixg. 1-1- h. At 48 h, blood glucose was similar to controls in fed rats (7.5 + 0.4 vs 6.1 + 0.5 mmol/1, NS) but increased in fasted rats (5.9_+ 0.3 vs 4.5 + 0.1 retool/l, p<0.01); 3 h clamp insulin was still depressed (3.4+ 0.3 ~g/1) as was AUCins (9.4+l.01xg'l-a'h)- By 120h after withdrawal, blood glucose and clamp insulin responses did not differ from controls. Pancreatic insulin contents were decreased 1 h (6.4 + 0.9 gg/g) and 48 h (53.6+_11.7 Ixg/g) but not 120 h (221.0+_30.1 gg. l/g) after withdrawal (controls, 303.3 ___28.8 l-tg/g), and were strongly

Abstracts correlated with the clamp insulin responses. Thus, overinsulinisation with prolonged periods of low blood glucose concentrations impairs B-cell function, albeit reversibly. We suggest that overinsulinisation should be avoided in patients in whom preservation of residual B-cell function is deemed important. 301. Islet cell antibodies and insulin autoantibodies in first degree relatives of patients with Type I (insulin-dependent) diabetes B. Kuglin and J. Bertrams. Diabetes Research Institute, University of Diisseldorf, Diisseldorf, and Elisabeth-Krankenhaus, Essen, FRG

The presence of cytoplasmatic islet cell antibodies (ICA) and IgG insulin autoantibodies (IgG-IAA) may confer increased risk for diabetes. To obtain further information about the prevalence of these markers in subgroups with known high risk for diabetes, we analysed sera from 1029 first degree relatives (1 ~ Rel)of 483 Type 1 (insulin-dependent) diabetic patients. Islet cell antibodies were present in 33/961=3.4% 1 ~ Rel versus 1/250=0.4% in control subjects (p< 0.05). Islet cell antibody prevalence was similar in parents (12/420=2.9%) and siblings (11/313 =3.5%). However, in children (n= 68) of diabetic patients ICA were not detected. Prevalence of ICA was increased in HLA identical siblings (4/71 = 5.6%) and in HLA DR3/4 + 1 ~ Rel (5/96 = 5.2%), but decreased in HLA DR2/X + 1 ~ Rel (3/167=1.8%). A considerable increase of IgG-IAA was observed in 1 ~ Rel (33/292 = 11.3% versus 2/138 = 1.4% in control subjects, p < 0.001). IgG-IAA were present in 11/124 = 8.9% of parents, in 16/114= 14.0% of siblings and in 0/18 of children. IgG-IAA prevalence was similar in HLA identical (4/26=15.4%), half identical (6/45 =13.3%) and nonidentical (3/24=12.5%) siblings. In conclusion, prevalence of IgG-IAA is higher in siblings than in parents but is not affected by HLA DR type. Prevalence of ICA is similar in siblings and parents but is increased in HLA identical or HLA DR3/4 + 1 ~ relatives. 302. Changes in glycosylated haemoglobin levels after a diabetes tare teaching course are not predicted by questionnaire evaluation of patient's knowledge F. Kuntschen, Ch. Piot, E.Temler, L. Locatelli, M.-O. Ranson~, J. Laure 1 and F. Gomez. Division of Endocrinology, Department of Medicine, and 1Dietetics Unit, University Hospital, Lausanne, Switzerland

Diabetes care teaching programs evaluation based on diabetic knowledge cf patients may not correlate with metabolic changes. We evaluated a 3-day group course in 48 unselected Type 1 (insulin-dependent) and Type 2 (non-insulin-dependent) diabetic patients, using a 50-question multiple choice questionnaire on theoretical and practical aspects of diabetes and compared the results with glycosylated hemoglobin levels (HbAlc, normal range 3.7-5.7%), before and 2 months after the course. The 2-month evaluation could not be performed in 14 patients. The remaining 34 were distributed according to HbA~c changes into 3 groups: (A)= decrease (HbAac 8.8+1.5 vs 6.6+1.2, mean+SD, n=21), (B)=stable (8.7+1.1 vs 8.1• n=8), and (C) = increase (7.0 +_1.2 vs 8.0_+ 1.3, n = 5). Patient knowledge was not significantly different (ANOVA) among the 3 groups, before (number of wrong answers in group A=16.1+9.8, B=16.7+14.9, and C=10A+8.0) or after the course (A=6.5+6.2, B=4.6+1.9, and C = 1.4 + 1.7). Group A included more patients with Type 2 diabetes (A = 72%, B = 24%, and C = 40%), whereas group C included patients with longer duration of diabetes (A=6.1_+6.2, B=3.2+3A and C = 16.7+7.1 years) and significantly more patients with long-term diabetic complications (A=10%, B=0%, and C=60%, p<0.005). These results suggest that improvement of diabetes knowledge scale does not necessarily result in better metabolic control. In addition, diabetic patients with short disease duration take more advantage of group teaching than patients with long-term complications. The latter may require a different educational approach to obtain further benefit in diabetes management. 303. Anti-IL-2 receptor targeted therapy of islet allografts in rats B.Kuttler, A.Dunger, I.Kl6ting, S.Lucke, H.D.Volk, R.v. Baehr, T. Diamantstein and H.J. Hahn. Central Institute of Diabetes "Gerhardt Katsch', Karlsburg, GDR The clonal expansion of activated T-cells depends on the expression of interleukin-2 receptor (IL-2R) which binds the T-cell growth factor interleukin-2 (IL-2). Consequently, the treatment of graft recipients with anti-interleukin-2 receptor antibodies (ART-18) leads to prolongation of heart and islet allograft survival. In the present study we investigated the efficiency of different doses and various immunoglobu-

545 A lin subclasses of ART-18 on pancreatic islet allograft survival. If pancreatic islets obtained from LEW.1A rats (RTla) were grafted under kidney capsules of streptozotocin (50mg/kg bw) diabetic LEWAW rats (RTIu), the recipients relapsed into hyperglycaemia within 11 days. Treatment of the recipients with 100 ~tg/kg or 300 ~tg/ kg ART-18 (IgG1 or IgG2b) for 10 days together with 2.5 mg/kg Cyclosporin-A (CsA) prevented allograft rejection during the treatment period, whereas the treatment of recipients with 1000 ~Lg/kg ART-18 plus CsA for 10 days markedly prolonged allograft acceptance, partly up to permanent survival ( > 120 days). The results underline the pivotal role of sensitised T-cells in allograft rejection, which could be blocked by this form of treatment. 304. Clinical, immunological and genetic profiles in 150 Type 1 (insulindependent) diabetic patients with extra-pancreatic auto-immunity C.Laborie 1, C.Lhomme 1, C.Boitard 2, P.Vexiau3, J.C.Homberg 4, J. Hors 3, J. F. Bach 2 and R.Assan 1. 1Bichat, 2Necker, 3Saint-Louis and 4Saint-Antoine Hospitals, Paris, France Early detection of autoimmunity in individuals and families at risk of diabetes is clinically relevant. We compared 150 Type l b patients (with clinical extra-pancreatic auto-immunity; EAI) with 108 control subjects and 261 Type l a (without EAI) patients. Associated EAI were endocrinopathies (80), vitiligo (35), chronic active hepatitis (CAH, 6), pernicious anaemia (7), pemphigus (5), other (17). Feminine predominance (100/150), late onset of diabetes (mean age: 40 + 4 years) and delayed onset of insulin-dependency (m = 3 years) prevailed in Type 1 b diabetics. Clinical onsets of diabetes and EAI were time-related (y=0.88x +6.14; r=0.83) although not concomitant. Diabetes preceded EAI in most cases (49%). The patient' lamphocytes blocked the insulin release from normal islets in vitro in 87% (Type 1 a: 62%), sera did so in 51% (Type l a : 50%). The insulin suppressive potency was associated with immunoglobulins and could be diluted out. Most Type 1 b sera also blocked the arginine-stimulated glucagon release, not that of somatostatin. Islet cell antibodies were detected in 32% of Type l b patients, irrespective of the duration of the disease. Other tissue antibodies were detected in 45% (thyroid microsomes), 29% (stomach) and 6% (adrenal) of Type 1 b sera. The genetic marker HLAB8 prevailed: 36% (controls: 18%; Type 1 a: 22% p<0.01 and p<0.02). DR3 was positive in 49% (controls: 21%; T y p e l a : 53%); DR4 in 34% (controls: 13%; T y p e l a : 62%); DR3/DR4 in 13% (controls: zero; Type l a : 34%). A familial history of diabetes was present in 39, of EAI in 20. A particular clinical, immunologic and genetic profile can help to trace early the subjects with a strong tendency to auto-immunity and diabetes. 305. Posthypoglycaemic insulin resistance is caused by at least two distinct mechanisms I. Lager, J. Fowelin, S. Attvall, H. von Schenck and U. Smith. Department of Medicine II, University of Gothenburg, and Department of Clinical Chemistry, University of Link6ping, Sweden The effect of insulin on glucose turnover was studied before and at different times after an insulin-induced hypoglycaemia with the euglycaemic clamp technique (insulin infusion 0.5 mU/kg per min). Two groups of 7 healthy subjects were studied before and 1-3 h or 6-8 h after the hypoglycaemia. Glucose turnover was evaluated with 3-3H-glucose. Each subject was studied three times: during placebo or propranolol infusion and in a control study where hypoglycaemia was prevented by increased glucose infusion. Glucose counterregulation was similar during placebo and propranolol (nadir 1.9• 0.2 mmol/1). The insulin effect (glucose infusion rate) was significantly impaired both 1-3 h (40+_9%, p<0.05) and 6-8 h (27+6%, p < 0.02) after the hypoglycaemia in the placebo group when compared to the control study. The posthypoglycaemic insulin resistance was seen on both inhibition of hepatic glucose production (Ra) and stimulation of peripheral glucose utilisation (Rd). Propranolol completely prevented the early (1-3 h) posthypoglycaemic insulin resistance but was without effect on the late phase 6-8 h). Thus, hypoglycaemia is followed by a prolonged period (at least 8 h) of insulin resistance. The early phase is completely accounted for by fl-adrenergic stimulation while the late phase is due to other, as yet unclear mechanisms. 306. Suppression of lymphocyte mediated anti-B cell cytotoxicity in Type I (insulin-dependent) diabetes by allogeneic lymphocytes in vitro and in vivo E. Lampeter, J. Krug, B. Bierwolf, H.-J. Verlohren, U. Nietzschmann, M. Borte, R. Schille, B. Haustein and D. Lohmann. City Hospital of Leipzig, GDR

546 A Incubation of isolated islets with mononuclear blood cells from Type 1 (insulin-dependent) diabetic patients result in a cytotoxic insulin release. The addition of lymphocytes from healthy subjects could inhibit this effect in vitro. Likewise the transfusion of lymphocytes entails a decrease of the anti-B cell cytotoxicity in the diabetic recipient. Frequency and correlation of the in vitro and in vivo results should be evaluated. The in vitro addition of lymphocytes from non-related healthy subjects diminished the cytotoxic insulin release significantly in 13 of 15 cases but in only 15 of 26 cases using lymphocytes from first degree relatives. The transfusion of lymphocytes from relatives in donor/recipient pairs with preceding positive in vitro suppression was effective in 12 of 15 cases. If no suppression was induced by relatives in vitro, also no effect could be observed in vivo (3 cases). The transfusion was also without suppressive effect in 3 cases of non-related donors despite significant in vitro suppression. The different efficiencies of lymphocytes from relatives and non-relatives in vitro and in vivo seem to reflect a different importance of HLA-mismatch and diabetes-related genetic background in both systems. However, in relatives with suppressive effect in vitro a suppression could be expected also after transfusion. 307. Islet cell antibodies in newly-diagnosed Type I (insulin-dependent) diabetic children - a case-reference study in Sweden during one year M. Landin O1sson, L. Blom, G. Dahlqvist, A. Lernmark and G. Sundkvist. Dept. of Medicine, University of Lund, Malm6 General Hospital, Malmr, Sweden and Dept. of Pediatrics, Sachs' Children Hospital, Stockholm, Sweden Each year nearly 400 Swedish children (0-14 years) develop Type 1 (insulin-dependent) diabetes. The prevalence, 0.2%, is one of the highest known. Autoimmune phenomena, including islet cell antibodies (ICA), are connected to the development of the disease. The aim of this study was to determine the prevalence and titres of ICA in a total population of Type 1 diabetic patients. Sera were collected from all Swedish children developing Type i diabetes during one year. Serum samples were also obtained from age-, sex- and geographically-matched reference subjects. Islet cell antibodies were analysed with a prolonged two-colour immunofluorescence assay. The prevalence of ICA among the Type I diabetic patients was 318/392 (81%). The median ICA titre was 1:128 (range 1:2-1:260,000). Neither the prevalence nor the ICA titres correlated to age, sex, month of birth, month of onset or geographical location of the Type I diabetic children. In the reference group 10/327 (3.0%) of the children were found ICA positive (median titre 1 : 64, range 1 : 64-1 : 16,000) and one developed Type 1 diabetes within one year. In conclusion, ICA was uniformly present in this total population of Type I diabetic children. The prevalence of ICA among the reference subjects was higher than the prevalence of Type I diabetes in this age group. 308. Newest spectrometric capabilities in the characterisation of brownproducts A. Lapolla, T. Poli, D. Fedele, G. Crepaldi, B. Pelli1, A. Sturaro ~ and P.Traldi 1. Istituto di Medicina Interna, Patologia Medica I ~ Policl. Universitario Padova, and 1CNR-Area di Ricerca, Padova, Italy The building up of brown-products on long-life proteins could be an important factor in determining long-term diabetic complications. A brown product 2-(2-furoyl)-4(5)-(2-furanyl)-lH-imidazole (FFI) was identified on acid hydrolysis product of brown-albumin (B-albumin) and brown-polylysine (B-polylysine). We developed an easier method to identify FFI in complex matrices. Brown-products were obtained incubating polylysine (1 g) and albumin (1 g), respectively, with glucose (50 g) for 28 days at 37 ~ After dialysis, lyophilisation, hydrolysis (HC1 6M for 10 h, at 110~ neutralisation (NaOH), evaporation, the samples were then analysed by 70 eV El and collisional spectrometry. Pure FFI was used as model compound. Its detection limit was found of 100 pg. The 70 eV EI mass spectrum of B-polylysine and B-albumin show peaks at every mass value up to m / r 300. Selection and collision of ions at m / r 228 from B-albumin and B-polylysine lead to a daughter spectra which perfectly matches that of M + of FII. Thus without any sample pretreatment or chromatographic separation it was easy to substantiate the presence of FFI in hydrolysed brown-products. 309. The effect of low Cyclosporin doses on rat pancreatic B cells in vivo R.Laube, S.Lucke, I.K16ting, W.Besch, R.Warzok and H.J.Hahn. Central Institute of Diabetes, Karlsburg, Department of Pathology, University Greifswald, G D R

Abstracts Organ or tissue transplantation across major histocompatibility bartiers demands chronic immunosuppression for which Cyclosporin (CS-A) is widely used. In rats the application of CS-A doses > 20 mg/kg b.w. daily leads to glucose intolerance and hyperglycaemia < 1 week of treatment. We investigated the daily application of 1.25, 2.5 or 5.0 mg CS-A/kg b.w. over 12 weeks on body weight, plasma glucose, glucose tolerance, pancreatic insulin content, pancreatic Bcell morphology, bilirubin, creatinine, liver and kidney morphology, and insulin secretion of isolated islets in vitro. By the doses used neither the liver nor the kidney were altered. The treatment of rats with 1.25 mg CS-A/kg b.w. resulted in a serum CS-A concentration of 100 mg/ml, which did neither prolong the islet allograft acceptance nor modify the parameters investigated. CS-A serum concentrations of 200 mg/ml resulted in an impairment of glucose tolerance from 6th week onwards, whereas animals treated with 5.0 mg CS-A/kg b.w. had developed a glucose intolerance within 4 weeks accompanied by a decreased pancreatic insulin content and pancreatic B-cell volume density. The results demonstrated the toxicity of CS-A on pancreatic B-cells in rats with serum concentrations recommended for organ or tissue transplantation.

310. Brockmann bodies for islet transplantation with special respect to tolerance of mammalian temperature and regulation of insulin secretion C. H. Laue, J.Schrezenmeir, J. Boddin, D.Gtbel, W. Stiirmer, M. Miintefering, U. Krause and J. Beyer. Dep. of Endocrinology and Metabolism, Medical School, Univ. of Mainz, FRG

The unique anatomical situation of certain teleost fishes may solve availability problems of islet transplantation. Formerly we have shown that survival of Brockmann Bodies (BB's) in long-term culture is limited by cultivation temperature (35 ~ By using 1) Trichogaster microlepis and 2)Osphromenus gourami, we investigated tropical species adapted to higher temperatures. Glucagon secretion of (1) after 1 day at 37 ~ cultivation temperature was 1098 + 405 pg/ml and was maintained for 14 days (895 _+298 pg/ml). BB's of (2) even survive for 28 days at 37~ increasing their secretion capacity up to 110% of the starting levels (351+92 after 1 day, 613_+232 after 14 days and 386 + 149 pg/ml after 28 days). Studies in regulation of hormone secretion show a glucose-dependent stimulation of insulin and inhibition of glucagon release: insulin (l.tU/24 h): 364-+170 at 1.66 mmol/1, 514-+ 323 at 3.31 mmol/1, 729 + 570 at 4.98 mmol/1 glucose; glucagon (pg/ml): 412+353 at 1.66mmol/1, 234+163 at 3.31 mmol/1, 153 + 118 at 4.98 mmol/1 glucose, (n = 27). These results show the long-term function at mammalian temperature and the glucose-controlled insulin secretion of BB's, which is most important with regard to their clinical application.

311. Long term regulation by insulin of glucose transporter concentration in membranes of differentiating 3T3-F442A adipose cells M. Lavau, M. Guerre-Millo, B. Hainque, N. Moustaid and k Wardzala. INSERM, Institut Biomedical des Cordeliers, Paris, France

Recent studies have suggested a role for chronic hyperinsulinaemia in the increase in the number of adipocyte glucose transporters (GT). We have addressed this question by using a cultured cell system: the 3T3-F442A preadipocytes. Cells grown to confluence were differentiated in DMEM containing 10% FCS and 20 retool/1 glucose, under 3 different conditions: 1)no insulin ( A - ) ; 2)10nmol/1 insulin ( A + ) ; 3)insulin deprivation for 24hours before the experiment (A + - ) . At day 15 after confluence, the concentration of GT in plasma membranes and low density microsomes, assessed by cytochalasin B binding, and 2 deoxyglucose uptake in intact cells, were measured. GT concentration in plasma membranes was 63 vs 18 pmol/ mg protein (p< 0.05; 5 experiments) in ( A + ) vs ( A - ) , paralleled by a 4-fold increased 2 deoxyglucose uptake. GT concentration in low density microsomes was also higher in ( A + ) than in ( A - ) : 29 vs 18 pmol/mg protein (p<0.05). The KD for cytochalasin B binding was not affected by insulin. The deprivation of insulin for the last 24 hours ( A + - ) changed very significantly GT concentration in both plasma (40 pmol/mg protein) and microsomal (51 pmol/mg protein) membranes. This reverse GT translocation indicates that in 3T3-F442A adipose ceils, as in human and rat adipocytes, GT partitioning between plasma and intracellular membranes is modulated by insulin. The present data clearly demonstrate that chronic exposure of differentiating 3T3-F442A cells to insulin led to marked increase in GT concentration, supporting a direct long term regulatory role of this hormone on GT synthesis.

Abstracts 312. Taste impairment in Type I (insulin-dependent) diabetes mellitus: relationship with peripheral neuropathy J. P. Le Floch, L. Perlemuter, G. Le Lievre, J. Sadoun, R. Peynegre and J. Hazard. Department of Diabetology and Department of Otolaryngology, CHU Henri Mondor, Creteil, France In order to specify their taste disorders, 57 diabetic patients (mean follow-up ll.4 _+0.9 years) and 38control subjects were studied. None of them had diseases or took drugs known to impair taste. Tobacco and alcohol consumption were recorded. Taste was studied using electrogustometry. HbAlc, retinopathy, nephropathy, peripheral and autonomic neuropathy were studied in the diabetic group. Data (mean_+ SEM) were compared with Student's t test or chi-square. Correlations were analysed using a linear regression and multiple correlations using a stepwise method. Diabetic patients and control subjects were not different for age, sex, blood pressure, tobacco and alcohol consumption. The mean electrogustometric threshold was 184.3 _+15.8 ~tA for diabetic patients vs 58.7 +9.2 for control subjects (p < 0.001). In the whole population, the electrogustometrlc threshold was correlated with age (r=0.23, p<0.05) and tobacco (r=0.25, p < 0.02). The stepwise multiple regression found 3 predictors of the threshold: diabetes mellitus, tobacco and alcohol (R2=0.42). In the diabetic group, the electrogustometric threshold was higher in case of peripheral neuropathy (225.2+21.3 ~tA vs 135.6_+20.0, p<0.01). It was correlated with age (r= 0.37, p < 0.01) and age of diabetes mellitus (r=0.35, p<0.01). No significant correlation was found with HbAlc. The multiple regression found the peripheral neuropathy as the only one significant predictor (R2=0.18). Taste impairment in Type1 (insulin-dependent) diabetes mellitus is associated with peripheral neuropathy. 313. Weight and waist/hip ratio in relation to glucose tolerance in pregnancy M. E. J. Lean, F. Sutherland and H. W. Sutherland. Diabetic Clinic and Combined Antenatal Diabetic Clinic, Aberdeen Royal Infirmary, Scotland, U K This study aimed to relate three factors - glucose tolerance in pregnancy, non-pregnant body mass index (BMI) and circumferential waist/hip ratio (WHR) - which may all predict the later development of diabetes. One-hundred-one randomly selected pregnant women, aged 27+4years, had a standard 75 g glucose tolerance test at 26-28 weeks gestation. Body weight (62 + 11 kg), BMI (24 + 4 kg/m 2) and WHR (0.92+0.05; umbilicus and anterior superior iliac spine) were measured 30_+ 5 weeks after delivery. Fasting plasma glucose correlated with body weight (pregnant p=0.02; nonpregnant p=0.014) and with BM! (p=0.008 and 0.004), but was not related to WHR, although WHR was significantly correlated with body weight and BMI (p<0.002). Waist/hip ratio but not body weight or BMI, was significantly correlated (p<0.05) with plasma glucose at 60 m and 90 m. After controlling for the effects of BMI, age and height, WHR showed an independent correlation (p< 0.05) with the incremental area under the blood glucose curves. Body mass index also correlated significantly with birthweight and WHR with gestational age at delivery. In conclusion, fatness (BMI) appears to determine fasting plasma glucose, but fat distribution (WHR), and not body weight or BMI, is related to indices of carbohydrate tolerance in normal pregnancy. 314. Incidence of acute metabolic episodes in Type t (insulin-dependent) diabetic patients treated by continuous subcutaneous insulin infusion or by conventional insulin treatment H. Leblanc, B. Billault, A.Thote, M. Marre and Ph. Passa. Service de Diab6tologie, H6pital Saint Louis, Paris, France Results are discordant concerning the incidence of hypoglycaemic and ketotic episodes in Type I (insulin-dependent) diabetic patients treated by continuous subcutaneous insulin infusion (CSII) compared to conventional insulin treatment (CIT). This study was designed to compare the incidence of these acute metabolic episodes in two populations of Type I diabetic patients followed in our clinic during 15months, one treated by CSII (n=42) and the other by CIT (n=468). The mean ages of patients on CSII and CIT were 38.6+ 13.3 and 49.0 + 16.7 years, respectively, (p< 0.002), durations of diabetes were 17.4_+ 8.3 and 14.0 + 9.8 years, (p< 0.05). Frequency of symptomatic hypoglycaemias, ketonurias and hypoglycaemic comas during the month preceding every consultation was checked. The monthly incidence of ketonuria was not significantly different during CSII and CIT. The incidence of symptomatic hypoglycaemias was higher in the patients on CSII. There was no significant difference in

547 A the frequence of hypoglycaemic coma during CSII and CIT (0.024 + 0.16 and 0.027-+0.13). Mean glycosylated haemoglobin was lower during CSII compared to CIT (7.8+0.1 and 8.8_+1.8, p<0.005). In the conditions of this study, the risk of ketonuria and hypoglycaemic coma was not higher with CSII; however, symptomatic hypoglycaemias were more frequent during CSII. Careful instruction and supervision of the pump-treated Type ] diabetic patient is still mandatory. 315. Encapsulation of a human insulin gene containing plasmid into liposomes I. Leibiger, D. Sarrach, R. Walther and H. Zfihlke. Department of Biochemistry, University of Greifswald, G D R One possibility of transfering genetic material (DNA) into mammalian cells is the use of liposomes as a cartier system. Reverse-phase evaporated vesicles (REV) are most suitable. Using ultrasonification, REV's of different sizes can be achieved, although the problem of DNA-fragmentation arises. We studied the influence of sonification time on the structure of a recombinant DNA-plasmid (5.0 kb) containing the human insulin gene during REV formation. Reverse-phase evaporated vesicles were produced using egg lecithin, phosphatic acid, and cholesterol (2:1:l/tool:tool) and varying the sonification time. Liposomes were separated from nonencapsulated D N A by isopycnic ultracentrifugation using a Ficoll-gradient. D N A was extracted from liposomes with chloroform: methanol (2:1/v:v), and precipitated by isopropanol. The structural integrity of the genetic material was examined by electrophoresis on a one percent agarose gel. Sonification for one minute showed no significant structural changes of the encapsulated plasmid-DNA, while certain amounts of fragments were detectable in the nonencapsulated fraction. Prolonged sonification periods led to a greater extent of fragmentation, both in encapsulated plasmid as well as in nonencapsulated DNA. The liposome fraction obtained is now under investigation to test its usefulness for the transfer of the insulin gene into mammalian cells. 316. A clinical trial of BAYmI099 in Type 2 (non-insulin-dependent) diabetic patients with secondary drug failure P. Leigh 1, V. Fonseca, J. Weerakoon and P. Dandona. Royal Free Hospital and School of Medicine, London, NW3, and 1Bayer U.K. Ltd., Pharmaceutical Division, Newbury, Berks, U K We carried out a double-blind, placebo-controlled trial of BAYm1099 (an alpha-glucosidase inhibitor) in 11 patients with Type 2 (non-insulin-dependent) diabetes who were poorly controlled (HbAl>9%; postprandial blood glucose > 10 mmol/1) on diet and maximal doses of oral hypoglycaemic agents (glibenclamide 20 mg daily and metformin 1.5 g daily). Patients continued with their usual medication and, in addition, had either BAYm1099 (50 mg tds) or placebo for 4 weeks. After a 2-week washout period, they were 'crossed over' to the other therapy. Before and after each treatment period we measured HbAa and blood glucose before and 20, 60, 120 and 240 min after a standard breakfast and lunch. Blood glucose was significantly lower on BAYm1099 when compared with placebo at 60 min after breakfast (10.6+0.91 vs 13.6+1.11 mmol/1, p<0.01), 60min after lunch (8.2_+0.59 vs 11.2+1.38 mmol/l, p<0.03), and 120 min after lunch (7.7 _+1.45 vs 10.0 ___0.90 mmol/1, p < 0.03). The HbA1 (mean +__ SD) fell from 10.8_+2.3% to 9.75+1.9% after BAYm1099, and to 10.33 -+2.1% after placebo (p< 0.04 BAYm1099 vs pre-treatment and, p<0.05 vs placebo). Only one patient suffered mild flatulence. We conclude that BAYm1099 is effective in controlling postprandial hyperglycaemia in patients with Type 2 diabetes and secondary drug failure. It may be used in conjunction with sulphonylureas and metformin. 317. A point of no return in chronic symptomatic sensory diabetic polyueuropatby (cssDN)? H. H. P. J. Lemkes, J. M. A. van Gerven and R.J.H.M. Arts. Depts. of Endocrinology and Metab. Dis. and Clinical Electroneurophysiology, Univ. Hospital Leyden, The Netherlands The effect of glycaemic control on cssDN is controversial. We examined the relationships between HbA1 and nerve function in 38 patients with cssDN, stratified according to severity of cssDN. Stratification was based on nerve excitability during measurements of nerve conduction velocity (NCV) - I: median and sural nerve excitable (n= 12, least severe); II: sural nerve not excitable (n= 14); III: median nor sural nerve excitable (n= 12, most severe). Groups were similar in HbA1, age, and diabetes- or cssDN-duration. Besides NCV, Vibration thresholds (VTs) were assessed by Biothesiometry at 3 loci. Central Conduction Time (CCT) was calculated from Somato Senso-

548 A ry Spinal Evoked Potentials (N14-N20-interval). Central conduction time was prolonged in Group I (6.38 _+0.54 ms, p < 0.05), but not in Groups II (5.97_+0.75ms) or III (5.99+0.65ms) (normal: 5.70_+ 0.56 ms). In Group I, HbA1 correlated significantly with - median NCV: r = - 0 . 8 2 , p<0.002; sural NCV: r=-0.67, p<0.02; CCT: r=0.57, p<0.05; VTs: r=0.36 to 0.52, NS (cf. Groups II and III: r = - 0 . 2 3 to 0.11). In Groups II and III, no significant correlations were found between HbA1 and any of these nervous parameters. These data suggest that, once excitability of a single nerve disappears in cssDN, relationships are lost between glycaemic control and impairment of the remaining nervous parameters. In less severe stages of cssDN, abnormalities of several central and peripheral nervous parameters correlate with glycaemic control. Stratification for severity is important for prospective trials of cssDN. 318. Alloxan inhibits glucokinase in pancreatic B cells S. Lenzen, M. Tiedge, F.-H. Brand and S. Freytag. Institute of Pharmacology and Toxicology, University of Grttingen, FRG

Glucokinase in B cells is the glucose recognition enzyme and plays a central role in the signal generation for insulin secretion. The inhibition of insulin secretion by alloxan is intimately related to the mechanism of glucose-induced insulin secretion. Alloxan inhibited glucokinase but not hexokinase activity in cytoplasmic fractions of pancreatic B cells from ob/ob mice. The half maximal inhibitory concentration was 5 gmol/1. Glucokinase was protected from inhibition by alloxan by D-glucose and D-mannose and the inhibitory sugar Dmannoheptulose. L-glucose, D-fructose, and D-galactose did not protect. 3-O-methyl-D-glucose provided protection only when whole cells were incubated with alloxan. Dithiothreitol also reversed the inhibitory action of alloxan. Thus, glucose provides protection of glucokinase against inhibition by alloxan through competition with alloxan at a SH-reactive binding site of the enzyme, while 3-O-methyl-D-glucose protects through inhibition of alloxan uptake into the cell. 319. Glycation and increased function of platelets exposed to high glucose concentrations in vitro S. Lenzi, T. Sampietro, W. Bernini and P. Cecchetti. C.N.R. Institute of Clinical Physiology, Pisa, Italy

Increased platelet functions are well documented in hyperglycaemic state. We have previously shown that human platelet membrane proteins (PMP) undergo glycation in vitro and that PMP from diabetic patients are excessively glycated compared to non-diabetic subjects. In order to demonstrate the causal relation between PMP glycation and increased platelet functions, platelets obtained from healthy volunteers were incubated on physiologic and high glucose (20-80 mmol/1) concentrations for 6 h. After this time the platelets are viable; the PMP from platelets exposed to high glucose concentration showed glycation 2-fold higher than PMP from platelets in physiologic conditions. The glycation was measured as 5-HMF liberated after acid hydrolysis. Its value was 0.19 + 0.05 nmol/mg of proteins for physiologic incubations, 0.36 + 0.08 nmol/mg of proteins for high glucose incubations. The response to thrombin, measured as mean height of aggregation curve, was significantly higher for platelets exposed to high glucose concentrations (t paired test: p<0.02). Since glucose addition immediately or 10-30 rain before thrombin stimulation had no effect on aggregation response, glucose does not act per se. We suggest that the irreversible modification of platelet proteins by glycation is the primitive molecular alteration leading to platelet disfunctions observed in diabetes mellitus. 320. The effect of xamoterol on lipolysis and fatty acid esterification in isolated human adipocytes P. J. Leslie, L. Wilson, E. T. Young and D. G. Johnston. Ashington Hospital, Northumberland and University of Newcastle upon Tyne, U K

Xamoterot (XAM), a fl~ partial agonist used for the treatment of cardiac failure, may also have potential use as an anti-obesity agent. This study has investigated the effect of XAM on fatty acid mobilisation and its influence on noradrenaline (NA) induced lipolysis and fatty acid esterification in isolated human adipocytes obtained during elective surgery. Lipolysis was assessed by glycerol release and fatty acid esterification by the non-isotopic method (NEFA:glycerol ratio). Xamoterol significantly increased lipolysis above basal in a dose-dependent manner (glycerol release: 0A 3 __0.04 to 0.11 + 0.04 (XAM 10 -0 to 10 .7 tool/l) vs 0.08 +__0.02 (basal) p~mol/10 s cells/4 h, p < 0.05) inhibited NA (10- 5 to 10- 7 mol/1) induced lipolysis (glycerol release: 0.23+0.08 (NA 10-6 mol/l) vs 0.10+0.04 (NA 1 0 - ~ + X A M 10 -6) ~xmol glycerol/10 -s cells. Concentrations of 10 .5 and 10 .6 XAM re-

Abstracts sulted in a significant increase in fatty acid esterification above basal [0.08 + 0.03 (basal) vs 0.14 + 0.05 (XAM 10--6), p < 0.05]. These data demonstrate fll mediated lipolysis by XAM alone but the lipolytic response to NA is significantly inhibited at therapeutic concentrations of XAM, confirming partial fl~ agonist properties of this agent which may be of clinical significance. 321. Early changes in immunity and metabolism before the diagnosis of Type I (insulin-dependent) diabetes R. D. G. Leslie, B.A. Millward, D.A. Heaton, R. Gledhill, M. Hussain, EJ.Hoskins, P.Gray, N.Hales, G.F.Bottazzo, D.Vergani and D.A. Pyke. Diabetic Department, King's College Hospital, Denmark Hill, London, U K

In order to determine whether changes could occur before the onset of Type 1 (insulin-dependent) diabetes we studied prospectively 7 non-diabetic identical twins of Type 1 diabetic patients who subsequently developed diabetes. The twins were examined for the presence of autoantibodies to islet cells and insulin, activation of T-lymphocytes expressing the HLA DR antigen, glucose, insulin and proinsulin responses to oral (75 g or 1.75 g/kg) and intravenous (0.5 g/kg) glucose. The results were compared with suitable control subjects. We conclude from these observations: 1)that all the twins had cytoplasmic and complement-fixing islet cell antibodies when first tested 0.5 to 11 years before the diagnosis and they persisted up to the time of diagnosis. 2) Insulin antibodies were present in 3 twins but the levels declined before diabetes developed. 3) All the twins had increased levels of activated T-lymphocytes over 4.1% (i.e. above the control range) and these changes persisted until diabetes was diagnosed. 4) The twins had increased levels of basal proinsulin (29.3 + 19.3 vs 12.5 +4.5 pmol/1, p < 0.02) with normal fasting glucose when first seen. Changes in glucose tolerance and insulin responses were only found subsequently. In summary certain changes in immunity and metabolism may precede the onset of Type 1 diabetes by many months, even years. 322. Cortical magnetic stimulation: a new noninvasive method for measuring central motor conduction in diabetic neuropathy D. M. Levy, R. R. Abraham, R. M. Abraham, J. Adams and S. McHardy Young. Departments of Endocrinology, Neurology and Neurophysiology, Central Middlesex Hospital, London, UK

Central sensory conduction deficits in diabetic neurnpathy have previously been demonstrated using somatosensory evoked potentials (SSEP), visual evoked potentials, and brainstem auditory evoked potentials. We have studied central motor conduction noninvasively using a new and painless method - a magnetic neural tissue stimulator, developed at Sheffield University, U K - in 8 Type 2 (non-insulin-dependent) diabetic patients, median age 63 years (range 50-69), median duration of diabetes 9 years (range 2-23). All had neurophysiological evidence of lower limb neuropathy. Magnetic latency was measured at each abductor pollicis brevis by stimulating right and left motor cortex in turn, and recording the shortest of 10 latencies. Median motor nerve action potential amplitude (MAP), conduction velocity (MCV) and 'F' wave latencies were measured. N13 and N20 SSEP latencies were measured using standard techniques. Magnetic latencies were prolonged in 6 patients, while their median MAP, MCV and 'F' latencies were within normal limits. N13 and N20 latencies were prolonged in 7 and 5 patients respectively, but N13-N20 interwave latency, a measure of central sensory conduction, was normal in all but 2. A central motor conduction deficit may accompany diabetic neuropathy in the absence of abnormal conduction in the corresponding upper limb sensory pathway. 323. Glucose and free fatty acid turnover and assessment of insulin sensitivity by a constant infusion of glucose test J.C.Levy, G.Brown, C.Sketch, D.R. Matthews, S.Manley and R.C.Turner. Diabetes Research Laboratories, Radcliffe Infirmary, Oxford, U K Insulin sensitivity (S), assessed in terms of the glucose response, and B-cell function (B) can be assessed by a continuous infusion of glucose with model assessment (CIGMA). We have determined whether additional metabolic variables may elucidate S. Seventeen Type 2 (non-insulin-dependent) diabetic patients and 10 normal subjects had glucose turnover (primed continuous infusion of 3H glucose) and free fatty acid (FFA) turnover (continuous infusion of 14C palmitate) assessed during 90 rain CIG (5 mg/kg bw per rain) followed by 12 mmol/1 hyperglycaemic damp. Diabetic patients had increased basal glucose appearance (Ra) and after CIG impaired Ra suppres-

Abstracts sion but normally increased disappearance (Rd). This was driven by high glucose, as diabetic patients had reduced Rd compared with normal subjects at 12 mmol/l clamp. Diabetic patients had normal basal FFA but increased FFA Ra and impaired suppression of FFA by CIG (% decrease normal subjects 60 + 6%, diabetic patients 23 ___ 9%) and this correlated with the severity of diabetes (r=0.69, p < 0.001 with basal glucose). After 1 h CIG plasma G correlated with FFA concentration (r= 0.65, p < 0.001), but the increase in G during CIG did not correlate with plasma FFA, and a major role for the glucose/FFA cycle was thus not apparent. Suppression of FFA did not correlate with suppression of glucose Ra by CIG or with S assessed by CIGMA. In conclusion: CIG allows an assessment of FFA as well as glucose metabolism. Lack of correlation between FFA and G responses to CIG suggests there are separate controlling factors of FFA and G metabolism. 324. Dose-related effects of Cyclosporine A (CsA) in extension of the remission period in Type I (insulin-dependent) diabetic children C. Levy-Marchal, N.Tubiana-Rufi and P. Czernichow. Unit of Pediatric Endocrinology and Diabetes, Hrpital des Enfants Malades, Paris, France A non-randomised pilot study using CsA in Type 1 (insulin-dependent) diabetic children included 63 patients treated for at least six months, soon after diagnosis (13 + 6 days). Group I (n=23) received 7-10 mg C s A / k g / d aiming at a trough plasma level of 100 ng/ml. Group II (n= 14) received a higher dosage (15 mg/kg per day) for a target of 200 ng/ml. Group III (n = 26) received the higher dosage for 2-3 months and the lower one thereafter. The rate of total remissions (TR= no insulin; HbA1C < 6%) was 49% at six months, being more extensive in Groups II (57%) and III (62%) than in Group I (30%). Among 12 children treated for 1 year (7 mg/kg per day), 7 are still not insulin-treated. The glucagon stimulated C-peptide was more elevated at 6 months in Groups II and IlI (2.85+0.36 ng/ml) than in Group I (1.84_+ 0.36 ng/ml, p < 0.05). The children with TR, isolated from the 3 groups, exhibited a higher C-peptide level than the rest of the patients (3.30 +0.4 vs 2.3_+ 0.35 ng/ml, p<0.01). Four children demonstrated a reversible increase in BP in Group II only. The creatinine level increased above 150% of the baseline value in 13 children receiving the higher CsA dosage, but returned to normal values (82 + 6 ~tmol/1) after CsA reduction. This study demonstrates a doserelated effect of CsA on the extension of the remission period in diabetic children. The benefits may be balanced by the nephrotoxicity of the drug. 325. Modified fat diet can achieve cholesterol lowering in diabetic patients over 10 years N. J. Lewis-Barned, J.I. Mann, R.C. Carter and T.D.R. Hockaday. Diabetes Research Laboratories and Sheik Rashid Diabetes Unit, Radcliffe Infirmary, Oxford, U K Two-hundred forty-seven Type 2 (non-insulin-dependent) diabetic patients were randomised at diagnosis to receive low carbohydrate (LC) or modified fat (MF) dietary advice. One-hundred twenty-eight were reviewed at 5 and 10 years after diagnosis. Triglycerides and HbAlc did not differ significantly. Total cholesterol was significantly lower at 5 and 10years than at entry with MF (mean_+SEM: 0 years = 5.3 + 0.14, 5 years = 5.0 +_0.133, 10 years = 5.1 + 0.16 m m o l / 1). With LC, cholesterol tended to increase with time (0 years = 5.2+0.15, 5years=5.6+0.12, 10years=5.6+0.15 mmol/1). Direct comparison of the groups at both 5 and 10 years showed that MF had mean cholesterol 0.6 mmol/1 lower than LC (p= 0.001 and 0.008 respectively). The changes were accounted for by differences in LDLcholesterol between the groups (at 10 years L C = 3.6 + 0.14, MF = 3.1 _ 0.12 mmol/1, p = 0.006). Differences were greater in women than in men. Mean triglyceride linoleic acid at 10 years was higher on MF than LC (10 years MF = 16%, LC = 12%), signifying long term compliance with dietary advice. Advice on modifying dietary fat influences total and LDL-cholesterol over 10 years, and may reduce the risk of coronary heart disease in diabetic patients. 326. Regulation of thermogenesis by glucose and triiodothyronine J. Leymann and A.von zur Mtihlen. Abt. Klinische Endokrinologie, Medizinische Hochschule Hannover, FRG

Type 2 (non-insulin-dependent) diabetes is associated with low triiodothyronine levels. This study investigated the regulation of thermogenesis by glucose and triiodothyronine in human T-lymphocytes. Methods: T-lymphocytes were prepared from the blood of healthy donors and cultivated under standardised conditions. Direct men-

549 A surement of heat production (p) was performed by a Bio Activity Monitor 2277, LKB. Data are expressed as mean _+SD. Results: Basal p was 5.5 +0.8 ~tW/106 cells. Cultivation with triiodothyronine-depleted serum decreased p to 2.9 + 0.21xW/106 cells. Glucose (10 mmol/1) increased p to 11.1 +0.9 txW/106 cells. This effect was dose-dependent and significant for 0.1 mmol/1 glucose (KM = 0.5 mmol/1 glucose). Ouabain inhibited glucose-induced thermogenesis by 19%. Triiodothyronine (10nmol/1) increased p to 5.4___0.8 ~tW/106 cells. This effect was dose-dependent with KM = 0.1 nmol/l T3. Up to a glucose concentration of 0.5 retool/1 triiodothyronine stimulated glucose-induced p, whereas at maximum glucose-induced p no further stimulation by triiodothyronine was observed. Conclusion: Triiodothyronine and glucose are both regulators of T-lymphocyte thermogenesis. These effects are not additive. The action of triiodothyronine is independent of Na/K-ATPase activity which on the other hand contributes to about 20% of glucose-induced heat production. 327. Early changes in hepatic glucose production in response to exercise in the dog H. L. A. Lickley, P. D. G. Miles, D.T. Finegood and M. Vranic. Departments of Physiology, Medicine and Surgery and Women's College Hospital, University of Toronto, Ontario, Canada Current tracer methodology cannot accurately assess early changes in glucose turnover during exercise. We examined this problem in exercising dogs (60 min, 100 m/min, 12% slope, n= 15) by comparing a constant glucose tracer infusion (3-3H-glucose, 0.25 gCi/min) with a step increase in tracer (1.5-5-fold) at the onset of exercise to minimise fluctuations in specific activity. Arterial blood was collected throughout, with samples every 1-2 min for the first 20 min. Glucose production (Ra) was determined using a one-compartment model with an assumed pool fraction (P= 0.65) for both constant and step infusion methods. Using a regression method with a time-varying P, the tracer equation could be represented by a straight line ( R a = y intercept, P = slope). This requires a range of tracer infusions for the linear estimate. During the first 20 min of exercise, Ra (constant tracer infusion, P=0.65) rose progressively to a plateau 3-4 X basal. In contrast, Ra (step infusion tracer, P = 0.65) demonstrated a biphasic response with a steep rise 5-10 rain into exercise, reaching a similar but earlier plateau than with the constant infusion. The regression method gave equivalent values for Ra to the step increase method (P= 0.65). Thus, the one-compartment model, using an assumed P = 0.65 is valid for calculating Ra during early nonsteady-state exercise, provided a step increase tracer infusion is implemented at the onset of exercise. 328. Panereastatin: studies on insulin and glucagon secretion in vivo and on 4SCaZ+-efflux in isolated islets S.Lindskog, B.Ahr~n, G.Skoglund and S.Efendir. Department of Pharmacology, University of Lund and Department of Endocrinology, Karolinska Institute, Stockholm, Sweden Pancreastatin is a recently discovered intrapancreatic peptide that contains 49 aminoacids. It shows a structural similarity to chromogranin A. It inhibits glucose-stimulated insulin secretion in the perfused rat pancreas. We investigated the effects of pancreastatin on basal and stimulated insulin and glucagon secretion in vivo. The peptide was injected intravenously in fed unanaesthetized mice either alone or together with glucose (2.8 mmol/kg) or the cholinergic agonist carbachol (0.16 lxmol/kg). In addition, we studied the influence of pancreastatin on glucose-stimulated 45CaZ+-effiux from preloaded isolated rat islets. Pancreastatin (4.0 nmol/kg) lowered basal plasma insulin levels from 55 + 8 ~tU/ml in controls to 21 + 7 ~tU/ml after 6 m i n (p<0.01) and increased the plasma glucagon levels from 190+ 12 pg/ml in controls to 301 + 19 pg/ml after 2 min (p<0.001). Pancreastatin also inhibited the plasma insulin response to both glucose (by 44%, p<0.01) and to carbachol (by 32%, p<0.05). The peptide did not affect the carbachol-induced plasma glucagon response. In vitro, pancreastatin decreased the integrated 10min glucose (13.3 mmol/1)-induced 45Ca2+-effiux by 45% (p< 0.01) in perifused islets compared to controls. We conclude that pancreastatin inhibits insulin and stimulates glucagon secretion in vivo and impairs glucoseinduced 45CaZ+-effiux in isolated islets in vitro. 329. Time relationships for calcium, phosphate, and insulin translocations and nucleotide content in rat islets P. Lindstr/)m, L.-A. Idahl and N. Freinkel. Department of Histology and Cell Biology, University of Ume~, Umefi, Sweden and Center for Endocrinology, Metabolism and Nutrition, Northwestern University Med. School, Chicago, Ill, USA

550 A Knowledge of the temporal hierarchy of cellular events associated with maximal glycaemic stimulation of pancreatic islets increases the understanding of insulin secretion. Accordingly we devised a perifusion system in which the transition from basal to maximal glucose challenge was achieved in 15 s. The initial decrease in 45Ca outflow occurred within 15 s of achieving stimulatory glucose, followed by simultaneously heightened insulin and 32p outflow and, 45 s later, a rise in 45Ca efflux. There was no initial decrease in ATP levels and the rise in ATP did not precede insulin release. AMP and ADP levels were unaffected the first 60 s. Equimolar rnannose was substituted for glucose to get a slower hexose metabolism. The decrease of 45Ca outflow again occurred within 15 s, but insulin release and 32p outflow were delayed. After one week culture in RPMI insulin release occurred already within 15 s whereas 32p efflux was retarded. A net change in islet ATP levels does not seem to be an early event, and the dissociability by experimental manipulation between insulin release and calcium and phosphate efflux suggests that their interdependencies may be loose and that each may be mediated by separate metabolic signal(s).

330. Relationship between circadian blood pressure and heart rate patterns and diabetic cardiovascular autonomic neuropathy C. Linig~r, L. Favre, R. Adamec, A. Pernet and J.-Ph. Assal. Diabetes Treatment and Teaching Unit, Endocrinology Division, Medical Policlinic, University Cantonal Hospital, Geneva, Switzerland To assess the effect of diabetic cardiovascular autonomic neuropathy on circadian blood pressure (BP) and heart rate (HR) patterns, BP was measured automatically every 15 rain and HR recorded continuously for 24 h in 15 ambulatory diabetic patients, 12 of whom had at least one defective cardiovascular reflex, and in 11 non-diabetic control subjects. Using HR variation during deep breathing and systolic BP response to standing after lying as yardsticks of autonomic failure, the disturbance of the circadian BP profiles was proportional to the gravity of the neuropathy. The magnitude of the nocturnal change in BPs correlated with HR variation (systolic: r= 0.63, p = 0.011 ; diastolic: r=0.79, p=0.001) and with BP response (systolic: r=0.69, p = 0.006; diastolic: r= 0.71, p = 0.005). Surprisingly, nocturnal HR change was independent of both HR variation (r= 0.004, p = 0.960) and BP response (r=0.294, p = 0.308). However, the coefficient of variation of 24 h HR (CV-HR%) decreased in proportion to the severity of the neuropathy (CV.HR% vs HR variation: r=0.69, p=0.005; CV. HR% vs BP response: r=0.85, p = 0.000). Although the circadian BP pattern is disrupted in diabetic autonomic neuropathy, HR may slow down normally at night despite an overall reduction in HR variability. This apparent dissociation highlights the complexity of the mechanisms controlling circadian haemodynamic rhythms.

331. Effect of chronic hyperglucagonaemia on glucose tolerance, insulin resistance and insulin action M. Linkesch, R. Prager, R. Klauser, W. Linkesch, H.Gisslinger and G. Schernthaner. Medical Department II, University of Vienna, Austria Recently it has been shown that danazol (14-ethinyl-testosteron) induces hyperglucagonaemia. In order to investigate the effect of chronic glucagon excess on carbohydrate metabolism we studied 5 patients, who were treated with danazol for immunothrombopenia. Before and after a 3-month danazol therapy we measured glucose tolerance and insulin secretion during an oral glucose tolerance test (OGTY) as well as insulin action by means of the euglycaemic clamp technique (40 m U / m 2. rain). Overall glucose turnover (Rd) was assessed radioisotopically. 1) Plasma glucagon levels rose significantly from 88 pg/ml before to 707 pg/ml after therapy (p<0.01). 2) During the OGTT glucose levels did not change significantly before and after therapy. 3)Glucose-induced insulin secretion, however, was significantly lower before danazol treatment than after therapy (81 vs 135, 77 vs 112 and 68 vs 100 IxU/ml at 60, 120 and 180 rain, p<0.05). Steady state Rd at the end of the euglycaemic clamp showed a significant decay from before to after therapy (5.3 vs 3.8 mg/kg, min, p < 0.05). 4) Basal hepatic glucose output did not differ significantly before and after therapy (1.74 vs 1.45 mg/kg, NS), whereas hepatic glucose output during the clamp was significantly higher under therapy (0.23 vs 0.48, p<0.05). We conclude that chronic glucagon excess leads to a decay in peripheral and hepatic insulin action which are counterbalanced by an increase in insulin secretion.

Abstracts 332. Immunregulatory effect of n-3 and n-6 polyunsaturated fatty acids (PFA) in the low dose streptozotociu (LDS) induced diabetes model Th. Linn, M. Noke, H.U. Kl6r, R.G. Bretzel and K. Federlin. III. Medizinische Klinik und Poliklinik am Zentrnm flir Innere Medizin der Justus-Liebig Universit/it, GieBen, FRG PFA have been reported to have effects on autoimmune models, such as experimental allergic encephalitis. We report here the effects of diets high in PFA on the development of diabetes in the LDS model. CD-1 or C57B16 mice were fed diets containing less than 6% total lipid in all groups. N-3 PFA were given in the form of a fish oil with 30% eicosapentaenoic acid; the source of n-6 PFA was thistle oil starting 3 weeks prior to 5 • 40 mg/kg per day streptozotocin (SZ). Repeated subcutaneous injection of SZ pretreated islet collagenase digest as a source of antigen resulted in the accumulation of sensitised mononuclear cells measured by popliteal lymph node weight and footpad swelling test. Suppression of cell-mediated immune response at doses of PFA ranging from 1-3 g/kg was demonstrated. Histologic examination showed that PFA protected from insulitis. Both n-3 and n-6 PFA enriched diets resulted in lower blood glucose concentrations and higher pancreatic insulin content. Additional administration of indomethacine abrogated diet induced suppression of diabetes. PFA did not influence diabetic disease when a single toxic dose of SZ was used. Further studies concerning the mode of action of PFA in this model are presently being performed. 333. Hepatic action of glicazide treatment in Type 2 (non-insulin-dependent) diabetes mellitus G.Lisato, A. Riccio, S.Vigili de Kreutzenberg, A.Tiengo and S.Del Prato. Cattedra di Malattie del Ricambio, University of Padova, Italy The mechanism(s) of hypoglycaemic action of glicazide was evaluated in 8 diet-treated Type 2 (non-insulin-dependent) diabetic patients (age =47 + 4 years; BMI =27.7 _+0.5 kg/m~). Five patients received a 240 iron glucose infusion (11A mmol/1/kg-rain) along with 3-3H-glu cose infusion with glicazide (240 mg PO) and placebo. In 3 patients glicazide and placebo were taken while keeping insulin and glucagon constant at basal level (somatostatin) and no glucose infusion. Basal plasma glucose (6.1 + 0.4 vs 6.3 _+0.5 retool/l), insulin (10 + 1.2 vs 10.9 + 1.5 mU/1), C-peptide (0.54_+ 0.06 vs 0.54-+ 0.05 pmol/1), glucagon (30 • 7 vs 27 + 7 pmol/l) and FFA (422 + 28 vs 438 + 25 ~mol/1) were similar in both occasions. Following glucose infusion, plasma glucose (120-240 min=7.0• vs 7.9_+0.4 mmol/1) and its incremental area under the curve (352 • 42 vs 461 _+52 retool/l;/7< 0.05) were lower with glicazide. Plasma insulin ( + 9 • vs +9_+I mU/l) and C-peptide level (+0.40_+ 0.07 vs 0.43 _+0.09 pmol/1) increased equally with glicazide and placebo. Plasma glucagon ( - 5 _ + 2 vs - 6_+ 2 pmol/1) and FFA (214_+ 39 vs 204 • 57 ~mol/1) were equally inhibited. Basal hepatic glucose production (HGP) and utilisation (15.0_+1.6 vs 14.4• -~) were comparable in both occasions. With glicazide, glucose infusion resulted in a larger HGP suppression (-10.7+1.1 vs -6.7_+1.5 ~ m o l / l . k g - l . m i n - l ; p < 0.05) but had no effect on glucose utilisation ( + 7.4_+ 1.4 vs 6.8 -+ 0.9 ~mol/l. kg -1. rain 1). HGP decremental area correlated with the plasma glucose incremental area (r= 0.63; p < 0.05). In the presence of constant insulin concentration (10_+1 vs 1 2 • both glicazide and placebo lowered glucose (5.2 • 0.5 vs 5.2 _+0.4 retool/l) and HGP (12.0_+ 1.3 vs 11.1 _+2.0 umol/1.kg -1.rain-I). In conclusion, glicazide: 1) exerts a primary extrapancreatic effect on the liver, and 2) increases liver sensitivity to insulin suppressive effect on HGP. 334. Influence of Na+-K+-ATPase blockade on insulin action in man L. Locatelli, G. Buzzigoli, E.Dipede, S.Taddei, R. Pedrinelli and E. Ferrannini. C.N.R. Institute of Clinical Physiology, and 1st and 2nd Medical Clinic, University of Pisa, Pisa, Italy Insulin is a potent stimulus for Na+-K+-ATPase activity in vitro; a defect in such activity has been postulated in insulin-resistant states (obesity, diabetes). To test whether insulin action on metabolic pathways is linked with its effect on the Na+-K § pump, we infused insulin into the forearms of healthy volunteers to raise local arterial concentrations by ca, 100 mU/l. During the final 80 rain of the 3-h infusion, ouabain, a known inhibitor of the pump, was co-infused (to achieve arterial levels of ca. 4.10 -8 tool/l). Insulin alone markedly stimulated glucose uptake (from 0.82+0.02 to 4.51 +0.48 ~mol/min per 100 ml, p<0.01), led to a positive net exchange of FFA (0.07+ 0.04 to 0.19+0.05 ~mol/min per 100 ml, p<0.01) and K + ( - 0 . 1 _+0.1 to 0.96 +0.11 ~mol/min per 100 ml, p<0.01), and enhanced lactate (0.45+0.12 to 0.73+0.12 gmol/min per 100 ml, p<0.02) and pyrurate (0,001 _+0.001 to 0.029 + 0.008 ~mol/min per 100 ml, p < 0.01) re-

Abstracts lease by deep forearm tissues. Addition of ouabain decreased (by ca. 40%, all p<0.01): total forearm blood flow, glucose and FFA uptake, lactate and pyruvate output, while net K + exchange was reversed to a release of 0.90_+ 0.08 gmol/min per 100 ml. We conclude that, in human forearm muscle tissue, pharmacologic blockade of Na+-K+-AT Pase acutely impairs insulin action, in part through a vasoconstrictive response. 335. Regenerative B-cell hyperplasia in the remaining pancreas after partial pancreatectomy in pigs: immunocytochemical and functional investigations M. L6hr, G. Kloppel, J. Ltibbesmeyer, B. Otremba, R. Klapdor and D.Grossner. Institute of Pathology, Departments of Internal Medicine and General Surgery, University of Hamburg, FRG The regenerative and functional capacity of B cells remaining in pancreatic remnants after partial pancreatectomy was examined after surgical resection of 40% (I), 60% (II) and 80% (III) of the pancreas of 3-month-old pigs. Prior to resection and at the end of a 120-day observation period, basal and glucose stimulated levels of insulin were determined and the values compared with those in non-resected controls. For quantitative morphology, the volume of the resected specimen and the residual pancreatic tissue was determined and sections evaluated by immunocytochemistry (insulin, glucagon, somatostatin, HPP) combined with morphometry. The volume density of the B cells showed a 20% (I and II) and 60% (III) increase of the B cells in the remaining pancreas when compared with the resected portion and the controls. This lead to an increment of the total endocrine volume from 0.028 ml/ml pancreatic parenchyma (controls) to 0.034 ml/ml pancreatic parenchyma (III). The absolute volume of B cells in the remaining pancreas was 0.35 ml (I), 0.27 ml (II) and 0.13 ml (III) in 2 comparison with 0.57 ml (controls). The number of B cells/mm did not change. These results suggest a B-cell hyperplasia in the residual pancreas after resection. With regard to the functional parameters, there was no significant change after 40% and 60% resection. In III (80% resection) impaired glucose tolerance was noted despite the marked B-cell hyperplasia; however, manifest diabetes was absent. 336. Effects of high glucose concentrations on in vitro aggregation of human blood platelets W. Loesche, S. Heptinstall 1, J. May 1, E. Michel and A. Burchardt. Institute of Pathological Biochemistry, Med. Acad. Erfurt, GDR, and 1Department of Medicine, University Hospital Nottingham, U K To prove whether high blood glucose levels may contribute to platelet hyperreactivity frequently found in diabetic patients, we studied platelet aggregation after incubating whole blood (WB) or plateletrich plasma (PRP) with 25 and 50 mmol/1 glucose for 1 h at 37 ~ Glucose (25 and 50 mmol/1) increased spontaneous aggregation in WB (+42%, p<0.02), but not in PRP. The effect in WB was completely prevented by the ADP-destroying enzyme apyrase. The idea that the glucose effect in WB was due to liberation of ADP (a strong aggregation stimulus) was supported by the finding that glucose increased red cell membrane permeability as evaluated by determining haemoglobin liberation. Aggregation in PRP induced by arachidonic acid and PAF-acether was inhibited by glucose in a dose-dependent manner. Measurements of malondialdehyde production indicated that platelet cyclooxygenase was inhibited under these conditions, which is believed to be due to glucose-induced changes in cellular levels of reduced co-enzymes. 337. Follow-up of cell mediated anti-B cell cytotoxicity in Type 1 (insulin-dependent) diabetes after lymphocyte transfusion D. Lohmann, B. Bierwolf, J. Krug, E. Lampeter and H.J. Verlohren. City Hospital of Leipzig, G D R In 21 newly-diagnosed Type 1 (insulin-dependent) diabetic patients a cytotoxic effect of blood mononuclear cells (BMC) against B cells was demonstrated by an increased non-stimulated insulin release from rat islets during an incubation of 20 h; this could be inhibited by the addition of lymphocytes (1:4) from healthy subjects in 18 cases (14.6--~4.7 ng IRI/islet). Under the presumption of a suppressor cell defect in the diabetic patients, lymphocytes from the same healthy subjects were transfused in a high number (2.0 x 109-1.5 x 101~cells). The cytotoxic effect of the BMC of the recipients was controlled after 6, 26 and 52 weeks. After 6 weeks a significant decrease was observed in 12 of 18 cases (insulin release: 14.6---~5.7 ng/islet). In 7 of 8 cases the effect was also demonstrable after 26 weeks. An increase between the 26nd and 52nd week was observed in 3 out of 7 patients. In a control group (n= 14) the cytotoxic effect remained constant over a peri-

551 A od of 6-9 months (insulin release: 11.1---10.7 ng/islet). The alterations of the cytotoxicity were correlated with the course of the stimulated C-peptide secretion (100 g glucose orally and 1.0 mg glucagon intravenously). A correlation between cytotoxicity and C-peptide secretion was demonstrable. 338. Combination of insulin and sulphonylurea (SU) in the treatment of Type 2 (non-insulin-dependent) diabetes with "secondary failure of SU therapy (SF)" - long-term results N. Lotz, T. Ladik, P. Rupp, W. Bachmann I and H. Mehnert. III. Med. Abt., St~idt. Krankenhaus Mtinchen-Schwabing und Forschergruppe Diabetes e.V.; aKreiskrankenhaus Kronach, FRG The combination of insulin plus SU was shown to be the most physiological way of treating SF. Avoiding hyperinsulinaemia, similar or even better metabolic control could be achieved compared to the common therapy with only insulin. This study investigated whether this benefit would persist. In a random order 16 Type 2 (non-insulindependent) diabetic patients with SF were treated either with insulin (G I) or with glibenclamide (7 rag/day) plus small amounts of insulin (G II). After an in-patient period metabolic control (basal and postprandial blood glucose, HbA1, body weight, plasma insulin and Cpeptide) was performed monthly. Clinical and biochemical parameters of both groups were comparable. After 34+4.8 (mean+SEM) months of treatment there were no significant differences in blood glucose (basal G I: 11.5 _+1.6 mmol/1, G II: 12.7 _+1.7; postprandial - G I: 13.1 +2.1, G 1I: 12.8+__1.8) and HbA1 (G 1: 10.5_+ 1.4%, G II: 10.4+ 1.3). Also no significant differences were seen in body weight, plasma insulin, C-peptide and lipids. However, exogenous insulin dosage differed significantly between the groups (G I: 44_+7 U/d, G II: 19.5 _+3). In conclusion, the combination of insulin and SU in the treatment of SF is still efficient after 34 months in spite of tess than a half of the insulin dosage given in G I. 339. The effect of first, second and third trimester maternal glycaemic control in established diabetic women on their babies' birth length and weight C. Lowy, D.C. Garvie, S. Till and T. E. T. West. Unit of Endocrinology and Diabetes, Department of Medicine, St Thomas' Hospital Medical School, U K Since 1980, 51 of 76 consecutive pregnancies in established diabetic women (EDM) were monitored from mid frist trimester onwards. Of the 47 early attenders (4 delivered prematurely), 21 achieved normal HbA1 levels in the first trimester, but 19 gave birth to babies in excess of the 90th centile for weight. To explore this unexpected trend in these 47 pregnancies, maternal variables including mean first, second and third trimester HbAI were related to birth weight and length. Control and gestational diabetic babies (GDM) were compared. The mean birth weight and length of EDM GDM and control babies were 3.8 kg, 53.9 cm (n=47) 3.6 kg, 52.8 cm (n=39) and 3.2 kg, 51.6 cm (n = 44) respectively. Birth length of the EDM babies was inversely related to the mean maternal first trimester but: not the second or third trimester HbA1 level. The EDM babies were divided into two groups according to their birth length, 'Short' <53.5 cm and 'Long' > 53.5 cm. The 'Long' babies were significantly heavier and their mothers first and second trimester HbAI levels were lower; (first trimester 8.2% versus 9.9% p<0.005 second trimester 7.4% versus 8.4% p < 0.05), EDM babies were long and heavy. These results suggest that early fetal growth is inhibited and late growth promoted by maternal hyperglycaemia. 340. Permanent acceptance of syngeneic islets grafted into freshly diagnosed diabetic BB-rats without immunosuppression S. Lucke, A. Dunger, B.Ziegler, B. Hehmke, W. Besch, I. K16ting, C.Kauert and H.J.Hahn. Zentralinstitut ffir Diabetes "Gerhardt Katsch" Karlsburg, G D R The BB-rat offers the opportunity to investigate the autoimmune process leading to pancreatic B-cell destruction when syngeneic islets were grafted into nonimmunosuppressed diabetic recipients. Twentytwo freshly-diagnosed (development of hyperglycaemia < 2 days) diabetic BB-rats were grafted beneath the kidney capsules with 1000 BB-rat islets obtained from 8-12 day old donors. Fifteen grafted BB-rats relapsed into hyperglycaemia <3 weeks and 7 grafted animals maintained normoglycaemia >120 days. The grafted BB-rats were retrospectively analysed. At the time of diabetes onset and transplantation the two groups of BB-rats did not show any significant difference of body weight, sex, age, plasma glucose, islet volume density, degree of insulitis or complement-mediated cytotoxicity (C'AMC).

552 A Three weeks later, the hyperglycaemic BB-rats were characterised by an enhanced C'AMC, which remained unchanged in normoglycaemic grafted animals, despite a further significant loss of pancreatic Bcell volume density to values also found in hyperglycaemic BB-rats. The permanent acceptance of BB-rat islets in a part of recipients underlines a heterogeneous response to a syngeneic stimulus and demonstrates that the autoimmune B-cell destruction is not a persisting phenomenon in all BB-rats. 341. Islet cell surface antibodies (ICSA) determined by a protein-A method J. Ludvigsson, S. Bergman and A. Lernmark. Dept. of Pediatrics, University Hospital, Linkrping, Sweden and Hagedorn Research Lab, Gentofte, Denmark Aims: To define specificity (Sp), sensitivity (Se) and limit of positivity of an ICSA-method. Subjects: A)45 healthy schoolchildren, 4 blood samples/child. B)161 1st degree relatives of diabetic children, C) 69 diabetic children, D) 10 schoolchildren from A and 26 diabetic patients. Methods: Rat B cells, RIN-5F, were cultured in monolayer and then fixed. Serum was heat inactivated and precipitated, then dissolved, dialysed and filtered. Fixed cells were incubated with serum dilutions at 0 ~ then washed; 12sI-Protein A was added. After further incubation and washing a 7~-counter was used. Each series included 14 negative controls. Binding was calculated in % of the mean of these controls. The D-samples were analysed 10 times to calculate the Se and Sp. Results: To get reproducible results we had to add Tween 20 to the washing buffer and to transfer the cells to glass tubes before counting. In A the binding percentages have been described in percentiles (P). With positivity above 1792(196% of the negative controls) Sp was 99% and Se 48%. With the limit Ps5 (175%) Sp remained 98% but Se raised to 66%. With pos > 196%, we found 8% pos samples in A, 18% in B and 36% in C p<0.01. Conclusions: Our Protein-A method has a good specificity but a low sensitivity. ICSA occurs more often in diabetic patients and their first degree relatives than in healthy subjects. 342. No effect of low-dose eaptopril therapy on heavy proteinuria in diabetic patients: a follow-up study of 6 months B. Ludvik, U. Hay and G. Schernthaner. Medical Department II, University of Vienna, Austria A positive effect of the angiotensin-converting enzyme inhibitor captopril (37.5 rag/day) on heavy proteinuria in diabetic patients has recently been reported. Since the observed study period lasted only 8 weeks, we investigated the long term effect of an identical dosage of captopril (37.5 mg/day) on renal function and proteinuria in 10 insulin-treated diabetic patients. The patients were studied before, 4, 8, 12 and 24 weeks after onset of therapy. Urinary protein excretion increased from 3706+1491 rag/day before to 5405+2699 after 6 months of treatment (/9<0.05); serum creatinine rose from 0.16• 0.10mmol/1 to 0.18_+0.12 (p<0.01), whereas the blood pressure showed no significant change before (150/80 mmHg-+23/6) and after therapy (150/90 mmHg + 23/10). HbAI~ - as a marker of metabolic long-term control decreased from 8.3+1.0% to 7.3+1.0% (p< 0.05). These findings indicate that there are no beneficial long-term effects of low-dose angiotensin enzyme inhibition on heavy proteinuria in patients with advanced diabetic nephropathy. In particular, the progressive deterioration of kidney function could not be influenced by the captopril treatment. 343. Results of a 10-year prospective study in Type 2 (non-insulin-dependent) diabetic patients - vascular complications and risk variables R. Lundershausen, D. Pissarek, S. Orban and G. Panzram. Outpatient Department of Internal Medicine, Medical Academy, Erfurt, G D R The frequency of vascular complications and associated risk variables were investigated in 150 newly-diagnosed Type 2 (non-insulin-dependent) diabetic patients (60 M and 90 F, range of age 24-60 years) during a 10-year follow-up. The prospective study is concerned with coronary heart disease (CHD, Minnesota Code), peripheral vascular disease (PVD, electronic oscillography and X-ray findings), retinopathy (ophthalmoscopy) and proteinuria. There were 19 deaths (12 patients due to cardiovascular disease) during the observation period. The prevalence of CHD increased from 37.0% at baseline to 52.8%, that of PVD from 9.3% to 62%. After the 10-year follow-up the frequency of retinopathy amounts to 17.0%, that of proteinuria to 22.1%. The multivariate analyses included age, body weight, cholesterol, triglycerides, blood glucose and IRI (fasting and 2h values after an oral glucose load), blood pressure and smoking habits. For CHD age, hy-

Abstracts pertension and fasting hyperglycaemia could be identifed as most powerful predictors in both sexes. In PVD age, hypertension and obesity proved to be independent risk variables for males and females. There were some sex-related differences in the pattern of significant risk factors. No potent predictor for retinopathy and proteinuria could be found. 344. Defective crosslinking of fibrina-monomers in poorly-controlled diabetic patients A.Lutjens 1, T.W.Jonkhoff-Slok 1, E.A.v.d.Veen 2 and J.v.d.Meer 2. 1Department of Clinical Chemistry, Andreas Hospital, Amsterdam and 2Department of Internal Medicine, Free University Hospital, Amsterdam, The Netherlands During the coagulation process fibrin monomers are covalently bound (crosslinked) under the influence of activated clotting factor XIII and Ca 2+. The influence of increased glycation of fibrin on the crosslinking is investigated. To plasma of 10 healthy volunteers, 6 poorly-controlled diabetic patients and 1 well-controlled patient (HbAlc respectively 3.8 + 0.2%, 11.0 + 0.8% and 7.0%, mean + SEM), thrombin and Ca 2+ were added to start the clotting process. At different time intervals the reaction was stopped and the clots were solved in 8 mol/l urea/SDS/dithiotreitol. Polyacrylamidegel electrophoresis was performed to separate the fibrin polymers. Crosslinldng was followed by measuring the appearance of the y- y-dimer, the disappearance of the a-monomer and the appearance of the an-polymers, yChain crosslinking did not show any difference between the two groups, a-Chain crosslinking in poorly-controlled diabetic patients, however, showed formation of incomplete a-polymers. Disappearance of the a-monomer was less complete compared to control subjects (a24h: 10.68+1.58% in the diabetic patients and 6.17+0.70%, (mean+SEM) in the control subjects, p<0.005), less completely crosslinked an-polymer was formed and more intermediate a-polymers could be shown. Values for the well-controlled patient fell well within the normal range. It is suggested that increased glycation of fibrinogen is the cause of the defective crosslinking of the a-chains of fibrin. 345. Effect of insulin and aminoacids on leucine kinetics in Type 2 (noninsulin-dependen0 diabetic patients L. Luzi, A. Battezzati, G. Pozza and R.A. DeFronzo. Department of Medicine, Yale University, New Haven, Connecticut, USA, and Department of Medicine, Istitnto Scientifico San Raffaele, Milan, Italy Type 2 (non-insulin-dependent) diabetic patients are characterised by insulin resistance and impaired glucose tolerance. To examine whether the defect in glucose utilisation extends to aminoacid metabolism, 6 Type 2 diabetic patients and 7 control subjects (age=61 + 4 vs 54+5years; I B W = 1 0 7 + 3 vs 105+2%; HbAlc=9.6 vs 5.5%; F G = 1 6 0 + 1 0 vs 80+2 mg%) were studied with the insulin clamp combined with 14C-l-leucine. All subjects participated in two studies: Study I: 3 h euglycaemic insulin ( 4 0 m U / m z per min) clamp; Study II: insulin clamp plus balanced aminoacid infusion to increase plasma aminoacids 2- to 3-fold. Insulin-mediated glucose uptake was reduced in both Study I (4.0_+0.5 vs 5.9+_0.4 mg/kg per min, p < 0.01) and Study II (3.6 _+0.5 vs 5.7 _+0.5, p < 0.01) in Type 2 patients vs control subjects. Basal plasma leucine (120_+ 12 vs 119_+ 12 gmol/1), endogenous leucine flux (ELF) (40 + 2 vs 41 _+2 ~tmol/m 2 per min), leucine oxidation (5.8 _+0.7 vs 6.0_+ 1.5 ~tmol/m2 per rain), and nonoxidative leucine disposal (NOLD) (34_+2 vs 35_+2 ]xmol/m2 per min) were similar in both groups. In Study I, the decrement in plasma leucine (54 + 3 vs 57 _+4 I,tmol/1), ELF (13 _+2 vs 14 _+2 l.tmol/m2 per rain), leucine oxidation (1.6_+ 0.2 vs 2.1 + 0.3 p,mol/m 2 per rain), and NOLD (11 _+1 vs 12_+1 gmol/m 2 per rain) was comparable in both groups. In Study II (plasma leucine 198_+40 vs 190_+20), the increment in leucine oxidation (8.2 + 0.9 vs 7.5 _+0.7 ~mol/m 2 per min) and NOLD (15 _+3 vs 12 + 2 umol/m 2 per min) was similar in Type 2 patients and control subjects. Conclusions: insulin-mediated glucose disposal is significantly impaired in Type 2 diabetic patients vs control subjects; nonetheless, the effects of insulin on ELF (protein degradation), NOLD (protein synthesis), and Ieucine oxidation are similar in Type 2 patients and control subjects. These results demonstrate a clearcut dissociation between insulin effect on aminoacids and glucose metabolism. 346. Glycation of low density lipoprotein in Type 2 (non-insulin-dependent) diabetes mellitus: correlations with other indices of glycaemic control T. J. Lyons ~, R.L. Klein2 and M. F. Lopes-Virella2. 1Attnagelvin Hospital, Londonderry, N.Ireland, 2Medical University of S.Carolina, Charleston, SC, USA

Abstracts Glycation of low density lipoprotein (LDL) was measured by boronate affinity chromatography in 16 patients with Type 2 (non-insulin-dependent) diabetes mellitus and in 16 age-, sex- and racematched non-diabetic control subjects. The diabetic patients were treated with diet alone, were in good glycaemic control (HbAI: diabetic patients 7.3 _+0.4%, controls 5.5 + 0.1%, mean + SEM) and had similar plasma cholesterol (5.21+0.16 vs 5.06+0.16mmol/1) and triglyceride levels (1.28 -+ 0.14 vs 1.11 vs 0.11 mmol/1) to control subjects. Glycation of LDL was significantly increased in the diabetic patients (93 + 5 vs 73 _+3 cpm (tritiated hexitol-amino-acid)/ixg protein, p<0.01) and correlated with HbA1 (n=16, r=0.65, p<0.01). Glycation of plasma proteins was also increased (166+ 11 vs 142_+ 7 cpm/~tg protein, p<0.01) and correlated with both LDL glycation (n=16, r=0.55, p<0.05) and with HbAI (n=16, r=0.55, p<0.05). These data show that in well-controlled, normolipaemic patients with Type 2 diabetes, LDL glycation is significantly increased and correlates with other indices of glycaemic control. Since LDL glycation may enhance foam cell formation in macrophages, the results may help explain why diabetes is an independent risk factor for atherosclerosis. They also suggest that, in Type 2 diabetes, "tight" glycaemic control should be sought in order to minimise the adverse metabolic effects of LDL glycation.

347. Relationship of altered haemostasis to metabolic control, serum lipids and retinal microangiopathy in diabetic children L. Madficsy, A. Feh~r, L. Kassay, L. Orosz and Z. Boda. II. Dept. of Pediatrics, Postgraduate Med. School, Miskolc and II. Dept. of Medicine, University of Debrecen, Hungary To study the relationship of haemostasis-relevant parameters to metabolic control, serum lipids and retinal microangiopathy in Type 1 (insulin-dependent) diabetes, plasma beta-thromboglobulin (BTG), factor VIII-related antigen, factor VIII-doagulant activity (VIIIC), lipid and lipoprotein levels were determined in 59 diabetic and in 30 agematched control children. Early microangiopathic signs in diabetics were evaluated by fluorescein angiography, a though metabolic control by measurement of serum glycosylated haemoglobin and fructosamine, respectively. Diabetic children with and without retinal changes had significantly higher BTG levels compared to control subjects (p< 0.01). Patients with poor metabolic control had significantly higher BTG values than those with good metabolic control (p < 0.05). There has been a significant positive correlation between triglyceride and BTG levels in diabetic patients. VIIIC levels were significantly higher in patients with poor actual control based on fructosamine levels (p<0.02). A significant positive correlation has been found between LDL-cholesterol and VIIIC levels in diabetic patients. Early retinal changes were not related to factor VIII-related antigen levels. Our results suggest that 1) an increased platelet turnover occurs during poor metabolic control and in case of hypertriglyceridaemia; 2) factor VIII coagulant activity correlates with preceding short-term hyperglycaemia and with LDL-cholesterol levels in diabetic children. 348. Effects of 6 weeks of strict metabolic control on glucose counterregulation and recognition of hypoglycaemic symptoms in Type I (insulin-dependent) diabetic patients S. Madsbad, B. Edsberg, M. Bahnsen, T. Krarup, J. Hilsted, N. J. Christensen and C. Ktihl. Hvidore Hospital, Medical Dept. F, Herlev Sygehus and Med. Dept. F Glostrup Sygehus, Denmark To assess the effect of strict metabolic control on glucose counterregulation 9 Type I (insulin-dependent) diabetic patients were studied before (Hbalc: 8.3 + 0.2%) and after (HbA1c: 6.8-+ 0.3%) 6 weeks of insulin pump treatment (overall mean blood glucose: 6.9-+ 0.1 mmol/1). No patients have neuropathy or B-cell function. After maintenance of euglycaemia overnight hypoglycaemia was induced by i.v. insulin (0.15 U. kg 1. h-1 for 45 min). The blood glucose nadir was 2.0-+0.2 vs 1.6+0.1 retool/l, p<0.07 before and after pump treatment, and significantly lower blood glucose concentrations were found after pump treatment from 15 to 45 min after stop of i.v. insulin. Responses of counterregulatory hormones were (area under curves): epinephrine (70.0+0.4 vs 48.8_+ 8.0 ng/ml per 150 min, p < 0.07), norepinephrine (p: NS), glucagon (p: NS), cortisol (69400-+ 4094 vs 58 365 _+2583 mmol/1 per 150 min, p < 0.03), growth hormone (p: NS) before and after pump treatment, respectively. Furthermore, the response of epinephrine was delayed after pump treatment. Awareness of hypoglycaemia occurred at 2.4 -+ 0.1 mmol/l before and at 2.0 + 0.2 mmol/1 after pump treatment (p< 0.05). Maximal systolic blood pressure during hypoglycaemia was 136 -+ 8 vs 125 + 5 mmHg, p<0.05. Conclusion: six weeks of strict metabolic control reduces glucose counterregulation and diminish the threshold for perception of hypoglycaemia.

553 A

349. Relationship between low density lipoprotein cholesterol and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in monocytes from Type 2 (non-insulin-dependent) diabetic patients V. Maher, R.A.M. Devery, P. B. Collins, A. H. Johnson and G. H. Tomkin. Department of Metabolic Medicine, The Adelaide Hospital, Dublin 8 and Department of Biochemistry, The Royal College of Surgeons in Ireland, Dublin, Ireland This study examined the effect of low density lipoprotein composition on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase, the key enzyme regulating cholesterol biosynthesis in monocytes from Type 2 (non-insulin-dependent) diabetic patients. Fasting venous blood was obtained from 10 diabetic patients and 6 age-matched control subjects. The results of this study demonstrated that the activity of H M G C o A reductase in monocytes was significantly elevated (p< 0.05) in diabetic patients (1.17_+ 0.18 nmol. m i n - l . m g 1, m e a n + S E M ) relative to control subjects (0.72+ 0.08 nmol-min-l.mg-1, m e a n _ SEM). Low density lipoprotein (LDL) composition with respect to cholesterol, triglyceride and phospholipid was similar in both groups. A significant negative correlation (r= -0.67, p < 0.05) was observed between the activity of HMGCoA reductase in monocytes and the ratio of cholesterol:protein in the LDL particles from diabetic patients. This study suggests that LDLcholesterol may be an important factor regulating the activity of H M G C o A reductase in monocytes from Type 2 diabetic patients and thereby may be implicated in the genesis of cholesterol-laden cells characteristic of the athernsclerotic process.

350. Effect of glibenclamide on carbohydrate levels in the haemolymph of the honeybee (Apis mellifica) V. Maier, J. Fuchs, M. Mezger, H. Herlitzius 1 and E.F. Pfeiffer. Dept. of Internal Medicine I and Biology III 1, University of Ulm, FRG In the honeybee insulin is localised (produced?) in the brain. The material is biologically active using the model of the isolated epididymal rat fat cell. Immunologically it seems to be very similar to porcine insulin. The insect haemolymph contains as the major form of circulating carbohydrates trehalose. Smaller amounts of sucrose, glucose and other monosaccharides also occur. Our study was designed to determine if administered glibenclamide (18 ~tg/kg) influences carbohydrate levels. The animals are living in a special bee-house under constant conditions. They are anesthesized by CO2 and the drug applied through T E R G U M III using a Hamilton syringe. After 2 h the animals are punctured using a 1 p.1 capillary and sugars of the haemolymph were determined in form of their trimethyl-silylderivatives by means of gaschromatography. The animals showed a dramatic breakdown of concentrations fructose and glucose as early as after a 2.5 m walk. After 7.5 m walk in addition trehalose declined. In vitro 5 brains were perifused according the model of perifused pancreatic islets and stimulated by glibenclamide. Insulin was released with a delay of 30 rain. Conclusion: an insulin-like substance might regulate carbohydrate metabolism in the bee. Glibenclamide is able to release the hormone from brain tissue. 351. Fructose 6-phosphate channelling in normal and tumoral islet cells W. J. Malaisse, F. Malaisse-Lagae and A. Sener. Laboratory of Experimental Medicine, Brussels Free University, Brussels, Belgium In mouse and rat islets, D-fructose is a weak insulin secretagogue, no metabolic explanation being yet available to account for such a situation. The metabolic and secretory responses to D-fructose were compared, therefore, in normal and tumoural islet cells. In rat islets, the secretory response to D-fructose, relative to that evoked by D-glucose, was much higher in the presence of the latter aldose than in islets exposed to D-mannose, non-carbohydrate nutrients or nonnutrient secretagogues. The magnitude of the secretory response was always commensurate, however, with the ketose-induced increase in ATP production. In glucose-deprived islets, fructose 6-phosphate was channelled into the pentose shunt, the ratio of D-(IJ4C)/D-(UJ4C) fructose oxidation averaging 1.44+0.06. In the presence of D-glucose, the latter ratio was decreased to 0.83 _+0.06 and, hence, similar to that found with labelled D-glucose. Likewise, in RINm5F cells, the ratio of D-(1-a4C)/D-(UJ4C)fructoseoxidation averaged 2.74-+0.13 and 1.66 -+ 0.19, respectively, in the absence and presence of D-glucose. In both normal and tumoural islet cells, the apparent Km for Dfructose (4-6 retool/l). These converging data illustrate the participation, in the control of hexose metabolism, of regulatory steps located distally to the site of phosphorylation.

554 A 352. Combined therapy with sulphonylurea (gliclazide) and insulin for Type I (insulin-dependent) diabetic patients: one year follow-up A. Maldonato, D. Bloise, M. Pittaluga, F. Barbetti, F. Marani, O. Falla, P.Gandolfo, P.D'Ottavi, E.Sciullo and F.Fallucca. Diabetes Unit, "La Sapienza" University, Rome, Italy We have recently described that the association of gliclazide (SU) for six months to the usual insulin treatment (INS) in Type 1 (insulin-dependent) diabetic patients was accompanied by a manifold improvement of diabetes control. To evaluate the persistence of such effects, we continued the study for six more months. The same dose of SU (160 mg/24 h) was left in association with INS in 8 Type 1 diabetic volunteers and plasma glucose, other intermediate metabolites, Cpeptide (CPR), glucagon (IRG) and growth hormone (GH) were measured in basal conditions and after arginine challenge (ATT). Moreover, the insulin need, HbAlc, and the erythrocyte filtration index were evaluated. At the end of the 12-month trial, the plasma glucose values were maintained at the 6-month levels (mean: 12.10_+2.21 at time 0 vs increased (deRa area: -0.14 at time 0 vs 0.93 at 6 months vs 1.18 at 6 months. Insulin need and HbAlc were significantly decreased. Red cell deformability was improved. In conclusion, the association of SU to INS in Type 1 diabetic patients induced metabolic, hormonal and haemorheological improvements, and a reduction of insulin need, which persisted after 12 months. 353. Nerve microangiopathy in human diabetic neuropathy of increasing severity: pathogenesis and adaptation R.A. Malik1, P.G. Newrick2, A.K.Ah-See 1, A.K. Sharma 1 and J.D.Ward 2. Department of Anatomy and Surgery, University of Aberdeen1, UK, Diabetic Unit, Royal Hallamshire Hospital, Sheffield2, UK

Abstracts release or potentiate IL-1 action. In conclusion: Only the MO-product TNF potentiates IL-1 mediated B-cell cytotoxicity. This study supports the role of IL-1 and activated macrophages in the pathogenesis of insulin-dependent diabetes. 355. Urinary neopterin level as a marker of autoimmune activity in Type I (insulin-dependent) diabetes mellitus R. Manna, A. Tafuri, G. Papa, M. Salvatore, E Pozzilli, B. Giannattasio, E. Ferrara, A. Cittadini, G. Marietti, A.V. Greco and G. Ghirlanda. Rome, Italy

Urinary neopterin levels have been found significantly increased in many conditions - viral diseases, allograft rejection, autoimmune disorders, etc. - characterised by the activation of T lymphocyte-macrophage axis; to investigate urinary neopterin variations in Type 1 (insulin-dependent) diabetic patients we have studied 18 newly-diagnosed patients, 24 long-lasting diabetic patients ( > 3 months after diagnosis), 9 of which with persistent islet cell antibodies (ICA), 15 ICA negative and 12 healthy control subjects. None of them underwent viral or other infectious diseases during the observation period. Urinary neopterin levels are significantly higher in newly-diagnosed diabetic patients than in control subjects (p< 0.003). Results obtained in longlasting diabetic patients are as follows: while ICA positive patients have neopterin values similar to newly-diagnosed diabetic patients, ICA negative ones show levels markedly lower than diabetic patients at diagnosis (p< 0.003). No significant difference has been found between control subjects and long-lasting ICA negative patients. We speculate that increased neopterin levels in newly-diagnosed diabetic patients are related to activation of T lymphocyte-macrophage axis due to autoimmune insulitis; high neopterin levels in long-lasting ICA positive patients should be determined by the activity of autoaggressive process, exhausted, in contrast, in ICA negative patients.

To determine the role of microangiopathy in the development and progression of diabetic neuropathy, sural nerve endoneurial capillaries were analysed from normal control subjects (n= 6), diabetic patients with mild sensory neuropathy (n= 5) and chronic sensori-motor neuropathy (n= 11), determined from a full clinical, electrophysiological and morphological assessment. All three groups had the same number of capillaries/fascicle. However capillary density was significantly reduced in both groups of neuropaths (p< 0.05) due to an increase in mean fasicular area (p< 0.05) when compared with controls. Both neuropathic groups displayed an increased basement membrane and total diffusion barrier (endothelial cell, pericyte and basement membrane) thickness with a reduced capillary oxygen diffusing capacity. Only in the severe neuropaths was this difference significant (p< 0.005), and they also showed a non-significant trend for reduced endoneurial oxygen levels. Endothelial nuclei/capillary, endothelial cell profiles/capillary, luminal perimeter, endothelial cell external perimeter and vessel perimeter were significantly increased in both groups of neuropaths (p< 0.05). Conclusions: (1) Increased resistance to capillary oxygen diffusion and reduced nerve perfusion exists in diabetic neuropaths. (2)Nerve hypoxia was inferred and demonstrated implicating it in the pathogenesis of diabetic neuropathy. (3) Endothelial cell hyperplasia and small vessel hypertrophy appears to be an adaptive measure to compensate for developing hypoxia, and it appears early in the diabetic state.

356. Relationship between microalbuminuria and diabetic retinopathy D. Manservigi, R.Graziani, F.Dal Pane and A.Zagatti. Department for Diabetes and Metabolic Diseases, St. Anna Hospital, Ferrara, Italy To study the relationship between retinopathy and preclinical nephropathy, microalbuminuria (RIA) was measured in 61 Type 1 (insulin-dependent) diabetic patients (D) (age 28.2_+ 9.8 years, duration of diabetes 15 + 6.2 years) and compared with the presence of retinopathy (assessed by fluorescein angiography). In the 41 D with background retinopathy, microalbuminuria was 153.9_+83.7 mg/24 h, vs 39.5 _+33.2 of those (20 D) with normal fundus (/9< 0.001). Age, duration of diabetes, HbA1 and sisto-diastolic pressure did not differ in the two groups. Microalbuminuric D (15-300 Ixg/min) were then compared with non-microalbuminuricD (15 ~g/min) with regard to retinopathy. Retinopathy was found in 72.4% (21 out of 29) of microalbuminuric D and in 62.5% (20 out of 32) of non-microalbuminuric D (x2=0.303, NS). Here, too, there were no significant differences in age, duration of diabetes, HbA1 and blood pressure between the two groups. In conclusion, background retinopathy is present in statistically significant association with microalbuminuria. However, microalbuminuria does not seem to be an adequately sensitive predictor of retinopathy.

354. Human tumour necrosis factor potentiates interleukin-1 mediated B-cell cytotoxicity T. Mandrup-Poulsen~, K. Bendtzen2, C.A. Dinarello3 and J. NerupL 1Steno Memorial Hospital, Gentofte, 2Laboratory of Medical Immunology, University Hospital of Copenhagen, Denmark, 3Tufts University School of Medicine, Boston, Mass, USA Interleukin-1 (IL-1) is selectively cytotoxic to B cells in vitro. Possible potentiating effects of other mononuclear cell products (cytokines) on the B-cell cytotoxic action of IL-1 have not been studied. The ability of combinations of human recombinant cytokines (the macrophage (MO)-products IL-la, -fl, and tumour necrosis factor (TNF) and the lymphocyte products lymphotoxin (LT) and interferon (7/(IFNT)) to inhibit dose-dependently the glucose stimulated insulin-release from isolated rat islets in 6-day cultures was tested. One hundred pg/ml of IL-lfl inhibited the insulin release by 50%, whereas 1000 pg/ml of ILl a was necessary to obtain a similar effect. 2.5-25 ng/ml of TNF, but not 40 ng/ml of LT or 25 ng/ml IFN~" markedly potentiated the effect of IL-la and @ TNF did not potentiate the lymphocyte activating activity of IL-1. To investigate whether other cytokines than TNF, LT, and IFN~" potentiate IL-1 effects, B-cell cytotoxic crude cytokine preparations (CCP) without TNF were depleted of IL-1 by anti-IL-1 column chromatography. The neutralised CCP did not inhibit insulin-

357. Adoptive transfer of Type I (insulin-dependent) diabetes in human. An unusual complication of bone marrow transplantation D. Maraninchi, B.Vialettes, M.San Marco and M.H.Gaspard. Bone Marrow Transplantation, Diabetology and Immunology Departments, University of Marseille, France Singular case-reports may illuminate pathogeny of Type 1 (insulin-dependent) diabetes. We report an observation of diabetes induced after adoptive transfer of immunocytes. Mrs B., 17 years with relapsing acute lymphoblastic leukemia, received bone marrow graft from her non-diabetic HLA-identical sister (a: Aw 32 B 37 DR, c: AI A8 DR3) after conditioning with cyclophosphamide and total body irradiation. One brother (a/d:Aw33 B27DR3) had Type I diabetes, mother (c/d) hyperthyroidism. Mrs B. developed acute and chronic GVH treated by methotrexate (month 0-month 5); azathioprine (month 5-month 15). Chimerism was confirmed by blood group ((~A). Islet cell antibodies were negative before and after the graft. On and after month 27, they became positive. Glucose tolerance was normal until month 39 when Type 1 diabetes abruptly occurred. Anti-insulin antibodies were undetectable during the entire follow-up. Other anti antibodies were investigated: antithyroid Ab were detected at month 6 (thyroglobulin: 1/5120, microsome 1/600) and thereafter with a progressive decline of titre (month 45 - Tg: 1/40, microsome: 1/100),

Abstracts acetylcholine receptor and stomach Ab were permanently found at low titre, intercellular substance, mitochondrial and smooth muscle Ab (low titre
555 A CT, r= 0.66 and 0.58; vs CSA, r= 0.66 and 0.65; vs E-S, r= 0.56 and 0.48 respectively). Abnormally low values (more than 2 SD of the normal mean) were found by single photon absorptiometry in 17 and 8 patients distally and proximally respectively, while the corresponding numbers were 7 patients for CT, 4 for E-S and 4 for CSA. It is concluded that both techniques of bone mass evaluation are highly correlated in diabetic patients, single photon absorptiometry being more sensitive in determining bone mass reduction. 361. Prevention of clinical diabetic nephropathy by chronic converting enzyme inhibition in patients with persistent microalbuminuria but no hypertension M. Marre ~, H. Leblanc1, J. Menard 2 and Ph. PassaL 1Service de Diabrtologie, H6pital Saint Louis, Paris, France, 2INSERM U36, Paris, France To study if chronic converting enzyme inhibition may prevent clinical diabetic nephrophathy, 20 diabetic patients with persistent microalbuminutia (albumin excretion rate (AER) 30-300 mg/24 h 2-3 times over 3 months) but no hypertension ( < 160-95 mmHg) were randomly allocated by blocks of two to Enalapril 20 mg/day or placebo. After a 3-month inclusion period, treatment lasted one year. Albumin excretion rate and mean arterial pressure (MAP) were measured monthly, and glomerular filtration rate, renal plasma flow, total renal resistances at 0, 6 and 12 months. Clinical diabetic nephropathy was assessed by persistent macroalbuminuria (AER> 300 mg/24 h 2-3 times over 3 months). Enalapril and placebo groups were comparable for MAP (100 + 8 vs 99 + 6 mmHg), AER (median (range): 124 (37-280) m~/24 h vs 81 (32-300) mg/24 h~, glomerular filtration rate (130+23 vs 133+26ml/min per 1.73 m-), and renal plasma flow (526+ 113 vs 597+ 124 ml/min per 1.73 m2). Mean arterial pressure fell with Enalapril to 88+ 5 mmHg throughout treatment, not with placebo: 100+ 8 mmHg (p<0.001). At 12 months, AER on Enalapril was 30 (16-231) mg/24 h, compared to 278 (25-1240) mg/24 h (p< 0.001). Persistent macroalbuminuria occurred in no patient on Enalapril, vs 3 of 10 on placebo (p< 0.05). With Enalapril, glomerular filtration rate increased to 141 +24 ml/min per 1.73 m2 at 6 months (p< 0.005) and remained steady afterwards, while renal plasma flow rose (/7<0.02) and total renal resistances declined with time (p<0.02). Chronic converting enzyme inhibition may modify the course of diabetic nephropathy. 362. The prevalence and correlates of albumin excretion in Type I (insulin-dependent) diabetes S.M.Marshall and D.R.Appleton. Department of Medicine and Medical Statistics, University of Newcastle upon Tyne, UK Diabetic nephropathy is an important cause of morbidity and mortality in Type I (insulin-dependent) diabetes. Albumin excretion rates (AER) of 30-150 lxg/min are predicative of clinical nephropathy. We have investigated the prevalence and correlates of albumin excretion in a clinic population of 416 Type 1 diabetic patients. Albumin was measured by radioimmunoassay in timed overnight urine samples and AER calculated. Mean age of the study population was 45 years (range 14-87), duration of diabetes 18.2 years (1-55), HbA1 10.0% (5.5-19.0); 30% had retinopathy, 7.5% peripheral neuropathy, 6.8% peripheral vascular disease (PVD) and 8.2% ischaemic heart disease. Albumin excretion rate was outside the normal range in 22.4%, 6.7% having microalbuminuria and 4.8% clinical nephropathy. Log AER correlated with duration of diabetes (r=0.21, p < 0.001) and systolic blood pressure (r=0.18, p<0.001) and was higher in males (T=2.45, p<0.02), those with PVD (T=3.49, p<0.001) and with peripheral neuropathy (T=2.51, p<0.02). Stepwise multiple regression analysis showed that only duration of diabetes and presence of retinopathy were significant (multiple r= 0.28, p < 0.001), but these accounted for only a small proportion of the variability in AER. 363. Pirenzepine blunts the nocturnal growth hormone release in Type 1 (insulin-dependent) diabetes mellitns V. Martina, M. Tagliabue, M. Maccario, P. Corno, E. Ghigo and F. Camanni. Dipartimento di Biomedicina Endocrinometabolica e Gastroenterologica, Universit~i di Torino, Italy Since antagonists of cholinergic muscarinic receptor were shown to abolish spontaneous and induced growth hromone (GH) increases in normal subjects and GH is implicated in some diabetic complications, we investigated whether nocturnal GH release in diabetic patients is inhibited by pirenzepine, a muscarinic antagonist. Eight patients with Type 1 (insulin-dependent) diabetes, made euglycaemic by Biostator and then infused overnight with 0 . 1 5 m U - k g -1. r a i n - 1 ,

556 A were studied from 23.00 to 8.00 hours during saline or pirenzepine infusion. Furthermore, 5 out of 8 patients were studied during oral treatment with pirenzepine 100 m g a day for 30 days. GH nocturnal secretion was studied on 1st and 30th day of treatment. When compared with saline infusion a marked reduction of nocturnal GH secretion was observed during intravenous pirenzepine infusion (area under curve, mean_+ SEM: 128.4_+ 35.4 vs 363.5 _+63.6 ng. mg-a- h-1; p<0.02) and after oral pirenzepine treatment both on 1st and 30th day (126.3+28.1 and 187.0-+38.3 vs 299.7+66.6 ng-ml-~-h -~, p < 0.005 and p < 0.05 respectively). These data clearly show that muscarinic blockade blunts the nocturnal GH secretion in Type 1 diabetes and this inhibitory effect is maintained during prolonged oral treatment. 364. Reduced urinary excretion of epidermal growth factor in incipient and overt diabetic nephropathy E.R. Mathiesen, E. Nexo, E. Hommel and H.-H. Parving. Hvidrre Hospital, Klampenborg and Department of Clinical Chemistry, Central Hospital, Hillerrd, Denmark To study the relationship between glomerular and tubular function we investigated urinary albumin excretion, glomerular filtration rate (GFR), and urinary excretion of epidermal growth factor (EGF, a mitogenic peptide synthesised in the renal tubular cells) in normal subjects (group I, n= 7) and in the following groups of Type 1 (insulindependent) diabetic patients: diabetic patients with normal albumin excretion rate (group II, n= 11); with incipient diabetic nephropathy (III, n= 11); with overt diabetic nephropathy and normal GFR (IV, n= 12); with reduced GFR (V, n= 10). Epidermal growth factor values are given in nmol/24 h, GFR values in ml/min per 1.73 m 2. P-values denote significant difference from the control group. Group I EGF: 7.9 (4.1-10.5), GFR: 100 (87-116); II - EGF: 6.7 (1.3-9.2), GFR: 129 (111-150); III - EGF: 5.0 (3.6-7.4), p<0.02, GFR: 131 (95-136); IV - EGF: 4.1 (2.5-15.1), p<0.10, GFR: 116 (90-132); V EGF: 1.4 (0.8-5.3), p<0.01, GFR: 39 (13-50). There was a significant correlation between the excretion of EGF and glomerular filtration rate, p < 0.001, and a negative correlation between the urinary excretion of EGF and albumin, p < 0.01. Our study demonstrates that urinary excretion of epidermal growth factor diminishes with increasing glomerular involvement, and that renal tubular function (EGF) is reduced early in the development of diabetic renal disease. 365. Low acute insulin response, a sensitive but non-specific marker of early stages of Type I (insulin-dependent) diabetes C.Mattei-Zevacco, B. Vialettes, G. Ramahandridona, C.Badier, V. Lassmann and Ph.Vague. Department of Diabetology, University of Marseille, France Low acute insulin response after IV glucose (LAIR) is an early marker of Type 1 (insulin-dependent) diabetes and could facilitate selection of genetically susceptible glucose tolerant or mildly hyperglycaemic subjects for etiological treatment. But LAIR may also characterise Type 2 (non-insulin-dependent) diabetes. Acute insulin response (sum of plasma insulin at 2 and 5 rain) after an intravenous glucose load (0.3 g/kg) was assessed in young ( < 40 years) non-diabetic control subjects (n=68, 22.9-+3 years), first degree relatives of either Type 1 diabetic patients (n=90, 17.5 _+0.9 years) or Type 2 diabetic patients (n=31, 20-+2years) and 38mildly hyperglycaemic ( < FBG < ) diabetic patients. Among diabetic patients, 8 developed insulin dependency (pre Type 1), 3 (ICA positive) and 27 (ICA negative) remained non-insulin-dependent. According to insulin response of control subjects (121 + 7.6 l.tU/ml, 51-300) LAIR was defined by a response < 50 ktU/ml. Insulin response of Type 1 relatives was similar (117.7+7.5 ~tU/ml)but 10/90 exibited LAIR. Among 10positive ICA subjects, one had LAIR and became diabetic. In Type 2 relatives, response was lower (101.6+10.3 ~tU/ml, p<0.01) and LAIR found in 2/31. Among diabetic patients, LAIR was constantly found in "pre-Type 1" (8/8, 34.4+ 2.4 ~tU/ml) and "positive ICA-Type 2" (3/3, 37.3 -+ 3.4 ~U/ml) it was also observed while less frequently in 11/27 "negative ICA-Type 2". Individually LAIR is sensitive but non specific of early stages of Type I diabetes as present in subjects at risk for or suffering from Type 2 diabetes. It must be interpreted in conjunction with immunological markers. 366. Cycloheximide decreases glucose transporters in rat adipocyte plasma membranes without affecting insulin-stimulated glucose transport S. Matthaei,t E. Karnieli 2 and J.M.Olefsky.2 1Department of Medicine, University of Hamburg, FRG; 2Department of Medicine, UCSD, La Jolla, Calif, USA

Abstracts This study describes the effects of cycloheximide (C) on glucose transport (GT) and glucose transporters (GTer) in rat adipocyte subcellular membrane fractions. Isolated adipocytes were incubated under the following conditions: 1)basal (B), cells were incubated for 90 rain at 37 ~ with no additions, 2) insulin (I), as above for 60 min then I for 30 min, and 3) C, cells were incubated with C for 60 min and then with I for 30 min. C had no effect on I-stimulated GT-activity, measured by 3-O-methyl-glucose uptake, which was 9-fold increased over B (Vm~x B : I : C = 9 : 83 : 77, n = 3). The affinity didn't change either (Km B: 5.9; I: 6.3; C: 6.1). In complete contrast, when subcellular membranes, plasma membranes (PM) and low-density microsomes (LDM) were prepared from fat cells and assayed for GTer, C blocked the I-induced increase in GTer in the PM: BPM 8, IPM 23 and CPM 12 (pmol/mg membrane protein), C also decreased GTer numbers in LDM by around 35%, BLDM 44, ILDM 23, CLDM 15 (pmol/mg protein). In conclusion: (1) C neither affected Vm,x nor Km of I-stimulated 3-O-methyl glucose uptake. (2) C decreases GTer numbers in PM and LDM derived from isolated rat adipocytes. (3)The observed discrepancy between I-stimulated GT and I-induced translocation of GTer may indicate that a still unknown mechanism is involved in I action on glucose transport. 367. Participation of Type t (insulin-dependent) diabetic patients in an intensive diabetes treatment and teaching programme immediately after manifestation of the disease G.Mauricio L., M. Bockholt, D. Hemmann, J. Grtindler, K. Haas, I. M~ihlhauser and M. Bergen Department of Metabolism and Nutrition, University of Diisseldorf, FRG At this department, all newly-diagnosed Type 1 (insulin-dependent) diabetic patients participate in a 5-day in-patient diabetes treatment and teaching programme (DTTP), in groups together with patients with long standing diabetes. The DTFP aims at normalisation of glycaemia and is based upon intensified insulin therapy. After discharge from the hospital, the patients are treated by their family physicians. In order to evaluate the efficacy of this procedure, 56 consecutively admitted newly-diagnosed Type I diabetic patients (age 15-40 years, 26 males, no prior insulin therapy) were included in a prospective study. Initial HbAlc (normal _<5.6%) was 9.2 + 1.8%, body mass index 20-+_2kg/m 2, and daily insulin dosage 0.56+0.33 U/kg body weight (after metabolic restabilisation). After a median of 18 (8-40) months, 55 patients were re-examined: the median HbAlc level was 6.5%; 29% of the patients had normal HbAlc levels. Three patients had experienced one severe hypoglycaemia episode each (incidence rate 0.03 per patient per year). Medium random C-peptide level was 0.95 ng/ml. Eighty-two % of the patients had C-peptide levels > 0.3 ng/ml. Body mass index was 22_+2 and daily insulin dosage 0.50_+0.27. Ten patients injected <0.25 U / k g body weight (2 patients, no insulin); median HbAlc level of these 10 patients was 5.7 (4.7-7.1) %. Conclusions: these data prove that excellent degrees of glycaemic control can be achieved in the majority of newly-manifest Type 1 diabetic patients by immediately subjecting the patients to an intensive DTTP. Any potentially hazardous therapy, such as immunosuppressive intervention, must be judged against such standards. 368. Effects of angiotensin converting enzyme inhibition on diabetic hypertension J. C. Mbanya, T. H. Thomas, R. Taylor, R: Wilkinson and K. G. M. M. Atberti. Department of Medicine, University of Newcastle upon Tyne, NE2 4HH and Freeman Hospital, Newcastle upon Tyne, U K The mechanism of diabetic hypertension has been investigated by comparing the hormonal, renal and electrolyte status of normotensive and hypertensive (HT) Type 2 (non-insulin-dependent) diabetic patients with essential hypertensives (EH) and by studying the effect of treatment with captopril on blood pressure and hormonal state. Twelve HT Type 2 patients with normal renal function were compared with 10 age- and weight-matched normotensive Type 2 patients and 8 untreated EH. Both Type 2 groups had reduced plasma renin activity compared to EH: EH 9.2 + 2.3 vs normotensive Type 2 6.2 -+ 1.4, p < 0.01 ; HT Type 2 5.3 + 2.3 pmol/l/min, p < 0.01. Exchangeable sodium was lower in HT Type 2 patients than in EH (46.6-+3.12 vs 53.5 + 1.1 mmol/kg LBM, p < 0.05), but in normotensive Type 2 patients was not different from HT Type 2 patients or EH. Fractional lithium clearance was significantly lower in the HT than in the normotensive Type 2 patients (0.185 _+0.014 vs 0.253 -4-0.016, p < 0.01). Other parameters were similar in the 3 groups. In HT Type 2 patients after 8 weeks captopril, plasma angiotensin II was reduced (2.0 +_0.14 to 1.4+_0.29pmol/1, p<0.001) and aldosterone increased from 270 + 3.2 to 352 ___4.1 pmol/1, p < 0.0001. Recumbent blood pressure

Abstracts fell from 166+_3.4/99+1.0 to 144_+2.0/83 _+2.0 mmHg, p<0.001. Blood glucose and insulin responses to oral glucose, and microalbuminutia did not change although glycosylated haemoglobin fell from 9.0_+ 0.36 to 8.1 +_0.3%, p < 0.01. HT Type 2 differ from EH having reduced plasma renin, exchangeable sodium and increased proximal sodium reabsorption. Captopril reduced blood pressure effectively with no adverse effects. 369. Clinical application of a screening protocol for gestational diabetes E. Meriggi~, G.F.Trossarelli~, G. Menato 1, M.A. Porta 1, R. Bordon 1, G. L. Gregoti 1, G. Molina1, Q. Carta 2 and A.Vitelli2. qnstitute of Obstettics and Gynaecology, University of Turin, Italy, 2Antidiabetic Center, San Giovanni Hospital, Turin, Italy The study goal was the evaluation of gestational diabetes screening protocol efficacy and the comparison of venous plasma (GOD/POD method) and capillary blood (Reflocheck Boehringer Mannheim, FRG) glycaemic values. Three-hundred and sixty pregnant women, with and without gestational diabetes risk factors, underwent at 12th-16th and 24th-28th week 50g oral glucose challenge test (OGCT) (0 and 60 glycaemia). A subsequent diagnostic 3-h oral glucose tolerance test (OGTY) (according to National Diabetes Data Group) was performed on patients with abnormal OGCT (1-h plasmatic glycaemia >_7.46 mmol/1). OGCT was again performed on 95 women with risk factors, positive OGCT but negative OGTT, between 24th and 28th week. One hundred twenty-two women (26.8%) showed positive OGCT; 20 of them positive OGqT (4.4%), according to literature. One-hour capillary glycaemia values, obtained by reflectance-meter, had good correlation to those obtained on venous plasma (p<0.001). 92.4% of the subjects presented glucose capillary blood values higher than plasma ones. We evaluated OGTT diagnostic accuracy, for 7.46-8.29 mol/1 glycaemic thresholds, and plasmatic and capillary optimal glycaemic threshold by receiver operator characteristic curve. Cut-off point for positivity was >_7.73 mol/1. As for blood glucose-meter values, OGCT threshold should be fixed at >_ 8.84 tool/1 being sensitivity and specificity values equivalent to those obtained with >_7.73 mol/1 plasmatic glycaemia. 370. An ultrastructural and morphometric study of A and D cells in the endocrine pancreas of bio-breeding Wistar and streptozocin-treated Wistar rats (a comparative study) V. Mezzogiorno and G. Papaccio. Department of Anatomy; I School of Medicine, University of Naples, Italy We observed the ultrastructural changes involving A and D cells of bio-breeding Wistar and streptozocin-treated Wistar rat pancreas. Diabetes was ascertained after determination of glycaemia and glycosuria levels and after urinary C-peptide excretion evaluations. Rat pancreases were removed and processed for transmission electron microscopy. Micrographs were subsequently analysed at a M.O.P. Videoplan videoanalyzer (Kontron-K.Zeiss-W.G.). Results showed that: a)in the bio-breeding Wistar rats D cells decrease in number and volume significantly (t7< 0.01), their cytoplasm showing vacuolated areas. In some cases one can observe "mixed" cells with different types of granules; A cells appeared unmodified with regard to numbers and volume; their cytoplasm bears evidence of numerous granules and a rich endoplasmic reticulum; b) in the streptozocin-treated Wistar rats D cells showed a notable increase in number and volume (/7< 0.001); granules fill their cytoplasm; A cell numbers, however, increased slightly. In conclusion, our observations give further evidence that changes affecting A and cells in spontaneous diabetes differ to those observed in the streptozocin-induced type. 371. Risk of diabetic nephropathy in Type I (insulin-dependent) diabetic patients with and without retinopathy R. Miccoli, O. Giampietro, G. Odello, G. Penno, L. Di Palma, R. Anichini, A. Clerico, G. Gregori and R. Navalesi. Cattedra di Malattie del Ricambio, Istituto di Clinica Medica II, Istituto di Fisiologia Clinica del CNR, University of Pisa, Italy While diabetic retinopathy (DR) is frequently associated with clinical nephropathy, little is known about its correlation with microalbuminuria (MA) in Type 1 (insulin-dependent) diabetic patients. We studied the prevalence of MA (Albumin Excretion Rate - AER - > 15 ~g/ min; RIA) and the correlation with DR (fluorangiography) in 113 A1bustix-negative Type 1 patients (age 31 +_13 years, disease duration (DD) 11 +7 years). DR was more frequent than MA (40% vs 22%). Patients with DR (n = 45) had higher age and DD than those with Absent Retinopathy (AI~ n=68; p<0.05). DD correlated with DR severity (p< 0.01) but not with MA. Blood pressure, creatinine clear-

557 A ance and HbAlc were superimposable in patients with AR, background retinopathy (BI~ n=31), proliferative retinopathy (PR, n = 14). The rate of MA increased with the severity of DR (p< 0.05); however, 15% of patients with AR or BR showed MA and 29% of those with PR had AER <15 ~tg/min. Among 16 patients with both DR and MA, 14 had more than 10 years of DD. 1) Among diabetic patients with retinopathy, a sub-group has increased risk of nephropathy; 2) after 10 years of DD, MA may predict microangiopathy even in non-renal districts (predictive positive value for DR: 76%); 3)microalbuminuria requires accurate detection and tight control of other risk factors for microangiopathy. 372. Epidemiological trends of diabetes duration in the diabetic population of the G. D. R. D.Michaelis and E.Jutzi. Central Institute for Diabetes "Gerhardt Katsch", Karisburg, GDR Based upon the National Diabetes Registry, which yearly records all known, all newly-diagnosed and deceased diabetic patients according to sex, age, and nature of treatment, diabetes prevalence has risen 5.3-fold between 1961 and 1985. In order to prove whether a prolongation of diabetes duration has contributed to this increase, its change was estimated over a 25-year time period by calculating the difference between mean age at diabetes onset and death. The rise of the diabetes incidence from 148/10 s to 374/105 was associated with an increase of the mean incidence age from 60 to 63 years, that of age at death from 69 to 73 years. It implies an enhancement of the overall diabetes duration by 1 year only. But taking into consideration diabetes type related differences, insulin-treated diabetic patients exhibited a time interval between manifestation and death of 14 years in 1961 and of 25 years in 1985. In Type 2 (non-insulin-dependent) diabetic patients this difference remained constant at 9 years. Although registered data do not permit an estimation of the "true" diabetes duration, the population-based results support the view of a prolongation of the survival time after diabetes onset during the past 25 years, especially in the insulin-treated diabetic patients. 373. Efficacy of the glucosidase inhibitor acarbose compared to the sulphonylnrea glisoxepid on metabolic control of Type 2 (non-insulin-dependent) diabetes R. Mies and M. Spengler. Department of Internal Medicine, Trinity Hospital, Wesseling and Bayer AG, Pharma Research Centre, Wuppertal, FRG a-glucosidase inhibition is a new principle in the treatment of diabetes mellitus. In order to compare this new principle with the well-established sulphonylurea therapy of Type 2 (non-insulin-dependent) diabetes, we treated 20 Type 2 diabetic patients with the a-glucosidase inhibitor acarbose or the sulphonylurea glisoxepid in a double dummy double-blind cross over study with 4 weeks drug-free pretreatment, 2 treatment periods of 8 weeks, with 4 weeks wash out. Dosage/day: acarbose 3 x 50 mg in week 1, 3 x 100 mg in week 2, 3 x200 mg in week 3-8, glisoxepid 2 x4 mg. Nine male and 11 female patients were included, age 62.4 + 9.0 years, BMI 25.7 + 1.6, duration of diabetes 6.7 _+3.5 years. Results: prevalues blood glucose (mmol/1) fasting (1)7.5+_0.5, l h pp (2) 10.7+_1.0, HbA1 (%) (3)9.3+_0.6; after 8weeks glisoxepid (1)6.9+_0.5, (2)8.9+_0.7, (3)9.0+0.5; after 8weeks acarbose (1)7.2+_0.6, (2)8.9_+1.2, (3) 9.1 +_0.6; after wash out (1) 7.8+_0.8, (2) 10.9+0.8, (3) 9.1 +0.6. Eight weeks treatment with glisoxepid and acarbose improved (1), (2) and the pp blood glucose increase significantly to the same extent; (3) stayed unchanged. Under glisoxepid 2patients (10%) reported moderate gastrointestinal side-effects, under acarbose 6 (30%) reported mild to moderate effects. The results indicate that glisoxepid and acarbose have the same efficacy (no significant difference for (1) and (2) between the treatments) on metabolic control of Type 2 diabetic patients after 8 weeks treatment. 374. Dose response relationship between plasma glucose and pancreatic insulin secretion K. J. Mikines, P.A. Farrell, B. Sonne, B.Tronier and H. Galbo. Dept. Med. Physiol.B, Panum Inst., Univ. of Copenhagen; NOVO Research Inst., Bagsvzerd, Denmark; and Univ. Wisconsin-Milwaukee, Wis, USA A sigmoidal relationship between insulin and glucose concentrations has been found during brief periods of glucose stimulation. To characterise the B-cell response to prolonged glucose stimulation in 7 young healthy men, we performed 4 sequential hyperglycaemic 90 minute clamps at plasma glucose concentrations of 7, 11, 20 and

558 A 35 mmol/1. Concentrations of insulin, C-peptide and proinsulin were measured in arterialised hand vein blood. We found A) a progressive increase in insulin concentrations with increasing glucose levels. Very high insulin levels were reached (1092 + 135 ~U/ml). B) A linear relationship between glucose concentrations and concentrations of Cpeptide (r=0.931+0.008) and proinsulin (r=0.952_+0.009). C)At high insulin concentrations insulin clearance, estimated from C-peptide/insulin ratios had decreased 5-fold. Conclusions: the B cell has a very large secretory potential and supra maximal plasma insulin levels may be found. In contrast to findings during brief glucose stimulation, B-cell secretion responds linearly to prolonged graded hyperglycaemia. The processing of insulin is essentially unchanged during prolonged B-cell stimulation. Insulin clearance decreases markedly at high secretion rates.

375. Production of interleukin-2 by lymphocytes from patients with Type I (insulin-dependen0 diabetes mellitus Z. Mili~evi6, J.Gabrilovac, K. Paveli6, M. Grani6, Z.Skrabalo and S.Marugi6. Vuk Vrhovac Institute of Diabetes, Endocrinology and Metabolic Diseases, Zagreb School of Medicine, University of Zagreb, Yugoslavia Frequent infections in diabetic patients indicate that their immune systems are often compromised. However, it is not clear whether this is an intrinsic feature of the disease, or a secondary complication. Normal production of interleukin-2 (IL-2) by T-lymphocytes is essential for both cellular and humoral immune response. We, therefore, investigated whether the production of IL-2 is normal in patients with Type 1 (insulin-dependent) diabetes mellitus. Nine newly-diagnosed patients and 8 patients treated for more than 15 years were compared with their age- and sex-matched controls for IL-2 production upon concavalin A (conA) stimulation of peripheral blood lymphocytes. Upon stimulation with an optimal concentration of conA (10 ~g/ml), the diabetic patients' lymphocytes produced normal amounts of IL-2. IL-2 production was significantly lower after the lymphocytes had been stimulated with limited concentrations of conA (0.5 gg/ml and 1 gg/ml); i.e. 3H-thymidine incorporation into the DNA of IL-2 dependent cultured T-cells was less than 50% with preparation of IL-2 of diabetic patients compared with control subjects. The same results were observed in both newly-diagnosed and long-term-treated diabetic patients. We suppose that the lymphocytes of diabetic patients have a higher trigger-dose for IL-2 production, resulting in decreased synthesis of this lymphokine upon weak immunological stimuli.

376. Miglitol as an additive to glibenclamide and chlorpropamide in the treatment of Type 2 (non-insulin-dependent) diabetic patients during a 12-week period. Double-blind comparison with placebo I. Militia, V. Profozi6, M.Grani6, H. Schultz, I.Hillebrandt and Z. Skrabalo. Vuk Vrhovac Institute of Diabetes, Endocrinology and Metabolic Diseases, School of Medicine University of Zagreb, Yugoslavia A total of 41 Type 2 (non-insulin-dependent) diabetic patients were treated with Miglitol or a placebo addition to the sulphonylurea therapy (Group 1: glibenclamide, n=21; Group 2: chlorpropamide) in a double-blind comparison over a period of 12 weeks. This was followed by a 4-week follow-up period (placebo). Under the basic therapy of glibenclamide (5-20 mg/day) and Miglitol (300-600 rag/day), there was a marked reduction in the morning postprandial rise in blood glucose in comparison with the placebo by the 12th week of treatment (placebo subgroup - pretreatment: + 5.8 + 1 mmol/1; week 12: +3.7+2.7 mmol/1; Miglitol subgroup - pretreatment: + 3.8 + 2.4 mmol/1; week 12: + 2.1 + 3.2 mmol/1). The corresponding postprandial rises under the basic therapy of chlorpropamide (125-375 rag/day) and Miglitol (150-600 rag/day) were - placebo subgroup: pretreatment: + 4.4_ 1.6 mmol/1; week 12: + 2.0 + 2.3 mmol/1; Miglitol subgroup: pretreatment +4.8_+ 2.7 mmol/1; week 12: + 1.0_+ 1.6 mmol/1. The values rose again in the follow-up period (placebo) in both main groups. There was no identifiable effect of the treatment with Miglitol on mean insulin levels. Body weight remained unchanged. Intestinal side-effects occurred in 42% of cases in the glibenclamide group and in 92% of cases in the chlorpropamide group. Objective tolerance was good. Miglitol as a supplement to both the glibenclamide and the chlorpropamide therapy led to a marked reduction in the postprandial rises in blood glucose in Type 2 diabetic patients.

Abstracts 377. Two year follow-up of the Bucharest-Diisseldorf study: effect of an intensive treatment and teaching program for Type 1 (insulin-dependent) diabetes on metabolic control and other parameters of diabetes care I.Mincu, I. Miihlhauser, I. Bruckner, D.Cheta, C.Ionescu-Tirgoviste, V. Scholz, V.J6rgens and M.Berger. Departments of Nutrition and Metabolism, Universities Bucharest, Rumania, and DOsseldorf, FRG

The study presents a prospective controlled trial on 300 Type I (insulin-dependent) diabetic patients carried out in the centralised health care system of Bucharest. One hundred patients (Group A, HbA1 12.5%) were treated according to standard therapy (individual-teaching, strict diet, no metabolic-self-monitoring) and followed for I year. Thereafter Group A participated in an intensive diabetes treatment and teaching program (DTFP, 5-day in-patient group-teaching, blood-glucose-self-monitoring, adaptation of insulin therapy by the patient, liberalised diet) and was followed for a 2nd year; 100 patients (Group B, HbA1 12.3%) participated in the intensive DTFP at the beginning of the study and were followed for 2 years; and 100 patients (Group C, HbA1 11.7%) participated in a "basic" DTTP (4-day group-teaching, self-monitoring of glucosuria and acetonuria) and were followed for 1 year. Mean HbA1 remained unchanged after standard treatment (12.8%), but decreased during intensified therapy (Group A: 10.1% at 24 months; Group B: 9.3% at 12 months, 9.5% at 24 months, p<0.0001). In Group C, no change was found compared to standard treatment. During standard therapy 6 patients experienced severe hypoglycaemia, during intensified therapy 3 patients (Group A), 12 patients (Group B, 1st year) and 4 patients (Group B, 2nd year), and 5 patients in Group C. Incidence rates of ketoacidosis were 0.16 episodes/patient/year during standard treatment, 0.01 during intensified treatment (p<0.01) and 0.04 in Group C (p<0.025). These data demonstrate the efficacy of an intensive D T f P to substantially lower HbA1 levels without increasing the risk of severe hypoglycaemia and to almost eliminate ketoacidosis. 378. Degradation of A14 and B26 t25I-Monoiodoinsulin by an insulindegrading enzyme isolated from human erythrocytes R. I. Misbin and E.C. Almira. University of Florida School of Medicine, Gainesville, Fla, USA

Insulin-degrading enzyme was isolated from human erythrocytes and purified to homogeneity. Insulin degradation was studied using A14 and B26 ~2SI-monoiodoinsulin. Using either label, a peptide was observed early in the incubation which eluted after insulin from Sephadex G50 with an apparent molecular weight of 2500. As the incubation proceeded the height of this peak decreased and final degradation products were observed. Using A14 l:sI-insulin a degradation product was observed with an apparent molecular weight of 1500 which was precipitable with 5% trichloroacetic acid but which had little or no immunoreactivity or receptor binding activity. Reduction of this material yielded a labelled peptide with apparent molecular weight less than 1000. Using B26 125I-insulin a degradation product was observed with an apparent molecular weight of 1000 whose elution pattern was not changed by reduction. This fragment was not precipitated by 5% trichloroacetic acid and had little or no immunoreactivity or receptor binding activity. We conclude that this human erythrocyte enzyme degrades insulin through an intermediate to two peptides, one of which is composed of the carbosy-terminus of the B chain and the other of which is composed of portions of both A and B chains. 379. Mononuclear blood cells: a model for the study of enzyme activities of glucose metabolism in man P. Moghetti, M. Querena, E. Bonora, V.Vacciatori, M. Cigolini and M. Muggeo. Chair of Metabolic Diseases and Institute of Clinical Medicine, University of Verona, Italy

To evaluate whether enzyme activities in mononuclear blood cells (lymphomonocytes) reflect those of the insulin target tissues, hexokinase, glucose-6-phosphate dehydrogenase, gluconate-6-phosphate dehydrogenase, and phosphofructokinase activities were measured in lymphomonocytes and in adipose tissue from 15 subjects. In addition, the same enzyme activities were studied in lymphomonocytes from 42 obese (24 non-diabetic, 18 diabetic) patients, and 27 control subjects. In 20 of these subjects 12sI-insulin binding to monocytes was also measured. Hypogastric subcutaneous adipose tissue samples were taken by needle biopsy, while lymphomonocytes were separated from 80 ml of blood. Cells were homogenised and centrifuged, and the supernatant was used for enzyme assays. A statistically significant relationship was found between hexokinase (r= 0.53, p < 0.05), gluconate-6-phosphate dehydrogenase (r=0.72, p<0.01), and phospho-

Abstracts fructokinase (r=0.85, p<0.01) activities measured in adipose tissue and in lymphomonocytes. As previously demonstrated in insulin target tissues, also in lymphomonocytes hexokinase and gluconate6-phosphate dehydrogenase activities were higher (p< 0.025) in nondiabetic obese patients than in diabetic obese subjects and control subjects. In lymphomonocytes hexokinase activity correlated with maximum insulin binding (r= 0.56, p < 0.05). These data suggest that lymphomonocytes may be suitable as a model for studying glycometabolic enzyme activities in man. 380. Incidence of Type 1 (insulin-dependen 0 diabetes mellitus in Denmark - evidence of secular trend

A. G. Molbak1, K. Norgaard 1, B. Christau 2, M. Kja~r3 and J. NerupL 1Steno Memorial Hospital, 2KAS Glostrnp, Dept. of Medicine, Glostrup, 3Holb0ek Centralsygehus, Dept. of Medicine, Holbaek, Denmark This retrospective epidemiological study was designed to estimate the incidence of Type 1 (insulin-dependent) diabetes mellitus for the period 1980-84 in the age group 0-29 years, and to compare it with results from a similar study from the period 1970-74 describing the see ular changes in the incidence of Type 1 diabetes mellitus in Denmark. The incidence was estimated for Caucasians of Danish ethnical origin and for the whole population. North Sealand was chosen as research area (606,000 persons at risk, 28% of the Danish population between 0 and 29 years). Cases were identified through the Danish Central Diagnosis for Hospital Admissions. Ascertainment was qualified by several independent methods. Four hundred sixty-two persons were included in the study (282M, 180F) giving an incidence of 16.4/100,000/year (males 19.7, females 13.0/100,000/year). Male/female ratio was 1.5. If patients of non-Danish origin were included the incidence was 15.6/100,000/year. Degree of ascertainment >95%. (The incidence of insulin-dependent diabetes mellitus in 1970-74 was found to be 13.2/100,000/year. Male/female ratio 1.22). Thus, incidence has increased significantly (p<0.01), mainly due to an excess of male cases in 1980-81 in the age group 15-29 to levels of 25/100,000/year. The occurrence of such incidence peaks points towards influences of environmental factors on the incidence of Type 1 diabetes mellitus. 381. HLA-DR 2 positive individuals demonstrate low interleukin-I secretion of lipopolysaccharide stimulated monocyte cultures

J. Molvig, L. B~ek, P. Platz, P. Christensen and J. Nerup. Steno Memorial Hospital, Gentofte, and Endotoxin Laboratory and Tissue Typing Laboratory, University Hospital of Copenhagen, and University Institute for Experimental Medicine, Copenhagen, Denmark Intefleukln 1 (IL-1) secretion from monocytes (MO), macrophages or natural killer cells in the insulitis process of Type 1 (insulin-dependent) diabetes may cause destruction of the insulin-producing B cells. It has been hypothesised that the HLA-DR association of Type 1 diabetes may reflect HLA-linked differences in the IL-1 production. To look for the existence of interindividual differences in MO IL-1 production, lipopolysaccharide (LPS) stimulated MO-cultures from 12 healthy men were prepared. Determination of IL-1 content in the culture-supernatants were performed by the mouse lymphocyte activation factor assay. Three individuals, of whom two were DR 1/2 positive, demonstrated low IL-1 responses. Three DR-identical family members to one of the low-responders demonstrated similarly low levels of MO IL-1 production. MO-cultures from 4 DR-different men were prepared simultaneously 4 times with one week's interval. Stable individual patterns of reactivity were observed. The only DR 2/2 positive individual demonstrated a very low IL-1 response. Conclusion: stable interindividual differences in extracellular production of IL-1 in LPS stimulated human MO-cultures were observed. DR 2 positive individuals demonstrate low IL-1 responses. The low relative risk of contracting Type 1 diabetes for DR 2 positive individuals may be associated with this phenomenon. 382. Effect of thymic hormone and azathioprine administration on remission rate and insular function of 35 recent diagnosed Type i (insulindependent) diabetic patients. A one-year follow-up study

E. Moncada, M.L. Subira, J.J. Barberia, A. Sanchez-Ibarrola, J. Salvador, F. Gofii, A. Oleaga and I. Cano. Department of Endocrinology. Metabolic Unit. University Clinic. University of Navarra, Pamplona, Spain Thymic hormones have been reported atoxic and useful in the treatment of several immunodeficiencies probably by modulating T-lymphocyte response. Increase in remission rate with immunosuppression and deficiency of T-lymphocyte suppressor activity in Type 1

559 A (insulin-dependent) diabetic patients have been documented. A trial with thymic hormone (TP-1 ; 1.5 mg/kg twice a week) and Azathioprine (2.5 mg-kg -1. day -c) was started in combined and separate administration in three similar groups of recent diagnosed Type 1 diabetic patients. Rate (R. R.) and length (R. L.) of remission, serial glucagon stimulated C-peptide levels and immunological data (inhibition of plaque forming cells by patient's lymphocytes stimulated with Con.A and 7/-IFN), were assessed. Remission was defined by mean weekly glycaemic profile <7.0 mmol/1 and insulin requirements of 0.1 units/kg for not less than two consecutive months. Eleven out of 13 patients treated with TP-1 plus Azathioprine went into remission (R.R: 73.3%; R.L: 6.7+3.3 months) vs 2 out of 10 patients on Azathioprine alone (R. R: 20%; K L: 2.7 _+0.25 months) and vs 2 out of 12cases on TP-1 alone (R.R: 16.6%; R.L: 2.2+ 0.2 months). C-peptide levels of combined group were higher than Azathioprine group (0.65+0.36pmol/ml vs 0.31+0.27pmol/ml p < 0.025) and group on TP-1 (0.65+_0.36pmol/ml vs 0.43_+ 0.23 pmol/ml, p < 0.05) at nine months of observation. Immunoanalysis suggested defective suppressor activity in all groups. Only mild and transient leucopoenia in 20% of the patients on Azathioprine alone was detected. 383. The "Parcours du Diab": the contribution of game-playing and group participation in the education of diabetic patients

k Monvoisin, J. R. Attali, C. Quinio, P. Valensi and J. Sebaoun. Unit~ de Diab6tologie, H6pital Avicenne, Universit6 Paris-Nord, Bobigny, France Teaching techniques used in educating diabetic patients have not given proof of their effectiveness, as patients' everyday behaviour is too often poorly adapted despite satisfactory theoretical knowledge. After a classic educational course a game based on "snakes and ladders", a board game played with dice, was proposed to 40 non-obese diabetic patients (27 M, 13 F); 35 were Type 1 (insulin-dependent) diabetic patients, 27 of French origin and 13 foreign, mainly from North-Africa; the mean age was 45 years (range: 15-76 years); there were 12 recorded sessions with 3 to 5 patients and a member of the medical staff. The "Parcours de Diab" elicited active participation on the patients' part as well as lively discussions on the entire range of theoretics and practical knowledge. The sessions in which recently-diagnosed diabetic patients participated made the whole group more dynamic. Thirty-two patients were re-assessed 24 __1.6 months (SEM) after playing the game. Metabolic control had significantly improved (HbAlc=7.8-+0.33% vs 10.1_+0.52%, p<0.05); knowledge and behaviour, assessed by the attending physician according to the patient's notebook and regular consultations, significantly improved more often when participation in the game-playing had been active than when it had been mediocre or poor (17 cases/18 vs 5 cases~14, Z2= 12.672, p < 0.001). This study shows the usefulness of incorporating group game-playing into diabetic education programmes. 384. Urinary IgG4: an additional parameter in characterising patients with stage III diabetic nephropathy

S. Morano, U. Di Mario, A. Cancelli, S. Frontoni, S. Bacci, M. Mancuso, P. Pietravalle, S. Gambardella and D. Andreani. Dept. of Endocrinology, University of Rome, Italy In the natural history of diabetic nephropathy an important aspect is the progressive impairment of protein permselectivity with the initial loss of charge selectivity and then of size selectivity. IgG4 has the same molecular weight of the other immunoglobulin subclasses but has an isoelectric point of about 5. Its possible selective urinary elimination could indicate a charge selectivity impairment in the so-called pre-clinical stage III diabetic nephropathy. To verify this hypothesis 28 Type 1 (insulin-dependent) diabetic patients with different albuminuric values and 15 normal subjects have been tested for urinary IgG4, total IgG, and other immunological and metabolic parameters. Urinary IgG4 and IgG have been detected with solid phase (ELISA and RIA respectively) methods deviced in our laboratory. The median (and range) in normal subjects and in normo-, micro-, and macroalbuminuric patients of urinary IgG4 were respectively 0.65 (0.58-2.65), 0.58 (0.58-75), 3.8 (0.58-28), 42 (7-160) ng/ml, of total IgG were 1.6 (0.6-5.25), 1.7 (0.9-625), 1.6 (0.3-4.8), 8.4 (5.04-315) ~tg/ ml and of the urinary IgG4/IgG ( x 103) ratio were 0.36 (0.19-3.11), 0.34 (0.08-0.72), 2.54 (1.1-5.83), 4.06 (0.47-8.88). Thus in microalbuminuric patients, whereas IgG values are within the normal range, IgG4 values are clearly elevated demonstrating a selective elimination of this acid medium-size protein. In conclusion, urinary IgG4 could be an additional parameter to further characterise and subgroup microalbuminuric patients.

560 A 385. Two colour flowcytometry analysis of lymphocyte subset and activated T cell and immunohistochemicai study of T cell in pancreatic islets of NOD mice Y. Moil, J. Yokoyama, I. Matsuba, A.Tsuruoka, M. Nishimura and Y. Ikeda. Third Department of Internal Medicine, The Jikei University School of Medicine, Japan

We examined changes in the subsets of splenic lymphocyte and activated T cell by two color flowcytometry analysis, and immunohistochemical study of T cell in pancreatic islets was performed to investigate the role of T cell in pathogenesis of NOD mice. In the spleen, the percentages of Thyl.2 +, L3T4 + and Lyt2+ cells were markedly increased from the initial stage of insulitis throughout the onset of diabetes as compared with control BALB/c mice. As for activated T cell, neither IL-2 R + T cell (anti-mouse interleukin 2 receptor +, Thyl.2 + cell) nor Ia + T cell (anti-mouse I-Ad+, Thyl.2 + cell) were increased in peripheral blood and spleen of NOD mice. Immunohistochemical examination using ABC method revealed predominant infiltration of Thyl.2 + cells in the pancreatic islet and as for T-cell subset, both of L3T4 + and Lyt2+ cells were seen. Moreover, IL-2 receptor and Ia antigen expression were also observed on these T cells. Thus, we conclude that the marked splenic T-cell proliferation indicates a state of activation of the immune system, and activated T cell in the pancreatic islets play an important role in the destruction of pancreatic B cells. 386. Lipolytic agents stimulate glucose uptake by a cAMP-dependent mechanism in 3T3-11 adipocytes R.J. Moss, H.C.M. Sips and H.M.J. Krans. Department of Endocrinology & Metabolic Diseases, University Hospital Leiden, The Netherlands

During our studies on the long term (24 h) effects of lipolytic agents on the regulation of insulin dependent glucose transport in 3T3-La adipocytes, we found an increase in glucose consumption in the presence of these agents. We investigated the effect of lipolytic agents on the stimulation of lipolysis and glucose uptake. Standard medium (Dulbecco's MEM, 25 mmol/1 D-glucose, 10% foetal calf serum) with lipolytic agents in increasing concentrations was added to fully differentiated cells. Lipolysis was assessed by glycerol release into the medium. At set times during 24 h of incubation the glycerol and glucose concentrations in the medium were measured. Results: lipolysis was stimulated at all times in a dose related manner by fenoterol (EDs0=10- 8 mol/l, max: 10- 4 mol/1), forskolin (EDs0=10- 5 mol/1, max: 10- 4 mol/1) and 8Br-cAMP (EDs0=5.10- 4 , max: 10- 3 mol/1). Simultaneously, the increase in glycerol release by these lipolytic agents was paralleled by a dose dependent decrease in glucose concentration (p<0.05). Glucose consumption in control cells was 1 1.4 p~mol,h - . There was a significant increase in glucose consumption compared to control cells at high concentrations of fenoterol 4 (10-mol/l:2.51xmol per h; p<0.05), forskoline ( 1 0 -4t o o l / l : 3.4 p~mol per h, p<0.05) and 8Br-cAMP (10- 3 mol/1:3.4 lxmol per h, p < 0.05). We conclude: lipolytic agents stimulate glucose uptake by a cAMP-dependent mechanism in 3T3-La adipocytes. The increased glucose uptake parallels the stimulation of lipolysis. 387. Psychological disturbances in diabetic patients with erectile dysfunction M. C. Mota-Diniz and J. Pereira da Silva. Associagfio Protectora dos Diab&icos de Portugal (Portuguese Diabetic Association), Lisbon, Porgugal

We studied psychologically 66 diabetic patients with erectile dysfunction (ages 25 to 70): 89.4% were married; 75.7% lived in urban areas; 71.2% were still professionally active; prevalent educational level was low or middle. We used Rorschad's and Mira y Lopes' Psychodiagnostic and Ilness Behaviour Questionnaires (I.Pilowsky and N. D. Spence) and when possible evaluation of sexual partner's behaviour before and after sexual dysfunction. Two groups emerged with different characteristics. In the first group (34.8% of the patients) depressive traits preceded diabetes and erectile dysfunction: these patients showed depression (100%), and anxiety (82.6%) and inhibition for fear of sexual failure (56.5%). In the second group (65.2% of the patients) psychological disturbances were directly related to erectile dysfunction: these patients showed depression (97.0%) and ideoaffective inhibition (58%). The differential diagnosis between the 2 situations proved important in advising psychiatric support and psychotherapy, chemical or surgical prothesis and in the outcome of the therapeutic intervention. In the first group 10 (43.5%) patients improved, 7 (30.5%) remained stable, 3 (13.0%) worsened and 3 (13.0%)

Abstracts are still under observation. In the second group 21 (48.8%) improved, 8 (18.6%) remained stable and no one worsened; 14 (32.6%) are still under observation. 388. Morbidity and mortality of diabetic uremic patients on CAPD V. Mrzljak, M. Grani6, K. Seidl and Z. Skrabalo. Vuk Vrhovac Institute of Diabetes, Endocrinology and Metabolic Diseases, Zagreb School of Medicine, University of Zagreb, Yugoslavia

Twenty uraemic Type 1 (insulin-dependent) diabetic patients (average age 49.8 + 10.8, range 26-70 years) were on CAPD over 6-65 months (average period of treatment 39.9_+ 8.7). Peritonitis was the most frequently observed complication in every patient. The incidence of peritonitis was one episode per 14.8 patient-month. Malnutrition as a result of poor appetite, decreased nutrient intake and increased loss of nutrients via the dialysate was observed in 2 patients - 10% incidence. Cardiovascular complications were frequent. Four patients (30%) suffered myocardial infarction, and in 2 patients (10%) amputation of lower extremities due to peripheral vascular disease was performed. The acuity of 4 eyes with good vision at the beginning of the CAPD treatment did not essentially deteriorate, and in 10 eyes with significantly impaired acuity, deterioration leading to blindness was observed. The walking capacity of 50% of our patients diminished, and decreased vascular patency was observed in 25% of the patients (5 patients). After 5.5 years, the survival rate of the patients was 30%, and only 25% of the devices applied in CAPD could be used. The main causes of death were cardiovascular incidents 45% (9 patients), peritonitis 14% (2 patients), sudden death 14% (2 patients) and cerebrovascular insult 7% (1 patient). Due to complications, the patients had to be hospitalised for an average 45 days a year. Peritonitis accounted for half of the hospital days. Since CAPD maintains considerably good glycaemic control, good control of hypertension and good stable uramic control, special attention should be paid to the control of complications not related to dialysis to improve the rehabilitation of patients. 389. Glucoregulatory function of thyroid hormones is independent of insulin and glucagou M. J. Mueller, A. G. Burger, E. Jequier and K.J. Acheson. Medizinische Hochschule Hannover; West Germany; H6pital Cantonal, University Geneve, Inst. Physiol., Univ. Lausanne and Nestle Res. Dep., Switzerland

The glucoregulatory function of thyroid hormones was investigated in 6 male volunteers before and 14 days after 300 gg T4/d (T4:82 vs 161 nmol/1; T3:1.6 vs 2.4 nmol/1; TSH 2.3 vs 0.1 mU/1) following a sequential clamp protocol: during somatostatin infusion (500 ~xg/h for 6 h) and euglycaemia (0-2.5 h), at hyperglycaemia (9.12 retool/l; 2.5-4.5 h) and during additional insulin infusion (1.0 mU/kg per min; 4.5-6 h). Parameters of glucose metabolism were measured by isotope dilution (3H3-glucose) and indirect calorimetry. (1)T4-treatment increased basal hepatic glucose output ( = R a ; 2.60 vs 1.97 mg/kg, min), the metabolic clearance ( = MCRg; 208 vs 161 ml/ min), the utilisation ( = Rd; 177 vs 135 mg/min) and the oxidation of glucose ( = glucox; 129 vs 69 mg/min), whereas its non-oxid, metabolism ( = gluc.nonox.) was slightly reduced (46 vs 66 mg/min). (2) During somatostatin and euglycaemia Ra fell by 2/3 in control subjects but remained at basal after T4 (2.04 vs 0.70) despite glucagon deficiency. Concomitantly MCRg (181 vs 109), Rd (168 vs 97) and glucox (97 vs 55) were enhanced after T4 despite insulin deficiency. (3) MCRg and Rd remained elevated at hyperglycaemia. (4) Insulin further enhanced MCRg, Rd and glucox in control subjects; this effect was more pronounced after T4 with a disproportionate increase in MCRg. We conclude that T4 increases Ra independent of glucagon and stimulates insulin-independent as well as insulin-dependent glucose uptake. 390. Effect of hyperglycaemia and hyperglycaemia plus somatostatin on plasma glucagon in human obesity M. Muggeo, E. Bonora, P. Moghetti, M.Querena, M.Cigolini and V.Cacciatori. Chair of Metabolic Diseases and Institute of Clinical Medicine, University of Verona, Italy Aim of this study was to evaluate whether an impaired inhibition of pancreatic A cell by glucose, insulin and/or somatostatin contributes to hyperglucagonaemia of obesity. For this purpose plasma glucagon was measured in 8 obese and 10 non-obese non-diabetic subjects during a 4 h hyperglycaemic clamp (11.05 retool/l). During the third hour of the clamp, somatostatin was infused (250 meg/h) in order to further inhibit A cell. Fasting plasma glucagon was higher in obese

Abstracts than in non-obese subjects (242_+32 vs 167+13 pg/ml, p<0.05) (mean_+ SEM). In the last 20 min of the glucose infusion period without somatostatin (100-120min) plasma glucagon averaged 199_+ 25 pg/ml in obese patients and 127_+11 pg/ml in control subjects (p<0.05), with a reduction of 17_+4% in the former and 24_+4% in the latter (t7= NS). In both groups somatostatin infusion did not result in a further decrease in plasma glucagon, which averaged 191 _+ 26 pg/ml in obese subjects and 127_+ 14 pg/ml in controls (p< 0.05) in the 160-180 rain period. In the two groups plasma insulin was similar at fasting, increased similarly during hyperglycaemia and was inhibited to the same extent by somatostatin. These results suggest that 1) hyperglycaemia plus endogenous hyperinsulinaemia has a normal inhibitory effect on A cell in obese patients; 2) somatostatin has no additive inhibitory effect on A cell in both non-obese and obese subjects. 391. Tumour-promoting phorbol esters stimulate the translocation of glucose transporters in isolated rat adipocytes C.MOhlbacher, P. Schaff, B.Obermaier and H. H~ring. Forschergruppe Diabetes e.V., Institut for Diabetesforschung, Munich, FRG The effects of the tumour-promoting phorbol ester tetradecanoylphorbolacetate (TPA) and of insulin on 3-O-methylglucose transport and on the distribution of D-glucose inhibitable cytocalasin B binding sites was studied in isolated rat adipocytes. Insulin (1000 ~U/ml) stimulated the 3-O-methylglucose uptake 9 fold while TPA (10 -9 tool/l) stimulated the uptake only 3-fold (mean values of 5 experiments given as percent of equilibrium reached after 4 s: basal 7 + 1.3%, insulin 60 -+ 3.1%, TPA 22 _+2.3%). In contrast both agents stimulate the glucose transporter translocation to the same extent (cytochalasin B binding sites, pmol/mg protein, n=7, PM: basal: 6.2 _+1.0, insulin 13.4 _+2.0, TPA 12.7 _+2.7. LDM: basal 12.8 + 2.1, insulin 6.3 _+0.9, TPA 8.9 +_0.7. HDM: 6.9 + 1.1, insulin 12.5 + 1.0, TPA 8.1 +0.9). In conclusion: (1)TPA stimulates the glucose transport by stimulation of glucose carrier translocation. (2) Insulin and TPA stimulate the carrier translocation to the same extent while the stimulation of glucose uptake is 3-fold higher with insulin, suggesting that the insulin effect on glucose transport activity involves in addition other mechanisms than carrier translocation. 392. Efficacy of a teaching and treatment programme for hypertension in patients with Type I (insulin-dependent) diabetes I.Miihlhauser, P.Sawicki, U.Didjurgeit, V.J6rgens, V.Scholz and M.Berger. Department of Nutrition and Metabolism, University of DiJsseldorf, FRG Normalisation of elevated blood pressure (BP) is of crucial importance for the prognosis of long-standing Type I (insulin-dependent) diabetes. However, control of hypertension is rarely achieved. In order to improve hypertension care and patient compliance, we have developed a structured outpatient hypertension treatment and teaching programme (HTFP) for groups of 5-8 patients consisting of 4 educational sessions in weekly intervals: general information about hypertension, BP self monitoring, dietary treatment, drug therapy, exercise, stress and smoking. The efficacy of this HTFP was evaluated in 38 consecutive, normal weight Type I diabetic patients recruited from our diabetes clinic (13 females, mean age 38 years, diabetes duration 24 years, HbAlc 6.7+1.2%, serum creatinine <200 umol/1) with a known duration of hypertension of at least 6 months. Thirtyfour patients were re-evaluated 1-2 years after the HTI'R Blood pressure (measured using a random-zero-sphygmomanometer, means of 2-4measurements) fell from 148_+20/90_+13 to 135_+18/82_+ 7 mmHg (p<0.01). The percentage of patients on antihypertensive drugs remained unchanged (74% versus 79%), and 24 h urinary sodium excretion fell only slightly from 207+100 to 174+68 mmol. However, in patients who were "on" beta-blocker therapy both before and after the HTFP (n= 16), pulse rates decreased from 83-+ 17 to 69+11/rain (p<0.001), indicating increased patient compliance to drug treatment. Conclusions: long-term quality of hypertension care in Type 1 diabetic patients can be improved by a structured HTTR 393. Long-term follow-up of polyneuropathy in patients after pancreas and kidney transplantation W. Mtiller, R. Landgraf, M. M. C. Landgraf-Leurs, D. Burg, L.A. Castro, A. Abendroth, W. D. Illner and W. Land. Medizinische Klinik Innenstadt der Universit~it Mtlnchen, FRG In order to investigate the influence of successful pancreas and kidney transplantation on sensorymotor polyneuropathy we followed 22 patients with longstanding Type I (insulin-dependent) diabetes for

561 A a mean observation time of 24 months postoperatively. Before transplantation 17 patients complained of neuropathic symptoms like paresthesia, sensory loss or gait disturbance. At our first examination clinical signs of polyneuropathy were found in 19 patients and pathological electrophysiological tests in all patients. After transplantation paresthesias and painful sensations improved in 16 patients within a few months. Clinical signs of polyneuropathy improved in 11 patients. Peroneal, sural and median nerve conduction velocities increased significantly (p<0.05) during the follow-up. On the other hand 5 patients developed symptoms of a carpaltunnelsyndrome; in 2 patients distal latencies of median nerve increased without clinical symptoms. Surgery was necessary in 1 patient. Seven patients developed trophic ulcers with toe amputation necessary in 5 of them. Following kidney graft rejection a decrease in nerve conduction velocity was seen in 2 patients; no change was observed in the other 2 patients. There was no correlation between serum creatinine levels and nerve conduction velocity. In conclusion, even progressed diabetic neuropathy can be improved by normalisation of glucose metabolism and kidney function. 394. Effect of hypertension on blood viscosity in Type I (insulin-dependent) and Type 2 (non-insulin-dependent) diabetic patients S. MacRury, M. Small, J. Anderson, C. D. Forbes, A. C. MacCuish and G. D. O. Lowe. Diabetic Clinic and University Department of Medicine, Glasgow Royal Infirmary, Glasgow, Scotland, UK Hypertension in diabetic patients may be an independent risk factor for the development of microvascular complications and in non-diabetic subjects is associated with increased blood viscosity. We have measured plasma and whole blood viscosity and its determinants (mean+ SD) in 66 diabetic patients (27 Type I and 39 Type 2) with (n=23) and without (n=43) hypertension, compared with 52 nondiabetic control subjects to assess if hypertension was an additive and adverse effect on blood viscosity. Plasma viscosity, high-shear blood viscosity, 94.5 s - 1 (corrected for haematocrit), low-shear blood viscosity, 0.945 s - 1 (uncorrected and corrected) were all significantly higher in diabetic patients (1.84+0.17; 5.33+0.42; 19.96+4.91; 21.11 _+2.42) compared to control subjects (1.67 _+0.11 ; 4.89 + 0.55 ; 17.55+3.60; 17.39-+2.59), p<0.01. Corrected high and low-shear blood viscosities were significantly higher in Type 2 (5.45+0.77; 21.96+2.06) compared to Type1 diabetic patients (5.16+0.38; 20.03 _+2.37), p < 0.05 and p < 0.002. Type 2 diabetic patients were significantly older (55 + 7 years; mean_+ SD) than both Type 1 patients (38 + 13 years) and control subjects (45 _+4 years). Plasma viscosity and corrected high and low shear blood viscosities were significantly higher in hypertensive (1.86 + 0.13; 5.57 + 0.43; 21.96 + 2.45) compared to non-hypertensive diabetic patients (1.83 + 0.19; 5.21 + 0.35; 20.76 + 2.25), p < 0.01. Plasma viscosity in diabetic patients correlated with systolic blood pressure (r= 0.51) and fibrinogen level (r= 0.43), but there were no significant correlations with age, body mass index, duration of diabetes or glycaemic control. Hypertension may therefore aggravate diabetic complications by increasing plasma and blood viscosity. 395. Insulinotropic effect of endogenous and exogenous cholecystokinin in man M. Nauck, J. Reimers, R. Ebert, P. Cantor and W. Creutzfeldt. Division of Gastroenterology and Endocrinology, Department of Medicine, University of G6ttingen, FRG The insulinotropic activity of cholecystokinin (CCK) in humans is controversial. Intraduodenal phenylalanine administration (333 mg/ rain over 60 rain) released endogenous CCK in five healthy young subjects as demonstrated radioimmunologically and by intraduodenal bilirubin and pancreatic enzyme output. Concomitantly, there was an increase of only 35-90% over basal in circulating IR-insulin and IRC-peptide concentrations. In eight healthy volunteers intraduodenal infusions with saline (10 ml/min), glucose (333 mg/min), or phenylalanine (333 mg/min) were performed during hyperglycaemic clamp (8 mmol/1) experiments. Phenylalanine enhanced IR-insulin and IRC-peptide responses three-fold more than did the same amount of glucose. IR-gastric inhibitory polypeptide (GIP) responses were small and not different after glucose and phenylalanine administration. IRCCK was significantly stimulated to 9.4+1.4 pmol/1 only by intraduodenal phenylalanine. Plasma phenylalanine concentrations increased into the supraphysiological range (1.2 mmol/1). Intravenous infusions of synthetic sulphated CCK-8 led to plasma concentrations of CCK in the upper physiological range (approximately 12 pmol/l) and did not augment the IR-insulin or IR-C-peptide levels during the hyperglycaemic clamp. It is concluded that the augmentation of glu-

562 A cose-induced insulin release by intraduodenal infusions of phenylalanine cannot be explained by CCK release alone. For explanation, a combined action of insulinotropic gastrointestinal hormones (GIP, CCK, unknown incretion hormones) and of amino acids has to be considered. 396. The effect of hypertension and proteinuria on mortality in Type 2 (non-insulin-dependent) diabetes R. G. Nelson, W.C. Knowler and D.J. Pettitt. Diabetes and Arthritis Epidemiology Section, National Institutes of Health, USA The impact on mortality of hypertension (systolic blood pressure > 140 mmHg) and proteinuria (urine protein (g/1)/urine creatinine (~tmol/l) >l.13.10-4g/lxmol) was assessed in Pima Indians > 45 years old. Among the 1461 subjects, 48% with Type 2 (non-insulindependent) diabetes at the beginning of follow-up, there were 518 deaths in 13,576 person-years of follow-up. The age-sex-adjusted death rate (-+ SE) in diabetic patients without hypertension or proteinuria was 31.2 + 5.7/1000 person-years, similar to the death rate of 27.5 + 3.7 in non-diabetic subjects without hypertension or proteinuria. Among diabetic subjects, death rates were 34.6+3.0 in those without proteinuria but with hypertension, 93.4 + 41.1 in those with proteinuria but without hypertension, and 115.5 + 11.7 in those with both proteinuria and hypertension. Most of the excess deaths associated with diabetes in Pima Indians were found in subjects with proteinuria. Hypertension had a slight additional effect. Among diabetic patients without hypertension or proteinuria, the death rate was not appreciably greater than in non-diabetic subjects. 397. Fasting insulin-dependent and independent glucose economy and hepatic glucose balance in diabetic retinopathy L. L. Ng 1, T. D. R. Hockaday ~and J. Howard-Williams 2. 1Sheikh Rashid Diabetes Unit, Radcliffe Infirmary, Oxford, UK, 2Oxford Eye Hospital, Oxford, U K Eighty members of a cohort of Type 2 (non-insulin-dependent) diabetic patients first diagnosed in 1973-76 had fundal photography at 7 years from diagnosis. All had a 20 g / m z surface area intravenous glucose tolerance test at diagnosis and 5 years later. Applying Bergman's minimal model, we derived insulin sensitivity, non-insulin-mediated glucose uptake rate constant, fasting hepatic glucose balance, insulin and non-insulin-mediated glucose fluxes. Patients with retinopathy had higher fasting glucose levels at diagnosis (mean 13.2 + SD 3.9 vs 10.9+3.8 retool/l, p<0.001), and higher fasting hepatic glucose balance (4.2 +3.0 vs 2.9-+ 1.2 x 10 -2 mmol/kg per min, p < 0.003). Retinopaths had similar non-insulin-mediated glucose fluxes to non-retinopaths, but had higher insulin-mediated glucose fluxes (2.0 + 2.4 vs 0.9 _+0.8 x 10-2 mmol/kg per min, p < 0.05). Progressively higher values of fasting plasma glucose and basal hepatic glucose balance at diagnosis were associated with progressively increasing prevalence of retinopathy at 7 years (p<0.01 and 0.002 respectively). All measures of glucose flux at the fifth year were similar in retinopaths and non-retinopaths. We conclude that the prevalence of retinopathy is associated with a higher fasting glucose at diagnosis. This is due to a higher basal hepatic glucose production rather than differences in insulin sensitivity or non-insulin-mediated glucose uptake. Such mechanisms assume lesser importance later in the disease. 398. Metformin mimics insulin action on cholesterol biosynthesis in cultured intestinal rat cells A. M. Ni Neill, U. Moore, P. B. Collins, A. H. Johnson and G. H. Tomkin. Department of Biochemistry, The Royal College of Surgeons in Ireland, Dublin and Department of Metabolic Medicine, The Adelaide Hospital, Dublin, Ireland This study examines the effects of insulin and metformin (a hypoglycaemic agent) on the expressed (dephosphorylated) and total (dephosphorylated + phosphorylated) activities of HMG Co A reductase in intestinal cells from normal and alloxan diabetic rats. Isolated intestinal cells were incubated for 2 h in tissue culture medium and in the presence of insulin (70 nmol/1) or metformin (1 txmol/1). Total HMG Co A reductase activity was significantly (p< 0.0005) higher in cells from the diabetic rats (377 + 34 pmol product/min per mg protein, mean+ SEM) compared with cells from the non-diabetic rats (83 + 11 pmol product/min per mg protein, mean + SEM). Incubation of cells from diabetic rats with insulin reduced the total activity of HMG Co A reductase by 50% (p< 0.0005), while incubation with metformin caused 40% reduction in total enzyme activity (p< 0.005). Neither metformin nor insulin influenced the total activity of HMG Co A reductase in normal cells. Expressed activity of HMG Co A re-

Abstracts ductase was unaltered by diabetes (68 +9.7% in normal cells, 62+ 8.4% in diabetic cells). However, incubation with either insulin or metformin resulted in greater than 90% dephosphorylation of H M G Co A reductase in both normal and diabetic cells. Insulin appears to effect both the dephosphorylation of HMG Co A reductase (in normal and diabetic cells) and the enzyme mass (in diabetic cells). Metformin mimics the action of insulin under these conditions. 399. Comparison of hepatic glycogen synthesis following a glucose or a fructose load in diabetic rats C. B. Niewoehner and F.Q. Nuttall. Minneapolis Veterans Administration Medical Center and Department of Medicine, University of Minnesota, USA Diabetic rats are thought to be in a maximal gluconeogenic mode and yet store glycogen after glucose or fructose loads. Therefore we wished to determine whether glycogen synthesis was due to diminished glucose production and diversion of the ingested nutrients into glycogen. Alloxan-diabetic 24-h fasted rats were given glucose or fructose (4 g/kg) by gavage and killed at intervals for 120 rain. More glucose was absorbed than fructose (3134 vs 2239/3334 p,mol). After glucose and fructose the maximal liver glucose concentrations were 42 + 4 vs 22 + 2 ~tmol/ml intracellular water. In glucose-treated rats glycogen synthesis (277 ~tmol) was rapid but transient. In fructosetreated rats it was much slower, but the amount of glycogen stored was similar (311 ~tmol). Liver glucose output was slightly diminished after glucose and was unchanged after fructose (1366 vs 1433 ~tmol). Circulating lactate increased and was extracted by the liver after both glucose and fructose (293 vs 277 p.mol). Conclusion: in spite of markedly different circulating and hepatic glucose concentrations the glucose produced, lactate extracted and glycogen stored by the liver were remarkably similar after glucose and fructose administration. Glycogen storage was not due to decreased glucose output and could not be completely accounted for by lactate extraction. 400. Differential course of insulin resistance in newly-diagnosed Type 1 (insulin-dependent) diabetic patients H. G. T. Nijs, J. K. Radder, M. Fr61ich and H. M. J. Krans. Department of Endocrinology and Metabolic Diseases, University Hospital, Leiden, The Netherlands To examine the course of insulin action in newly-diagnosed Type 1 (insulin-dependent) diabetic patients, we studied 8 patients (P) 89 3 and 6 months (t89 t3, t6) after starting insulin treatment and 8 control subjects. Sequential euglycaemic (5.0 mmol/l) glucose clamps were performed with insulin infusion rates of 0.5, 1.0, 2.0 and 5.0 mU/kg per min in 2 h-periods. Results: at t89insulin sensitivity was decreased in P, whereas insulin responsiveness was normal. At t3 and t6 insulin action was normal. However, based on observed individual changes in insulin action from t89 to t3, P could be divided into: 'early responders' (P-ER, n=4), in which insulin sensitivity normalised at t3, and 'late responders' (P-LP,, n= 4), in which it improved, but did not normalise, at t6. Coinciding with this, in P-ER HbA1 was nearly normal (vs controls) at t3 (t89 t3, t6 resp.: 12.2___1.2 (SEM, p<0.01), 8.2 + 0.6, 8.3 +_1.0%), but in P-LR HbA1 was only improved at t6 [t89 t3, t6 respectively: 12.3+0.3 (/9<0.001), 10.2_+1.5 (/7<0.05), 9.5_+ 1.5%]. Higher insulin doses were needed in P-LR than in P-ER to achieve this, while the B-cell reserve was equally decreased. In conclusion: 1)insulin sensitivity was decreased in newly-diagnosed Type 1 diabetic patients after two weeks of treatment, which normalised after 3 or improved after 6 months; this coincided with near normalisation of glycaemic control; 2) late improvement of insulin action required more insulin than early normalisation, while the B-cell reserve was equally decreased. These findings suggest a differential course for insulin resistance, caused by other factors than glycaemic control alone. 401. A DP-alpha gene related polymorphism associates with coeliac disease, but not Type 1 (insulin-dependent) diabetes M. J. Niven 1, M. Madi 1, J.A. Sachs 2, P. Kumar 3, J. Awad e, H. Festenstein 2 and G.A. Hitman 1. Medical Unit I and Department of Immunology,2 The London Hospital Whitechapel, London and Dept. of Gastroenterology, St. Bartholomew's Hospital,3 London, UK Genes on the short arm of chromosome 6 are important in determining susceptibility to Type 1 (insulin-dependent) diabetes and coeliac disease. Associations with HLA-DR antigens are well described, and more recent studies have shown stronger associations with the DQ subregion. The aim of this study was to use Southern blotting to examine loci located centromeric to DQ, in these autoimmune diseases. Using D N A from 55 diabetic patients, 58 coeliac patients and

Abstracts 80 healthy control subjects, restriction fragment length polymorphism (RFLP) was found for DPa (Bgl II sized 3.5 and 2.2 kb) and DOff (Barn HI 8.6 kb) genes. There was no correlation between DPa/DOfl RFLPs and DR-serotypes. The D P a - 3.5 kb allele was found in 35% of controls, 47% of diabetics (NS) and 74% of coeliacs (t7< 0.0001). The DOff RFLP was found in 67% controls, 58% of diabetics and 54% of coeliacs. We therefore conclude that the genetic susceptibility to Type 1 diabetes is encoded by genes in the D R / D Q region, whereas in coeliac disease independent associations are also found in the DP region. 402. Morphometry of Rambourg-positive and Rambourg-negative Bcell granules after culture at low and high glucose concentrations L. Norlund, R. Norlund and I.-B. TNjedal. Department of Histology and Cell Biology, University of Umefi, Ume~t, Sweden We have earlier shown that silver-stained B cells exhibited two distinct classes of granules. Therefore a stereological analysis of the granules was performed in cultured B cells to investigate if these types of granules are physiologically relevant subsets. B cells from ob/ob mice were cultured with 3 or 20 mmol/1 D-glucose and silverstained according to Rambourg. Two tinctorial subsets of dark and light granules were analysed by morphometry at the ultrastructural level. The two types of granules were similar in size and shape. However, at 3 mmol/1 glucose the dark granule cores were surrounded by larger vesicles than the light granules. During culture at 20 mmol/1 glucose, both types of granule vesicles and cores became smaller, and the dark granule cores more rounded, as compared with cultures at 3 mmol/l glucose. The high glucose concentration also induced a marked decrease in the number ( - 8 4 % ) and volume density ( - 90%) of dark granules. In contrast, the number of light granules increased ( + 60%) with maintenance of their volume density. In conclusion, the morphometric analysis disclosed several ultrastructural differences between Rambourg-positive and Rambourg-negative granules in B cells. These differences suggest that the two subsets of granules play distinct roles. The dark granules are probably engaged in insulin discharge. 403. Quantitative energy dispersive X-ray micro-analysis of eight elements in pancreatic endocrine and exocrine cells after ultrafast freezefixation R. Norlund, N. Roos, I.-B.T~iljedal and L. Norlund. Department of Histology and Cell Biology, University of UmeS, Umeft, Sweden and EMLB, University of Oslo, Oslo, Norway The accurate measurement of elements in various cell compartments is not a simple task. Different methods are affected by various sources of artifacts. X-ray micro-analysis in combination with rapid cryo-fixation without additional chemical treatment seems to be the best way. We therefore report on the subcellular distribution of 8 elements as measured by quantitative X-ray micro-analysis on ultrathin, freeze-dried sections of islets and pancreas pieces from ob/ob mice. Diffusion of elements was reduced to a minimum by rapidly freezing the tissue samples between nitrogen-cooled polished copper surfaces and avoiding the use of chemical fixatives and stains. The ultrastructural morphology was adequately maintained to allow measurement on secretory granules, mitochondria, cell nuclei, and cytoplasm free of these organelles. The distribution of the various elements between cellular compartments was similar in islet B cells and exocrine pancreas cells. However, the insulin secretory granules were outstanding in exhibiting the highest concentration of zinc and calcium. In comparison with cytoplasm in the B cells, the insulin granules accumulated calcium 2-fold and zinc as much as 40-fold. As no correlation could be made for endoplasmatic reticulum in the cytoplasmatic measurements areas, the true accumulations above cytosol are likely to be even higher. 404. Cholesterol metabolism in diabetes N. M. G. O'Meara, R.A.M. Devery, D: Owens, P.B. Collins, A.H.Johnson and G.H.Tomkin. Department of Metabolic Medicine, The Adelaide Hospital, Dublin and Department of Biochemistry, The Royal College of Surgeons, Dublin, Ireland The effects of diabetic control on the activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA reductase), acal coenzyme A cholesterol-O-acyltransferase (ACAT) and cholesterol 7ahydroxylase, the major enzymes regulating cholesterol metabolism were determined in alloxan-diabetic rabbits. The results obtained were correlated with lipid and lipoprotein levels. While intestinal HMGCoA reductase activity was significantly increased (p< 0.001)

563 A in poorly-controlled diabetic animals compared with well-controlled animals ( + 122%), there was a significant reduction (p< 0.05) in the activities of intestinal ACAT ( - 74%), hepatic H M G C o A reductase (-51%), hepatic ACAT ( - 6 4 % ) and cholesterol 7a-hydroxylase (-43%). The poorly-controlled animals were hypercholesterolaemic and this was reflected in the very low density lipoprotein (VLDL) fraction (p<0.01). A significant negative correlation (p<0.05) was observed between 1) intestinal ACAT and total cholesterol ( r = - 0 . 5 4 ) , 2) cholesterol 7a-hydroxylase and total cholesterol (r= -0.73), and 3) cholesterol 7a-hydroxylase and VLDL cholesterol ( r = - 0 . 6 7 ) . Thus, hypercholesterolaemia in diabetic rabbits would appear to result from increased intestinal sterologenesis, decreased cholesterol storage and decreased cholesterol catabolism. In addition, the increased cholesterol available to the liver may be utilised in the formation of new VLDL particles.

405. Exclusion of close linkage between Type 2 (non-insulin-dependent) diabetes and the pro-opiomelanocortin (POMC) gene S.O'Rahilly, P.Patel, R.S.Spivey, J.S.Wainscoat and R.C.Turner. Diabetes Research Laboratories and Department of Haematology, John Radcliffe Hospital, Oxford, UK Two products of the pro-opiomelanocortin gene (Beta-endorphin and B-cell trophin or ACTH 22-29) are known to modulate insulin secretion. Alterations of endorphin levels have been found in murine genetic diabetes and in human Type 2 (non-insulin-dependent) diabetes. The possible role of the POMC gene in Type 2 diabetes has been investigated by tracing the segregation of POMC alleles in large diabetic pedigrees with an autosomal dominant mode of inheritance. Three families with Type 2 diabetes and 2 families with MODY were studied. MODY is defined as Type 2 diabetes presenting at age < 25 years in the majority of family members. Non-diabetic family members of all pedigrees have been studied by a glucose infusion test. D N A was extracted from blood, digested with RSA1, subjected to agarose gel electrophoresis, blotted onto nylon and hybridised with 32e labelled pLambda p2 which contains the first exon of the POMC gene (courtesy of Dr. Delphine Chang, Stanford University). Results: the probe gave the expected pattern of a constant band at 2.5 kb and two allelic bands at 1.8 (A) and 0.9 kb (B). One Type 2 and one MODY family were uninformative. Family 1 had 13 available Type 2 diabetic members over 3 generations, 8 informative meoises and a minimum of 2 obligate crossovers between diabetes and the POMC locus. Family 2 had 5 available diabetic members over 2 generations, 3 informative meoises and 1 obligate crossover. Family 3 had 9 available MODY members over 3 generations, 2 informative meoises and 1 obligate crossover. Summary: We have excluded close linkage between the POMC locus and Type 2 diabetes using linkage analysis of a common RSA1 polymorphism. The use of large, well characterised, diabetic pedigrees will allow rapid testing of other candidate genes as they become available.

406. Defective insulin receptor tyrosine kinase in the skeletal muscle of two patients with Type 2 (non-insulin-dependent) diabetes B. Obermaier, Z.Su, C. Mtihlbacher and H. H~iring. Forschergruppe Diabetes e.V., Institut for Diabetesforschung, Mfinchen, FRG Insulin receptor kinase was isolated from the skeletal muscle of two Type 2 (non-insulin-dependent) diabetic patients as well as five nondiabetic control subjects. After in vitro phosphorylation of the receptor /qsubunit from normal control subjects, tryptic digestion and HPLC separation of phosphopeptides insulin stimulated 32p-incorporation was found into 6 peptide peaks as well as non-stimulated P-incorporation into 1 peak. Stimulation of sensitive phosphorylation sites at 10 10mol/1, 10 -9 tool/1 and 10 -7 mol/1 insulin was 2-fold, 4-fold and 7-fold. In contrast no insulin effect was detectable up to 10 -9 tool/1 in the receptor kinase isolated from the diabetic patients, only higher insulin concentrations stimulated the 32p-incorporation reaching the normal maximum at 10 -7 mol/1. Lower insulin binding or differences in the amount of fl subunit as determined by the 32p label of the non-stimulated HPLC peak which was used as an internal standard was not detectable. After maximal insulin stimulation (10 -7 mol/1) the normal HPLC profile was found in one diabetic patient, while in the second patient two of the insulin stimulated peaks were not detectable at all. The data show that defects of the insulin receptor kinase in the skeletal muscle of Type 2 diabetic patients exist which might contribute to the pathogenesis of insulin resistance in these patients.

564 A 407. Biochemical mechanisms of hyperlipaemia in Type 2 (non-insulindependent) diabetes: studies in db/db mice I. G. Obrosova and N.D. Tronko. Department of Diabetology, Kiev Institute of Endocrinology and Metabolism, USSR

Accumulation of very low density (VLDL) and low density lipoproteins in Type 2 (non-insulin-dependent) diabetes mellitus is a causative factor of atherosclerosis and diabetic microangiopathy. In studies on db/db mice - model of Type 2 diabetes - we found VLDL hyperproduction induced by increased rate of hepatic biosynthesis of lipids forming VLDL (triglycerides, cholesterol, cholesterol esters, phospholipids) from acetate and pyruvate. As lipogenesis rate is the determinant of hepatic VLDL production, regulatory mechanisms of the former in diabetic mice were studied in detail. Increased total and free CoA levels in absence of any changes of short-chain acyl-CoA and long-chain fatty acyl-CoA promote to increased carbon flux through pyruvate dehydrogenase, acetyl-CoA synthetase, acetyl-CoA carboxylase and fatty acid synthetase. Alterations in CoA content may be caused by 4.3-fold rise of plasma corticosterone level which stimulate mitochondrial steps of CoA biosynthesis in liver. Increase of cytosolic phosphate potential (ATP/ADP. Pi) and oxidative properties of NAD(P)-couples enhance energetic supply of lipogenesis and potential ability of NADPH utilisation by fatty acid synthetase. These findings may serve as theoretical basis for application of drugs acting on biochemical mechanisms of hepatic lipogenesis regulation in therapy of Type 2 diabetes. 408. Hyperglycaemia and endothelium: differences between capillaries and large vessels B.Olgem611er and S.Schwaabe. Klinisch-chemisches Institut und Forschergruppe Diabetes, Krankenhaus Mtinchen-Schwabing, Miinchen, FRG

The interaction of blood glucose and endothelium is of basic importance for the pathogenesis of diabetic angiopathies. The endothelium is generally regarded to be insulin-independent since glucose uptake is mediated by insulin-independentfacilitated diffusion analogous to glucose uptake into the erythrocyte. We have shown formerly that transport of glucose into cultured endothelial cells from big vessels (porcine aorta and human umbilical vein) is the slowest step of glucose utilisation. In contrast to the erythrocyte the intracellular glucose concentration is independent of the extracellular glucose level. We have now demonstrated a different situation in endothelial cells from retinal capillaries (pig). The glucose-cartier has the same affinity for glucose (kM approx 0.8 mmol/1) as the carrier in endothelial cells from large vessels but total glucose utilisation is already saturated at lower glucose (kM approx < 0.2 mmol/l). Consequently, glucose uptake is not the rate-limiting step for glucose metabolism in capillary endothelium. Accordingly - again in contrast to large vessels - a retardation of the uptake by glucocorticoids does not affect overall glucose metabolism. Obviously, hyperglycaemia is linked to an elevation of cytosolic glucose in retinal endothelium whereas the glucose concentration in large vessels is unaffected. These findings may help to explain differences in the pathogenesis of micro- and macroangiopathies. 409. Overnight insulin substitution with a bedtime injection or continuous subcutaneous infusion in Type I (insulin-dependent) diabetic patients EO.Olsson, H.Arnqvist and H.von Schenck. Departments of Internal Medicine and Clinical Chemistry, University of Link6ping, Sweden Ten Type 1 (insulin-dependent) diabetic patients were investigated from 21.00 to 07.00 hours with a bedtime injection (BI) of intermediate-acting insulin or continuous subcutaneous insulin infusion (CSII) at constant rate. In the morning fasting blood glucose was 5.7 (4.2-7.1, median and interquartile ranges) mmol/1 with BI and 5.4 (4.6-5.8) mmol/1 with CSII (NS) without any rise. Blood glucose was higher in the evening (p< 0.05) and it decreased more during the night (p<0.02) with BI compared to CSII. Hypoglycaemia ( < 3.0 mmol/1) was observed at about 20% of the measurements with BI and 10% with CSII (NS). Correspondingly we found hyperinsulinaemia during the night with BI compared to CSII (p< 0.02). Free insulin rose to a maximum at midnight during BI (p< 0.05 compared to CSII) and then slowly decreased. Differences in blood glucose and free insulin were found between those using lente and NPH insulin for BI. With CSII free insulin decreased continuously after the last bolus at 18.00hours until steady state was reached at 04.00-05.00 hours. We conclude that although the same metabolic

Abstracts control in the morning is achievable, CSII is superior to BI for overnight metabolic control due to less pronounced hyperinsulinaemia during the night and a steady state of free insulin in the morning. 410. The use of 123I-insulin for the study of hepatic insulin binding in diabetic patients and non-diabetic subjects M.Oolbekkink, R.J. Heine and E.A.van der Veen. Department of Endocrinology, Free University Hospital Amsterdam, The Netherlands

We studied the hepatic binding characteristics of insulin in 6 healthy control subjects and 6 Type 2 (non-insulin-dependent) diabetic patients by intravenous bolus administration of 1 mCi 123Ilabelled insulin in the fasting state. External counting of ~'-emission over the liver region did not reveal significant differences in peak binding (10.4 (SEM +_1.03) vs 9.7 (+_ 1.40)% of administered radioactivity) or time to peak (7.5 (+0.89) vs 7.4 (_+1.21)min), respectively, for control subjects and Type 2 diabetic patients. The relationship between hepatic 123I-insulin binding, plasma insulin level and hepatic glucose production (HGP) was investigated in 3 control subjects and 1 Type 2 diabetic patient by constructing insulin dose response curves at plasma insulin levels of 15 (_+4.08), 38 (_+ 6.45), 55 (_+2.16) and 84 ( + 14.2)mU/1. At the end of each 2 h insulin infusion, 0.3 mCi 123I-insulin was administered as intravenous bolus. Hepatic 123I-insulin binding was not influenced by the insulin levels and did not correlate with HGP, in either group of subjects. Finally, we assessed the effects of supraphysiological plasma insulin levels (app. 1000 and 2000 mU/1) during 2 h infusion periods on hepatic 123I-insulin binding in 3 control subjects. At these insulin levels only a small decrease, as compared to the fasting state, in peak radioactivity was measured after intravenous 123I-insulin bolus administration: 10 vs 12% and 8 vs 12% at 1000 and 2000 mU/l respectively. In conclusion, hepatic ~23I-insulin binding does not correlate with insulin action on the liver and can only be affected by supraphysiological insulin levels. 41t. Glycaemic thresholds for counterregulatory hormones in Type 1 (insulin-dependent) diabetes mellitus H. R. Oosten, W.J. Sluiter, W. D. Reitsma and H. Doorenbos. Department of Clinical Endocrinology, University ttospital Groningen, The Netherlands

In Type 1 (insulin-dependent) diabetes mellitus diminished counterregulation frequently occurs. It is uncertain whether glycaemic threshold or release capacity is depressed. We studied the glycaemic threshold in 35 Type 1 diabetic patients (median diabetes duration 15 years) in whom a 2 h glucose clamp (median 8.34 retool/l) was performed using 30 m U / m 2 per rain human insulin. Thereafter glucose infusion (median 144 mg/m 2 per min) was abruptly discontinued while insulin infusion was continued, inducing hypoglycaemia. We measured adrenaline, noradrenaline, glucagun, cortisol, growth hormone and glucose every 30 min. Based on the variance of the hormone levels in the steady state, a hormonal increase was considered to be significant during an interval if the rise was more than: 25% (adrenaline, noradrenaline), 20% (glucagon, growth hormone), 15% (cortisol). Glycaemic threshold was defined as the average of glucose levels in such an interval. Using ANOVA different thresholds (median) were obtained: glucagon: 4.47 mmol/1 (versus noradrenaline, p<0.01, growth hormone, p<0.01, cortisol, p<0.01); adrenaline 4.25 mmol/1 (versus noradrenaline, p < 0.05, growth hormone, p < 0.05, cortisol, p < 0.05); noradrenaline 3.26 mmol/1 (versus glucagon, p < 0.01, adrenaline, p < 0.05); growth hormone 3.26 mmol/l (versus glucagon, p < 0.01, adrenaline, p < 0.05); cortisol 3.09 mmol/1 (versus glucagon, p < 0.01, adrenaline, p < 0.05). In conclusion: The glycaemic thresholds for counterregulatory hormones do not seem to be depressed. 412. Curvilinearity in Scatchard plots of rat adipocyte insulin receptors is due to Chloroquine sensitive insulin degrading pathways E. C. Opara 1, T.W. van Haeften 2, J. E. Gerich 3. Bethesda 1 Md, USA, Amsterdam2, Pittsburgh3, Pa, USA Insulin degradation can lead to underestimation of internalised insulin in insulin binding experiments. We assessed the effects of the use of the combination of the insulin degradation inhibitors Bacitracin and Chloroquine with the use of Bacitracin alone on insulin equilibrium binding kinetics. Isolated rat adipocytes were incubated with 12sI-insulin, Bacitracin (0.5 g/l), various insulin concentrations with and without Chloroquine (0.2 mmol/1) (37 ~ 60 min). Chloroquine reduced insulin degradation as assessed by immunoprecipitation

Abstracts (5.5 +_0.4% with vs 9.5 + 0.3% without Chloroquine, p < 0.01). Internalised insulin was increased by use of Chloroquine (43.5 _+1.2% vs 20 + 4%, p < 0.01). After correction for internalisation control studies resulted in a curvilinear Scatchard plot. Addition of Chloroquine resulted in a lower number of binding sites (9.4_+ 1.6 x 104 vs 21.9 + 4.5xl04sites/cell, p<0.01) and a linear Scatchard plot (r=0.98, p<0.001). Addition of Chloroquine did not significantly change binding site affinity as reflected by comparable EDs0 (insulin concentration reducing 12sI-insulin binding with 50%) (25+4 ng/ml without vs 20+3.8 ng/ml with Chloroquine). Conclusion: Rat adipocytes possess one class of insulin receptors without negative cooperativity. Curvilinearity in Scatchard plots is due to (presumably lysosomal) insulin degrading enzymes (in rat adipocytes). 413. Glucagon-like peptide-2 in pig and human small intestine C.Orskov, T.Buhl, J.J.Holst, H.Kofod, L.Thim, F.Baldissera and O.V.Nielsen. Department of Medical Physiology C, University of Copenhagen, The Hagedorn Research Center, Dep. of Surgery, Rigshospitalet, Novo Research Institute, Denmark

Recent studies in our laboratories have shown that the glucagon gene is expressed in the small intestine, where it codes for enteroglucagon (proglucagon 1-69) and proglucagon 78-107. The glucagon precursor contains another glucagon-like sequence (proglucagon 126-160) plus an intervening sequence of 13 amino acids. To investigate the occurrence and molecular nature of these 50 C-terminal amino acids of proglucagon, we developed radioimmunoassays for proglucagon 111-123 and proglucagon 126-160. We then isolated the naturally occurring peptides from acid ethanol extracts of pig ileal mucosa, and sequenced them on an Applied Biosystems Gasphase Sequencer. Two peptides were found: one corresponding to proglucagon111-123 (spacer peptide-2) and one to proglucason 126-160 (GLP-2). Similar products were found in human intestinal extracts. By comparison of the pig peptide sequences and human proglucagon we found the following differences: mlall7, Ilei3s, Ala144, Ilels2 and Glnls3 in the pig proglucagon were substituted with Thr, Val, Thr, Leu and His respectively. We also found that GLP-2 is released from pig ileum in response to luminal isotonic glucose and that GLP-2 immunoreactivity in plasma increases after a mixed meal. This suggests that GLP-2 is a hormone of the small intestine. 414. Effects of pancreastatin on insulin secretion and insulin sensitivity in vitro C. G. Ostenson and S. Efendic. Department of Endocrinology, Karolinska Hospital, Stockholm, Sweden

Pancreastatin is a novel 49-residue peptide isolated from the porcine pancreas. We studied the effects of pancreastatin on insulin secretion from isolated peffused pancreas and pancreatic islets of the rat, on glucose oxidation of isolated islets, and on glucose uptake and insulin sensitivity of isolated rat adipocytes. In the perfused pancreas, pancreastatin (1 and 10 nmol/1) suppressed, by at least 50%, the first phase of insulin response to 16.7 mmol/1 glucose and to 20 mmol/l arginine. In isolated islets, 100 nmol/1 pancreastatin inhibited significantly, by 32-52%, insulin response to 27, 16.7, 11 and 5.5 mmol/1 but not to 50 mmol/l glucose, whereas 10 nmol/1 pancreastatin inhibited the response to 11 and 5.5 mmol/1 glucose only. Pancreastatin did not affect glucose oxidation (formation of 14CO2 from U- 14Cglucose) in islets. Basal glucose incorporation into adipocytes was slightly but significantly enhanced by 1-100 nmol/1 pancreastatin, whereas lipogenesis induced by insulin (25-250 ~tU/ml) was decreased by 15% in the presence of the peptide. In conclusion, pancreastatin suppresses the insulinogenic effects of glucose and arginine without affecting glucose metabolism of islet cells. Pancreastatin also modulates basal and insulin-stimulated glucose uptake in adipocytes. Hence, the peptide may influence glucose homeostasis, interfering with both the secretion and action of insulin. 415. Effects of growth hormone and somatomedin C/insulin-like growth factor I on human fetal pancreatic islet-like cell clusters in culture T. Otonkoski, M. Knip, I.Wong and O. Simell. Children's Hospital, University of Helsinki, Finland and Children's Hospital, The McGill University, Montreal, Canada Growth hormone (GH) stimulates insulin production and B-cell replication of rat islets. It is unclear whether these effects are direct or mediated via the somatomedins. We studied the effects of human GH (1 pog/ml) or somatomedin C/insulin-like growth factor (IGF I) (0.2 ~tg/ml) on the production of insulin and glucagon in human re-

565 A tal islet-like cell clusters ('islets') in tissue culture. Islets were derived after coUagenase digestion of pancreas from 20 fetuses (mean gestational age 15.6 weeks). They were cultured in RPMI 1640+ 10% human serum for 7 or 31 days. Media were changed at 2-3 day intervals and analysed for insulin (IRI) and glucagon (IRG). No effects were observed during the first 17 days of culture. However, the release of both IRI and IRG were significantly augmented by GH during culture days 18 to 31 (IRI: 32.9_+4.2 vs 17.7_+2.1; and IRG: 2.8_+ 0.35 vs 1.6_+0.16 pg/islet/24 h, p<0.01). A delayed effect on the IRI release was also induced by IGF I (59.1 +4.4 vs 42.8+3.8, p<0.01). Islets cultured for 7 days in the presence of GH secreted significantly more insulin when incubated for 120 min in 20 as compared with 2 mmol/1 glucose (1.19+0.26 vs 0.58 +0.08 ng/t.tg DNA, p<0.05) in contrast to control islets (0.68_+0.11 vs 0.58+0.08, NS). GH may be involved in functional maturation of the human fetal B cell. 416. Retinopathy in newly presenting Type 2 (non-insulin-dependent) diabetic patients D. R. Owens, D. Jones, A.G. Shannon, I.R. Jones, J. Vora, S. Luzio and J. Dolben. Diabetes Research Unit, University of Wales College of Medicine, Cardiff, U K

One hundred twenty-nine (97 M, 32 F) previously untreated Type 2 (non-insulin-dependent) diabetic patients were studied. Glucose, Cpeptide and insulin levels, fasting (FPG) and post glucose (75 g: OGTY) and standard test meal (MTT) were determined. Clinical evaluation included detailed ophthalmological examination with fundus photography. Patient characteristics (mean_+SEM): age (years) 53 _+0.9; body mass index ( W / H 2 = BMI) 28.7 + 0.5; FPG (mmol/1) 11.9 _+0.3; HbA1 (%) 11.6 + 0.2; creatinine (ixmol/1) 88.9 _+ 1.4; BP (mmHg) S. 145 + 1.8, D. 87.7 _+1.0. Differences between retinopaths n=21 (R +) ( R = 1 7 B D R , 4PPDR) and non retinopaths n=108 (R-): FPG 13.7 vs 11.6 (p<0.01); HbA1 12.9 vs 11.3 (p< 0.01); BMI (25.2 vs 29.4 (p<0.001); age 56.8 vs 52.4 (NS); creatinine 91.2 vs 88.4 (NS); systolic blood pressure 152.4 vs 143.9 mmHg (NS); diastolic blood pressure 87.9 vs 87.7 mmHg (NS). Multivariate logistic analysis revealed that systolic blood pressure in conjunction with the insulin response gave the most significant correlation with retinopathy. The insulin during MTT is better correlated with retinopathy than during OGTr. No significant correlation was observed with age, sex, diastolic blood pressure, creatinine, family history or smoking. The effect of disease duration could not be evaluated. Bcell function is central to microvascular complications in Type 2 diabetes mellitus. 417. Measurement of pre-hepatic insulin secretion and its liver extraction in man by using minimal models of C-peptide and insulin kinetics G. Pacini and C.Cobelli. Bioengineering Dept. LADSEB-CNtL Padova, Italy

Methods for measuring pre-hepatic insulin secretion (IS) and its liver extraction (IE) in vivo are invasive. We introduce a non-invasive method for measuring in each individual subject IS and IE during 0.3 g/kg frequently sampled intravenous glucose tolerance test (IVGTI). Concentrations of glucose, insulin and C-peptide were measured in 11 young healthy volunteers. A minimal model of Cpeptide secretion/kinetics was developed and used in conjunction with insulin minimal model. Results: pre-hepatic measure of IS was obtained as first (61 4_11 pmol/1/min- 1 mg/dl) and second phase (0.0154+0.0034pmol/min/1/mg/dlmin) sensitivity to glucose. IE pattern showed an initial decrement (30-50 min) (from fasting value: 63 + 8% to nadir: 53 _+8%), then 62% steady value was re-established and maintained. Validity of IVGTF-based C-peptide model was assessed comparing its fractional clearance estimate (0.063_+ 0.007 min -1) with that found in experiments where biosynthetic human C-peptide was given. Conclusions: Minimal modelling of Cpeptide IVGTI'-data allows estimation in each individual of IS and IE. Due to its non-invasiyeness, this method should prove useful in clinical investigations of many pathophysiological states. 418. In Type 2 (non-insulin-dependent) diabetic patients in secondary failure peripheral sensitivity to insulin improves after low-dose insulin addition more than hepatic level or B-cell function G. Pagano, E. Pisu, D. Debenedictis, C. Baggiore, A. Diana, C. Marengo, C. Bozzo and A. Renzetti. Institute of Internal Medicine, University of Turin, Italy

Low dose (0.2-0.4 U / k s bw) insulin addition to oral antidiabetic drugs has been used to ameliorate blood glucose control in 9 Type 2

566 A (non-insulin-dependent) diabetic patients in secondary failure to oral treatment (mean diurnal plasma glucose of 12mmol/1). After 3 months treatment the following results were checked: significant decrease of fasting blood glucose (9.3_+ 0.1 vs 11.8 -+0.8 mmol/1, p<0.01); no variation of C-peptide peak level during oral glucose tolerance test (75 g: 1.27 + 0.3 vs 1.4 + 0.2 pmol/ml, NS); significant improvement of peripheral sensitivity to insulin assessed by euglycaemic hyperinsulinaemic insulin clamp at three sequential steadystate plasma insulin levels (81 -+7 - 351 -+30 - 3044-+191 txU/ml: M-coefficient 0.26 _+0.05 vs 0.16 + 0.03 mmol. kg-1. min-1, p < 0.05 at first level; 0.60 _+0.02 vs 0.50 _+0.03 mmol- kg-1. min-l, p < 0.01 at second level; 0.74 + 0.03 vs 0.65 + 0.04 mmol. kg -a. rain -a, p < 0.025 at third level); a not significant reduction of endogenous glucose production (3H-glucose infusion: 0.15+0.28%, NS). These results indicate that the metabolic improvement due to low-dose insulin treatment takes place principally on peripheral target tissues at a post-binding level and at least in part at the hepatic level. A recovery of B-cell function, as proposed by others, cannot be demonstrated. 419. Immunogenetic study of the familial accumulation of Type I (insulin-dependent) diabetes mellitus and rheumatoid arthritis P. Pfincz~l, K. Mer&ey, Gy. Illyrs, A. Falus, L. Romics, I~. Gyrdi 1, Ch. Alper2, U. Brhm, Gy. Petrhnyi a and P. Vrrtes. National Institute of Rheumatology and Physiotherapy, Budapest, Hungary, National Institute of Haematology and Blood Transfusion, Budapest, Hungary ~, Center for Blood Research, Harvard Medical School, Boston, USA2 The association of HLA DR3 and/or DR4 histocompatibility antigens in addition to the presence of a "silent" C4 allele has been shown both in Type I (insulin-dependent) diabetes and rheumatoid arthritis (RA). If these immunogenetic traits are of importance in the pathogenesis of these autoimmune diseases, an associated family accumulation of these illnesses can be expected. The authors observed 310 patients suffering from RA. Among the first grade relatives of 15 RA patients there was at least 1 member who was suffering from Type 1 diabetes. Eighteen RA patients had first degree relatives with Type 2 (non-insulin-dependent) diabetes. Among the first grade relatives of the members of a control osteoarthritic group none had either Type 1 diabetes or RA. In contrast 33 cases of Type 2 diabetes occurred among them. The authors accomplished HLA A, B, C, DR typing, complotyping, determination of the serum level of complement factor-4 (C4) in addition to screening of autoantibodies in 14 out of the 15 discovered families (55 persons in all) in which the RA patients had a first degree relative suffering from Type 1 diabetes. Based on the presented data a common immunogenetic interrelationship might be in the background of the familial accumulation of the two diseases. 420. Greater inhibition of endogenous glucose production by pulsatile insulin delivery at basal glucagon levels in normal man G. Paolisso, A.J. Scheen and P.J. Lefebvre. Division of Diabetes, Institute of Medicine, University of Lirge, Lirge, Belgium In order to study the metabolic effects of pulsatile insulin delivery in the presence of basal levels of glucagon, glucose kinetics parameters were studied using 3-3H-glucose during a 325 min euglycaemic glucose clamp in 6 normal volunteers receiving at random either continuous (C: 0.2 m U . k g - a . m i n -1) or pulsatile (P: 1.3 mU.kg 1.min-l, 11 min out of 13) intravenous infusion of human insulin (Actrapid HM, Novo). In both conditions, endogenous pancreatic secretion was suppressed by a somatostatin intravenous infusion (125 ~tg. h -a) and glucagon replaced by an exogenous continuous intravenous infusion (Glucagon Novo, 4 ~tg. h-C), maintaining mean plasma levels q_ (166_+67 p g . m l - 1 during C versus 171_71 p g . m l - 1 during P, NS) close to basal values. Plasma insulin levels remained stable at 14 mU.1 -a during C but fluctuated between 7 and 33 m U . l -~ during P. During the first 65 min, blood glucose and endogenous glucose production (EGP: 2.22_+0.24 versus 2.66+0.38mg.kg-l-min -1, p < 0.05) were significantly lower during P rather than C. During the last 65 min, blood glucose (clamped at 5.2+0.2 mmol.1-1) and glucose metabolic clearance rate were similar in both conditions whereas EGP was lower (0.92_+0.16 versus 1.76+0.22 m g . k g - l . m i n -1, p < 0.01) and Biostator glucose infusion rate significantly higher during P rather than C. In conclusion, pulsatile insulin delivery is more efficient than continuous infusion in decreasing both initial glucagon-stimulated and late glucagon-maintained EGP, providing that glucagon is adjusted at basal circulating levels.

Abstracts 421. Recovery of B-cell function and its relation to islet cell antibodies (ICA) in newly-diagnosed Type I (insulin-dependent) diabetes G. Papa, G. Ghirlanda, A.V. Greco, M. Salvatore, A. Tafuri, P. Fasciani, C. Manuppelli, A. Migliore, F. Scuderi and R. Manna. Rome, Italy A group of 27 newly-diagnosed Type I (insulin-dependent) diabetic patients have been studied for a period of 6 months: at diagnosis and later on, 17 were ICA positive (10/17 M, 7/17 F; mean age 15+8 years), 10 were ICA negative (6/10 M, 4 / 1 0 F ; mean age 19 + 4 years). At the time of the diagnosis the first and the second group had similar insulin requirement (0.7 +0.35 versus 0.58 +0.2, NS) and B-cell function studied through C-peptide values. Six months after diabetes onset, insulin requirement to keep metabolic control was significantly higher in ICA positive patients than in ICA negative ones (0.55 +_0.2 U/kg versus 0.22 +_0.15 U/kg, p < 0.001). Only one remission has been noted in ICA positive group (insulin requirement <0.25 U/kg), while 4/10 ICA negative patients had complete remission and 3/10 partial remission. C-peptide values seem to confirm previous data: in ICA positive patients fasting Cpeptide was 0.5 _+0.28 ng/ml versus 1.4 + 0.5 ng/ml (p< 0.001); after stimulus 1.1+0.6 ng/ml versus 2.6_+1 ng/ml (p<0.05). Our study suggests that ICA presence at the time of diagnosis and persistence in the first period of the disease is an early and predictive marker for a worse B-cell function recovery and a higher insulin requirement. 422. Defects of insulin action and insulin receptors in a new animal model of Type 2 (non-insulin-dependent) diabetes M. earillo, Y. D. I. Chen and G. M. Reaven. Institute of Internal Medicine and Metabolic Disease 2nd Medical School, University of Naples, Naples, Italy Aim of this study was to produce a form of experimental diabetes in rats which simulates the metabolic characteristics of Type 2 (non-insulin-dependent) diabetes and quantify the insulin-stimulated glucose transport, insulin binding (IB) and internalisation on this rat model. Eight-month-old rats were injected with small amounts of streptozotocin (25 mg/kg). The rats became diabetic but not catabolic. They were hyperglycaemic, blood glucose (23.1 +1.1 vs 6.5+ 0.1 mmol/1) with respect to control rats, but there were no significant differences in body weight (473 _+9 vs 453 + 9 g) and fasting plasma insulin (200.9 + 28.7 vs 165.0-+ 14.3 pmol/1) between the two groups. All the studies were carried out in isolated adipocytes from the epididimal pad. Glucose uptake was significantly lower in adipocytes from diabetic rats, both in basal (28.5 vs 86-+ 10 t / c e l l per sec, p < 0.001) and insulin stimulated (58-+ 7 vs 205 _+36 t / c e l l per sec, p < 0.001) state. Specific i25I-IB w as significantly lower in adipocytes from diabetic rats (1.53_+0.41 vs 2.72_+0.33 pg/10Scells, p<0.05) and less insulin was internalised in adipocytes from diabetic rats (0.68 _+0.06 vs 1.14_+ 0.18 pg/10 s cells, p < 0.01). In conclusion, glucose transport, IB, insulin internalisation are reduced in adipocytes of this type of experimental diabetes which resembles Type 2 diabetes. As such, this rat model may be a useful experimental tool in attempts to define the mechanism of the cellular insulin resistance in Type 2 diabetes mellitus. 423. Can the timing of end-stage renal failure be predicted in diabetic patients with renal disease? A. D. Paterson and T. L. Dornan. City and University Hospitals, Nottingham, U K With progressive nephron loss, reciprocal serum creatinine decreases linearly with time. We applied a least squares fit to this relationship to study patterns of deterioration in all 75 patients with raised serum creatinine attending our clinic. Thirty-nine had linear deterioration (r>0.85) but the rate was highly variable: predicted time for creatinine to rise from 150 to 500~mol/l ranged from 4months to 23 years (median 5 years). Much of the variance in rate of deterioration was unexplained although it correlated with age at diagnosis of diabetes, the presence of persistent proteinuria (p< 0.05) and diastolic blood pressure (0.1 > p > 0.05). Excluding patients with heart failure, recurrent urinary infection and hypertensive nephropathy, only mean diastolic blood pressure correlated with rate of deterioration (p<0.05). Ten patients had non-linear deterioration (r<0.85). Ten had non-progressive renal impairment (4 with heart failure and/or hypertensive nephropathy and 4 with primary renal diseases other than diabetic nephropathy). Sixteen had insufficient data for analysis. Conclusions: When creatinine was first raised, the subsequent rate of deterioration could not be predicted. Once serial data were available, prediction was possible for many but not all patients. Blood pressure and other factors influenced the rate of deterioration but much of its variability was unexplained.

Abstracts 424. Comparison of intravenous glucagon and dextrose in the treatment of severe hypoglycaemia in an Accident and Emergency Department A. W. Patrick 1, A. Collier1, D.J. Steedman 2, G. R. Nimmo ~, D.M. Matthews I, C. C. A. Macintyre 3, K. Little2 and B. F. ClarkeL 1Diabetic and Dietetic Department, Royal Infirmary and 2Accident and Emergency Department, Royal Infirmary, Edinburgh, 3Medical Statistics Unit, Edinburgh University, U K Hypoglycaemia is a serious problem in insulin-treated diabetic patients. In this study the efficacy of intravenous glucagon (1 rag) was compared with intravenous dextrose (25 g) in the treatment of hypoglycaemia in insulin-treated (n=52) patients attending an Accident and Emergency Department. In addition the prevailing glycaemic control of these patients was compared with insulin-treated patients routinely attending the Diabetic Out-Patient Clinic. The glucagon- (n=26) and dextrose-treated (n=26) groups were comparable in terms of initial plasma glucose, prevailing glycaemic control (HbA0, age, duration of diabetes and duration of hypoglycaemic coma. Both intravenous glucagon and dextrose were effective in the treatment of hypoglycaemic coma. There was a difference in the glycaemic profile following intravenous glucagon compared with intravenous dextrose and recovery of normal conscious level after glucagon was slower than after dextrose (4.0 vs 6.5 min, p<0.01), although the average duration of hypoglycaemic coma was 1.4 h. The patients presenting with hypoglycaemia had significantly lower HbA1 than comparable patients routinely attending the clinic (9.5 _+ 0.8 vs 12.0• p<0.01). In view of the ease of administration and the small risk of vascular or extravascular complications, intravenous glucagon appears to be a useful alternative to intravenous dextrose in the treatment of severe hypoglycaemia. 425. Body weight reduction improves renal haemodynamic function in long-term diabetic patients with overt nephropathy A. L. Patti, S.B. Solerte, M. Fioravanti, N. Schifino, M.G.Zanoletti, D.Mancin and E.Ferrari. Department of Internal Medicine, Medical Clinic II, University of Pavia, Italy Obesity is often associated with vascular and metabolic changes in diabetic patients. Twenty-four Type 1 (insulin-dependent) and Type 2 (non-insulin-dependent) diabetic patients with obesity (BMI above 30) and overt nephropathy (proteinuria persistent at rest > 550 rag/ 24 h) were followed for 12 months after diet modification from 1870 (mean value) to 1410 kcal/die (without changes of protein/carbohydrate ratio). Several parameters were evaluated throughout the study, i.e.: arterial blood pressure, HbAlc, proteinuria, albuminuria, glomerular filtration rate (GFR), creatinine clearance, triglycerides and cholesterol. A significant reduction of body weight (BMI from 33 • 1.6 to 26 + 1.8, p<0.001), concomitantly with a decrease of blood pressure levels, was reported during the study. An improvement of GFR (from 66_+]3 to 81_+11 ml/min per 1.73m 2, p<0.01) and creatinine clearance (from 79 + 14 to 91 _+ 13 ml/min, p < 0.001) was also demonstrated after 12 months. A reduction of triglycerides (p< 0.05) and cholesterol levels (p<0.01) were found during the study in our patients, whereas no changes in HbAlo concentrations were observed. A marked decrease of both proteinuria (from 1280• 511 to 923_+ 307 mg/24 h, p < 0.01) and albuminuria (from 723 • 388 to 492 _+170 ~tg/min, p<0.01) was demonstrated at the end of the study. The decrease of albuminuria following diet therapy was correlated with the initial urinary albumin excretion (r=0.71, p<0.001). Body weight reduction by hypocaloric diet may therefore delay the progression of advanced nephropathy in diabetic patients. 426. Regulatory action of basic amino acids on prohormone biosynthesis in pancreatic islets C.Pazelt. Department of Physiological Chemistry, University of Wuerzburg, FRG Basic amino acids (arginine, lysine) are by-products of the intracellular conversion of prohormones. Unlike peptide conversion products, these amino acids may pass the membrane of secretory vesicles leading to elevated intracellular levels. In the present study, the effect of L-arginine and L-lysine on synthesis and coversion of prohormones was studied in isolated rat islets of Langerhans. Both amino acids inhibit the synthesis of proinsulin and proglucagon, but not that of prosomatostatin. The effect can be demonstrated within a few minutes and is rapidly reversible. The efficient amino acid concentrations are in the range of 1 to 10 mmol/l. In contrast to the inhibitory action of arginine on proglucagon formation, the inhibition of proinsulin biosynthesis is counteracted by glucose. D-arginine and D-ly-

567 A sine are less efficient, whereas other L-amino acids as well as analogues of arginine do not show this effect. The intracellular conversion of prohormones is not influenced by basic amino acids. It is concluded that arginine and lysine control to a variable degree the biosynthesis of individual prohormones. With respect to insulin formation, a model for negative feed-back control by basic amino acids is suggested which is interrupted by the action of glucose. 427. Control of Type I (insulin-dependent) diabetes by insulin pump treatment and energy utilisation J. Pfiv, J. Kabrt, Z. Magek and S. Sva~ina, 3rd Medical Clinic, Faculty of General Medicine, Charles University, Prague, Czechoslovakia, Prof. J. Pfiv, M.D.,3rd Medical Clinic, Faculty of General Medicine, Prague, Czechoslovakia Nineteen hospitalised Type 1 (insulin-dependent) diabetic patient 19-55 years old (102+12% ideal weight) were studied, when they were unsatisfactorily controlled (mean value 55_+41) and after a good control (mean value 9.5 + 8.0) by insulin pump treatment had been achieved. Average values of three (before breakfast, lunch and supper) 15 rain-measurements of 02 consumption and CO2 production using indirect calorimetry were assessed. A 24-h excretion of nitrogen was also established. Significant increase of RQ from 0.79_+ 0.06 to 0.83 + 0.07 and of non-protein RQ from 0.80_+ 0.08 to 0.85 + 0.09 were noted. Carbohydrate utilisation rose and fat utilisation decreased significantly from 25% to 36% and from 52% to 42% of total energy requirement respectively. Catabolic nitrogen and energy expenditure decreased insignificantly from 19.3+6.8 to 17.6+ 4.3 g/ 24 h and from 8.96 -+ 1.38 MJ to 8.67 -+ 1.17 MJ respectively. Conclusion: Diabetes control by insulin pump resulted in a significant improvement of carbohydrate utilisation and in a restriction of fat utilisation. No significant decrease of protein catabolism and of energy expenditure were registered. (Mean values -+ SEM are given; for statistic computation paired t-test was used). 428. The impact of dietary fat on metabolic control and adipocyte insulin action in Type I (insulin-dependent) diabetic patients O. Pedersen, O. Hother Nielsen and J. Bak. Medical Department III, Aarhus Amtssygehus, Denmark In recent years it has become almost a dogma that high-fat-dieting induces a state of insulin resistance. The experimental evidences are, however, based upon studies in rodents. In 8 Type 1 (insulin-dependent) diabetic patients (mean age 31 _+5 year's, mean duration of diabetes 12+ 3 years, mean daily insulin dosage 48 • 8 units) we have determined the effect of a fat-enriched diet on metabolic control and in vitro insulin action on isolated adipocytes. The diabetic patients were in random order and for periods of 2 x 8 weeks given isoenergic diets containing either 55% fat, 15% protein and 30% carbohydrates (HFD) or 30% fat, 15% protein and 55% carbohydrates (LFD). Every month diet compliance and metabolic control were checked. Comparing results from HFD- and LFD-perinds no significant changes could be found in mean values of body weight, daily insulin dosage, HbA1c, fasting or day-long (08.00-16.00 hours) plasma values of insulin, glucose, FFA or ketones. 125I-insulin binding to adipocytes and basal, half-maximal or maximal insulin-stimulated 14C-D-glucose transport and metabolism were totally unaffected by the dietary modifications. In conclusion: High-fat-dieting for 8 weeks does not induce insulin resistance of adipose tissue or aggravate metabolic control in Type 1 diabetic patients. 429. Insulin action and phospholipid fatty acid pattern T. Pelikfinov~, M. Kohout, J. Bage, J. Hilgertov~, J.Vfilek and K.Vondra. Institute for Clinical and Experimental Medicine, Prague, Czechoslovakia The cell membrane of target tissue plays an important role in the mechanism of insulin action. It comprises insulin receptors and is the medium through which glucose transport is effected. The fatty acid pattern of the phospholipid bilayer affects the properties of cell membranes in in vivo experiments. It can be reasonably assumed that phospholipid fatty acid composition might relate to insulin action at receptor and postreceptor levels. For this reason we have examined in eleven healthy volunteers the relationship between insulin action (hyperinsulinaemic euglycaemic clamp technique) and serum phospholipid fatty acid pattern. At the same time erythrocyte membrane phospholipid fatty acid composition and erythrocyte insulin receptor characteristics were evaluated. It was found that increased availability of essential fatty acids (especially linoleic acid) in serum phospholipids was associated with enhanced responsiveness and de-

568 A creased sensitivity to insulin. There was also a significant negative correlation between insulin binding to erythrocytes and relative amount of essential fatty acids in erythrocyte phospholipids. These results suggest that the level of essential fatty acids in phospholipids may play a role in the pathogenesis of insulin resistence. 430. Papaverine intracavernons auto-injection in diabetic impotent males J.Pereira da Silva. Associag~o Protectora dos Diabrticos de Portugal, Lisbon, Portugal

We studied 39 diabetic patients with sexual impotence, 19 Type 1 (insulin-dependent), 20Type2 (non-insulin-dependent), aged between 31 and 68 years (51.6+ 8.7), with duration of impotence between 6 and 192 months, average 36. Bidirectional ultrasonography Doppler, determination of Penile-Braquial Index and Intracavernous Papaverine chloridrate injections were performed as steps of our male sexual impotence protocol. After Papaverine injection, 16 of these patients got and maintained erections sufficient for sexual intercourse (9 Type 1, 7 Type 2 patients); 4 of them are now on the auto-injection method (Pharmacological prosthesis) with satisfactory results on sexual intercourse. No relationship was found between the conclusions of Doppler evaluation or the Penile-Braquial Index and the results of intracavernous papaverine injections in Type I or Type 2 diabetic patients. 431. Nocturnal hypoglycaemia deteriorates morning plasma glucose control in diabetic patients treated with continuous subcutaneous insulin infusion (CSII) by inducing hepatic as well as extrahepatic insulin resistance G. Perriello, G. Crispino, G. Basta, S. Di Santo, C. Cruciani, F. Santeusanio and G. B. Bolli. Istituto Patologia Medica, Universitfi di Perugia, Italy Six diabetic patients on continuous subcutaneous insulin infusion (CSII) were studied to assess the effects of nocturnal hypoglycaemia on morning plasma glucose (PG) control. Insulin (0.5 mU.kg -1. min -1) was infused intravenously from 22.00 to 02.00 hours, but hypoglycaemia (PG nadir 2.5 + 0.22 mmol/1) was allowed to occur only on one occasion and prevented on the other (euglycaemic clamping). On both occasions the patients were given identical insulin (overnight CSII 0.65_+0.1 U/h, subcutaneous insulin bolus 4 + 1 U 30 min before breakfast). Plasma free insulin (FIRI) was identical on both occasions, but counterregulatory hormones (not glucagon) only increased after hypoglycaemia. Fasting PG was greater after hypoglycaemia than after euglycaemia (7.16 +_0.38 vs 6.22 + 0.27 mmol/1, p<0.05) due to greater glucose production (Ra, 3-3H-glucose) (14.5 + 1.16 vs 12.6 + 1.00 I.tmol.kg ~-min -1, p < 0.05). Post-breakfast PG was greater after hypoglycaemia than after euglycaemia (13.1 +-0.61 vs 9.38+0.5 mmol/1, p<0.01) due to greater Ra (21.0+ 2.27 vs 16.7 +_1.61 i.tmol, kg -1- min -~, p < 0.001) and failure of glucose utilisation corrected for glycosuria to increase appropriately for prevailing FIRI and PG (16.6+1.22 vs 15.6+l.221xmol.kg -1. rain -a, p < 0.05). In four patients where the individual roles of counterregulatory hormones were assessed, adrenaline accounted for app. 50%, cortisol app. 20% and growth hormone app. 30% of post-hypoglycaemic hyperglycaemia. Conclusions: nocturnal hypoglycaemia induces hepatic and extra-hepatic insulin resistance and deteriorates fasting and to a greater extent post-breakfast hyperglycaemia in diabetic patients even when insulin deficiency is prevented. 432. Effects of pretreatment with phorbol esters on insulin secretion from electrically permeabilised islets of Langerhans S. J. Persaud, P. M. Jones, C. S. T. Hii and S. L. Howell. Department of Physiology, King's College London, London, UK Prolonged pretreatment of some cell types with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) decreases their protein kinase C activity. We have studied the effects of PMA pretreatment on the exocytotic release of insulin from rat islets of Langerhans permeabilised by high voltage discharge. Collagenase-digested islets were cultured for 24 h in the presence of either 200 nmol/1 PMA or 200 nmol/1 4a-phorbol 12, 13 didecanoate (PDD), an inactive phorbol ester. Pretreated islets were permeabilised by 5 exposures (200 us) to an electric field (3.4 kV/cm) in a glutamate-based Ca 2 + /EGTA buffer. Incubation of PDD-pretreated permeabilised islets with 500 nmol/l PMA at a substimulatory Ca 2 + concentration (50 nmol/l) produced significant increases in insulin secretion (50 nmol/1 Ca 2 + , 236+_31 pg/islet per h; +500 nmol/1 PMA, 547 +_60, mean+ SEM, n=19, p<0.001) but PMA-pretreated islets

Abstracts failed to respond to subsequent exposure to PMA (50 nmol/1 Ca 2+, 269 __16 pg/islet per h; + 500 nmol/1 PMA, 305 _+20, n = 22, p < 0.2). PMA pretreatment did not affect the secretory response ofpermeabilised islets to Ca 2+ alone ~PDD-pretreated 50 nmol/1 Ca :+, 220+ 28, n=18; 10p~mol/1 Ca +, 707+78, n=19; PMA-pretreated, 50 nmol/1 Ca 2+, 269+16, n=22; 10 ~mol/1 Ca 2+, 613+_43, n=23). In addition both PDD-pretreated and PMA-pretreated islets responded to 100 mol/1 cAMP after permeabilisation. These results suggest that protein kinase C does not play an essential role in Ca 2+ and cAMP-induced insulin secretion from permeabilisied B cells. 433. Nonenzymatic glyeosylation of membrane phospholipids: a new model of cellular damage in insulin-insensitive tissues G. P. Pescarmona, L. Mannuzzo, O. DavidI and A. Bosia. Department of Genetics, Biology and Medical Chemistry, University of Torino, Italy. ~Istituto di Clinica Pediatrica, Italy

In the past nonenzymatic glycosylation of proteins in diabetic patients has been thoroughly studied; the binding sites have been identified as the aminoacid free aminogroups. As glucose binding is not specific other non-protein amino groups should react in the same way; between structural molecules phosphatidylethanolamine (PE) and phosphatidylserine (PS) have free amino groups. Experiment: red cell ghosts have been incubated with 2, 5, 10, 20 and 50 retool/1 labelled glucose at 37 ~ for 2.5 h. At the end of incubation ghosts were treated with sodium borohydride. Aliquots of ghosts were removed, dissolved with SDS 2% and counted for total glucose binding to the membranes; the remainder was extracted with chloroform: methanol (2 : 1). 0.05 ml of extract were counted (glucose bound to phospholipids); the remainder was used for a bidimensional TLC run. After development plates were autoradiographed, spots were scraped and counted for specific labelling of single phospholipids. Conclusions: a substantial amount of membrane-bound glucose was recovered from phospholipids fraction; PE, lyso-PE, PS, lyso-PS incorporated, respectively, about 40, 10, 20 and 30% of the phospholipids radioactivity. Glucose binding was proportional to glucose concentration up to 10 retool/l, reached maximum at 20 mmol/1 and decreased slightly at 50 retool/1. The linear correlation between glucose concentration (under 10 retool/l) and glucose bound to phospholipids makes PE and PS adducts with glucose possible candidates in the genesis of red cell membrane rigidity in well controlled diabetic patients. 434. Serum lipid concentrations in normal and diabetic Ethiopians W.-H. Peters, F. T. Lester, K. Brachmann and H.J. Rose. Gondar College Medical Sciences and Yekatit 12 Hosp., Addis Ababa, Ethiopia; Inst. Pathol. & Clinical Biochem., Greifswald, GDR

Diabetes mellitus and hyperlipoproteinaemia represent an increased risk for cardiovascular diseases. However, arteriosclerosis seems to be absent in native African populations and there are few reports on serum lipids and other risk factors. In a hospital-based survey from Ethiopia we found high rates of microvascular complications (retinopathy in 20%, nephropathy in 21%) strongly related to the duration of diabetes. Hypertension and cigarette smoking (males) were more prevalent in Type 2 (non-insulin-dependent) patients, who were more obese in contrast to the underweight Type 1 (insulin-dependent) diabetic patients. However, we found no electrocardiographic changes suggestive of coronary heart disease. The aim of this study is to present serum lipid concentrations in Ethiopian diabetics. Total cholesterol (TC) and HDL-C were chemically determined and triglyceride (TG) was measured enzymatically. Data was available on 90 control subjects and 137 diabetic patients of different age groups classified according to the WHO criteria. Type I diabetic children and males aged 18-29 years had higher TC values (p<0.01). HDL-C was higher in the latter, whereas the HDL-C/TC ratio was lower (03p<0.01). TC was not significantly different in older Type 1 or Type 2 diabetic men and women. HDL-C was unchanged in these patients. A significant hypertriglyceridaemia was found in all diabetic patients. 435. Beta-2 adrenergic stimulation as a cause of posthypoglycaemic hyperglycaemia K.-G. Petersen and L. Kerp. University of Freiburg, FRG Insulin-induced hypoglycaemia is followed by hyperglycaemia and insulin resistance in patients. Healthy subjects develop glucose intolerance. Eight healthy male volunteers received propranolol 160 mg, metoprolol 200 mg, or placebo 2 h prior to insulin-induced hyperglycaemia (0.1 U/kg body weight). Alternatively they were infused with

Abstracts the Beta-2 stimulator fenoterol (2 gg/min) simulating the timing of the adrenergic response to hypoglycaemia. Beta-2-stimulation with fenoterol for 25 rain caused glucose intolerance and insulin resistance for 4 h. Posthypoglycaemic glucose intolerance was prevented by propranolol (Beta1- and Beta2-adrenoceptor blocker) but not by metoprolol (Betal-adrenoceptor blocker). Posthypoglycaemic hyperglycaemia (the Somogyi phenomenon) is caused by Beta2-adrenergic stimulation. It is a lasting effect of the short-acting hormone epinephrine. 436. In vitro cyclosporine pretreatment of splenocytes prevents diabetes when reinfused in female N O D mice C. M. Peterson and B. Formby. Sansum Medical Research Foundation Santa Barbara, California, USA

Previous experiments in our laboratories have documented that incubation of leukocytes or islet-like cell clusters with cyclosporine A (CSA) results in specific immunosuppression in vitro. Our NOD mouse colony is characterised by development of Type I (insulin-dependent) diabetes (DM) in 80% of females by 30 weeks of age. To test whether CSA pretreatment of leukocytes influenced the development of DM in the NOD mouse, 18 females were randomised into three groups at 12 weeks of age. All mice had splenectomy. Group A underwent only splenectomy. Group B received 5 x 107 autologous splenocytes intraperitoneally (IP) following incubation in phosphate buffered saline (PBS) at 37~ for 40min. Group C received 5 x 107 autologous splenocytes IP incubated in CSA 50 ng/ml in PBS at 37 ~ for 40 min. Mice were monitored weekly. A reading of + + + glucosuria was considered consistent with DM and confirmed with a blood glucose determination greater than 27.6 mmol/1. Colony controls showed 80% DM by 32 weeks. In Group A, 5 of 6 mice had DM by 32 weeks. In Group B, 2 of 6 had DM by 32 weeks. In Group C, 0/6 mice had DM by 32 weeks. Splenocytes are important in the immunomodulation resulting in DM in the NOD mouse. In vitro CSA pretreatment of these cells abrogates the development of DM while avoiding CSA toxicity. 437. Lower erythrocyte oleate levels in Asian diabetic patients may predispose to coronary heart diesease D. B. Peterson, K. Fisher, R. D. Carter, P. G. Frost and J. I. Mann. Diabetes Research Laboratories and Department of Community Medicine and General Practice, Radcliffe Infirmary, Oxford, UK

The high prevalence of coronary heart disease (CHD) in Indian Asians may be related to factors other than serum lipid levels. One factor may be the fatty acid composition of erythrocyte and platelet membrane phospholipids, which act as prostaglandin precursors and are influenced by diet. We conducted a cross-sectional study comparing 36 predominantly vegetarian Gujarati Asians with Type 2 (non-insulin-dependent) diabetes, with 24 white Europeans of similar age, glycaemic control and mode of treatment. There were significant differences in erythrocyte phospholipid fatty acid composition, expressed as mean + SEM percentage of total fatty acids. (The proportions of most fatty acids in erythrocytes reflect those in platelets). Asian subjects had higher proportions of linoleate (C18:2n6), 15.6 + 0.4 vs 12.1 + 0.5, p < 0.005; arachidonate (C 20: 3n6), 15.1 +- 0.4 vs 12.2 +- 0.8, p < 0.01 ; and dihomo-gamma-linolenate (20: 3n6), 1.33+0.05 vs 1.07+0.07, p<0.001. In contrast, levels of oleate (C 18:1n9) were lower in Asians, 16.8+_0.2 vs 20.3+0.6, p<0.001. Mean serum lipids were similar in both groups: total cholesterol in Asians 5.7 + 0.2 mmol/1 vs Europeans 5.8 +- 0.2 mmol/1, NS; HDLcholesterol 1.3 _+0.1 mmol/1 vs 1.2 + 0.1 retool/l, NS; total triglycerides 1.7 +_0.1 mmol/1 vs 1.5 _+0.2 mmol/1, NS. High linoleate levels do not protect Asian diabetic patients (or non-diabetic subjects) from CHD. Lower levels of oleate may account for their higher risk, particularly as oleate levels are high in Mediterranean populations with a low prevalence of CHD. 438. Leucine metabolism in Type 1 (insulin-dependent) diabetes: the role of insulin and substrate availability A. S. Petrides, L. Luzi, P.Castellino, M.C. Rocco, G. Pozza and R.A.DeFronzo. Department of Medicine, Yale University, New Haven, Connecticut, USA, and Department of Medicine, Istituto Scientifico San Raffaele, Milan, Italy

The effect of insulin and plasma amino acid concentration on leucine metabolism was studied in 8 Type 1 (insulin-dependent) diabetic patients using the euglycaemic insulin (1 m U - k g - 1 -min- 1) clamp technique in combination with 1-14C-leucine. Type 1 patients were

569A studied while in good metabolic control (7.46 + 0.55 mmol/1 mg/dl; HbA1c = 8%). Insulin-mediated total body glucose uptake in the diabetic patients (4.8 _+0.5 mg. kg-1- rain-l) was reduced respect to control subjects (7.6+0.5, p<0.01). In the basal state, plasma leucine concentration (I 10 +_2 vs 105 -+_3 lxmol/l), endogenous leucine flux (ELF) (39 +- 1 vs 41 + 2 gmol/m 2- rain-l) leucine oxidation (8 + 1 vs 7 + 1) non-oxidative leucine disposal (NOLD) (31 +- 2 vs 34 _+1) were similar in Type 1 patients and control subjects respectively. During the insulin clamp ELF, leucine oxidation and NOLD declined to 22 + 2, 4 + 1 and 18 _+2 txmol/m2. min -1 respectively in Type 1 patients (p= NS vs controls). All the diabetic patients and control subjects received a repeat euglycaemic insulin clamp combined with an amino acid infusion designed to increase plasma amino acids levels two-fold. Under these conditions NOLD was enhanced above baseline in both control subjects and diabetic patients (58_+ 3 vs 62+ 4 gmol/m2.min-1), while ELF was inhibited to a greater extent (p< 0.01) than during the insulin clamp performed without amino acid infusion. Conclusions: insulin-mediated glucose metabolism is impaired in Type 1 diabetic patients, while the effect of acute insulin infusion on leucine turnover is normal; both increased plasma insulin and amino acid concentrations are required to stimulate protein synthesis in healthy and diabetic individuals.

439. Effects of gastrin releasing peptide on stimulated insulin and glucagon secretion in the rat M. Pettersson and B. Ahr~n. Department of Pharmacology, University of Lund, Lund, Sweden Gastrin releasing peptide (GRP) is a peptide with 27 amino acids that originally was isolated from the porcine gastrointestinal tract. Later on, however, GRP was shown to be an intrapancreatic neuropeptide. Since GRP has been shown to be released from the pancreas during vagal nerve activation, it may be of importance for the neutral control of islet function. Therefore, we investigated if GRP affects basal and stimulated insulin and glucagon secretion in the rat. GRP was infused intravenously for 30 rain in fed, tiopenthal anaesthetized rats, either alone or as addition after infusion for 30 min with either glucose alone or arginine alone. Infusion of GRP (17pmol/min) elevated basal plasma insulin levels by 60+ 18 p~U/ml (p<0.01) and basal plasma glucose levels by 2.1+ 0.38 mmol/l (p< 0.001). The glucose-induced elevation of plasma insulin levels was moderately augmented (p<0.05). Basal plasma glucagon levels were not significantly altered by GRP, whereas GRP was able to potentiate the arginine-induced elevation of plasma glucagon levels (p< 0.05). The increase in plasma glucose levels following arginine infusion was not affected by GRP. We conclude, that in the rat GRP 1) stimulates basal insulin secretion, 2) augments insulin secretion induced by glucose and 3)potentiates glucagon secretion induced by arginine.

440. Immunobistochemical evidence and chemical characterisation of an IGF Mike peptide in the vessel wall B. Pfeifle, D. Moday, D.Froesch 1 and H. Ditschuneit. University of Ulm, Internal Medicine II and Section of Electronmicroscopy 1, Ulm, FRG The insulin-like growth factor I (IGF I) has been shown to be an important growth factor for cultivated arterial smooth muscle cells. Cultures of these cells were able to release an IGF I-like peptide, which could be measured by an IGF I radioimmunoassay. The synthesis of this peptide was dependent on insulin. We used the peroxidase-antiperoxidase (PAP) technique for the localisation of IGF I in the vessel wall. For this study a rabbit anti-human IGF I was used in conjunction with a goat anti-rabbit antibody and a peroxidase-labelled sheep anti-goat antibody. The control experiments were carried out with rh-IGF I. The IGF I-like peptide, which has been produced by cultured smooth muscle cells was isolated and characterised by Superose and Mono Q (FPLC, Pharmacia) and finally by an analytical isoelectric focusing. Smooth muscle cells from the proliferative tissue of the vessel wall contained a large amount of granula with IGF I. The healthy vessel wall contained only a very small amount of IGF I. The IGF I from the serum-free medium in which the smooth muscle cells have been cultivated had a molecular weight between 5000-10,000 and an isoelectric point of 8.5. We assume that IGF I is involved in the growth control of arterial smooth muscle ceils and may act by paracrine or autocrine mechanisms.

570 A 441. Decreased serum osteocalcin levels in patients with Type 2 (non-insulin-dependent) diabetes mellitus P.Pietschmann, G.Schernthaner and W.Woloszczuk. 2nd Medical Department, University of Vienna, Austria Serum levels of osteocalcin, a 49 amino acid bone matrix protein, are assumed to be an indicator of bone formation. In diabetic patients a reduced bone mass has been observed. Thus, we measured serum osteocalcin levels in 28 Type I (insulin-dependent) diabetic patients (mean age: 34 _+4 years), 39 Type 2 (non-insulin-dependent) diabetic patients (mean age: 6 2 + 2 years) and two control groups, matched for Type 1 (n=14, mean age: 3 4 + 6 years) and Type 2 diabetic patients (n= 13, mean age: 60+3 years) by radioimmunoassay. Serum creatinine levels were in the normal range in all subjects. Serum osteocalcin levels in Type 1 diabetic patients (6.0 • 0.5 ng/ml) and control subjects (5.9 + 0.4 ng/ml) were comparable. Osteocalcin levels in Type I diabetic patients with proteinuria and/or advanced retinopathy were significantly lower than in patients without signs of late complications (5.0+__0.7 vs 7.1• p<0.025). Serum osteocalcin levels in Type 2 diabetic patients were significantly decreased when compared with control subjects (5.6+_0.4 vs 7.3 + 0.7 ng/ml, p<0.025). Osteocalcin levels in Type 2 diabetic patients without and with late complications were comparable (5.7 • 0.5 vs 5.4+0.5 ng/ml; NS). Our data suggest a decreased bone formation in Type 2 diabetes mellitus. In Type 1 diabetic patients bone formation seems to be influenced by the presence or absence of late complications. 442. Intraperitoneal insulin infusion by external pump in brittle Type 1 (insulin-dependent) diabetes M.Pinget, L.Thonnet, V. Sarafian and N.Jeandidier. Service d'Endocrinologie et des Maladies de la Nutrition, H6pital Central, C. H. R. U., Strasbourg, France The aim of the study was to examine the feasibility of long term intraperitoneal insulin infusion using an external pump and a neutral lyophilised U100 insulin preparation. Ten brittle Type 1 (insulin-dependent) patients poorly controlled by subcutaneous insulin infusion, were treated for three to ten months by intraperitoneal insulin infusion (total duration=70 patient-months). Intraperitoneal catheters from the Siemens Compagny (polyethylene-silicone) were implanted under local anesthesia and connected to the external pump (Disetronic MRS1 or NORDISK Infusor). Insulin preparation was realised extemporaneously by solubilisation of a lyophilised U100 insulin (Organon) in a basic solvant. Five catheters were removed following: abdominal pain (1 case), catheter blockage (1 case), subcutaneous infection (1 case), or dislocation of the catheter (2 cases). Other complications were: local infection (1 case) or inflammation (2 cases). Nine pump failures were observed due to unexpected battery discharges. Catheter blockage occurred in the external part of the catheter in three cases; section and reparation of this external part could prevent explanation in two of them. Glucose control was improved in all cases (HbA1=10.589+0.618% vs 9.000_0.549%, p<0.005) without change in lipidic profile (APOB=0.904+ 0.074 g/1 vs 0.902 +0.078 g/l). The intraperitoneal insulin infusion by external pump using neutral lyophilised U100 insulin is feasible and improves glucose control. 443. Destruction of pancreatic B cells increases the number of class IIexpressing islet cells with insulin immunoreactivity D. G. Pipeleers 1, P.A. In't Veld2, H. Markholst 3, S. Duys 1, W. Gepts 2 and M.Van De Winkel 1. Dept. of Metabolism & Endocrinology 1, Dept. of Pathology z, Vrije Universiteit Brussel, Belgium. Hagedorn Research Lab), Gentofte, Denmark In normal rat islets, only few cells express MHC class II or I a antigens. Within 24 h after injection of a diabetogenic dose of streptozotocin, the number of I a-positive cells increases 10-fold in islet tissue but not in the exocrine part of the gland. More Ia-expressing cells were also noted in islets of BB rats developing overt diabetes than in those of their normoglycaemic littermates. When Ia-positive islet cells were isolated by fluorescence-activated cell sorting, they were identified as non-endocrine cells, most of them exhibiting myeloid features and many containing large vacuoles filled with insulin immunoreactive granules. When examined in situ, the Ia-positlve cells were recognised in the islet interstitial space. The presence of insulin immunoreactive material was also their prominent feature in situ, whether in conditions of streptozotocin-induced or of autoimmunerelated B-cell destruction. These results indicate that cytotoxic events in the pancreatic B-cell population can lead to an increased number

Abstracts of myeloid I a-positive islet cells which exhibit the capacity to ingest cellular remnants such as insulin secretory vesicles. 444. Trans-cutaneous oximetry in morpho-functional study of diabetic microcircle G. L. Pizzi, A. Aldeghi, F. Favales, E. Mazzola, B. Spelta, A. Mastropasqua and E. Faglia. Centro di Diabetologia e Mal. Metaboliche Ospedale Niguarda Ca'Granda, Milano, Italy In order to detect early microvascular damage we studied peripheric tissues oxigenation and microvascular response to thermic stimulus using trans-cutaneous oximetry (Microgas-Kontron) in 80 Type I (insulin-dependent) diabetic patients (average age 28 • 8 years, range 14-45 years, disease length 8 + 7 years, range 1-25 years) comparing data with the ones obtained in 66 control subjects of similar ages. Those 80 diabetic patients examined in good metabolic control (HbAlc 7 +2, range 5-9) presented no microangiopathy (Group 1: FAG neg, microalbuminuria <15 m g / 2 4 h ; 40 of those were also tested for neuroautonomic disease, resulting unaffected by this complication (Group 2 : 5 standard tests for NAD with score =0). We found a significant difference in 02 peripheric level between the two groups and control subjects: (Group 1 = 02 tc 58 + 5 mmHg, Group 2 = 0 2 tc 58___9 mmHg, controls=02 tc 68+7 mmHg, p < 0.001. The oximetric curve of adaptation to thermic stimulus (Clark elektrode with T = 44 C) given to arteriolise microcircle shows in all diabetic patients a different morphology with prolonged falling time (Group 1 = 1.8 + 1A min, Group 2 = 2.2 • 1.4 min, control subjects =1.1+0.3 min, p<0.01, p<0.001 respectively). Results obtained show this method as capable detecting an early microvascular damage and an initial vasal pathologic reaction probably depending on morphologic alteration in the vasal wall and/or on an initial suffering in nervous fibres even in diabetic patients without evidence of neuroautonomic derangement and incipient microangiopathy. 445. Properties of sodium and calcium currents in cultured mouse pancreatic B cells T. D. Plant. I. Physiologisches Institut der Universit~it des Saarlandes, Homburg/Saar, FRG The whole-cell mode of the patch-clamp technique was used to study inward currents in mouse pancreatic B cells at 20-22~ B cells, kept in culture for 1-4 days, were identified by their response to glucose or their electrophysiological properties. Under conditions where K-currents were blocked, dihydropyridine- and Co-sensitive inward Ca-currents were observed on depolarisation to potentials more positive than - 50 mV from a holding potential of - 70 inV. These currents partially inactivated during 150 ms depolarisations. In activation, measured in 2-pulse experiments, was Ca-dependent and was almost completely abolished when Ba replaced Ca externally. Na also carried large currents through these Ca-channels when divalent cations were omitted from the external solution and the chelator EGTA was added. Evidence for the existence of voltage-dependent Na-channels in 80% of B cells was obtained when holding potentials more negative than - 80 mV were used. Under these conditions, depolarisation activated a fast transient inward current which inactivated completely and was blocked by tetrodotoxin (TrX). In conclusion, mouse B cells have both Na- and Ca-currents, but it is likely that only Ca-currents play a role in glucose-induced electrical activity. Na-currents are almost completely inactivated at the B cell resting potential. 446. Stimulation of glucose transport in skeletal muscle by insulin and contractions is caused by redistribution of glucose transporters in the plasma membrane T. Ploug, H.Galbo and J.Vinten. Dept. Medical Physiology B, Panum Institute, Copenhagen, Denmark To study the cellular mechanisms involved in activation of glucose transport in skeletal muscle, a fraction highly enriched in plasma membrane vesicles was prepared from leg muscle from groups of rats that were controls (C) or had been stimulated in vivo by insulin (I), contractions (E), or by both insulin and contractions (I + E). Time course of specific D-glucose uptake in plasma membrane vesicles showed that stimulation increased equilibrium distribution space for D-glucose and lowered the half time for equilibration: Distribution spaces were 394+16 lxl/mg membrane protein (C-rats), 822 + 44 (I-rats), 107 +_44 (E-rats) and 1347 +_61 (E + I-rats). Quantitative immunoblotting showed that the concentrations of glucose transporter protein in plasma membrane vesicles from C-rats and I + E-rats were identical. Furthermore, all glucose transporters were

Abstracts inaccessible to trypsin in vesicles from any group of rats. D-glucose inhibitable equilibrium binding of 3H-cytochalasin B to plasma membrane vesicles showed positive cooperativity in E-rats, and no cooperativity in I + E-rats. As judged from glucose transporter insensitivity to trypsin, no vesicles from an intracellular transporter depot were present in the preparation, and the transporter number remained constant despite stimulation. Nevertheless, stimulation increased glucose transport and cytochalasin B-binding was changed. Thus, stimulation induces a redistribution of transporters within the plasma membrane. 447. A cytotoxic islet cell surface monoelonal antibody from the NOD mouse O. Pontesilli, P. Carotenuto and S.J. Prowse. Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver Co, USA Autoantibodies against the islet cell surface have been described in non-obese diabetic (NOD) mice. In order to obtain monoclonal antibodies (mAb) of this type to be used to investigate the in vivo function and the biochemical specificity of anti-islet autoantibodies, spleen cells fi'om diabetic NOD mice were fused with P3X63-Ag8.653 myeloma cells within a week from the appearance of glycosuria. In two out of five fusions, seven hybrids secreting cytotoxic antibodies were detected by complement mediated lysis of chromium labelled rat insulinoma (RIN5F) cells. One stable antibody producing clone was established and termed 1.93B7. The antibody is of the IgM class and its binding to RIN5F celt surface, but not to a rat pituitary tumor line (GH3), was confirmed by indirect immunofluorescence. Although showing a lower fluorescence intensity than that obtained with RIN5F cells, approximately 30% of dispersed islet cells from mouse or rat pancreas were specifically stained. Trypsin treatment of RIN5F completely abolished the specific staining suggesting the protein nature of the antigen recognised by 1.93B7. The complement fixing capacity and the binding to primary islet cells make this mAb a potentially useful reagent in investigating the B-cell destruction mechanisms in the NOD mouse. 448. Effect of dietary fish oil on erythrocyte composition and insulin sensitivity in obese and non-obese subjects C. Popp-Snijders, R.J. Heine and E. A. van der Veen. Department of Endocrinology, Free University Hospital, Amsterdam, The Netherlands We studied the effect of an 8 weeks daily supplement of 3 g of 0)3 polyunsaturated fatty acids (PUFA) on plasma triglyceride levels, plasma and erythrocyte phospholipid composition and on in vivo insulin sensitivity in 8 obese insulin resistant subjects with a body mass index (BMI) of 33.4_+ 0.8 (mean_+ SEM) kg/m 2 and in 9 non-obese healthy subjects with a BMI of 22.'1 _+0.2 kg/m 2. Plasma triglycerlde levels fell in the obese group from 2.4+ 0.7 to '1.3 -+ 0.2 mmol/1 (p< 0.02) and in the non-obese group from 1.4-+0.2 to 0.8+0.1 mmol/l (p<0.01). The net incorporation of 0)3 PUFA in erythrocyte phosphatidyl ethanolamine was 3.4_+ 0.5 vs 5.8 -+ 0.3 mol% (obese vs nonobese, p < 0.001); the change in plasma phospholipids however, was comparable in the obese and the non-obese group. No effect was seen on the metabolic clearance rate of glucose during insulin infusion at 5 0 m U . k g - l . h - ~ : in the obese group 4.5_+'1.0 vs 4.2_+ 0.8 ml- k g - 1 rain-1 and in the non-obese group '12.7 + 0.2 vs 1'1.7 _+ 0.5 ml-kg -1 .rain -1 (before vs after o3 PUFA). This absence of an effect on insulin resistance in the obese group is in contrast with our finding in a previous study with non-obese Type 2 (non-insulin-dependent) diabetic patients. Possible explanations for this discrepancy are the diminished net incorporation of 0)3 PUFA into membranes of obese subjects, and/or the difference in nature of insulin resistance in obese subjects and non-obese subjects with Type 2 diabetes. 449. Reduced antibody titre to B-hepatitis following vaccination in patients with Type I (insulin-dependent) diabetes P. Pozzilli, N. Visalli, P. Arduini, P. Pezzella, M. Galli, E.A.M. Gale and D. Andreani. Endocrinologia (I), Clinica Malattie Infettive, University of Rome; Department of Diabetes & Immunogenetics, St Bartholomew's Hospital, London, U K It has been reported that positivity for Australia antigen is seven times more frequent in diabetic patients compared to normal subjects, implying that diabetic patients may be predisposed to B-hepatitis infection. We have investigated the immune response of 2'1 patients with Type '1 (insulin-dependent) diabetes ('13 M and 8 F, mean duration of disease: 13 years) and of 47 normal subjects following

571 A immunisation with the B-hepatitis vaccine. The vaccine (Haevac B Pasteur) containing 5 ~tg of HBsAg, was given subcutaneously into the deltoid region on three occasions at I month intervals. If the subject was still negative for anti-HbsAg after a third injection, a fouth booster was administered. Patients were well controlled at the time of the study (mean HbAI: 7.2_+ 1.4). The antibody titre to HbsAg was measured by radioimmunoassay and lymphocyte phenotyping was performed with a panel of monoclonal antibodies. The median anti-HbsAg titre was 27 m U I / m l in diabetic patients compared to a median of 230 m U I / m l in normal subjects (p< 0.0001). 31% of patients had undetectable titres despite the fourth injection. The C D 4 / C D (helper/suppressor) lymphocyte ratio was significantly reduced in diabetic patients (1.6+ 0.4, p<0.00l). These data indicate that patients with Type 1 diabetes may be more susceptible to B-hepatitis infection compared to normal subjects. 450. In vitro inhibition of non-enzymatic glyeation of human serum albumin and glomerular basement membrane induced by D-Lysiue F. Pricci, M. Sensi, M.G. De Rossi, R. Bruno, L. Valente, V. Cirulli, U. Di Mario and D. Andreani. Dept. of Endocrinology, University of Rome, Italy Enhanced non-enzymatic glycation of body proteins might be involved in the pathogenesis of late diabetic complications. Thus inhibition of this process could interfere with the reactions leading to their appearance. Preliminary experiments have shown that D-lysine can inhibit in vitro glycation of human serum albumin (HSA) and isolated glomerular basement membrane (GBM). However the glucose concentrations used (25, 50 and 100 mmol/1) were non-physiological. We have therefore investigated the inhibitory effect of D-lysine at glucose levels (10 and 20 mmol/l) such as are found in diabetic patient sera. D-lysine concentrations were 1/10 and 1/20 those of glucose. The results show that glycation in the presence of 1/10 D-lysine is reduced by 22% and 25% for HSA and by 24% and 26% for GBM at 10 and 20 mmol/1 D-glucose by comparison with glycation without D-lysine. Significant inhibition was also present with 1/20 D-lysine. Differently from aspirin, which inhibit glycation by competing with glucose for the same amino group of the protein, D-lysine reacts directly with the sugar and therefore reduces the availability of the latter for other proteins. This form of non-enzymic glycation inhibition might lower the incidence of diabetic complications without altering the physical-chemical characteristics of body proteins. 451. Evidence of early increased hepatic glucose production in the New Zealand obese mouse J. Proietto, M.Veroni and R.G. Larkins. University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Australia The New Zealand obese mouse (NZO) is an inbred strain characterised by obesity, hyperglycaemia, hyperinsulinaemia and insulin resistance. Previous studies have shown that despite early hyperglycaemia and hyperinsulinaemia muscle insulin resistance was not present at 5 weeks but was well established by 1"1 weeks. The aim of the present study was to determine if hepatic glucose overproduction could account for the early onset of hyperglycaemia. Glucose turnover was measured in overnight fasted young (5 week) anaesthetised NZO (n= 10) and control NZC (n= 9) mice using a primed continuous infusion of [6-3Hlglucose. Fasting glycaemia was marginally higher in the obese mice (7.1 _+0.67 vs 5.9 -+ 0.4 mmol/1, p = 0A) despite significantly higher insulinaemia (77.5 _+24.5 vs 25.0 + 2.5 gU/ml, p < 0.05). Basal hepatic glucose production (HGP) expressed per body mass index (wt/length 2) was higher in the NZO mice (6.7 + 0.8 vs 4.9 _+ 0.5 ~tmol- c m2/ g m , p < 0.05). Metabolic clearance rate of glucose was the same in obese and lean animals (0.99_+ 0.05 vs 0.88 + 0.08 ml. cmZ/gm). There was a positive correlation between fasting glucose and basal HGP (r= 0.779, p < 0.001). It is concluded that hepatic glucose overproduction is an early defect in the NZO mouse. 452. Long-term galactose feeding and streptozotocin-induced diabetes produce similar renal haemodynamic and filtration function changes in rats G. Pugliese, R.G.Tilton, K. Chang, C. Kilo and J. R. Williamson. Department of Pathology, Washington University School of Medicine, St. Louis, Missouri, USA To investigate the role of increased hexose metabolism by aldo-keto reductase enzymes in the pathogenesis of diabetic kidney disease, we compared renal haemodynamics and filtration function in 3 groups of male Sprague-Dawley rats: controls (n= 9), streptozotocin treated

572 A

Abstracts

(60 mg/kg body weight) to induce diabetes (n=9), and a group fed 50% galactose (n=9). Eight months later, renal blood flow (ml/min per g kidney) was measured with 85Sr microspheres (15 p~m), GFR and albumin clearance were assessed by measuring STCo-EDTA and 125I-BSA plasma clearances (g plasma/g kidney per min), and 24 h urinary albumin excretion (UAE; mg/24 h per 100 g body weight) was quantified by radial immunodiffusion assay. In both galactosefed and diabetic rats, GFR did not differ from controls (1.00 + 0.16 (SD) and 0.93 +0.17, respectively, versus 0.87+0.07 for controls), while renal blood flow was decreased (4.2 _+0.4 and 4.0 _+0.4, versus 4.9 _+0.2, p < 0.001), and filtration fraction was increased (0.50_+ 0.14 and 0.45 _+0.10, versus 0.32 _+0.03, p < 0.005). 125I-BSA plasma clearance was increased for both galactose-fed and diabetic rats versus controls (0.0052 _+0.0021 and 0.0064 _+0.0032, versus 0.0017 + 0.0005, p<0.001), as was UAE (1.24+0.73 and 3.09_+2.65, versus 0.27+ 0.14, p < 0.005). These results indicate that long-term galactose feeding and diabetes produce similar renal functional and haemodynamic changes, and suggest that these changes are linked to increased metabolism of hexoses by aldo-keto reductase enzymes.

Reduced sodium pump activity and an elevated intracellular sodium has been reported in streptozotocin-induced diabetes. In previous works we observed an increase in intracellular Na and a decrease in the erythrocyte membrane Na, K-ATPase in Type 1 (insulin-dependent) diabetic patients with respect to control subjects. In the present study we analyse the plasma membrane cation transport in erythrocytes from 19 Type 2 (non-insulin-dependent) diabetic patients and 19 sex- and age-matched control subjects. The Na, K-ATPase was determined according to Kitao; Na content was measured by ion-selective electrodes. Sodium content in diabetic patients was 6.9_+ 1.2 mEq/l (controls: 5.8_+ 0.9, p < 0.05). The Na, K-ATPase activity was 1.13_+0.23 l.tmol/1 Pi/h per mg protein in Type 2 diabetic patients and 1.59 + 0.14 in the control group (p< 0.01). Our data suggest an alteration of ionic movements in both insulin- and non-insulindependent diabetes. The molecular mechanism underlying such phenomenon needs further investigation in order to analyse an involvement of membrane physical properties (e.g. fluidity, lipid moiety modifications) or the action of a plasmatic modulator.

453. In vitro induction of Class II expression in human islet and thyroid cells is independent of the HLA type of the donor R.Pujol-Borrell, I.Todd, B.A.S.Abdul-Karim, R.Sutton, D.Gray and G. F. Bottazzo. Department of Immunology, Middlesex Hospital Medical School, London, U K In several autoimmune diseases with known HLA associations, including Type 1 (insulin-dependent) diabetes and thyroid autoimmunity, inappropriate expression of HLA Class II molecules by target epithelial cells has been observed. We examined the possibility that the HLA-DR alleles associated with those diseases convey increased susceptibility to factors inducing HLA Class II expression in the target epithelial cells. Monolayers of islet-enriched pancreatic tissue were stimulated with interferon (IFN)-gamma plus tumour necrosis factor or lymphotoxin, and thyrocyte monolayers were stimulated with IFN-gamma alone. HLA Class II expression on the surface of the thyroid cells was assessed by staining the viable monolayers with MoAb(s) to non-polymorphic determinants of Class II molecules. Islet cultures were similarly processed and then fixed and counterstained with antibodies to insulin to identify the B cells. Cultures were examined blindly with a U.V. Zeiss Photomicroscope (X800) by one or two observers who scored them blindly. In all cases, similar maximal induction of HLA Class II was observed in islet cells and thyrocytes, regardless of whether they were positive or negative for HLA-DR3 and/or HLA-DR4.

456. Metabolic effects of high glucose concentrations: inhibition of liver pyruvate kinase A.M.Rabuazzo, S.Iannello, R.Campione, G.Volpicelli and F. Belfiore. Chair of Pathophysiology of Metabolism, Istituto Clinica Medica III, University of Catania, Ospedale Garibaldi, Catania, Italy We tested the in vitro effect of glucose (G) at supraphysiological concentrations on the activity of the key glycolytic enzyme pyruvate kinase (PK, E.C. 2.7.1.40) in mouse liver. In liver homogenates (n=6), high G levels (10 and 20 mmol/1) inhibited PK by 37% (p< 0.01) and 26% (p< 0.01), respectively, compared to 5 mmol/1 glucose (mean+SEM: 13.49_+2.69 and 15.82+3.09nmol.m-a.mg -1 protein versus 21.42 + 3.30). In the presence of the activator fructose1,6-P (F-1,6-P) at 2 ~tmol/1 concentration, PK was 20% (/0=0.05) and 24% (p< 0.05) at G concentrations of 10 and 20 mmol/1, respectively. In homogenates prepared from liver slices (n= 10) preincubated for 10 min with G, a similar effect on PK was observed, with inhibition of 28% (p< 0.01) and 21% (p= 0.05) at 10 and 20 mmol/1 G, respectively; addition of 10 -8 mol/1 insulin during slice incubation prevented the G effect. Insulin alone did not affect PK. Experiments (n= 8) with purified rabbit liver PK (obtained from SIGMA, St. Louis, Mo., USA) assayed in the presence of 15 lxmol/1 F-1,6-P confirmed a G inhibitory effect of 21% at 10 retool/1. A similar inhibition was observed when PK activity was assayed in the presence of 5 Ixmol/1 F-1,6-E G might inhibit PK by competing with the activator F-1,6-R Insulin might overcome the G effect by activating PK through the well known dephosphorylation mechanism. Thus, in decompensated diabetes the high level of G may contribute, together with counterregulatory hormones, to inhibit hepatic PK.

454. Mechanisms by which alpha-glucosidase inhibitors lower glycaemia following sucrose ingestion in man S. Pye1, I. Hillbrand 2 and J. Radziuk 1. 1Royal Victoria Hospital, Montreal, Canada, ZBayer, Wuppertal, FRG Alpha-glucosidase might lower glycaemia after meals by reducing the rate of carbohydrate absorption as glucose and by extending the absorption time. Glucose-3-H 3 was infused intravenously to monitor changing metabolic clearance rates of glucose. 50 g sucrose labelled . . . . with glucose-l-C-14 without mhlbator or with varying doses (25, 50 or 100 mg) BAYmI099 or (5, 10, 20 rag) BAY01248 were ingested. Simultaneously the gastric contents of Tc-99m sulfur-colloid mixed with the drink was monitored for 150 min. Rates of absorption were determined by measuring the systemic appearance of glucose-l-C 14, using the measured tracer concentrations and compartmental modelling of glucose kinetics. In subjects who did not receive an inhibitor 93 + 2% of the sucrose load appeared systemically within 198 -+ 1 rain. 71 -+ 4% and 29 _+11% of the load remained in the stomach at 60 rain and 150 min respectively. With BAYm1099 absorption was decreased to 42-+7% over 369 _+30 min. The gastric emptying pattern did not change. With BAY01248 (5 mg) when absorption was decreased to 56_+ 5% over 354 min, gastric contents fell to 66 _+9% (p> 0.1) in 60 min but only to 44 + 7% by 150 min indicating a markedly slowed emptying rate in the later period (p< 0.05). Thus slowed gastric emptying can contribute to the decrease in absorption rates and glycaemia when the digestion of complex sugars is inhibited. 455. Erythrocyte Na,K-ATPase activity and sodium content in Type 2 (non-insulin-dependent) diabetic patients R.A.Rabini, I.Testa, L.Mazzanti 1, E.Bertoli ~ and P.Fumelli 2. Department of Internal Medicine, University of Ancona, Italy; aDepartment of Biochemistry, University of Ancona, Italy; 2 INRCA Hospital, Ancona, Italy

457. The reduction of total and low density lipoprotein cholesterol by Metformin is maintained with long-term therapy in Type 2 (non-insulin-dependent) diabetes S.G.H. Rains, R.S. Elkeles, G.A.Wilson and W. Richmond. St. Mary's Hospital, Praed Street London, UK High concentrations of total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) are associated with the development of coronary heart disease in Type 2 (non-insulin-dependent) diabetes. Short term therapy with Mctformin lowers TC and LDL-C. To study whether this reduction in cholesterol is maintained with prolonged therapy, we have examined in a single blind study the effect of Metformin withdrawal and reintroduction on serum lipoproteins in 14 Type 2 diabetic patients on established Metformin therapy (duration 1 to 7 years; median 5 years). During an initial period on active Metformin, serum lipoproteins were unchanged. In the six-week placebo phase, there were increases in fasting blood glucose (10.0 + 2.5 to 13.6 _+5.8 retool/l, p<0.05), glycosylated haemoglobin (9.2 + 1.9 to 11.3_+3.1%, p<0.01), TC (6.19+1.18 to 6.76+1.53 mmol/l, p<0.05) and LDL-C (3.97+1.22 to 4.61_+1.49 retool/l, p<0.01). There were no changes in triglyceride and high density lipoprotein cholesterol. Reintroduction of active Metformin reversed these changes. Analysis of covariance showed the individual changes in TC and LDL-C to be independent of changes in glycaemic control. We conclude that the LDL-C lowering effect of Metformin is maintained with long term therapy and that it is independent of its hypoglycaemic action.

Abstracts 458. Prediction of glycaemic effects of mixed meals in Type I (insulindependent) diabetic patients: studies with constant insulin delivery by the artificial pancreas O.Rasmussen, J. Arnfred, E.Winther and K. Hermansen. Second University Clinic of Internal Medicine, Aarhus Kommunehospital, Aarhus, Denmark Recently, we reported that spaghetti caused a lower glycaemic response than an exchangeable amount of potato in Type 1 (insulindependent) diabetic patients made isoinsulinaemic by the artificial pancreas. The question arises, however, whether this difference is preserved if these carbohydrate-rich foods are taken as part of a mixed meal. To answer this question we measured blood glucose, free insulin, and glucagon responses to exchangeable amounts of spaghetti and potato when ingested together with Sauce Bolognese in 7 Type 1 diabetic patients who had attained euglycaemia by the artificial pancreas prior to the meal intake. The amount of potato (raw weight 200 g) with Sauce Bolognese (167 g) and spaghetti (raw weight 50 g) with Sauce Bolognese (167 g) had approximately identical caloric content (435 and 447 kcal respectively), as well as fat, protein and carbohydrate content. As compared to the absolute incremental blood glucose areas to potato plus meat sauce, those to spaghetti with Sauce Bolognese was reduced by 49_+6% (2 p < 0.001) after 180 rain and 45 + 8% (2 p < 0.001) after 240 min. Identical constant free plasma insulin levels and identical increments in glucagon concentrations were found. The results demonstrate that a difference in glycaemic responses is preserved when the carbohydrate foods are taken as part of a mixed meal. 459. Different changes of plasma magnesium and potassium after exogenous insulin in long-term normoglycaemic glucose-clamp G. W. Ratzmann and K.P. Ratzmann. Department of Paediatrics, Ernst-Moritz-Arndt-University, Greifswald, and Centre for Diabetes and Metabolic Diseases, Berlin, GDR The homeostasis of magnesium (Mg) and potassium (K) is influenced by hyperglycaemia and by insulin. Because of the importance for intensive care of diabetic patients long-term studies are of interest. We have investigated the course of plasma Mg and K after a medium-long-acting insulin (0.4U/kg body weight) over 16h in 12 healthy, non-obese volunteers under normoglycaemia maintained by means of the glucose controlled dextrose infusion system (Biostator). Mg was measured by atomic absorption spectrophotometry, K by flame photometry. In contrast to short-term studies we find a different behaviour of plasma Mg and K under influence of insulin. Both Mg and K show a significant fall within the first hour (p< 0.001). Contrasting to a persistent, significant hypokalaemia during the whole test the initial drop of Mg is followed by cyclic changes of the mean Mg levels within hypo- and normomagnesaemic ranges. The incidence of hypomagnesaemias (11.2% of 225 values) is markedly lower than that of hypokalaemias (73% of 294 values). These results confirm the assumption that K is more influenced by insulin than Mg and is not influenced by counterregulatory mechanisms which are responsible for the cyclic maintainance of the Mg homeostasis. Further, our study shows that hyperglycaemia is not a precondition for the decreasing effect on both plasma Mg and K produced by insulin. 460. Excretion of urinary enzymes in Type 1 (insulin-dependent) diabetic patients with and without nephropathy K. P. Ratzmann, E. Schimke, M. Pergande, A. Ilius and K.Jung. Centre of Diabetes and Metabolic Disorders GDR~ Berlin, GDR We investigated the urinary excretion of lysosomal and brushborder enzymes such as alanine aminopeptidase (AAP), alkaline phosphatase (AP), 7-glutamyltransferase (GGT), and N-acetyl-~-D-glucosaminidase (NAG) in 36 Type 1 (insulin-dependent) diabetic patients with (Group 1) and without nephropathy (Group 2) in comparison with a reference group of 20 body weight and sex-matched non-diabetic subjects without renal diseases. With the exception of GGT, all the urinary enzyme activities in diabetic patients were significantly increased. The AAP, AP and NAG activities of Group 2 were significantly higher than those of Group 1. NAG exceeded most frequently the upper standard limit (97.5 percentiles) in both groups. There was a positive correlation between NAG activity and protein as well as albumin excretion (r=0.93 and 0.95 respectively). In addition the NAG activity was correlated with the excretion of low-molecular proteins such as lysozyme and ribonuclease (r= 0.52 and 0.92 respectively). On the other hand, there was no relationship between urinary enzyme activities and parameters of metabolic control (HbAi and

573 A glucosuria). The results demonstrate: 1)increased urinary enzyme activities in Type 1 diabetic patients without renal microangiopathy; 2) increased NAG activity in patients having a diabetic nephropathy irrespective of normal creatinine levels; and 3) a close relationship between NAG activity and the degree of proteinuria. 461. Increased concentration of atrial natriuretic peptide in Type I (insulin-dependent) diabetic patients with glomerular hyperfiltration K. Rave, L. Heinemann, P. Sawicki, A. Hohmann and M. Berger. Dept. of Metabolic Diseases & Nutrition, University of Dfisseldorf, FRG Glomerular hyperfiltration has been described as an early finding in the development of diabetic nephropathy. Previously we have demonstrated elevated concentrations of atrial natriuretic peptide (ANP) in patients with overt and incipient nephropathy. In this study we have measured ANP concentrations in 22 normal subjects, 56 Type 1 (insulin-dependent) diabetic patients without any sign of nephropathy, (mean age 27 years; duration of diabetes 9 years; fasting blood glucose 9.8 mmol/1; HbAlc 7.7%) and 29 patients with hyperfiltration (creatinine clearance > 140 ml/min per 1.73 m2) (age 28 years; duration of diabetes 9years; fasting blood glucose 8.9mmol/l; HbAlc 7.1%). One patient of this group had macroproteinuria (> 500 mg/24 h) and six had microproteinuria (> 60 rag/24 h and < 500 rag/24 h). Two patients had hypertensive blood pressure values. All three groups were age-matched and the diabetic groups did not differ with regard to diabetes duration, glycaemia, HbAlc and insulin therapy. ANP concentrations were measured by a radioreceptor assay. Normal subjects and diabetic patients with normal glomerular filtration had comparable ANP concentrations (median: 8.8 and 4.7 ng/1). Patients with hyperfiltration had elevated ANP concentrations (23.5 ng/1, p<0.001). Our findings suggest a possible role of ANP in diabetic glomerular hyperfiltration and hence in the development of diabetic nephropathy. 462. The role of C-peptide in the choice of treatment for poorly controlled Type 2 (non-insulin-dependent) diabetic patients M. Ravnik-Oblak. University Medical Centre Ljubljana, Department of Endocrinology and Metabolism, Ljubljana, Yugoslavia One hundred Type 2 (non-insulin-dependent) diabetic patients, poorly controlled on diet and 20 mg glibenclamide daily were hospitalised because of suspected secondary drug failure. Seventy-seven were discharged with insulin (Group A), 23 with glibenclamide (Group B). The aim was to find differences among parameters, specially C-peptide (CP) for the outpatient distinction of groups without hospitalisation. The groups did not vary significantly (p< 0.05) in age (all parameters are given with mean_+SD) - (61.5_+10.6 years vs 59.0+11.7 years), but significantly in duration of diabetes (12.8_+ 5.7 years vs 10.0_+4.7 years), mean blood glucose values of the last three outpatient controls (16.2_+2.3 mmol/1 vs 15.0_+2.3 retool/l), -l- 2.2 oVo vs 12.1 + glycosylated haemoglobins (13.6_ _ 1.8og0), body mass index (BMI) 26.6_+4.3 kg/m 2 vs 30.0_+4.3 kg/m2). There were no significant differences in the amount of 24h urine CP (13.0_+ 7.3 nmol/day vs 17.0_+10.9 nmol/day), fasting blood CP (0.68_+ 0.36nmol/1 vs 0.75_+0.23 nmol/1) and index fasting blood CP/ BMI x 103 (25.8_+ 11.9 vs 26.1 _+8.0). CP 6' after 1 mg glucagon intravenously (0.98 _+0.38 nmol/1 vs 1.22 _+0.37 nmol/1) and index fasting blood CP/fasting blood glucose x103 (49.1_+27.8 vs 60.0_+18.3) were significantly different. We conclude that there are (non)significant differences; overlapping between groups is so high due to high variability of values that it is impossible to group patients without clinical trial. 463. Subcutaneous implanted glucose sensors in control of artificial B-cell function K. Rebrin, P. Abel, H. Fischer and E. Brunstein. Central Institute of Diabetes ,,Gerhardt Katsch", Karlsburg, GDR The real prognostic improvement of Type 1 (insulin-dependent) diabetes over the long-term requires optimised insulin supply, e.g. using feedback-controlled systems. The crucial part of such systems is the glucose sensor which is thought to show best function in interstitial fluids. In normal dogs we implantated electrochemical sensors (amperometric GOD/H2Oz-electrodes) to monitor glucose levels in subcutaneous (SC) tissue of the neck during short-term experiments. Under steady-state conditions there was a linear relationship between circulating and SC glucose concentration between 2 and 20 mmol/1. Also it was examined, whether the sensor output is suitable for continuous feedback-control of insulin adminstration de-

574 A spite the delay characteristics between glycaemia and sensor current. Using standard algorithms for intravenous insulin infusions on the basis of glucose measurement in circulating blood, normoglycaemia could be restored and maintained and oral glucose loads be compensated in chronically diabetic dogs. Nearly identical sensor characteristics before and after the in vivo period including response times T95 < 90 s were required to maintain stable conditions during artificial B-cell treatment. We conclude that the SC measured glucose signal is appropriate as an input of artificial B cells. 464. Ontogeny of islet antigen antibodies and insulitis in the non-obese diabetic mouse S.Reddy, N.J.Bibby and R.B.Etliott. Department of Paediatrics, School of Medicine, Auckland, New Zealand

The non-obese diabetic mouse develops spontaneous Type 1 (insulin-dependent) diabetes in 30% of females in our colony. The predictive value of serum islet cell antibodies (ICA) and insulitis was examined by studying their ontogeny in females cr0ss-sectionally at day 15 (n=8), 25 (n=8), 40 (n=12) and day 90 (n=13). Sera were studied longitudinally from either day 35 (n=4) or day 144-168 (n=9) until day 250 for ICA and insulin autoantibodies (IAA). Pancreatic sections were examined for insulitis. ICA were determined by immunofluorescence and IAA by elisa. Insulitis was observed at day 40 (50%) and day 90 (70%) while ICA were present in one animal at day 15 and at increased rates at day 25 (60%), 40 (40%) and 90 (54%). Concordance rates were 18% at day 40 and 50% at day 90. In longitudinal studies, ICA were present irrespective of diabetes, being coincident in several sera. In ICA procedure immunoreactive cells corresponded to insulin and/or glucagon cells. We conclude that the markers are present well before clinical diabetes. ICA preceded insulitis with the prevalence rate for each marker being almost twice as high than for diabetes. Cellular staining by ICA and insulin autoantibodies suggest multiple islet autoantigens. 465. Interaction of polyamines with the binding and action of insulin in rat adipocytes B. Richelsen, S.B. Pedersen and D. Hougaard. Division of Metabolism and Endocrinology, Medical Department III, Aarhus Amtssygehus, Aarhus, Denmark Spermine is released in high concentrations from the pancreatic B cells in association with insulin. Since it previously had been shown that spermine could enhance the receptorbinding of the polypeptide hormones such as FSH and hGH, the possibility of a permissive effect of spermine on insulin binding and action was investigated. Spermine (1 mmol/1) enhanced the insulinbinding to rat adipocytes with 44_+3% (p<0.001). The half-maximal inhibitory concentration (EDs0) of unlabelled insulin on the 125I-insulin binding was similar in control and spermine-treated cells (1.58 _+0.14 nmol/1 vs 1.14_+ 0.13 nmol/l, p>0.05), indicating that spermine enhanced the insulin receptor number without affecting the affinity. Other polyamines such as spermidine and putrescine had minor or no effects on the insulin binding. Spermine alone had no effects on glucose metabolism in adipocytes. The maximal insulin stimulated CO2-production (and lipogenesis) was similar in the absence and presence of spermine (1 mmol/1). However, the half-maximal effective concentration (EDs0) of insulin on CO2-production was reduced in spermine-treated cells (from 0.29_ 0.07 nmol to 0.21 • 0.06 nmol/1, p < 0.05), indicating that the enhanced insulin binding induced by spermine was accompanied by a slightly increased sensitivity of insulin on glucose metabolism. Thus, it is concluded that spermine possibly could be a physiological regulator of the binding between insulin and its receptor. 466. Heart beat variation under forced handgrip - a rapid test of the sympathetic part of the autonomous nervous system in Type I (insulindependent) diabetic patients R. Riel, K. Piweruetz, H. Dettmer, R. Renner and K.D. Hepp. 3rd Med. Department, Dept. for Metabolism and Endocrinology, Miinchen, FRG After having improved the methods for the investigation of the autonomous nervous systems (ANS), the understanding of the autonomous neuropathy (aN) and its importance for the instability of blood glucose control have increased markedly. Unfortunately most of the tests for the investigation of the aN are time consuming. Therefore a reliable method was developed, which renders information about the sympathetic part of aN rapidly. After a preperiod a handgrip had to be pressed while the RR-intervals were recorded with a program-

Abstracts mable computer; both time periods lasted 15 s. The force was adjusted such that the handgrip could be kept pressed for 30 s without exhaustion. At the end of a recovery of 30s the means of the RR-intervals within the periods were calculated. The relative decrease of the mean was interpreted as the reactibility of the sympathetic part of the ANS. Fifty-two patients with Type I (insulin-dependent) and 27 non-diabetic control subjects have been investigated. There were correlations 1) between the mean RR-interval before, during, and after the handgrip (p< 0.001) and 2) between relative decrease and increase of the RR-intervals (p< 0.001). The reproducibility was in the order of magnitude of comparable tests. Correlations of this Handgrip-Test with other tests of the ANS (heartbeat-variation under rest or by forced respiration, tilting and exposure to icewater) were significant (p<0.01). The icewater-test is introduced at the same meeting. 467. Analysis of low t3Cf12C ratio on nanomole glucose sample with on line gaz chromatograph isotope ratio mass spectrometer J.P.Riou, R.Guilluy, C.Pachiaudi and J.Jumeau. INSERM U 197 and LEACM - Lyon, France; VG Isogaz, Middlewich, U K

Turnover of 13C labelled compounds is commonly studied by gas chromatography mass spectrometry (GCMS) analysis. The potential value of this method applied to the study of glucose, lactate and fatty acids turnover in human is limited by the low sensitivity of the method: 0.5 0V0above the background. Higher sensitivity for 13 C assay can be attained by isotope ratio mass spectrometry (IRMS) where the purified compound is combusted and assayed as CO2. Since the required instrumentation to work on nanomoles sample size with an on line system were not widely available we designed a special GCfurnace-IRMS. Using a modified multidimensional GC (capillary) module, selected compound of the GC profile, from blood extract containing either glucose + glycerol or fatty acids were individually introduced in the furnace and the 13 C / 12 C ratio of the CO2 produced was monitored on the IRMS. In glucose, 0.0011 atm% excess were measured on nanomole sample size with a precision of 5.10 -4 atm% excess. Standard curves from 0.0011 to 0.0668 atm% excess were obtained with a correlation coefficient of 0.998. This method, which allows 13C tracer study at low tracer enrichment, should yield new insight in the study of glucose, lactate and fatty acids metabolism. This work was supported by a special grant from INSERM network and R. P. Santr. 468. Enzymatic differentiation of cardiac and skeletal muscle D. A. Robertson, K. Falholt, L.G. Heding, M. Nattrass and J. Flint. The General Hospital, Birmingham, UK, Novo Research Institute, Bagsvaerd, Denmark

Cardiac muscle differs from other striated muscle in preferred substrates. In normal heart energy is obtained primarily from lipid metabolism. It is not known whether differences in intracellular enzyme activities contribute to this. Eighteen patients undergoing cardiac surgery (10 coronary artery bypass graft; 8 aortic valve replacement) had perioperative left atrial and intercostal muscle biopsy. Known diabetic and hyperlipidaemic patients were excluded. Lipid content was significantly greater in atrial muscle; triglycerides (mean + SEM) 308_+67 vs 69 -+14 ~tmol/g, p<0.01; NEFA 0.84+0.10 vs 0.35+ 0.05 ~tmol/g, p < 0.001. In tissue homogenates enzyme activities (U/g wet weight) differed significantly. Glycolytic enzyme activity was lower in atrial muscle; hexokinase 0.96-+0.10 vs 1.92-+0.18; phosphofrnctokinase 0.45 • 0.04 vs 3.13 _+0.37; pyruvate kinase 7.9 + 0.5 vs 15.8-+1.4; glycerol 3-phosphate dehydrogenase 3.8_+0.2 vs 15.6+1.4 (all, p<0.001). Enzymes of fat metabolism had significantly greater activity in atrial muscle; glucose 6-phosphate dehydrogenase 0.43 • 0.05 vs 0.17_ 0.03 ; malic enzyme 0.14 • 0.02 vs 0.08 + 0.01 ; 3-hydroxybutyrate CoA dehydrogenase 8.4-+ 0.6 vs 4.2+0.3 (all, p < 0.001). These results support the increased importance of lipid metabolism and a reduced role for glycolysis in cardiac muscle compared with other striated muscle. 469. Long acting somatostatin analogue S M S 201-995 lowers endogenous insulin in Type 2 (non-insulin-dependent) diabetic patients without worsening blood glucose control M.Rodier, A.G. Harris, J.P. Daures, A.Orsetti, L.Monnier and J. Mirouze. Clinique des Maladies M&aboliques et Endocriniennes, H6pital Lapeyrnnie, Montpellier, France Obese Type 2 (non-insulin-dependent) diabetic patients suffer from peripheral insulin-resistance and circulatory hyperinsulinism poorly responsive to carbohydrates. The acute effects of the somatostatin

Abstracts analogue SMS 201-995 on blood glucose, plasma insulin and C-peptide were evaluated in 6 obese Type 2 diabetic patients (3 M, 3 F, BMI: 31.1+1.0, age: 49.8_+7.7years, diabetes duration: 16.2_+ 7.5 years) during a standard breakfast (450 cal., carbohydrates 45%, lipids 40%, protein 15%). Each patient was randomly allocated to receive placebo, 25 ~tg SMS subcutaneously and intravenously 30 min after the beginning of the meal and 8 mg SMS per os immediately before it. Biological parameters were measured before and 30, 60, 90, 120 min after the meal. Analysis of variance and covariance show: a similar blood glucose increase during SMS administrations compared to placebo. - a significant reduction (p< 0.01) of mean insulin and C-peptide levels during SMS administration; slope of regression lines became negative for C-peptide with intravenous and oral SMS only. The areas under the curve for C-peptide were significantly reduced (p< 0.05) during each SMS administration. Acute administration of SMS during a meal reduces strikingly insulin secretion without further deterioration of blood glucose control. This may be of benefit on weight reduction and macroangiopathy in the long term.

470. Effects of long-term physical exercise on serum lipids in Type 2 (non-insulin-dependent) diabetic patients. A controlled randomised study T. Rrnnemaa, J. Marniemi, P. Puukka and T. Kuusi. Rehabilitation Research Centre of the Social Insurance Institution, Turku and Third Department of Medicine, University of Helsinki, Finland The effects on 4 months' physical exercise on serum lipids, lipoproteins and lipid metabolizing enzymes were studied in 25 Type 2 (non-insulin-dependent) daibetic patients (15 men, 10 women) divided randomly into exercise (n=13) and control (n=12) groups. Their mean age was 53 years and mean duration of diabetes 7 years. Exercise consisted of 5-7 weekly, at least 45 rain periods of walking, jogging or skiing at 70% intensity of maximal aerobic capacity. Exercise induced a significant decrease in mean serum LDL-cholesterol (from 4.6 to 4.3 mmol/1, p<0.05) and an increase in serum HDLcholesterol (from 1.22 to 1.29 mmol/l, p<0.05) which was mainly due to an increase in HDI4-cholesterol. The ratio of HDL-cholesterol to LDL-cholesterol increased from 0.27 to 0.32 (p< 0.05). Serum triglycerides and apoprotein A~ and B were virtually unchanged. The mean activity of postheparin plasma lipoprotein lipase increased from 263 to 324 U/1 (p<0.01) during the exercise period, while no change occurred in the activities of hepatic lipase, adipose tissue lipoprotein lipase or serum lecithin:cholesterol acyltransferase. In the control group no significant changes occurred in any of the lipid variables. In conclusion, training had favourable effects on serum lipids regarding the prevention of atherosclerotic diseases in Type 2 diabetic patients.

471. Cionidine inhibition of glucose-stimulated insulin-release involves reduction of the Ca 2+ current P. Rorsman 1, P. Arkhammar, P.-O. Berggren and T. Nilsson. 1Department of Medical Biophysics, University of Grteborg, Sweden and Department of Medical Cell Biology, University of Uppsala, Sweden The effects of the aradrenergic agonist clonidine on insulin release, cytosolic free Ca 2 + concentration ([Ca2 + ]i), membrane potential and Ca 2§ currents (Ica) were investigated using pancreatic B cells isolated from obese-hyperglycaemic mice. Addition of 2 ~xmol/1 clonidine promptly inhibited glucose-stimulated (20 mmol/1) insulin release. The inhibition was associated with a hyperpolarisation and a decreased [caa+]i. The subsequent addition of 25 mmol/1 K + increased [Ca2+li and partially restored secretion. At low glucose, however, clonidine abolished K+-evoked secretion although there was still an increase of [Ca;+]i. The effects of clonidine were reversed by addition of 5 umol/1 yohimbine but not by increasing intracellular cAMP. The actions on membrane potential and [Ca2+]i were counteracted by increasing extracellular Ca a§ to 5 mmol/l. Direct measurements of Ica using the whole-cell configuration of the patchclamp technique revealed that it was effectively reduced by 10 p~mol/1 clonidine. The inhibitory action of clonidine on glucosestimulated insulin release can thus partly be attributed to a suppression of Ic~, However, as it was possible to dissociate an increase of [Ca2+]i from stimulation of insulin release in the presence of clonidine, it seems that the a2-adrenergic inhibitory action is exerted also at steps distal to the influx of Ca 2 + in secretory process.

575 A 472. What is the cause of coma in diabetic ketoacidosis? V.Rosival. Department of Clinical Biochemistry, County Hospital, Trnava, Czechoslovakia

In the ,,Annals of Clinical Research" (1987) it has been claimed that increased hydrogen ion concentration in the blood (i. e., low pH) is an important factor in the development of coma in diabetic ketoacidosis. We have therefore reviewed from this point of view 383 of our own patients with diabetes mellitus and ketone bodies in the urine who had 1) blood hydrogen ion concentration more than 43.6 nmol/ 1, i.e. pH less than 7.36 and 2) base excess less than - 2.5 mmol/l. Their mental status has been classified according to Plum and Posner (1980). Following mean blood hydrogen ion concentrations (mean-+ SEM) have been observed in respective categories of the mental status (number of patients in parentheses): alertness (295) 50.4 + 0.3 nmol/l (= pH 7.30), drowsiness (55) 70.9_+ 2.1 nmol/1 ( = p H 7.15), stupor (14) 101.1 _+6.2 nmol/1 ( = p H 7.00), coma (19) 106.7_+ 7.5 nmol/l ( = p H 6.97). Analysis of variance has shown that the variance between the four groups is greater than within the groups (p<0.001); the coefficient of correlation is 0.76 (p<0.001); the equation of the regression line is y = 1.96 x + 31.04. Our results confirm the claim that increased blood hydrogen ion concentration is an important factor in the development of coma in diabetic ketoacidosis. 473. Influence of the route of glucose administration and of the plasma glucose concentrations on hepatic glycogen repletion as assessed by NMR L. Rossetti and G. I. Shulman. Department of Internal Medicine, Section of Endocrinology, Yale University School of Medicine, New Haven, Connecticut, USA

In order to delineate the role of route of glucose administration and hyperglycaemia on liver glycogen synthesis, we administered 1-13Cglucose (99% enriched) either by intravenous or intraduodenal infusion to chronically catheterized 24h fasted conscious rats and performed: 1)Euglycaemic clamp (E) (n=8)-Steady-state plasma glucose (PG) = 6.39 + 0.11 lxmol/ml and steady-state plasma insulin ( I ) = 3 1 + 2 n g / m l ; 2)Hyperglycaemic clamp (H) (n=8)-PG=11.00+0.55 ~tmol/ml and I = 3 1 _ 4 n g / m l ; 3)Hyperglycaemic clamp (M) (n=6)-PG=11.22+0.17 ~tmol/ml and I = 18 + 2 ng/ml (portal vein) and 4) Intraduodenal hyperglycaemic infusion (D) (n=8)-PG=11.33_+0.83 ~tmol/ml (portal vein) and I = 1 7 + 2 n g / m l (portal vein). The glucose infusion rates were, respectively, 189_+6, 283_+6, 200___6 and 133+6~tmol/kgmin in groups 1 through 4. At the end of the studies (120 min) rats were anesthetised, portal vein blood obtained, liver freeze-clamped, and glycogen extracted and subjected to ~H and 13C-NMR analysis. Mean liver glycogenic rates were E = 0.14_+ 0.03, H = 0.33 + 0.05, M = 0.26 +_0.06 and D = 0.62-+ 0.07 ~tmol/g liver rain. From the enrichments of a3C in the C1 position of glucose in glycogen and portal vein, it could be calculated that the per cent of glycogen synthesized by the direct pathway was: E = 1 1 + 2 % , H=34_+7%, M=29-+8% and D=36-+2%. Conclusions: 1)Hyperglycaemia, per se, under conditions of hyperinsulinaemia, markedly increases the per cent of glycogen synthesized by the direct pathway; 2)The intraduodenal route of glucose administration (D), compared to the intravenous (H and M), markedly increases the total amount of glycogen synthesized without altering the pathways (direct vs indirect) by which liver glycogen has been replete& 474. Lowered content of glutathione and protein thiols in sciatic nerves of streptozotocin diabetic rats J. Rudel, B. Rudel and R. Warzok. Department of Pathology, Peripheral Nerve Study Group, University of Greifswald, G D R

It has been suggested that aldose reductase plays an important role in the pathogenesis of diabetic neuropathy. The mechanism is unclear. One explanation might be that aldose reductase competes with glutathione reductase for the same cofactor (NADPH) followed by decreased glutathione (GSH) under hyperglycaemic conditions. To test this hypothesis, GSH and protein thiols (P-SH) were determined according to Sedlak and Lindsay in sciatic nerves of 25 female Wistar rats at days 10, 12, 14, 16, 20 after the administration of streptozotocin. Age-matched rats served as controls. For statistical analysis ttest was used. Results were given in p,mol GSH or P-SH, respectively, per 10 g wet weight tissue. Both GSH and P-SH were found to be decreased at days 16 (GSH: 5.62 + 0.41; P-SH: 26.11 + 1.57) and 20 (GSH: 6.07_+0.13; P-SH: 25.91+1.24) as compared to controls (GSH: 6.50+ 0.34; P-SH: 28.85_+ 1.24). The possible role of an al-

576 A tered glutathione redox cycle with concomitant loss of protein thiols will be discussed. Thiol dependent mechanisms such as Na+,K+-ATPase activity and other glutathione related pathways might be disturbed by this way.

475. Removal of (t25I)glucagon-likepeptides (GLP-I and GLP-2) from rat plasma; role of the kidney C.Ruiz-Grande, J.Pintado, J.M.Lopez-Novoa and I.Valverde. Fundacion Jimenez Diaz, Madrid, Spain The disappearance from plasma of (12SI)GLP-1 and (~25I)GLP-2 after a single intravenous injection was studied in normal (C), bilaterally ureteral ligated (BUL) and binefrectomized (BNX) rats. Blood samples were obtained at different intervals during 60 min after injection, and total and TCA-precipitable radioactivity were measured in plasma aliquots and in urine samples. The logarithm of TCA-precipitable counts present in plasma from rats injected with either radioactive peptide followed a straight-line between 5 and 15 min after injection. The t89 of (125I)GLP-1 was 8.2 _+0.4 min (mean_+ SEM, n = 5) in C, 10.7_+1.2 in BUL (n= 6), p<0.01 vs (2) and 12.3 _+0.6 in BNX (n=6, p<0.001 vs C, p<0.05 vs BUL). The t5 of (~2SI)GLP-2 was 8.9_+0.6 rain in C (n=6), 11.2_+0.5 in BUL (n=6, p<0.001 vs C) and 11.0 + 0.6 in BNX (n= 6, p < 0.02 vs C, p < 0.05 vs BUL). Gel filtration of plasma samples showed mainly two radioactive peaks, one in the elution volume of the peptide and the other in that of the salts, in good correspondance to TCA-precipitable and TCA-soluble radioactivity, respectively. The radioactivity of the urine samples from C rats injected with either peptide was not TCA-precipitable. In conclusion: GLP-1 and GLP-2 are removed from circulation in normal rats with a mean t5 of 8.2 and 8.9 rain respectively. The kidney seems to play a role in the catabolism of these peptides via glomerular filtration. 476. Relationship between autonomic and somatosensory neuropathy in Type I (insulin-dependent) diabetic patients P. Rupp, S. Lautenbacher, F. Strian, H. Baldermann and M. Haslbeck. III. Med. Abt. Krankenhaus Mtinchen-Schwabing, Miinchen, FRG The relations between somatosensory and autonomic small fibre neuropathy (NP) have not yet been studied in detail. Thus this problem was addressed in 24 Type I (insulin-dependent) diabetic patients. The study included standard neurological tests, measurements of motor and sensory nerve conduction velocity, beat to beat heart rate variation (RSA), measures of temperature discrimination and pain thresholds. Patients were divided into two subgroups: A (n= 6, patients with no neurological dysfunction) and B (n=8, patients with abnormal neurological and electrophysiological findings). We found a significant impairment of cold perception of the feet (p< 0.03) and of the RSA (0<0.007) in Group B. There was a considerable but not significant difference in the duration of diabetes. There were no differences in cold thresholds of the hand, warm thresholds and pain thresholds. These results indicate a close correlation between small fibre NP in somatosensory and autonomic nervous system. In addition long fibres appear to be affected first. Vice-versa, a small fibre NP is not necessarily correlated to a clinically apparent NP. In conclusion, impairment of thermal discrimination is often found in advance of clinically significant symptoms such as dysaesthesias or pain. 47% Interactions between endoplasmic reticulum and mitochondria in the regulation of pancreatic B-cell calcium transport I.Rustenbeck, J.-K.G6rlich and S.Lenzen. Institute of Pharmacology and Toxicology, University of Grttingen, G6ttingen, FRG With a newly designed Ca 2+ sensitive minielectrode with a microincubation chamber we investigated the interactions between endoplasmic reticulum and mitochondria in the regulation of the free cytoplasmic Ca 2+ concentration using isolated subcellular organelles and permeabilised islet cells from mice. The polyamine spermine stimulated Ca 2+ uptake by mitochondria through activation of the mitochondrial Ca 2+ uniporter. Mg 2+ antagonised the stimulation of mitochondrial Ca 2+ uptake by spermine. Lysophosphatidylinositol (LPI) also inhibited Ca 2 + uptake by the mitochondria apparently through interaction with the Ca 2+ uniporter. Inositoltrisphosphate (IP3) increased the free Ca 2+ concentration through stimulation of Ca z+ efflux from the endoplasmic reticulum. It is concluded that different inositolphospholipids contribute to the elevation of the cytoplasmic C a 2+ concentration during stimulation of the pancreatic B cell through concordant mobilisation of Ca 2+ from different subcellular stores.

Abstracts

478. Hypoglycaemic unawareness and inadequate hypoglycaemic counterregulation are not due to autonomic neuropathy R.E.J.Ryder, J.P.Vora, J.A.Atiea, D.R.Owens and T.M.Hayes. University Hospital of Wales and University of Wales College of Medicine, Cardiff, Wales, UK Hypoglycaemic unawareness is said to be a feature of diabetic autonomic neuropathy (DAN). DAN has also been proposed as the cause of the hypoglycaemic counterregulatory defect found in some Type 1 (insulin-dependent) diabetic patients. Seven patients with advanced DAN, 9 patients with a serious clinical problem with hypoglycaemia, 7 diabetic control patients and 10 normal subjects were subjected to a previously described 40 mu. kg -1. h-1 insulin infusion test for inadequate hypoglycaemic counterregulation. Comprehensive testing for DAN (7 cardiovascular tests, a pupil test and a sensitive sweat test) was undertaken. None of the DAN patients had inadequate counterregulation. None of the 7 patients with inadequate counterregulation had DAN. Of 12 patients who had experienced hypoglycaemic unawareness none had DAN. None of the 7 patients with DAN had experienced hypoglycaemic unawareness. Seven of the 12 patients with hypoglycaemic unawareness had inadequate counterregulation and one other was borderline. We conclude that hypoglycaemic unawareness and the hypoglycaemic counterregulatory defect are related to each other but are not due to autonomic neuropathy.

479. Does diet influence glycated haemoglobin levels? A. Ryle, R.D. Forrest, C.A.Jackson and J.S. Yudkin. Dietetic Unit and Academic Diabetic Unit, Whittington Hospital, London, UK During a screening survey of 223 subjects aged over 40, 2 glucose tolerance tests were performed and glycated haemoglobin (GHb) levels estimated by 4 assay methods both fasting and at 2 h. The correlations between different GHb assays and 2 h blood-glucose (2hBG) were 0.42-0.57, but using multiple regression analysis, the multiple correlation coefficient of GHb level with 2hBG and another assay of GHb increased to 0.51-0.85 (Ar2=0.02-0.40). Thus factors other than the degree of glucose intolerance explain over half the variance of GHb level, subjects having high or low GHb by one method having similar levels by all methods. We identified 41 nondiabetic subjects whose mean decile for GHb was more than 3 deciies higher or lower than that for 2hBG to study factors which might be responsible for high or low glycosylation. Thirteen "low-glycosylators" and twelve "high-glycosylators" completed 7-day dietary diaries and interview. There was no difference in age, gendel, body mass index, alcohol intake or smoking between the groups. Intakes of energy, total carbohydrate, Sugars, starch and fibre did not differ between groups and nor did the proportion of energy provided by any nutrient (0<0.2 for all comparisons). We found no evidence for a contribution of diet to levels of glycated haemoglobin. 480. Upper digestive tract involvement in Type 2 (non-insulin-dependent) diabetes: pH-manometric and endoscopic study T. Salvatore 1, D. Cozzolino 1, R. Torella1, G. Amato 2, k Fei 2, G. Izzo2, N. Di Martino 2 and A. Del Genio 2. Institute of Internal Medicin0 and Institute of Chirurgic Esophagologia 2, First Faculty of Medicine, Naples, Italy Abnormal esophageal and/or gastric motor function are reported in diabetes. We evaluated the upper digestive tract abnormalities by manometry, gastroesophageal-24-h-pH-monitoring, and endoscopy in 18 Type 2 (non-insulin-dependent) diabetic patients (mean age: 56_+9.9 years; duration of disease: 1 to 30 years) with different degrees of metabolic control. Nine patients were receiving insulin, 7 0 H A , and 2 diet only. No patient had esophageal symptoms. Manometry at lower esophageal sphincter (LES) showed: resting pressure of 9.05_+4.03 mmHg (normal: 17.55_+4.65), LES mobility in 6patients, relaxation of 98.33_+5.14% (normal: 91.13_+8.36%). Esophageal body exhibited: waves amplitude of 19.83 + 5.29 mrriHg (normal: 25.75 _+4.26), hypotonic waves in all and hypertonic waves in 8 patients, post-deglutitive simultaneous waves in 5 patients, segmental hypotonic dyskinesia in 2, and hypertonic dyskinesia in 3 patients. The pharingeal pump was normal; 3 patients showed a marked weakness of the peristalsis start. The pH-metry documented a pathological duodenogastric and gastroesophageal reflux in 7 and 5 patients respectively. Endoscopy disclosed esophagitis in 8 patients; however, histological damage (esophagitis and gastritis) was always found. There was no correlation between gastroesophageal abnormalities and duration of diabetes, metabolic control or therapeutic management. Hypotonic dyskinesia we found in diabetic

Abstracts esophagus may result from vagal degeneration, while hypertonic manifestations are probably due to predegenerative nervous changes. The gastroduodenal alterations likely depend on dysfunction of the antropyloroduodenal complex. 481. Discrepancy between acute and long-term effects of BAYmI099 in Type 2 (non-insulin-dependent) diabetes A.H.B.Samad, T.S.Ty.Willing, K.G.M.M.Alberti and R.Taylor. Department of Medicine, University of Newcastle upon Tyne, UK To examine the clinical role of BAYm1099 fifteen diet-treated Type 2 (non-insulin-dependent) diabetic patients were randomised to start drug (50 mg TID) or placebo following a 4 week run-in period in a double-blind cross-over study. Treatment periods (4 weeks) were separated by a 2 week wash-out period. During the last week of each treatment period, three test meals were administered, 60 g starch (TM1), 25 g sucrose (TM2) and combined 60 g starch and 25 g sucrose (TM3). The peak postprandial blood glucose, lactate and pyruvate levels (mean + SEM) were significantly lower with active drug after all test meals, particularly TM2 (11.3 ___1.0 vs 14.3 + 1.4 mmol/1, p < 0.001, 1.53 + 0.20 vs 2.48 + 0.17 mmol/1, p < 0.001, 105.1 _+17.6 vs 147.6+ 11.1 Ixmol/1, p<0.05 respectively). Peak blood glucose levels were significantly delayed. However, fasting blood glucose, HbA~, fructosamine and cholesterol did not change during active treatment (10.0+0.1 vs 9.9+0.1 mmol/l, 10.0+-0.7 vs 9.4+0.7%, 2.44+1.0 vs 2.37 • 0.07 mmol/100 g protein, 6.7 + 0.1 vs 6.5 +-0.I mmol/l, p = NS). Flatulence and diarrhoea were severe in 2 patients, requiring termination of study. Thus, in Type 2 diabetes mellitus, BAYm1099 was effective in diminishing and delaying postprandial excursions of blood glucose, lactate and pyruvate after high and low sucrose meals, but overall metabolic control remained unchanged. The results of acute studies on food absorption cannot be extrapolated to predict long-term effects on glycaemic control in Type 2 diabetes mellitus. 482. Inhibitory effects of interleukin I on insulin secretion, insulin biosynthesis and oxidative metabolism of isolated rat pancreatic islets S. Sandler, A. Andersson and C. Hellerstrrm. Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden Recent observations suggest a role for interleukin 1 (IL-1), in the autoimmune B-cell destruction in Type 1 (insulin-dependent) diabetes. In the present study we have investigated the effects of IL-1 on the pancreatic B cell paying particular attention to insulin production and glucose metabolism. Isolated rat pancreatic islets were kept in tissue culture for 5 days. The islets were subsequently transferred to medium RPMI 1640 plus 0.5% human serum with or without human recombinant IL-1 (25 U/ml), and cultured for another 48 h. After culture, the islets were subjected to light microscopical examination and different, functional tests in short-term incubations in the absence of IL-1. IL-1 reduced insulin release in culture and totally inhibited glucose-stimulated insulin release in short-term incubations. Islet (pro)insulin biosynthesis, glucose oxidation and oxygen uptake at 16.7 mmol/1 glucose were partially inhibited by IL-1. The DNA content of islets cultured with IL-1 was decreased and may partly explain these latter findings. Inhibition of glucose oxidation was not observed in islets exposed to IL-1 in short-term experiments. Light microscopically there were marked signs of degeneration. The present results indicate that IL-1 may be cytotoxic to islet B cells. The primary toxic action of IL-1 seems to involve factors other than an impaired islet glucose metabolism. 483. Is co-transport of Na +, K +, and CI- involved in the function of the B cells? P.-E. Sandstrrm and J. Sehlin. Department of Histology and Cell Biology, University of Ume~., Ume~, Sweden Pancreatic B cells accumulate CI- against its electrochemical gradient and replacement of extracellular chloride by certain substitute anions inhibits insulin secretion. It is not known how C1- ions are accumulated in the B cells. Some cells accumulate C1- by loop-diuretic sensitive co-transport of Na +, K +, and C1-. To investigate whether this transport system is present in the B cells, we have studied the effects of furosemide or ion substitution on fluxes of 86Rb+ (K + analogue) and 36C1 in isolated mouse islets. Furosemide (0.1-1 mmol/1) or replacement of CI- by isethionate reduced the ouabain-resistant 86Rb+ influx, whereas the ouabain-sensitive influx (Na+/K+-pump) was not affected. The effiux of 86Rb+ was reduced by furosemide or CI-i deficiency in addition to the inhibitory effect of glucose, suggesting different mechanisms of action. Both the addi-

577 A tion of furosemide (0.1-1 mmol/l) and replacement of extracellular K + or Na + by choline reduced initial uptake (3 rain) and equilibrium concentration of 36C1-. The data suggest that the B cells are equipped with a loop-diurctic sensitive co-transport system for Na +, K + and CI-, that functions as a C1- pump. This mechanism might be of importance for normal insulin secretion, since blocking with furosemide is paralleled by reduced insulin secretion. 484. Metabolic responses to the combination therapy with bedtime insulin and daytime sulfonylureas in Type 2 (non-insulin-dependent) diabetes T.A.Sane, E.Helve and M.R.Taskinen. Third and Second Departments of Medicine, University of Helsinki, Finland

To study effects of bedtime insulin therapy on glucose metabolism in Type 2 (non-insulin-dependent)diabetes uncontrolled by oral agents we measured glycaemic control, response to test meal, overnight glucose kinetics using 3-(3H)-glucose and nocturnal profiles of plasma free fatty acids (FFA), lactate, glycerol, serum counterregulatory hormones and insulin before and after evening insulin. The group included 10 patients aged 57 + 2 years (mean___SEM; RBW 128 +4%, duration of diabetes 8.5 _ 2 years, fasting C-peptide 2.34 +_0.20 p~g/1). Long-acting insulin (dose 23 ___2 IU/day) injected at 21.00 hours was combined to the previous therapy. Glycaemic control improved by insulin as indicated by a fall in fasting blood glucose (13.8+0.8 vs 8.2+0.6mmol/1, p<0.001), HbAa (11.0_+0.5% vs 9.7+_0.4%, p < 0.01) and 1 h glucose afier-test meal (18.9 _+1.1 vs 14.0 +_0.6 mmol/l, p < 0.001). Hepatic glucose production (HGP) was reduced by 17.5% (p< 0.05) both at midnight and morning hours. Overnight levels of blood glucose, FFA and glycerol were significantly lowered by insulin therapy. Pre- and postinsulin profiles of counter-regulatory hormones and lactate were similar. Nocturnal and fasting insulin levels were higher after insulin than on oral therapy. In conclusion; bedtime insulin combined with oral therapy improves effectively glycaemic control in Type 2 diabetes by suppressing nocturnal HGE We suggest a causal link between the reduction of HGP and fall of plasma FFA and glycerol. 485. Insulin sensitivity in patients with idiopathic and alimentary reactive hypoglycaemia P. Sbraccia, F. Leonetti, A.Giaccari, N. Locuratolo, E. Pugliesi, S. Busco and G. Tamburrano. Endocrinology Unit 1, University "La Sapienza', Rome, Italy

It has been proposed that increased peripheral insulin sensitivity may be involved in the pathogenesis of reactive hypoglycaemia (RH). To test this hypothesis a euglycaemic-hyperinsulinaemic clamp (1 mU. kg -I. min -1) was performed in 12 subjects with symptoms suggestive of RH (Group A) and in 5 patients with RH developed after gastrojejunostomy (Group B); 7normal volunteers formed the control group. Each subject was previously investigated by a 5 h oral glucose tolerance test (OGTI'), measuring plasma glucose, insulin, glucagon and cortisol. On the basis of glycaemic nadir Group A was subdivided in patients with biochemical hypoglycaemia associated with symptoms (Group 1A; n=7) and patients who showed only symptoms (Group2A; n=5), (2.15+-0.17 vs 3.49+0.07 mmol/l respectively, 2 p<0.05). Group B showed nadir glucose similar to Group 1A, significantly lower when compared to normal subjects (2.12 +- 0.2 vs 3.69 + 0.22 mmol/l, 2 p < 0.05). Plasma insulin concentrations, during OGTT, were similar in Group and control subjects whereas higher levels were observed in Group B at 30, 60 and 90 rain (2p<0.05). After the glycaemic nadir, cortisol levels were higher (2 p<0.05) in Group 1A when compared to the other groups, whilst no significant difference occurred in plasma glucagon concentrations in all groups. During the euglycaemic clamp the metabolised glucose (M) appeared to be significantly higher in Group 1A when compared to the M value observed in Group 2A, Group B and normal subjects, (8.10+0.72 vs 6.94+0.5, 6.48+0.2, 6.29 + 0.41 rag. kg a. min-1 respectively, 2 p < 0.05). Thus, increased insulin sensitivity seems to be a peculiar pathogenetic feature of idiopathic RH. 486. Purine metabolism and antibody-dependent cellular cytotoxicity of mononuclear cells in individuals at increased risk to develop Type l (insulin-dependent) diabetes H. Sch~tfer, Th. Jira, F. Krhler, I. Rjasanowski and D. Michaelis. Central Institute of Diabetes "Gerhardt Katsch', Karlsburg, GDR Many reported interactions between purine metabolism and the reactivity of immune competent cells lead to the question of alter-

578 A ations of purine metabolism in mononuclear cells of subjects at risk of Type I (insulin-dependent) diabetes. The adenine incorporation rates were measured in the following sononuclear cell populations of peripheral venous blood of 35 children of diabetic mothers (risk persons) and 15 control subjects: sononuctear cells, macrophage-depleted, T lymphocyte-depleted and T lymphocyte- and macrophage-depleted mononuclear cells. In extracts of these cells, the incorporation of the adenine label was measured into the ATP, ADP, AMP and hypoxanthine fractions. In 16 risk persons adenine incorporation rates were 4.2-fold increased in the T lymphocyte- and macrophagedepleted cell population. In addition the percentage of adenine label incorporated into ATP and ADP was decreased by 47%. Seven out of 16 risk persons with increased adenine incorporation in the T lymphocyte- and macrophage-depleted cell population, an increased antibody-dependent cellular cytotoxicity against rat islets of Langerhans was exerted by the same cells. Immunological and pufine metabolic alterations within the same mononuclear cell population strengthen the suspicion of an increased risk of developing Type I diabetes. 487. Effect of glycaemic control on serum lipids and apoproteins in long-term diabetic patients U.J.W. Schauer, D. PiBarek, R. Lundershausen, K. Rtihling, L. WinNer and G. Panzram. Outpatient Clinic of Intern. Med. and Inst. of Path. Biochem. Med. Acad. of Erfurt, Inst. of Path. Biochem., Univ. of Jena, GDR Follow-up data of all 208 long-term diabetic patients (duration of the disease at least 20 years) living in the closed area of the Erfurt district in 1970 had demonstrated the importance of lipoprotein pattern for longevity. The influence of glycaemic control on lipids and apoproteins was examined in 44 of them still alive in 1985, that is, 35 or more years after the onset of diabetes. Control of hyperglycaemia was assessed by measuring the glycosylated haemoglobin colorimetrically. Serum lipids and apoproteins were determined enzymatically or immunoturbidimetrically. Poor metabolic control was associated with increased levels of cholesterol, LDL cholesterol, apo B, and triglycerides as well as a changed HDL composition indicated by a decreased HDL cholesterol/apo A-I ratio. Furthermore, higher cholesterol/HDL cholesterol and apo B/apo A-I ratios were found in poorly controlled long-term diabetic patients in comparison with well controlled ones. 86% of the well controlled long-term diabetic patients had normal values of LDL cholesterol, triglycerides, apo B, HDL cholesterol, and apo A-I but only 31% of the poorly controlled patients did so. Therefore it is concluded that good glycaemic control might help to delay atherosclerosis in long-term diabetic patients via its beneficial influence on lipoprotein metabolism. 488. Effect of metabolic regulation on the plasma levels of protein C and protein S in diabetic patients: a prospective follow-up study G. Schernthaner1, Th. Chr. Vukovich, P. Km3bl and U. Hay 1. aDepartment of Medicine II, University of Vienna, Institute of Medical Physiology, University of Vienna, Austria The protein C system is an important natural anticoagulant and profibrinolytic system consisting of protein C and protein S. Recently, we reported decreased plasma levels of protein C and protein S in Type 1 (insulin-dependent) diabetic patients. In this prospective follow-up study we investigated the effect of metabolic regulation on plasma levels of protein C and its cofactor protein S in 15 Type 1 diabetic patients, 6 newly-diagnosed. Patients were tested before and 1, 2, 4 and 8 weeks after onset of intensified insulin therapy. Metabolic control improved highly significantly as assessed by HbAlc (11.1% vs 6.8%, p<0.001) and fructosamine (4.8retool/1 vs 2.9 mmol/1, p<0.001) after the 8 week treatment period. Protein C was decreased at onset of study (mean: 0.88 U/ml) and could not be normalised by improved metabolic control. Protein S was elevated at onset of study (1.12 U/ml) and decreased significantly (p< 0.01) to normal values (0.96 U/ml) during normoglycaemia. Crosslinked fibrin degradation products (XFDP) were markedly elevated (360 ng/ ml) in the 6 newly diagnosed patients at onset of disease, indicating intra-vascular fibrin formation by thrombin. XFDP declined to normal values (150 ng/ml) after 4 weeks of therapy. These results clearly indicate a hypercoagulable state in newly diagnosed Type 1 diabetic patients. Since the decrease of protein C was most pronounced in these patients and correlated closely with XFDP levels, the observed decrease of protein C may be a result of enhanced intravascular activation of protein C.

Abstracts 489. Haemostatic and haemorheologic changes in long-standing Type 1 (insulin-dependen0 diabetic patients with peripheral and autonomic cardiovascular neuropathy N. Schifino, S.B. Solerte, M. Fioravanti, M.G. Zanoletti, V. Peveri, V. Inglese, G. Gamba and E. Ferrari. Department of Internal Medicine, Medical Clinic II, University of Pavia, Italy

Microvascular degenerative complications may be associated to the pathogenesis and progression of diabetic neuropathy. In 17 longstanding Type 1 (insulin-dependent) diabetic patients with peripheral and autonomic cardiovascular neuropathy, several haemostatic and haemorheological alterations were demonstrated compared to 13 matched Type 1 patients. In particular increased plasma Von Willebrand factor antigen and fibrinogen levels were found in neuropathic (208 +47% and 28.4+9.14 mmol/1 respectively) in comparison with non-neuropathic diabetic patients (127 + 21%, p < 0.001 and 18.84_+2.87mmol/1, p<0.001 respectively). Moreover negative correlations among these parameters and both motor and sensitive conduction velocity of sural, peroneal and radial nerves were demonstrated in diabetic patients with neuropathy. Higher blood viscosity (p< 0.01 at shear-rates of 450 and 225 s - l ; p < 0.001 at shear-rates of 4.5 and 2.25s-1), plasma viscosity (1.78+0.17 versus 1.51+ 0.07 mPas, p<0.001) and lower erythrocyte deformability (1.67+ 0.47 versus 2.97 + 0.78 ml/min, p < 0.001) were also found in diabetic patients with neuropathy (p< 0.01) and nephropathy (p< 0.001) was finally reported in diabetic patients with autonomic and peripheral neuropathy. Microvascular alterations may be involved in the progression of neurological disease in long-term Type 1 diabetic patients. 490. Evaluation of the fructosamine assay for determination of glycated serum proteins E. D. Schleicher, E. Wagner and K.D. Gerbitz. Klinisch-chemisches Institut und Forschergruppe Diabetes, Mtinchen, FRG

Baker et al. have proposed that glycation of serum proteins may be quantified by measuring the reducing activity of serum in alkaline solution. We have measured serum samples of normal subjects and diabetic patients as well as in vitro glycated albumin and serum samples with the fructosamine assay and the furosine method described by us. When e-fructose-lysine was used for calibration 7-fold lower fructosamine values were obtained when compared to calibration with deoxy-l-morpholino-fructose (DMF). Linear correlation of values from 50 diabetic sera measured by both methods, showed an intercept at half of normal range for fructosamine activity and again 7-fold higher values due to DMF calibration. After specific NaBI-I4 reduction of serum proteins no furosine was detectable, whereas the fructosamine value decreased to a value of about half the normal range indicating a reducing activity that is not due to glycation of proteins. Our results indicate that the furosine method determines specifically, glycation of proteins, whereas the fructosamine assay measures glycation of proteins but also an unknown component. This component is not dialysable, it is not albumin associated and it is not constant in sera from different patients. 49t. Biochemical characterisation of an ICA antigen: a protein similar to factor B of the complement system S. Schmidt, J. Sundsmo, D. Kawahara, A. Kershnar, B. Buckingham, S.John and M.A.Charles. University of California, Irvine, California; Children's Hospital of Orange County, Orange, California; Central Institute of Diabetes, Karlsburg, GDR

Factor B, a protease of the complement system and a biosynthetic product of macrophages, is associated with Type 1 (insulin-dependent) diabetes since a) it is a class III HLA gene, b) an increased frequency of Factor B alleles occurs and c) circulating levels of Factor B are increased all suggesting Factor B involvement in the autoimmune process of diabetes. We observed that islet antigens of 90-100 Kd and 58-64 Kd are recognised by Type 1 patient sera using western blots, and since Factor B is 90-93 Kd and its activation fragment Bb is 60-63 Kd, the possibility of Factor B autoantibodies was examined. Type 1 patient sera specifically binds purified Factor B and Bb, using western blotting and ELISA. Excess Factor B added to patient sera eliminated its reactivity on western blots. Addition of Factor B to homogenates of rat islets and control sera showed the immunoreactive antigens to be the same apparent molecular size as Factor B and Bb, and a marked decrease in amount after activation by cobra venom factor, Factor D and magnesium. These results indicate 1) rat islets may express an HLA protein similar to Factor B, 2) patients with Type 1 diabetes may have autoantibodies reactive

Abstracts with Factor B, and 3) Factor B, a protein with known inflammatory function, appears to be associated with the autoimmune process in Type 1 diabetes. 492. C-terminal fragments of pancreastatin inhibit GIP-induced insulin secretion in isolated rat pancreatic islets W. E. Schmidt, G. Binder, B. Gallwitz, E. G. Siegel and W. Creutzfeldt. Div. of Gastroenterology and Endocrinology, Dept. of Medicine, University of Grttingen0 Grttingen, FRG Pancreastatin, a novel 49 amino acid residues comprising C-terminally amidated peptide, was recently isolated from porcine pancreas. It shows striking sequence homology to a part of bovine chromogranin A suggesting that this protein is a prohormone precursor. Preliminary biological experiments revealed that pancreastatin may inhibit glucose-induced insulin secretion, but its physiological role remains to be established. We synthetised the C-terminal fragments pancreastatin (29-49), P-29/49, and pancreastatin (33-49), P-33/49, by automatic Fmoc solid-phase synthesis and purified the synthetic peptides by HPLC. In this study we characterised the influence of these peptides on GIP-induced insulin secretion in isolated rat pancreatic islets. Methods: Collagenase-isolated rat pancreatic islets, precultured for 24 h, were incubated in the presence of glucose, 2.8 or 16.7 retool/l, GIP, 10 or 100 ng/ml, and synthetic P-29/49 or P33/49, 1 ~tg/ml. Results: Insulin secretion (ng/10 islets/30 rain) was at 2.8 mmol/1 glucose 3.68 • 0.68 (n = 16), at 16.7 mmol/1 5.06 _+0.77 (n=16), at 16.7 retool/1 glucose+GIP 10 ng/ml 8.95 • (n=4), at 16.7 m m o l / l + GIP 10 ng/ml + P-33/49 1 ~g/ml 5.37 + 1.58 (n=4). At 16.7mmol/1 glucose+GIP 100ng/ml insulin secretion was 38.96 + 2.06 (n = 6), at 16.7 mmol/1 + GIP 100 ng/ml + P-29/49 1 ~tg/ ml 25.50---1.14 (n=6), all representing mean_+SEM. Conclusion: The synthetic C-terminal fragments P-29/49 and P-33/49 of the novel peptide pancreastatin inhibit glucose-dependent GIP-induced insulin secretion by 35%. Pancreastatin may take part in the regulation of endocrine pancreatic secretion. 493. Characterisation of the insulin resistance in adipocytes from patients with hyperthyroidism and hypothyroidism O. Schmitz, B. Richelsen, J. Arnfred and O. Pedersen. Medical Department III, Aarhus Amtssygehus and Medical Department C, Aarhus Kommunehospital, Denmark Previous studies have shown that disturbed functions of the thyroid gland cause abnormalities of carbohydrate metabolism. In the present investigation we have examined the insulin action of isolated fat cells from untreated patients with hyperthyroidism (10 patients) and untreated hypothyroidism (6 patients) and 10 healthy age- and sexmatched control subjects. In both groups of thyroid patients 125I-insnlin binding to adipocytes was decreased by on average 35% (p< 0.05). Studies of ~4C-D-glucose transport into adipocytes from patients with hyperthyroidism or hypothyroidism revealed no changes in basal uptake rate but a 3-fold shift to the right on the insulin-doseresponse curve (p<0.05) indicating an impaired insulin sensitivity. In fat cells from hyperthyroid patients the maximal insulin responsiveness of glucose transport was decreased by 65%, too (p<0.01). Additionally, the sensitivities of insulin on a4C-D-glucose conversions to total lipids and CO2 were several-fold diminished in adipocytes from patients with both types of thyroid disorder (p<0.05). In conclusion: In man both hyper- and hypothyroidism are associated with insulin resistance of glucose utilisation localized to insulin binding and postbinding steps in adipose tissue. 494. Prevalence of microalbuminuria in Type 2 (non-insulin-dependent) diabetes mellitus: effect of duration of disease, glycaemic control and blood pressure Ch. Schnack, W. Scheithauer, J. Winlder, Ch. Gisinger and G. Schernthaner. Department of Medicine lI, University of Vienna, Austria Since information concerning microalbuminuria in Type 2 (non-insulin-dependent) diabetes mellitus is very limited, we have analysed the prevalence of microalbuminuria and the nature of clinical manifestations associated with incipient renal dysfunction in 318 patients with Type 2 diabetes. Elevated (> 15 ~tg/min) 24 h urinary albumin excretion (AER) was found in 59%. The rate of microalbuminuria among 205 Type 1 (insulin-dependent) diabetic patients screened during the same interval was 43%. AER in Type 2 diabetes was significantly higher compared to Type 1 diabetes (median AER Type 1 : 11.3; Q1 5.3, Q3 39.5; Type 2: 19.7; Q1 7.4, Q2 62.5 ~tg/min, p < 0.0001). AER in Type 2 diabetes was found to be positively correlated with both duration of disease (p< 0.001) and metabolic control as

579 A evaluated by HbAlc (p< 0.01). There was only a weak relationship between AER and mean systemic blood pressure (p-0.06). An impaired renal function indicated by elevated serum ~2-microglobulin was observed in 18.5% of Type 2 diabetic patients. The observed correlation between AER and serum /7-2-microgtobulin (r=0.35, p<0.001) indicates a marked increase of AER in patients with impaired renal function. Promising results from early intervention of incipient diabetic nephropathy in Type 1 diabetic patients warrants prospective follow-up studies in Type 2 diabetes mellitus. 495. The diabetic patient dining out. Evaluation of photographs showing natural-sized meals used for educating insulin-treated diabetic patients V. Scholz, M.Gruesser, M. Kanz, P.Kronsbein and V.Joergens. University of Diisseldorf, Department of Metabolism and Nutrition, WHO Collaborating Center for Diabetes, FRG Diabetic patients should be able to enjoy the pleasure of dining out like everyone. In order to train patients to manage their diet when dining out, we developed a new teaching tool (24 natural-sized color photographs of various meals) for use during the diet-session of our five-day teaching program. The patients were trained to recognise carbohydrates in a complete meal, to differentiate between 'free' and 'calculated' carbohydrates and to estimate the different amounts of carbohydrates. Formative evaluation revealed that the material was well accepted. Summative evaluation was performed using a dietknowledge-test (21 items). Non-medical students without diabetesteaching (n=42): 33% correct answers. Dietitians (n=20) without special diabetes-training: 52% correct answers. Nurses (n= 13): 54% correct answers. A control-group of diabetic patients (n=41): 36% correct answers before and 49% after the teaching program. The intervention-group of diabetic patients (n=70): 40% before and 77% correct answers after the training program with the new teaching tool (p< 0.001 compared to the control group). Conclusion: We have developed a program based upon photographs of natural-sized meals which was proven to be of significant benefit in diabetes teaching. 496. Complement-dependent cytotoxicity to islet cells and lymphocytes in diabetes-prone BB/OK rats D. Schrrder, B. Hehmke, I. K16ting, W. Besch and H.J. Hahn. Central Institute of Diabetes "Gerhardt Katsch", Karlsburg, GDR In a cross-sectional study complement-dependent cytotoxicity against rat islet cells was detectable by 51Cr-release in serum of 45% of diabetic BB rats (n=44). Cytotoxicity to spleen lymphocytes was found in 24% of diabetic animals (n= 29). A comparable incidence (33-45%) of islet cell cytotoxicity appeared in normoglycaemic rats at an age of 50 (n=26), 100 (n=25) or even 200 days (n=30, age > 3 SD range of manifestation peak). In contrast, all investigated sera ( n = 9-13) of 20-, 30- and 40-day old animals exhibited islet cell cytotoxicity. Lymphocytotoxicity was found in all age groups but with an increased incidence at day 40 (72%) and 50 (92%). Islet cell suspensions preexposed (1 h) in vitro to cytotoxic BB rat serum showed an increased number of trypan blue-positive cells, elevated basal insulin release and a reduced insulin secretory response. (Pro)insulin biosynthesis can be affected but is still detectable. After syngeneic islet transplantation in diabetic BB rats the relapse into hyperglycaemia reflecting B-cell destructive processes is also accompanied by increased islet cell cytotoxicity. However, the appearance of humoralmediated cytotoxicity in normoglycaemic BB rats could be a sign of general immunological dysregulation but the relation to the development of an overt diabetic syndrome remains undefined. 497. Differences in protein and fat metabolism of lean compared to obese subjects during brief starvation I. N. Scobie, A.M. Umpleby, L. Beckwith and P.H. S6nksen. Department of Medicine, United Medical and Dental Schools of Guy's and St Thomas' Hospitals, London, UK We have previously noted leucine flux rates to be higher in lean [L] compared to obese [O] subjects on brief starvation. To assess whether L subjects became 'starved' earlier on fasting, blood or plasma concentrations of products of fat and protein metabolism and lactate, pyruvate, glucose, insulin, cortisol and glucagon were measured before and after a 60 h fast in 8 L and 14 O subjects. No difference basally or at 60 h was observed for lactate, pyruvate, glucose, cortisol or glucagon but, at 60 h acetoacetate (0.4 retool/l+0.03 SEM ILl; 0.3_+0.03 [O]; p<0.01), 3-hydroxybutyrate (2.6mmol/1_0.2 [L]; 1.7 _+0.2 [O]; p < 0.01), valine (403 ~tmol/1 _+25 ILl; 335 + 17 [O]; p < 0.05), iso-leucine (135 [.tmol/l• ILl; 100_+7 [O]; p<0.01) and leu-

580 A cine (230 l s m o l / l - 16 [L]; 202 + 9 [O]; p < 0.01) were all higher in the L subjects while insulin at 12 h (4.9 mu/l+_ 1.0 [L]; 11.3 -+ 1.3 [O]; p<0.005) and 60 h (3.1 -+0.5 [L]; 7.9+1.0 [O]; p<0.005) was lower. FFA rose in both L and O but were higher in the L (1.5 mmol/1_+ 0.1 [L]; 1.26 rnmol/1_+ 0.1 [O]; p=NS). A rise in glycerol at 60 h occurred in the L only. Proteolysis and lipolysis occur earlier on fasting in L subjects in keeping with increased leucine turnover. Higher insulin levels in the O subjects may be sufficient to inhibit proteolysis and lipolysis despite relative insulin resistance with respect to glucose metabolism. 498. High fibre diets do not improve control in Type 2 (non-insulin-dependent) diabetic patients already poorly controlled on maximum doses of sulphonylureas A. R. Scott, I. Peacock, Y. Attenborough, R.B. Tattersall and W. Jeffcoate. University Hospital, Queen's Medical Centre, Nottingham, UK High fibre diets (HFD) are recommended for all diabetic patients, even though most studies of their effects on blood glucose control have been limited to a few weeks. We have studied the effects of a HFD for 6 months in 31 Type 2 (non-insulin-dependent) diabetic patients with stable but poor control (HbA1, 13.4 + 0.5% - mean + SEM) on maximum doses of sulphonylureas. After an initial run-in period, patients had an individual consultation with a dietitian, followed by 3 group teaching sessions to increase the fibre in their diet to 50 g/day. Mean fibre intake rose from 20 + 1.3 to 37 + 2.2 g/day but only 10% achieved target intake. During the run-in period there was no change in blood glucose control but by the end of the HFD fasting blood glucose rose from 10.3_+0.6 to 12.4+_0.7 rnmol/l (p< 0.0001) and HbA1 from 13.1 _+0.5 to 14.1 +0.6% (p=0.05). We conclude that diets can be modified and fibre intakes substantially increased with appropriate teaching. However, in Type 2 diabetic patients with poor control who are already on maximum doses of sulphonylureas, a HFD has little to offer and may result in a deterioration in blood glucose. 499. Quantitative screening for microalbuminuria using a rapid immunoturbidimetric technique G. S. Scott, A. C. G. Collins, N. Ghansah and H. Keen. Unit for Metabolic Medicine, United Medical and Dental Schools, (Guy's Campus), London, U K The measurement of urinary albumin concentration between normal and albustix-positive levels has proven of value in predicting progressive kidney disease of diabetes rnellitus. Accurate measurement of urinary albumin concentration currently requires time-consuming radioimrnunoassay. We have evaluated a rapid, single reagent irnrnunoturbidirnetric assay (Orion Diagnostica), automated on the Cobas Mira random access analyser (Roche Diagnostica). The method is based on the enhancement of turbidity produced by a buffered polyethylene glycol solution with a specific antigen antibody complex. The reaction is complete in less than 5 rnin. On 193 albustix-negative urine samples measured by this new method and our standard radioirnmunoassay, there was good correlation between methods (r= 0.92; linear regression equation y (pg/rnl)= 0.9608 x + 1.11. The coefficients of variation within and between batch were 1.7 and 5.0% (immunoturbidimetry) and 7.3 and 11.0% (radioirnmunoassay) respectively. This methodology gives the potential to produce a serial profile of urinary albumin excretion rate for each diabetic patient and may well be of value in detecting a) risk of renal disease, b) estimation of the rate of progression of the nephropathic process and c) evaluating the response to corrective clinical manoeuvres. 500. Neonatal transplantation of MHC-compatible thymuses into diabetes-prone BB rats J.Scott, V.H.Engelhard, C.A.Scott, J.C.Herr and D.C.Benjamin. Departments of Pediatrics, Pharmacology, Anatomy, and Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia, USA Results of our earlier studies suggested a defect in the T-cell differentiative environment of BB rats. In an attempt to determine whether the thymus is the site of this defect, 92 diabetes-prone BB neonates were grafted with thymuses from neonatal Wistar-Furth (WF) rats; half of the recipients were thymectomized. Another 27 neonates were thyrnectomized only; 28 neonates received "sham" surgery (Shams). Diabetes incidence was predictably low in thymectomy-only rats: 0% (0/7) in completely-thymectomized, 15% (3/20) in partially-thymectomized. Thymus grafts in rats not thymectornized had

Abstracts no significant effect on diabetes incidence, leucocyte counts, T-cell subsets, or mixed lymphocyte response (MLR). Diabetes incidence in thymectomized/thymus-grafted rats was similar to that in Shams; reconstitution of leucocyte counts and T-cell subsets was only to levels seen in Shams. Paradoxically, MLRs in thymectornized/thymusgrafted rats were comparable to that in WFs. These results suggest that: 1) WF thymus grafts do not prevent diabetes in BB rats; 2) thymus grafts in thymectomized BB rats enable generation of T-cells necessary for expression of diabetes; 3) thymus grafts in the absence of BB thymuses restore lymphocyte function. The thymus is apparently not the site of the defect(s) leading to diabetes or T-lymphocytopenia, but may be involved in decreased lymphocyte function in BB rats. 501. Decreased left ventricular function and its relation to duration of diabetes P.Segal, Z.Bar-Sever, Z.Vered and A.Battler. Diabetes Unit and Heart Institute, Chaim Sheba Medical Center, Tel-Hashomer, Israel Exercise-induced left ventricular dysfunction is a slowly progressive condition not related to short-term glycaemic control or to diabetic microangiopathy. Radionuclide ventriculographic studies at rest and during exercise were performed in 30 young male diabetic patients, 25 of whom were re-examined four years later using identical methodology with the aim of studying the natural history of diabetic cardiomyopathy. Mean left ventricular ejection fraction (LVEF) at rest decreased from 65.4+6.5% to 59.8+__5.9% four years later. Mean exercise LVEF data were 71.4 + 5.9% and 65.7 + 6.4% accordingly. These time-related differences were statistically highly significant. The LVEF decrease was more marked in diabetic patients with exercised-induced left ventricular dysfunction compared to those with normal LVEF response to exercise. Tirne-related LVEF decreases of >~4% occurred in half of the diabetic patients at rest, and in twothirds of them during exercise. These results are in contrast to stable rest LVEF values by age found in non-diabetic subjects. Time-related LVEF changes in diabetic patients represent a progressive left ventricular dysfunction which may contribute, by itself or in conjunction with coronary heart disease, to the development of congestive heart failure, the prevalence of which is higher among diabetic patients. 502. Raised blood pressure: a risk factor for the simultaneous presence of both microalbuminuria and retinopathy in Type 2 (non-insulin-dependent) diabetes G.Seghieri, G.Bartolomei, L.Alviggi, P.Caselli, S.Vignoli and L.A.de Giorgio. Outpatient Clinic for Diabetes, Spedali Riuniti, Pistoia, Italy Increased values of blood pressure have been associated with a raised risk for retinopathy or rnicroalburninuria in patients with Type 1 (insulin-dependent) diabetes mellitus; whether this same relation would be present in Type 2 (non-insulin-dependent) diabetes is the question asked by the present study. We studied 105 Type 2 diabetic outpatients, affected by background retinopathy only, assessed by fluoroangiography ( R + ; n=12), by microalburninuria without retinopathy, expressed as a daily albumin excretion ranging between 18 and 300 mg/24 h evaluated by RIA in at least two samples (A+ ; n = 11), by both complications (R+ A + ; n = 14), as compared with diabetic patients without any complication (N; n= 68). Median values of systolic/diastolic blood pressure, measured three times in standard conditions, were 149/83 in N patients, 155/87 in R + patients, 155/89 in A + patients and 162/95 in the R + A + group (F=3.4, p=0.02; by Duncan test, p<0.05 for R + A + vs N). After a logistic regression analysis including age, duration of diabetes, sex, HbAlc, body weight and type of therapy (sulphonylureas, insulin) as independent variables, blood pressure remained significantly higher in the R + A + group (p=0.03). In conclusion blood pressure is not significantly raised in patients with background retinopathy or microalburninuria in Type 2 diabetes, its increase being an independent risk factor for the simultaneous presence of both. 503. Effect of a2-adrenoceptor blockage by RX 821037A on B-cell function J. Sehlin, J. Doxey and P. LindstrOm. Department of Histology and Cell Biology, University of Ume~, Sweden Stimulation of a2-receptors in the B cell inhibits insulin release, although the underlying mechanisms are ohscure. We have tested whether the highly selective inhibitor of a2-adrenoceptors

Abstracts RX821037A can stimulate insulin release in isolated mouse islets. Basal insulin release was not affected by RX821037A (10-~~ x 10 -5 tool/l) but release stimulated by 10 or 20 mmol/1 glucose was markedly and dose-dependently potentiated by 10 - 6 - 5 • 10 -5 tool/1 antagonist. The inhibition of glucose-induced insulin release caused by epinephrine or the a2-agonist UK14304 was reversed by RX821037A in a dose-dependent manner. The a2-antagonist did not measurably affect the islet glucose oxidation, cAMP content or 45Ca2+ uptake. S6Rb+ influx during 5 min in the absence of glucose was reduced by 10 - 8 - 5 x 1 0 -Smol/1 RX821037A and increased by 1 0 - 8 - 1 0 - 6 m o l / 1 UK14304. These effects were totally confined to the ouabain-resistant portion of the S6Rb+ influx, the ouabain-sensitive portion ( N a / K pump) was not affected. The stimulatory effect of UK14304 (10 -6 mol/l) was reversed by RX821037A (10 _5 mol/1). At this concentration RX821037A also decreased 86Rb+ effiux. The results indicate that inhibition of a2-receptors in the mouse B cells by RX821037A potentiates glucose-induced insulin release. The effect may be mediated by reduction in B-cell K + permeability.

504. Morning versus bedtime NPH insulin in Type 2 (non-insulin-dependent) diabetes mellitus D. E. Seigler, M. Olsson and J. S. Skyler. University of Miami School of Medicine, Miami, Florida, USA We compared morning versus bedtime administration of NPH insulin in 11 Type 2 (non-insulin-dependent) subjects (age 55 +3 years; duration 8.5 _+1.5 years; 99.5 +4.6% IBW). After diet education, subjects developed their own meal plans, averaging 1808 _+118 kilocalories daily (carbohydrate - 45.4___2.1%; protein - 27.8 + 1.1%; fat 27.6_+ 2.6%). Subjects were studied at baseline (diet alone) and after two months on each of the two insulin programs in a random crossover design, in which dosage was increased until at least one daily preprandial blood glucose was < 4 mmol/1. Comparing baseline (diet) to morning and to bedtime, there was no change in body weight. Insulin doses (units. kg -I. day -a) were equivalent on the two insulin regimens. Glycaemic control was improved on both insulin regimens, but was better on bedtime than morning insulin. Fasting plasma glucose (mmol/1) was 12.0 + 0.8 (baseline), 8.6 + 0.8 (morning), and 4.5 + 0.3 (bedtime), respectively. Mean 24 h plasma glucose (mmol/1) was 13.6_+ 1.3, 9.1 _+0.8, and 7.9_+ 0.7. Glycohaemoglobin (%) was 7.7 + 0.4, 6.3 _+0.3, and 5.8 + 0.4. There was no change in hepatic glucose output, C-peptide/glucose ratio, or insulin receptor binding. Glucose disposal rate, following intravenous insulin, was improved on insulin therapy, and better on bedtime insulin. This suggests that increased insulin action accounted for the improvement in glycaemic control.

505. The U.S. trial of an implantable programmable intraperitoneal insulin system in diabetic patients J. L. Selam, C.D. Saudek, K. Waxman, H.A. Pitt, C. Holleman, M. Rubio, R.E. Fischell and M.A.Charles. University of California, Irvine, California; Johns Hopkins University, Baltimore, Maryland, USA A method of insulin delivery more acceptable, safe and effective than subcutaneous multiple injections or external pumps is needed for Type 1 (insulin-dependent) diabetes. Thus, we are studying a pulsatile pumping mechanism (MiniMed Technologies) powered by a 5-year battery design. A memory can store 60 days of insulin doses. Patients can remotely control the delivery rates using a hand-held programmer. A 10 cc reservoir is refilled every 2 months using a buffered human U400 insulin stabilised by a surfactant (Hoechst-Roussel P.I.). The study includes a 3-month pre-pump implantation runin period where patients use routine intensive subcutaneous insulin regimens followed by a 2-year post-implant follow-up in up to 40 patients. To date, 10 C-peptide negative patients have been implanted for 21 patient-months. Patients noted an increase of freedom and found the programming unit much easier to use than currently available methods. No clinical or technical problems occurred using this system. Mean (_+ SD) fasting and post-prandial blood glucose levels were maintained in the near-normal range during the run-in and post-implantation periods: 7.4+1.1 and 7.7_1.6mmol/1 respectively. These preliminary results, if confirmed by ongoing further implants, suggest that this insulin delivery system represents an acceptable, safe and effective alternative to currently used insulin treatments.

581 A 506. Prolonged inhibition of ketogenesis in man by Etomoxir P. L. Selby, K. Bartlett, H.S.A. Sherratt and K.G.M.M. Alberti. Departments of Medicine/Child Health/Pharmacology, University of Newcastle upon Tyne, The Medical School, Framlington Place, Newcastle upon Tyne, U K Etomoxir is an oxirane carboxylic acid whose CoA ester specifically inhibits carnitine palmitoyl transferase I and hence the availability of fatty acids for fi-oxidation. It is a potent inhibitor of ketogenesis and is hypoglycaemic in fasted but not fed animals. To assess its activity in man and to establish its therapeutic potential, Etomoxir was given to 6 normal male volunteers aged 26-58 years. Subjects were studied after a 12 h fast which was continued for a further 12 h after the administration of either placebo or 50 mg or 150 mg of oral Etomoxir. Blood was taken hourly. Etomoxir (50 mg) caused marked lowering of blood ketone body levels by 6 h (mean 3-hydroxybutyrate = 30 ~mol/1 + 15 (mean ___S EM); control 107 Ilmol/1 _+21, p<0.05) which persisted after 12 h (mean 3-hydroxybutyrate = 37 ~tmol/1 _+12, control 419 ~mol/1 + 78, p < 0.01). This was accompanied by increased concentrations of non-esterified fatty acids and glycerol. Plasma glucose was not altered but by 12 h there were higher blood pyruvate and alanine concentrations in the treated subjects suggesting inhibition of gluconeogenesis. These results confirm that Etomoxir is capable of producing prolonged inhibition of ketogenesis in man and suggest that after a longer period of fasting or in diabetic patients, where plasma glucose concentrations are more dependent on gluconeogenesis, it may have a hypoglycaemic effect. 507. The influence of Dichloracetate on cardiac performance and metabolism of the diabetic rat heart M. Seng, D. Str6dter, M. Mogk and K. Federlin. IIIrd Medical Clinic and Policlinie, Univ. GieBen, FRG In the isolated working rat heart model diabetes leads to cardiomyopathy, caused by reduced carbohydrate utilisation, characterised by increased production of lactate and pyruvate, indicating a reduced activity of pyruvatdehydrogenase (PDH). The aim of the study is to explore whether an activation of PDH by Dichloracetate (DCA) is able to restore alterations in cardiac function and metabolism. Materials and Methods: After 2 weeks of diabetes duration (Streptozotozin diabetes, postprandial blood glucose > 500 rag%) hearts of Wistar rats (D) are perfused for 60 rain (preload 15 cm H20, afterload 110 m H20; Krebs Henseleit-Buffer). Another group of diabetic hearts is perfused with the same buffer, containing 10 mmol/1 DCA. Results: DCA reduces lactate and pyruvate production significantly. DCA has no effect on glucose uptake, O2-uptake myocardial ATPand CP-levels, though all these parameters are significantly reduced in diabetic hearts. According to reduced cardiac metabolism cardiac function is significantly reduced in diabetic hearts and shows no amelioration with DCA. Conclusion: DCA activates PDH in the diabetic heart, shown by significantly reduced lactate and pyruvate production. An amelioration of glucose uptake and glucose utilisafion is not seen, indicating that other steps of glycolysis are sisturbed, too. Therefore DCA isn't an agent for the prevention of diabetic cardiomyopathy. 508. In vitro non-enzymic glycation of isolated human glomerular basement membrane: relationship between the protein-glucose bond and spectrofluorescence M. Sensi, F. Pricci, M. G. de Rossi, E. Capuozzo, C. Crifo, U. di Mario and D.Andreani. Depts. of Endocrinology and Biochemistry, University of Rome, Italy Advanced glycation endproducts (AGE), believed to be involved in pathologic protein crosslinking and other protein alterations, which could lead to late diabetic complications, are revealed by their emission fluorescence at 440 nm when excited by light of 380 nm wavelength. We have shown that human glomerular basement membrane (GBM) - non-enzymatically glycated in vitro - can trap increased amounts of plasma proteins. It remains to be seen whether the enhanced reactivity is caused by AGE. In this study, GBM was glycated in vitro by incubation for 10 days in phosphate buffer containing 500 mmol/l D-glucose and tracer amounts of 14-C labelled glucose. Unreacted glucose was thoroughly washed out, and the radioactivity of a sample of GBM measured immediately after, and again 10 days later, following incubation in sugar-free buffer (thus allowing release of unstably-bound glucose). In parallel experiments, glycated GBM was enzymatically solubilised and its emission fluorescence measured by means of a spectrofluorimeter. Although the glucose incorporation fell from 1898 epm/mg to 1364 cpm/mg after 10days,

582 A there was a 20% increase in peak fluorescence in glycated GBM compared to native GBM. These data suggest that, given a sufficiently high glucose concentration, AGE can be formed even after a relative short incubation time. 509. Sparteine sulfate improves glucose homeostasis in Type 2 (non-insulin-dependent) diabetes mellitus S. Sgambato, G. Paolisso, N. Passariello, G. Pizza, P. Tesauro, M. Varricchio and F. D'Onofrio. Istituti di Medicina Generale, Terapia Medica e Malattie Metaboliche e Gerontologia e Geriatria, Univers. of. Naples, Naples, Italy Twelve moderately obese Type 2 (non-insulin-dependent) (Basal Cpeptide 0.231 _+0.067 pmol/ml) patients underwent sparteine sulfate infusion in order to investigate its metabolic effects. In one group (n=6) sparteine sulfate (0.96 mg/min) infused in basal condition increased in the last 20 rain insulin (11 _+2 vs 20+7 mU/1, p<0.001) and decreased plasma glucose (8.34+0.99 vs 7.07-+0.83 mmol/1, p < 0.01) and adrenaline (67 _+12 vs 41 _+11 pg/ml, p < 0.01) levels along 60 rain of infusion. No changes were detected in plasma glucagon, growth hormone, cortisol and noradrenaline levels. In the second group (n=6) an euglycaemic hyperinsulinaemic (insulin rate = 50 mU. kg -1. h -a) and a hyperglycaemic (plasma glucose + 11.05 mmol/1) clamp were performed along 120 min. In the presence of sparteine (0.96 rag/rain) while no differences were evidenced in glucose infusion rate (GIR) in the euglycaemic clamp, along the hyperglycaemic clamp GIR (26 _+4.2 vs 20_+2.8 mg- kg -1- rain -1, p < 0.01) and plasma insulin levels (48 + 11 vs 35 _+12 mU/1, p < 0.001) were significantly higher in the last 60 min compared to the control values. So in conclusion, as in normal man, sparteine sulfate is able to improve glucose homeostasis in Type 2 diabetic patients. 510. Effect of diet change on in vivo insulin action and liver glycogen repletion G.I.Shulman and L.Rossetti. Yale University, Dept. of Internal Medicine, New Haven, Connecticut, USA To investigate the influence of dietary manipulation on in vivo glucose metabolism, we pairfed for 10 days normal rats with three diets: 1) High Protein (Hi-PN) (n=15); 2)Normal (N) (n=6); High Carbohydrate (Hi-CHO) (n= 12). Fasting plasma glucose and postmeal glucose and insulin concentrations were: (*=p<0.01 vs Hi-CHO) 6.57_+0.11mmol/1, 7.35+0.16 retool/1 and 4.0_+0.3 ng/ml in Hi PN; 6.13+0.16, 8.23 +0.16 mmol/l and 5.0+0.4 ng/ml in N; 5.63 +_0.11, 9.23 -+0.27 mmol/l and 5.8 ng/ml in Hi-CHO. Basal hepatic glucose production (HGP) were 6.4 + 0.2* mg/kg per min in Hi-PN, 6.0 + 0.2 mg/kg per min in N and 5.4 + 0.1 mg/kg per rain in Hi-CHO. Insulin sensitivity was assessed with the euglycaemic clamp using 2 insulin infusions: 2 and 4 mU/kg per min. The "M" was 14.3+0.3" and 24.4+0.8* in Hi-PN, 15.2+0.4 and 26.1 -+0.5* in N and 16.2+0.4 and 30.6 _+0.4 mg/kg per min in Hi-CHO. HGP was suppressed by 90% in the 2 mU and by 98% in the 4 mU insulin clamp in the Hi-CHO, whereas HGP suppressed by 62%* and 89%* at the 2 steps in the Hi-PN group. In order to quantitate thep a t h ways by which liver glycogen is repleted we administered 1-a3Cglucose by constant intraduodenal infusion into awake 24 h fasted rats and determined the distribution of 13C label in glycogen by NMR. The amount of glycogen synthesized from the direct pathway was calculated to be 19+4%* in Hi-PN, 30+4% in N, and 38_+1% in Hi-CHO. In conclusion Hi-PN/Lo-CHO feeding leads to: 1) fasting hyperglycaemia, 2) excessive basal HGP, 3) peripheral and hepatic I resistance, 4) shift in liver glycogen repletion from the direct to indirect pathway. 5tL In vitro and in vivo detection of interleukin-2 receptor positive lympbocytes in the pancreas of non-obese diabetic mice A.Signore, P.Pozzilli, P.C.kBeverley, J.Xu and D.Andreani. H. T, I.G. University College Hospital, Department of Diabetes and Immunogenetics, St. Bartholomew's Hospital, London and Endocrinologia (I) University of Rome, Italy Activated lymphocytes with receptor for interleukin-2 (IL-2r+ cells) have been suggested to play a major role in the pathogenesis of Bcell destruction in Type 1 (insulin-dependent) diabetes. Detection of IL-2r+ cells in the pancreas may be then important for early diagnosis. Non-obese diabetic (NOD) mice (n= 30) were studied at different ages (6 to 36 weeks) and presence of IL-2r+ cells in the pancreas was investigated by immunoperoxidase staining using an anti-IL-2 receptor monoclonal antibody (AMT-13). All animals presenting with lymphocytic infiltration in the pancreas showed IL-2r +

Abstracts cells in the islets. In order to detect in vivo IL-2r+ cells in the pancreas as a marker for lymphocytic infiltration, we have injected intravenously 123-Iodine labelled recombinant IL-2 and/or 123-iodine labelled AMT-13 antibody to 10 NOD mice and to normal Balb/c animals. Gamma camera imaging showed higher radioactivity in the pancreas of NOD mice compared to control animals; combined autoradiography and immunoperoxidase have confirmed that radioactivity in the pancreas was associated with IL-2r+ cells. We conclude that IL-2r+ cells are present in the pancreas of NOD mice and can be detected in vivo by injecting 123-iodine labelled IL-2 or a labelled anti-IL-2 receptor antibody. This method may be useful for the diagnosis (non-invasive) of lymphocytic infiltration in the pancreas. 512. Microalbuminuria and diabetic retinopathy. A correlation study in 220 Type 1 (insulin-dependent) diabetic patients A. Silva-Graga, F.Carreiras, M.Marques Vinagre, V.Genro, R.Laires, J.Moita and L.Pinto Figueiredo. Associagao Protectora dos Diabtticos de Portugal (Portuguese Diabetic Association), Lisbon, Portugal Microalbuminuria was determined by RIA in 220 Type 1 (insulin-dependent) diabetic patients (age of onset ~<30years; present age 29.3 +_16.1) and negligible insulin reserve confirmed by determination of C-peptide in the same 24 h urine. Possible relation between microalbuminuria values and diabetic retinopathy diagnosed by fluoresceinic angiography was investigated. The results did not show a clearcut relationship between the two microangiopathic lesions. Of the 108 patients with retinopathy (49.1%; 19.6+ 10 years of evolution) we found microalbuminuria >130 Ftg/min only in 37 (34.2%; 21.1 +_11.3 years of evolution), and microalbuminuria < 30 lxg/min in 71 (65.8%; 18.6_+9.3 years of evolution). In the 20 patients with proliferative retinopathy, only 8 (40%) had microalbuminuria /> 30txg/min while 12 (60%) had microalbuminuria <30 ~g/min. However, of the 48 patients with microalbuminuria >130 I.tg/min (21.8%; 18.5+11.5 years of evolution) only 11 had no retinopathy (21.2%; 9.9+7.8 years of evolution), while 37 had retinopathy (79.8%; 21.1 _+11.3 years of evolution). The results indicate that the risk of nephropathy appears associated with the existence of retinopathy, while the existence of retinopathy seems dissociated of the risk of nephropathy. An earlier detection of the microangiopathic lesions in the retina by fluorphotometry might increase the correlation observed between the appearance of microalbuminuria and blood-retinal barrier alterations. This is now under investigation. 513. Extra-pancreatic effects of Giipizide in Type 2 (non-insulin-dependent) diabetic patients H. C. R. Simpson, C.A. Stifling and J. P. D. Reckless. Clinical Investigation Department, Royal United Hospital, Bath, UK Sulphonylureas stimulate pancreatic insulin secretion, but the effect may diminish with time. Despite this, improved glucose tolerance is usually sustained, suggesting additional extra-pancreatic mechanisms. This study measured the effects of the sulphonylurea Glipizide on insulin-mediated peripheral glucose disposal. Twenty inadequately controlled Type 2 (non-insulin-dependent) diabetic patients were converted to insulin. After 3 months, insulin-mediated glucose disposal was measured, using a hypergl2(caemic clamp technique (insulin infusion rate 40 milliunits/metre~ per minute). This was expressed as the M value, defined as glucose infusion rate (rag. min -1 .kg -1) over the second hour. They were then randomised to Glipizide (Farmitalia Carlo Erba) or placebo, and 8 weeks later had a second clamp. Results are medians (rag. min -1. kg -1) With ranges. M value of those on Glipizide rose from 2.46 (1.47 to 7.98) initially to 4.23 (2.32 to 8.43) at 8 weeks (p<0.01). In the placebo group M value rose on Glipizide by 1.12 (0.04 to 2.13) compared to -0.5 ( - 3 . 0 6 to 0.89) on placebo (p<0.01). Glycosylated haemoglobins did not change. Addition of the sulphonylurea Glipizide to insulin in secondary failure Type 2 diabetic patients leads to increased peripheral glucose disposal, without change in diabetic control. 514. Insulin resistance in glucose, fatty acid but not ketone metabolism in patients with impaired glucose tolerance B.M.Singh, A.J.Krentz and M.Nattrass. The General Hospital, Birmingham, UK To determine the contribution of insulin resistance in patients with impaired glucose tolerance (IGT), 5 patients with IGT (age 54.8 + 9.8years (mean_+SD), weight 69.7+10.6kg, ideal body weight (IBW) 105+6%) and 6subjects with normal glucose tolerance (NGT) (age 39.8 + 14.9 years, weight 70.7 + 11.9 kg, IBW 101 _+11%)

Abstracts were studied using an incremental insulin infusion technique comprising sequential hourly infusions of 0 (basal), 0.005, 0.01 and 0.05 U-kg -1. h -1. Glucose tolerance was determined by standard 75 g oral glucose tolerance tests. Basal plasma insulin (IGT 4.6 _+1.5, NGT 3.9+2.5 mU/1), blood glucose (5.2+0.5 vs 4.8+0.6 retool/l), blood glycerol (0.09+0.03 vs 0.06+0.02 mmol/l), plasma N E F A (0.79-+_ 0.16 vs 0.68 + 0.2 mmol/1), and total ketone bodies concentrations (TKB) (0.08 _+0.04 vs 0.09 + 0.05 mmol/1) were not significantly different. During insulin infusion, plasma insulin (10 g) and metabolite concentrations were linearly correlated. The slopes of these relationships were not significantly different but the relationships were displaced between groups. When metabolite concentrations were compared at an insulin concentration of 30 mU/1, patients with IGT had higher glucose (0.5 mmol/1, p<0.001), glycerol (0.02 mmol/l, p<0.001) and NEFA (0.14 retool/l, p<0.001) but not TKB (10g) concentrations. Patients with IGT have insulin resistance in NEFA and glucose metabolism but not in ketone body metabolism. 515. Increased insulin secretion and growth inhibition in rat insulinoma cells by inhibition of polyamine biosynthesis A. Sj6holm and N. Welsh. Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden The functional significance of polyamines in cultured rat insulinoma cells (RINm5F) was studied by the use of DL-a-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC), the rate limiting enzyme in the polyamine biosynthetic pathway. In cells exposed to 1 mmol/1 DFMO for 4 days ODC activity, and putrescine and spermidine levels were reduced whereas the addition of putrescine during culture elevated the intracellular level of putrescine and suppressed ODC activity. DFMO-exposure resulted in a drastic inhibition of the cellular proliferative activity and consequently increased the population doubling time. Moreover, it was found that the intracellular ATP content was increased in DFMO-treated cells. Further, the inhibitor caused an increase in the cellular insulin content whereas exogenous putrescine decreased the insulin content. DFMO also evoked an increase in the release of insulin and increased the fraction of cellular hormone content released. Putrescine, when added together with DFMO, completely counteracted the effects elicited by DFMO on all parameters studied, thus confirming the specificity of action of the inhibitor. We conclude that polyamines affect the proliferation of RINm5F cells and that inhibition of polyamine biosynthesis leads to an increased insulin release and content of these cells. 516. Inhibition of glucose-induced insulin secretion by clonidine: possible involvement of calcium mechanisms G. Skoglund, I. Lundquist I and B.Ahr6n. Department of Pharmacology, Lund University, Lund, Sweden and Department of Cellbiology,1 University of Link6ping, Link6ping, Sweden The mechanisms behind the inhibition by a-adrenoceptor stimulation on glucose-induced insulin secretion are still unclear. Therefore, we investigated the possible involvement of calcium mechanisms, on clonidine-induced inhibition of glucose-stimulated insulin secretion. Glucose (13.3 mmol/1)-induced insulin secretion from isolated rat islets incubated in 2.0 retool/1 calcium was inhibited by clonidine at 1.0 gmol/l (by 48%, p < 0.001). By raising the extracellular calcium concentration from 0.1-3.0 mmol/1, glucose-stimulated insulin secretion was potentiated and the inhibition by clonidine was counteracted. Also the calcium-channel agonist BayK8644 (1.0 ~mol/1) potentiated glucose-stimulated insulin secretion and counteracted the effect of clonidine. Glucose (13.3 mmol/1)-induced 4%alcium-efflux from preloaded perifused rat islets was inhibited by clonidine, during the first phase by 50% (p<0.001) and during the second phase by 25% (p< 0.001). Conclusions: The results show that a-adrenoceptor stimulation inhibits glucose-induced insulin secretion in isolated rat islets by an a2-adrenoceptor effect. Since raised extracellular calcium and the calcium-channel agonist BayK8644 counteracted the inhibition and clonidine reduced glucose-induced calcium-effiux, it is likely that calcium mechanisms are involved in the effect. 517. Acetazolamide reduces glomerular filtration rate and proximal tubular reabsorption in diabetic nephropathy P.Skott, E. Hommel, S.Amold-Larsen, N.E. Bruun and H.-H. Parving. Hvid6re Hospital, Klampenborg and Department of Clinical Physiology and Nuclear Medicine, Glostrup Hospital, Copenhagen, Denmark

583 A A double-blind placebo-controlled cross-over study of the renal effects of a 3-day course of acetazolamide 250 mg was performed in 7 healthy subjects (Group I) and 8 Type 1 (insulin-dependent) diabetic patients with diabetic nephropathy (Group II). Glomerular filtration rate (GFR) was measured with the single bolus 51Cr-EDTA technique, and fluid flow rate from the proximal tubules into the thin descending limb of Henle's loop by the renal lithium clearance. Results are shown as mean + SD. GFR (ml/min) diminished during acetazolamide (I: 108 (11) vs 82 (9), p<0.02, II: 71 (19) vs 54 (14), p < 0.01), while the renal lithium clearance (ml/min) was unchanged (I: 22 (6) vs 27 (8), II: 14 (5) vs 15 (4)). Absolute proximal reabsorption of water (ml/min) declined (I: 85 (11) vs 56 (7), p<0.02, II: 55 ('17) vs 37 (6), p < 0.02), as did fractional proximal reabsorption of water and sodium (%) during active treatment (I: 79 (5) vs 67 (8), p<0.02, II: 79 (5) vs 72 (6), p<0.02). Renal sodium clearance and distal fractional reabsorption of sodium was unchanged. Acetazolamide reduces GFR and proximal tubular reabsorption of sodium and water in parallel both in Type 1 patients with diabetic nephropathy and in control subjects, while the output of fluid from the proximal tubules is kept nearly constant.

518. Platelet carnitine metabolism in diabetes mellitus C. C. T. Smith, M.B. Cooper, L.D. Curtis, C.A. Forte, D.A. Jones, A. P. Wilson and D.J. Betteridge. Department of Medicine, University College London, London, U K Carnitine is an essential co-factor for the metabolism of fatty acids. Its function is as a transporter of fatty acids into the mitochondrial matrix for foxidation. We have measured carnitine and its sub-species (short-chain esters, including acetylcarnitine, and long-chain acyl esters) in platelets from normal subjects and Type I (insulin-dependent) and Type 2 (non-insulin-dependent) diabetic patients. The effect of thrombin stimulation on platelet carnitine metabolism was also studied. Camitine species were generally lower in platelets from Type 1 diabetic patients (n=15) than in age-matched normal subjects (n=12), short-chain esters being significantly reduced (50%, p < 0.05). In Type 2 diabetic patients (n= 8) platelet camitine species were generally higher than in normal subjects (n= 5), significant increases being recorded for total (85%, p<0.04), short-chain (147%, p < 0.03) and acetyl (123%, p= 0.04) camitine species. Washed platelets from normal subjects (n=7) showed an increase of 135% (/)=0.01) in long-chain acyl camitine content on thrombin stimulation. Platelets from Type 1 diabetic patients (n=6) showed an increase of 176% (p<0.05) in this species, a decrease of 50% (p<0.05) in short-chain ester concentration also being observed. These data may have important implications for platelet fatty acid metabolism and energy production in diabetes mellitus and hence platelet activity.

519. Increasing insulin resistance through puberty? C.P.Smith, H.R.Archibald, J.M.Thomas, A.C.Tarn, A.J.K.Williares, E.A.M.Gale and M.O.Savage. Queen Elizabeth Hosp. for Children, and Dept. of Diabetes & Immunogenetics, St. Bartholomew's Hosp., London, U K The effect of puberty and age on insulin secretion was studied. Intravenous glucose tolerance tests (0.5 g/kg, maximum 25 g) were performed on 53 islet-cell antibody negative siblings of diabetic children (29boys, 24girls, aged 5.6-16.9years) and 16adults (10men, 6 women, 21-39 years). Puberty was staged using Tanner's criteria and subjects divided into 4 groups: I - stage 1 (n= 19), II - stages 2 & 3 (n= 16), III - stages 4 & 5 (n= 18), IV - adults (n= 16). Children showed a progressively larger insulin response with increasing pubertal development. Spearman's rank correlation showed significant increase in fasting insulin (rho=0.6, p<0.001), 0-10min area (rho =0.5, p < 0.001) and 10-60 min area (rho =0.7, p < 0.001). There was positive correlation between insulin (fasting, 0-10 and 10-60 min areas) and age to 17 years but multiple regression analysis using logged data showed that this was due to puberty alone. Indeed pre-pubertal children and adults did not differ. We found no sex differences nor any relationship between relative body weight or height standard deviation scores and insulin levels. There was no correlation between glucose (fasting and 0-60 rain area) and puberty or age. This suggests an increasing insulin resistance during puberty which may contribute to the frequent presentation and worsening of control of Type 1 (insulin-dependent) diabetes at this time.

584 A 520. Anti-insulin antibodies. How should they be looked at? F. Sodoyez-Goffaux, M. Koch, N. Dozio and J.-C. Sodoyez. Laboratory of Experimental Nuclear Medicine, University of Liege, Li6ge, Belgium

Anti-insulin antibodies (AIA) of 60 insulin-treated patients were characterised by a variety of techniques. It was found that insulin displays several antigenic determinants, some of which contain a tyrosine residue. Iodination of tyrosine may alter the fit between the epitope and the paratope. Therefore, techniques based on the use of radioiodinated insulin may lead to incorrect antibody determination. Some epitopes (A chain loop and region of AI4) are "hidden" or conformationally altered when insulin is coated, introducing a bias in solid phase assay. In each patient, antibodies were directed against more than one determinant, allowing the formation of immune complexes of 2 or more IgG. AIA also varied with regard to affinity and IgG subclass composition. In conclusion: 1) In each patient, the number and properties of AIA form a unique mosaic. 2) Neither liquid nor solid phase assay is fully safe for AIA measurement. 3) Because of the numerous varieties of AIA, the concept of an international standard loses its rationale. 4) In view of their potential importance in insulin-treated diabetic patients, but also in Type 1 (insulin-dependent) diabetic patients before treatment, and in autoimmune hypoglycaemic syndrome, AIA should be searched for and characterised by a number of techniques rather than by a single one. 521. Plasma atrial natriuretic peptide, renal haemodynamics and microalbuminuria in short-term Type I (insulin-dependent) diabetic patients with glomerular hyperfiltration S.B.Solerte, M.Fioravanti, P.Spriano 1, C.Aprile 2, A.kPatti and E. Ferrari. Department of Internal Medicine, Medical Clinic II, University of Pavia, Italy, aDepartment of Nuclear Medicine, University of Pavia, eFondazione Clinica del Lavoro, Pavia, Italy

Atrial natriuretic peptide (ANP) has been involved in renal haemodynamic regulation. Glomernlar hyperfiltration in short-term diabetic patients might therefore be related to plasma ANP changes. In 14 short-term Type I (insulin-dependent) diabetic patients with glomernlar hypeffiltration (mean G F R = 155 ml/min per 1.73 m 2) and 20 matched Type 1 diabetic patients with normal GFR (105 ml/min per 1.73 m2), plasma ANP (09.00 hours, RIA Peninsula, Belmont, Calif., USA), plasma renin activity (PRA), aldosterone, urinary albumin excretion rate (UAER) and clearance of albumin were studied. Increased plasma ANP levels were found in hyperfiltering diabetic patients (mean + SD = 35 _+7 pg/ml) compared to patients with normal GFR (16.6___3 pg/ml, p<0.001) and to healthy subjects (21 + 6.7 pg/ml, p < 0.001). Higher PRA was demonstrated in diabetic patients with high GFR (4.1+0.8 n g . m l - a . h -a) than in diabetic patients with normal GFR (2.8 _+0.7 ng- ml-1. h - l , p < 0.001), whereas no variations in plasma aldosterone levels were found between the two groups of patients (141+53 vs 127+27 pg/ml). A significant positive correlation between ANP and PRA was demonstrated in our diabetic patients (r=0.56, p<0.001). Furthermore a positive correlation among ANP, GFR (r=0.60, p<0.001), U A E R (r=0.93, p < 0.001) and clearance of albumin (r= 0.81, p < 0.001) was observed in diabetic patients with glomerular hyperfiltration. Blood glucose levels seem to influence plasma ANP changes, as well as GFR, both in patients with high and normal GFR. Our data suggest a role for ANP in the occurrence of glomerular hyperfiltration and microalbuminutia in short-term Type 1 diabetic patients. 522. The effect of severe hypoglycaemia and diabetes on the EEG in diabetic children G.Solt6sz and G.Acsfidi. University Department of Paediatrics, Prcs, Hungary Insulin hypoglycaemia (H) is the most common form of H in childhood and severe and recurrent H can cause brain damage. Serial EEG recordings were made in 70 diabetic children (DC) (age I1.2 _+0.5 years, duration of diabetes (DD) 5.1 + 0.4 years, mean + SEM) and EEG findings were related to age (at EEG and at diagnosis), DD, daily insulin dose, long-term metabolic control assessed by HbM and severe H episodes. EEG-abnormalities (EEG-A) were found in 26% of DC. There was no relationship between EEG-A and DD, daily insulin dose or HbAv DC with EEG-A were younger (9.2_+ 0.7 vs 12.8 _+0.7 years, p < 0.01), had an earlier onset of diabetes (4.8 + 0.6 vs 7.6 + 0.6 years) and 78% of them had severe antecedent H, whereas EEG-A were found in only 22% of DC with no H (p< 0.001). All DC with H and convulsions had permanent EEG-A.

Abstracts The degree of metabolic control has no effect on the EEG during the early years of diabetes, but severe antecedent H, young age and early onset are important risk factors. 523. Potentiation of insulin release by earbamoylcholine depends on an early transient calcium entry B. Sofia, C. Ripoll and J. V. Sanchez-Andres. Department of Physiology, Faculty of Medicine, University of Alicante, Alicante, Spain

Muscarinic potentiation of glucose-induced insulin release depends on extracellular calcium. We herewith demonstrate that calcium is required during the first minute after the challenge with carbamoylcholine. Calcium channel activity was continuously monitored measuring: spike frequency, dV/dt of the spike depolarisation and the variance of the membrane potential fluctuations recorded intracellularly from microdissected mouse islets. Addition of carbamoylcholine (1-1000 lxmol/l) produces: 1) Calcium channel activity increase during 30-60 s. 2) Insulin secretion increase to about twice control values while carbamoylcholine is present (25 min). When carbamoylcholine is applied in a Ca-free medium (No Ca 2+, 1 mmol/l EGTA, 3.6 Mg 2+) no potentiation of insulin release is observed. However, when extracellular calcium (2.5 mmol/l) is present during the first minute of exposure to carbamoylcholine the stimulatory effects are not different from those in which Ca 2+ was present throughout the experiment. When carbamoylcholine and Ca 2+ are withdrawn after the first minute, insulin secretion increases during 4-5 min and then decay to calcium-free values. Conclusions: There is an early transient in calcium entry which triggers insulin secretion. After the first minute, carbamoylcholine effects on secretion are independent of extracellular calcium but require its early transient entry. 524. Interleuldn-1 exerts bimodal effects on insulin release from rat islets depending on dose, exposure time and ambient glucose concentration G.A. Spinas, J.P. Palmer, T. Mandrup-Poulsen, J.H. Nielsen, H. Andersen and J. Nerup. Steno Memorial Hospital, Gentofte, Denmark Low concentrations of Interleukin-1 (IL-1) have been shown to stimulate, and higher concentrations to inhibit insulin release (IRI) from isolated islets of Langerhans. To test whether this bimodal effect on IRI is related to IL-1 dose, duration of exposure and ambient glucose concentration (G), and whether glucagon release (IRG) is similarly affected, rat islets were exposed for 6 h to 6 days to IL-1 in doses ranging from 20-2000 pg/ml at G of 3.3, 5.5 and 11 retool/1. After 6 h all IL-1 doses stimulated IRI at all three G (maximally 272% at 5.5 mmol/1 G and 100 pg/ml IL-1, p<0.01). At 24 h exposure a biphasic effect on IRI was seen: stimulation (301% at 3.3 G and 200 pg/ml IL-1, p < 0.01) followed by inhibition directly related to higher G and higher IL-1 dose. After 6 days all IL-1 doses inhibited IRI irrespective of G. Effects on IRG were inconsistent: after 24h no effect on IRG was seen at 3.3 mmol/1 G, whereas at 11 mmol/1 G IL-1 doses which clearly inhibited IRI stimulated IRG (85%, p=0.01). Conclusions: IL-l's effect on IRI is biphasic: stimulation followed by inhibition, depending on IL-1 dose, duration of exposure and ambient glucose concentration. If IL-1 mediates the B-cell cytotoxicity of Type 1 (insulin-dependent) diabetes, this effect may be exacerbated by high blood glucose levels. 525. Prevalence and risk profile of incipient diabetic nephropathy in Type 2 (non-insulin-dependent) diabetes mellitus E. Standl, B. Rebell I, H. Stiegler1, A. Ziegler~, H. U. Janka I, K. Schulz2, R.Roth 2 and W. Lehmacher 2. aCity Hospital Munich-Schwabing; 2midis-Institut ftir Med. Informatik und Systemforschung, Munich, FRG Recently, major emphasis has been put on the stage of incipient diabetic nephropathy as defined by microalbuminuria, since it heralds overt nephropathy some years later and may still be accessible to secondary prevention. Most of the work, however, has been done in Type 1 (insulin-dependent) diabetes. The present study was designed to investigate a representative sample of 307 unselected Type 2 (noninsulin-dependent) diabetic patients (110 males, 197 females) in general practice with normal ereatinine, compensated cardiac function, and below age 75, for microalbuminuria (RIA; 30-200 mg/dl) and the associated risk profile. After accounting for urinary tract infection, prevalence of incipient diabetic nephropathy was 20.4% in males and 18.7% in females. There were peaks in prevalence associated with short known duration of diabetes ( < 4 years) and with

Abstracts long duration (> "13 years). Incipient nephropathy was closely correlated (p< 0.0"1) with systolic blood pressure, HbAlo, factor VIII R: Ag (v.Willebrand-factor) in blood, flz-microglobulin in serum, and peripheral artery disease (Ultrasound-Doppler-examination). Compared to Type 1 diabetes results indicate that incipient diabetic nephropathy in Type 2 diabetes is at least as common and especially frequent in patients with rather short known duration of the disease. In the possible context of prevention the association with elevated blood pressure and peripheral vascular disease seems particularly noteworthy.

526. The action profiles of human N P H insulin preparations A. A. R. Starke, L. Heinemann, A. Hohmann and M. Berger. Medical Department of Nutrition and Metabolic Diseases, Diisseldorf University, FRG We determined the action profiles of Protaphan HM (Novo/PN), Insulatard H (Nordisk/IN), and Huminsulin Basal (Lilly/HL) by means of the euglycaemic clamp technique during normoglycaemia and normoinsulinaemia. Seven normal subjects (age 28+"1 years; BMI 22.5 _+0.5) were connected to the Biostator on 4 occasions in randomised order using standard procedures. After 2 h, a stable insulin level of 10 + 0.8 .uU/ml was established by means of a low dose insulin infusion (0."1 m U . k g -1.min -~ by a separate pump). C-peptide fell by 30% below 0.8 ng/ml throughout the experiments. Blood glucose was clamped at 5.0 mmol/1 to establish basal glucose infusion rates (GIR) or 90 min. Twelve U of long acting insulin were injected subcutaneously into the abdominal wall at least 3 h after connection to the Biostator and GIR was monitored overnight for "19 h. In control studies, GIR rose slightly but not significantly compared to basal levels. Plasma free insulin peaked at 18.6+l.9p~U/ml (240min, PN), 18.7+1.4 (180min, IN), and 15.5+0.6 (180rain, HL); and returned to basal levels within 16 h. Maximal GIR's were comparable (3.6-3.9 + 0.6 mg. kg -~. min -1) and peaked at 330 min (PN, IN) and 420 rain (HL). They returned to basal rates within 19 h. After determination of insulin action - maximal GIR - the time ranges were calculated during which 90, 75, 50, and 25% of maximal action was present. No significant differences were observed between insulin preparations. Conclusion: The action profiles of Protaphan HM, Insulatard H, and Huminsulin Basal Lilly are indistinguishable.

527. Effect of mild hypoglycaemia on counter-regulatory responses and psychomotor performance in man A.B. Stevens, W.R. McKane, P.M. Bell, P. Bell, D.J. King and J. R. Hayes. Departments of Medicine & Therapeutics & Pharmacology, Queen's University of Belfast and Diabetic Clinic, Belfast City Hospital, Northern Ireland The effect of mild hypoglycaemia on counter-regulatory responses and psychomotor performance in "12 non-diabetic subjects, (age range 18-27 years) was studied using a double blind cross-over design. Each subject was studied on 2 separate days, plasma glucose being held at either fasting, euglycaemic (4.9 + 0.0 mmol/1, mean + SEM) levels or lowered to hypoglycaemic (3.4_+ 0.1 mmol/1) levels using the hyperinsulinaemic clamp technique. Blood was taken every 15 rain for hormone measurements. Psychomotor tests and a questionnaire assessing hypoglycaemic symptoms were administered before, and repeated during, each clamp period. Comparison of hormone concentrations for the 2 study periods (Wilcoxon Signed Rank Test) demonstrated higher plasma concentrations of adrenalin (median 567 ng/1 vs 28.5 ng/1, p<0.0'1), glucagon (255 ng/1 vs 85 ng/1, p < 0.01) and pancreatic polypeptide (390 ng/l vs 32.5 ng/l, p < 0.0'1) during hypoglycaemia. Results for psychomotor tests and symptom scores were taken as change from baseline. Analysis of symptoms questionnaires (Wilcoxon Signed Rank Test) showed higher scores during hypoglycaemia (median 2.5 (range -'1 to 11) vs 1 ( - 4 to 7), p < 0.01). Impaired performance during hypoglycaemia was demonstrated (Paired T Tests) on 2 psychomotor tests: Trailmaking, a test of fine motor performance ( + 1.2 + 4.8 targets/min, mean + SEM, vs -"19.32+4.2 targets/min, p=0.004) and Digit Symbol substitution, a test of cognitive function (27 + 4 symbols/min vs 18 + 3 symbols/ min, p=0.003). Thus significant, symptomatic counter-regulation and impaired psychomotor performance occurs in non-diabetic subjects at plasma glucose values of 3.4 mmol/1.

585 A 528. The islet of Langerhans: morphological and biochemical changes during Cyclosporin. A treatment in the rat F. St6ckmann, V. Steffens, B. Stobbe and W. Creutzfeldt. Med. Univ. Klinik, Abt. Gastroenterol. Endokrinol. G6ttingen, FRG

Treatment of rats with Cyclosporin A (CyA) in a dosage of 50 rag/ kg bw induces pancreatic insulin depletion, vacuolisation of B cells and hyperglycaemia after 7 days. The aim of this study was to evaluate the effect of therapeutical doses of CyA on morphological and biochemical parameters of rat islets. Male Wistar rats were tube fed once daily with CyA a)5 rag, b)10 mg and c)20 m g / k g b w for 6 weeks, controls (C) received solvent solution. At the end of the treatment period and after 4 weeks of recovery (without CyA-treatment) blood was collected during pentobarbital anaesthesia from the abdominal aorta, the pancreas was removed and either fixed in Bouin's solution or frozen for insulin extraction. Whole blood CyA levels, plasma insulin and GIP were determined by specific RIA, glucose with the hexokinase method. Islet B cells were stained for insulin by immunohistochemistry using the PAP-method, morphometric analysis was performed with a Zeiss morphomat. Weight gain was reduced significantly in rats fed 10 and 20 mg/kg bw CyA, but no hyperglycaemia was observed in the animals. Plasma insulin and pancreatic insulin content decreased dose-dependently. After 4 weeks recovery pancreatic insulin content was back to normal whereas plasma insulin levels were still lowered. Immunohistochemistry of the B cells revealed a decrease in volume density, in the recovery group the changes were reversible except for the 20 rag/ kg bw group. Feeding different doses of CyA in rats induces a dose-related decrease of plasma and pancreatic insulin. These effects coincide with the B-cell morphology. 529. Effects of ascorbic acid on nonenzymatic glycation of serum proteins in vitro and in vivo P. Stolba, K. Hfitle1, A. Krfifikovfi, M. Stgeda ~ and L. Stfirka. Research Institute of Endocrinology, Prague, Medical Faculty, Charles University, Prague 1, Czechoslovakia

We investigated whether redox system of ascorbic acid could influence the nonenzymatic glycation of serum proteins in vitro, on guinea pig model, and in diabetic patients. In vitro effects of various concentrations of ascorbic acid and dehydroascorbic acid on total glucose incorporation into human and bovine albumin and human serum proteins were investigated using D(u-agc)-glucose, fructosamine and thiobarbituric acid assays. The artificial glycations (n = 6) of serum albumin after 3 days incubation 'with 25 mmol/l glucose and 1 mmol/1 vitamin C were as follows: 60 .umol/g (glucose only), 49 p~mol/g (+ ascorbic acid) and 71 lxmol/g (+ dehydroascorbic acid). The same data for the whole serum: 161, 121, 136 p.mol/g. Effects of both forms of vitamin C were concentration dependent. The group of guinea pigs after 10 days on diet with reduced content of C vitamin showed significantly (p= 0.0023) higher glycation of serum proteins (102.5 _+_12.4 p~mol/g, n= 17) than controls (84.9 _+17.2, n=17). Twenty-one Type 1 (insulin-dependent) diabetic patients were treated with 3 x 500 mg vitamin C/day per os, for 3 weeks. Significant decrease of glycation (before 1.81+0.29 mmol/1, after 1.69 _+0.34 retool/l, p = 0.0076) was recorded. No significant changes of glycaemia were observed. Our results suggest the possibility to influence the nonenzymatic glycation of proteins by administration of vitamin C. 530. D-fenfluramine ameliorates fat-feeding induced insulin resistance L. H. Storlien, G.A. Smythe, A.W. Thorburn, A.B. Jenkins, E.W. Kraegen and D.J.Chisholm. Garvan Institute of Medical Research, St Vincent's Hospital, Sydney, Australia

There is some evidence that d-fenfluramine, an important anorectic and weight loss pharmaceutical, independently improves insulin action. Our recent results also show that chronic d-fenfluramine reduces hypothalamic noradrenergic tone which correlates highly with circulating glucose levels. We now report that chronic d-fenfluramine (5 mg-kg -1- day -t) ameliorates insulin resistance induced by high fat feeding. Insulin action was assessed in adult rats using the euglycaemic clamp (insulin 140 mU/1) combined with 3H-2-deoxyglucose and ~4C-glucose bolus administration. Food intake was matched between groups. Basal glucose turnover was reduced 29% (p<0.05) in fat-fed rats receiving d-fenfluramine (fat+FEN). The glucose infusion rate to maintain euglycaemia was 22.0+1.1 mgkg -~. min -1) in carbohydrate-fed rats, reduced to 8.2_+ 1.0 with fatfeeding but only to 15.1 + 0.5 in the fat + FEN group. Peripheral glucose disposal, reflecting measured skeletal muscle changes, was

586 A reduced by fat-feeding (from 23.5+1.0 to 13.8+0.6mg.kg -1. min -a) but improved by d-fenfluramine (16.9+0.5, p<0.05). Impaired suppression of liver glucose output by insulin, caused by fat feeding, was totally reversed by d-fenfluramine. Thus d-fenfluramine counteracted fat-feeding induced insulin resistance with major effects in liver. We hypothesize that d-fenfluramine improves insulin action through reducing hypothalamic noradrenergic tone which in turn reduces both basal hepatic glucose output and improves the hepatic response to insulin. 531. Diabetic cardiomyopathy - fact or fiction?

D.Stroedter, K.Schuster and K.Federlin. 3 ra Medical Clinic and Policlinic, Univ. Giessen, FRG The influence of diabetes on cardiac function and metabolism is examined with reference to the question of whether haemodynamic or metabolic changes show reversibility after islet transplantation. Material and methods: After 2-(D0 and 8-(D2) weeks of diabetes duration (Streptozotocin-diabetes, fasting blood glucose >350 rag%) hearts of Lewis-rats are perfused about 60 min in the working rat heart model. In addition, in 2 groups, prepared as D1 and D2 ( = T1 and T2) an islet transplantation is performed, leading to fasting blood glucose levels < 90 mg%. After 1 week of therapy these animals are killed (n=7/group). The groups are compared with controis (C). Results: Diabetes leads to a significant reduction of cardiac output (D1 11%, D2 28% lower than C). Islet transplantation normalises cardiac output. Whereas glucose uptake/h is reduced in diabetic hearts by 50%, lactate and pyruvate production is significantly increased. Islet transplantation leads to an increase in glucose uptake of 62% (D1), 64% (D2) respectively, and to normalisation of cardiac output, too. Conclusion: Diabetes leads to a cardiomyopathy, not only characterised by reduced cardiac function, but by reduced carbohydrate utilisation, too. Cardiac function is restored by islet transplantation, indicating that the cardiomyopathy is a result of insulin deficiency. 532. Development of fetal pre-chondrocytes in vitro is altered by increased levels of D-glucose and B-hydroxybutyrate

J.Styrud 1, E.Unger2 and U.J.Eriksson 1. aDepartment of Medical Cell Biology, Uppsala University, Uppsala, Sweden, 2Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences Biomedicum, Uppsala, Sweden The in vitro study of pre-chondrocytes and chondrocytes was used to elucidate the effects of a maternal diabetic environment on early chondrogenesis in the rat. On gestational day 12 fetal rat limb buds and mandibles were digested to single cells, suspended, plated and cultured for 5 days (2.0-2.5.10 7 cells/ml) under varied conditions (D-glucose: 5.5-44.4 mmol/1, B-hydroxybutyrate: 0.5-32.0 mmol/1). On culture day 5, the cells were incubated for 24 h with Na235SO4 and 3H-thymidine, harvested, their levels of radioactivity measured and correlated to DNA content. Limb bud cells showed decreased thymidine incorporation when the B-hydroxybutyrate levels were increased but not when D-glucose levels were raised. Gel chromat graphy analysis of the Guanidine-HC1 extracted proteoglycans produced by the cultures yielded a single peak of chondroitin-sulfate proteoglycan (Kav 0.41 on a CL-2B column), thus confirming the differentiation of cartilage in vitro. It was also possible to maintain fetal mandibular pre-chondrocytes in culture for six days. The mandibular (pre)chondrocytes showed decreased thymidine incorporation in response to both elevated glucose and B-hydroxybutyrate concentrations. Fetal mandibular pre-chondrocytes are thus more vulnerable than limb bud pre-chondrocytes to high levels of D-glucose and Bhydroxybutyrate. This may be of relevance for the understanding of skeletal malformations in expermintal diabetic pregnancy. 533. Insulin regulates the serum levels of low molecular weight insulinlike growth factor binding protein

A.-M. Suikkari, V.A. Koivisto, E.-M. Rutanen, H.Yki-J~irvinen and M. Sepp~il~i. Helsinki University Hospital, Department I of Obstetrics and Gynecology, and Department III of Medicine, Helsinki, Finland We measured serum 34K insulin-like growth factor (IGF)-binding protein in 88 Type '1 (insulin-dependent) diabetic patients, 9 Type 2 (non-insulin-dependent)diabetic patients, 7 patients with insulinoma and in 93 healthy control subjects. In addition, the effect of 2-3 h euglycaemic hyperinsulinaemia (1 mU insulin clamp) on IGF-binding protein levels was studied in each group. Compared with control

Abstracts subjects, IGF binding protein was elevated by 5-fold (p< 0.001) in Type 1 diabetic patients treated with conventional injections, by 3-fold (p<0.001) in patients treated with CSII, and by 2-fold (t9< 0.001) in Type 2 diabetic patients. In insulinoma patients, IGF binding protein level was 50% below normal (p< 0.001), but was normalised after removal of the tumour. IGF-binding protein level and insulin dose correlated inversely in diabetic patients ( r = - 0 . 2 2 , p < 0.05). During steady state hypefinsulinaemia, IGF-binding protein level fell by 40%-70% in each group (p< 0.001). The fall was closely related to the baseline binding protein levels (r=0.94, p<0.001). In conclusion: 1) 34K IGF binding protein levels are elevated in both Type '1 and Type 2 diabetic patients, and decreased in patients with insulinoma. 2) The binding protein level is reduced by acute hyperinsulinaemia. 3)These data suggest that serum insulin concentration is important in the regulation of serum 34K IGF-binding protein level.

534. Autonomic and peripheral nerve function before and after treatment with a novel aldose reductase inhibitor

G. Sundkvist, B. Lilja, I. Ros~n and C.-D.Agardh. Departments of Medicine and Clinical Physiology, Malta6 General Hospital and Departments of Clinical Neurophysiology and Medicine, University Hospital, Lund, University of Lund, Sweden In this study we compared autonomic and peripheral nerve function in 30male Type1 (insulin-dependent) diabetic patients (age 25-44 years, mean 34; duration of diabetes 10-20 years, mean 14). All patients demonstrated neurographic signs of peripheral neuropathy (PN) although none reported such symptoms. Autonomic neuropathy (AN) was established from the heart rate reaction to deep breathing (E/I ratio) and to tilt (acceleration and brake indices). The patients then took part in a 4-week double blind cross-over study where the effect on neuropathy of a novel aldose reductase inhibitor (ARI), Statil (300 mg daily), was evaluated. Twenty-nine patients, 13 with AN, completed the study. Among neurographic tests, only those of sural nerve function correlated with AN. Patients with AN showed significantly lower sensory action potential amplitudes sural, indicating axonal losses, than patients without AN (3.58 + 0.79 ~tV vs 7.34+'1.12 lmV, p<0.01). AN and not PN improved on ARI. After ARI treatment, the E/I ratio increased (1.33+0.03 vs 1.37+0.03, p<0.05) in all patients and the brake index (7.5 +0.7 vs 10.5 + 1.4, p < 0.05) in patients with AN. Moreover, among 12 patients with an abnormal brake index, 8 improved after ARI and 1 after placebo (p< 0.01). The lack of correlations between most neurographic tests and AN as well as the selective effect of ARI on AN suggest that AN not necessarily is a part of a generalised polyneuropathy.

535. Biochemical characterisation of islet protein antigens reactive with serum from Type I (insulin-dependent) diabetic patients

J. Sundsmo, S. Schmidt, J.M. Plunkett, A. Kershnar, B. Buckingham, D. Kawahara and M.A.Charles. University of California, Irvine, California; Children's Hospital of Orange County, Orange, California; Central Institute of Diabetes, Karlsburg, GDR The possibility of characterising a single islet antigen, e.g. a glycolipid or protein, having a pivotal role in the pathogenesis of Type 1 (insulin-dependent) diabetes has been emphasised in studies using monodonal antibodies. Because of the immunological heterogeneity of Type 1 diabetes, this possibility seems unlikely. The complexity of islet proteins reactive with serum from Type 1 patients was thus examined. Proteins isolated from rat islets, human insulinoma and RIN-cells were separated on sodiumdodecylsulfate polyacrylamide gel dectrophoresis, western blotted, reacted with patient (n= 10) or control (n= 5) sera and antibody binding detected using pcroxidaseconjugated rabbit anti-human IgG (Heaw and Light chain specific). Seven patients' sera were ICA positive whereas three patients and all controls were ICA negative. Rat islet proteins reacted with antibodies in control as well as diabetic patient sera, however, several unique proteins were identified which were diabetes specific. The apparent molecular weights of major antigens were 135:145 Kd; 90-100 Kd, and 58-64 Kd. The insulinoma had major antigens of 90-'100 Kd and 48-55 Kd, whereas RIN-cells showed 170-180 Kd, 110-125 Kd, 83-90 Kd, and 48-55 Kd. These results suggest that 1)ICA molecular heterogeneity is strongly determined by the origin of islet tissues used and 2)there are multiple islet protein antigens reactive to ICA positive sera that appear diabetes specific.

Abstracts 536. Myocardial biopsy pathology in asymptomatic Type I (insulin-dependent) diabetic patients C. G. G. Sutherland, B.M. Fisher, G.B. Lindop, I.A.R. More, H.J. Dargie and B.M. Frier. Department of Pathology, Royal Infirmary, Glasgow, UK, Departments of Pathology, Cardiology and Diabetes, Western Infirmary, Glasgow, UK Previously we have reported impaired ventricular ejection fraction response to exercise in asymptomatic middle aged Type 1 (insulindependent) diabetic patients with no evidence of coronary artery disease. To investigate the contribution of small vessel disease to the abnormality of ventricular function we compared endomyocardial fight ventricular biopsies from seven symptom-free Type 1 diabetic patients aged 30-50 years with biopsies from seven age and sex matched non-diabetic control subjects. The diabetic patients had normal coronary angiograms but all showed a reduced ventricular ejection fraction on exercise. Morphometry performed on electron micrographs showed no significant difference in myocardial capillary basement membrane thickness in diabetic patients (mean 90.3 nm) and control subjects (mean 85.3 nm). Electron microscopy of myocytes and interstitium showed no significant abnormality. Non-specific interstitial fibrosis in 5 diabetic patients and hyaline arteriolar thickening in 2 diabetic patients was seen on light microscopy. We conclude that the abnormality of cardiac function described in diabetes may not be associated with myocardial basement membrane changes and that endomyocardial biopsy does not provide morphological alterations specific for diabetes in asymptomatic patients. 537. Screening for diabetes: does serum fructosamine help? A. B. M. Swai, K.Harrison, L.M. Chuwa, E. Maro, V.Masih, W. Makene, D. McLarty and K. G. M. M, Alberti. Departments of Medicine, Universities of Newcastle upon Tyne, U K and Dar-es-Salaam, Tanzania A single blood test diagnostic for diabetes would be of value in screening programmes. We have compared the sensitivity and specificity of serum frnctosamine (frnctosamine/albumin ratio; ~mol/ 100 g albumin), and fasting venous blood glucose (FBG) with a 2 h 75 g oral glucose tolerance test (OGTT) (1985 WHO criteria) in 212 asymptomatic subjects (146 normal OGTT; 46 IGT; 20 diabetes) during a screening programme of adult Muslim Asians in Tanzania. Serum frnctosamine was 2.18 + 0.28 SD in normal tolerance subjects, 2.25 + 0.32 in IGT, and 2.60_+ 0.46 in newly detected diabetes (and 2.88 _+0.80 in those with previously known diabetes). Values above mean 2 SD normal had a specificity of 98% but a sensitivity of only 35% for diabetes. Predictive values were 88% (7/8) and 94% (191/204) for positive and negative results respectively. An FBG /> 6.7 retool/1 had a predictive value of 100% and a sensitivity of 30%. FBG >/4.9 mmol/l included all diabetic patients and 74% of those with IGT but 26/146 normal tolerance subjects. The specificity was thus 63% and sensitivity 67%. FBG ~>4.4 retool/1 had a specificity of only 48% but a sensitivity of 82% including all diabetic patients and 74% IGT. We conclude that both serum frnctosamine and FBG are poor screening and diagnostic tests for diabetes; and that glucose loading is required. 538. Impaired metabolic functions in human pancreatic islets following infection with Coxsackie 134 virus in vitro T.M.Szopa, T.Ward and K.W.Taylor. The London Hospital Medical College, London, UK Certain strains of Coxsackie B4 virus (CB4) cause biochemical changes in mouse islets in vitro. Here the in vitro effects of CB4 on human islet cell metabolism have been examined, since CB4 may be involved in the development of Type 1 (insulin-dependent) diabetes in man. Islets obtained by collagenase digestion of pancreatic tissue were inoculated with CB4 and cultured in RPM1 1640 medium. At 24 and 48 h post-infection the islets were incubated in bicarbonate buffer containing 2 mmol/1 or 20 mmol/1 glucose and 3H-leucine, for 2 h at 37 ~ Insulin release and islet insulin content were measured by radioimmunoassay. Incorporation of 3H-leucine into (pro)insulin and TCA-precipitable proteins was measured in islet sonicates. CB4 increased basal insulin release at both 24 and 48 h ( = 2-fold and 5-fold respectively). In some cases insulin release was decreased at high glucose. Neither basal or glucose-stimulated (pro)insulin biosynthesis was affected by CB4 infections after 24 h but after 48 h an inhibition occurred at both glucose concentrations. At 24 and 48 h there was a decrease in total protein synthesis at both high and low glucose. No significant changes in islet insulin content

587 A were found. These experiments show that CB4 virus infection can impair human islet cell metabolism in vitro. Significantly the changes occurred without the involvement of the immune system. 539. Palmitate accelerates 45Ca2+-turnover in a La3+-nondisplaceable pool of islets J.Tamarit-Rodriguez, E.Vara and O. Fernandez-Martin. Departamento de Bioquimica, Facultad de Medicina, Universidad, Madrid, Spain Stimulation of islets with 20 mmol/1 glucose and varying palmitate concentrations (0 to 1 mmol/1) induces closely correlated rates of insulin secretion and "de novo" phospholipid synthesis that are suppressed by 10 -6 mol/1 norepinephrine. We have now investigated whether palmitate affects 45Ca2+-uptake by isolated rat islets at 3 mmol/1- and 20 mmol/l-glucose. After incubation with the isotope (5, 10, 15, 60, 120 rain), islets were centrifuged through silicone-oil into a layer containing 2 mmol/1 La 3+, further incubated for l h, and its radioactivity measured. 20 mmol/l-Glucose induced an initial increase of 45Ca2+-uptake that was linear between min 5 and rain 15 and after 120 rain reached a 4-fold higher steady-state than that obtained at 3 mmol/1 glucose. Palmitate (0.25 and 1.0 mmol/1) increased the slope of the initial 45CaZ+-uptake (within 50% and 100%, respectively) at 20 mmol/1 glucose but did not modify the steadystate content of isotope that was reached earlier (after 15 and 60 rain respectively). Norepinephrine (10 -6 mol/l) almost completely suppressed the stimulation by glucose and palmitate of the initial uptake and steady-state content of 45Ca2+, an effect that was blocked by 10 -5 mol/1 yohimbine. These results support the existence of a functional link between insulin secretion, palmitate metabolism and 4SCa2+-uptake. 540. Human muscle insulin sensitivity: in vitro studies upon a metabolically relevant tissue R. Taylor, M.Argyraki and S. Chowdhury. Department of Medicine, University of Newcastle upon Tyne, U K Although human adipocytes have been extensively studied, muscle is quantitatively more important in glucose disposal in vivo. Regulation of insulin action differs between muscle and adipose tissue. An in vitro method for investigating human muscle has therefore been developed. Muscle samples were obtained during abdominal surgery, the sample being clamped at either end to maintain resting fibre length. Muscle strips (20-70 mg) were dissected without allowing change in fibre length and mounted in plastic clamps. Incubations were carried out at 37 ~ in oxygen-saturated Krebs-Henseleit buffer pH 7.4 containing 1% albumin. Prior to adopting the procedure for maintaining fibre length, no insulin effect upon human muscle in vitro could be detected. In the clamped strips, basal and insulin stimulated (10 -9 mol/1) rates of glycogen synthesis from 14C-glucose were 3.5 _+0.8 and 6.5 + 1.4 nmol glucosyl units/25 mg muscle/60 rain (n=7). Rates of glycogen synthesis were dependent upon medium glucose concentration (0.006, 0.7, 1.8 and 7.5 nmole glucosyl units/ 25 mg muscle/60 min at 0.004, 0.5, 1.0 and 5.5 mmol/1 respectively), but percent insulin responsiveness was constant at all glucose concentrations. Rates of glycolysis were not changed by insulin stimulation (lactate production 15.2 _+2.0 and a 5.0 _+2.3 : pyrnvate production 10.0+0.9 and 8.2+1.1 ~tmol/gram muscle/60 min, basal and stimulated respectively), in contrast to rat soleus muscle studied under identical conditions, emphasising the need to avoid generalisation of conclusions between species. The method described makes possible detailed metabolic studies upon human muscle in vitro. 541. Inverse relationship between alanine flux and ketone body concentrations in conscious dogs P. Tessari, M. Cattalini, B. Beaufrere and M. W. Haymond. Endocrine Research Unit, Mayo Clinic, Rochester Minn, USA, and Malattie del Ricambio, University of Padova, Italy An inverse relationship between alanine and ketone body (KB) concentrations has been demonstrated in a variety of conditions, either spontaneous or following exogenous infusions of alanine or KB. However, the effects of acute changes in "endogenous" KB concentrations, achieved by modulating the availability of their major precursors, i.e. free-fatty-acids (FFA), on alanine concentrations and flux, have not been investigated. Therefore, alanine flux (using the stable isotope L-[3,3,3-2H/Halanine) was determined at steady-state in conscious dogs before and following the infusions of: A) triglyceride and heparin (High FFA with no changes in KB); B)nicotinic acid (low FFA and low KB); C)trioctanoin (high FFA and high

588 A KB). Alanine concentration and flux did not change in group A, increased by 60% in B (/7< 0.01) and decreased by 70% in C (p< 0.01). Alanine flux correlated directly with alanine concentration, and indirectly with the changes in KB, FFA and 3-hydroxybntyrate to acetoacetate ratio. These data suggest that changes in endogenous ketogenesis may inversely modulate alanine entry into the plasma space. 542. Diabetes mellitus and impaired glucose tolerance in acromegaly N.Thalassinos, I.Tzavara, C.Augoustis, M. Kyriakou, P.Gania and D.Ikkos. Department of Endocrinology, Evangelismos Hospital, Athens, Greece The higher incidence of diabetes meUitus (DM) and impaired glucose tolerance (IGT) observed in acromegaly is attributed to the increased growth hormone (GH) secretion; but no agreement seems to exist on the relation between GH levels and deranged carbohydrate metabolism. To investigate this relationship we studied 89 untreated acromegalics (35 males, 54 females) aged 17 to 72 years by oral glucose tolerance test (OGTT) and simultaneous GH determination. Using the WHO criteria, patients were classified as DM (8 patients 9%) IGT (43 patients - 48.3%) or non-diabetic (38 patients - 42.7%); while according to the lowest GH value observed during the OGTT they were grouped as: GH > 10, GH > 5 < 10 and GH < 5 ng/ml. Of the 8 acromegalic patients with DM only 3 had G H > 1 0 n g / m l while the corresponding figure for the 38 non-diabetic subjects was 27 (42.7%). Of the 58 patients with GH > 10 ng/ml, 5.2% had DM, 48.3% had IGT and 46.5% were non-diabetic while the corresponding figures for the group with GH < 10 ng/ml were 22.7%, 40.9% and 36.4% respectively. These findings suggest that DM and IGT in acromegaly occur more frequently (57.3%) than previously reported but their incidence does not seem to be related to GH levels. 543. A role for protein kinase C in glucose-stimulated cAMP accumulation in mouse pancreatic islets P.Thams, K.Capito and C.J.Hedeskov. Department of Biochemistry A, University of Copenhagen, Denmark The influence of the protein kinase C activator 12-O-tetradecanoylphorbol-a3-acetate (TPA) on regulation of cAMP accumulation in mouse islets was studied. In the presence of IBMX (0.1 retool/l), TPA (1.6~tmol/l) enhanced cAMP accumulation from 0.20_+ 0.02 pmol/20 islets to 0.50 + 0.07 pmol/20 islets in 60 min incubations. In comparison glucose (16.7 retool/1 stimulated cAMP to 0.48+0.07 pmol/20 islets after 60 min of incubation. Both TPA (1.6 txmol/1)-stimulated and glucose (16.7 mmol/l)-stimulated cAMP accumulation were abolished by the omission of extracellular Ca 2+. The Ca 2+ ionophore A23187 (2.5 ~tmol/1) did not affect cAMP accumulation per se but affected the time course of TPA-induced cAMP accumulation, the effect of A23187 + T P A mimicking the time course for glucose. Both TPA-induced and glucose-induced cAMP production were abolished by glucagon antiserum, and after pretreatment with antiserum glucose stimulation was dependent on exogenous glucagon. Pretreatment of islets with TPA potentiated glucagon stimulation and impaired somatostatin inhibition of adenylate cyclase in a particulate fraction of islets, suggestive for a protein kinase C-mediated phosphorylation of the inhibitory GTP-binding regulatory component Ni of adenylate cyclase. It is suggested that glucose-stimulated cAMP accumulation is mediated by protein kinase C, potentiating the effect of endogenous glucagon on adenylate cyclase. 544. The effects of diabetes on placental glucose metabolism and transfer in the rat C. R. Thomas, G. L. Eriksson, I. Kihlstr6m and U.J. Eriksson. Department of Medical Cell Biology, Uppsala University, Biomedicum, Uppsala, Sweden Altered fetal growth in diabetic pregnancy may reflect perturbations in placental metabolism and transfer of glucose which still require full elucidation. We therefore perfused the fetal side of an in situ placenta of normal diabetic, glucose infused hyperglycaemic normal (HGN), and insulin infused normoglycaemic diabetic (NGD) rats, whilst infusing 3H D-glucose and 74C 3-O-methylglucose into the mother. The potential glucose transfers from mother to fetus, calculated from the 3-O-methylglucose data and maternal specific activities, were: 34 ~mol/h (normals), 105 ~mol/h (diabetics), 162 ~mol/h (HGN) and 17 p~mol/h (NGD). The difference between diabetics and HGN reflects lower (p< 0.001) uteroplacental blood flow in diabetics, their respective glycaemia being comparable. However, except the diabetics, all groups had lower 3H D-glucose transfer than 3-0-

Abstracts methylglucose transfer (p<0.001 throughout), indicating placental sequestration of glucose, which reduced the calculated transfers by: normals 12%, HGN 59% and NGD 73%. Perfusate glucose accumulation is however, the result of bidirectional glucose flux; and in diabetics an increased specific activity of transferred glucose indicated increased backflux to the mother. This resulted in lower net glucose transfer in diabetic patients than normal subjects despite an elevated maternal-fetal gradient. We thus conclude that uteroplacental blood flow, and the placental sequestration and transfer of glucose are all markedly altered by maternal diabetes. 545. Diabetes-induced changes of [125]] insulin binding sites in some peripheral tissues of the rat: an autoradiographic study C.S.Thompson and M.R. Dashwood. Department of Physiology, Royal Free Hospital School of Medicine, London, UK We recently described the distribution of specific [125I]insulin binding sites in the kidney, liver and heart of the rat using in vitro autoradiography. In the present study we have investigated whether [lzsI] insulin binding in these tissues is influenced by the insulin deficiency found following streptozotocin-induced diabetes. Tissues were removed from control and diabetic animals, frozen, and serial sections were cut in a cryostat and thaw-mounted on to microscope slides, which were incubated in Hepes buffer containing 0.1-0.5 nmol/1 [125I]-porcine insulin for 120 min. The degree of nonspecific binding was established by incubating paired slides in the presence of I ixmol/1 unlabelled insulin. Slides were apposed to Hyperfilm for 6 days at 4 ~ after which the films were processed and the autoradiographs examined. A regional distribution of insulin receptors was observed in each of the tissues examined (predominantly to the renal cortex, to ventricular myocardium and uniformly in the liver). In each of these tissues, diabetes appeared to induce a marked increase in receptor density. These results can be taken as further evidence that a lowered circulating hormonal level may cause an increase in the density of specific binding sites on target tissues. 546. Insulin clearance from plasma in Type 1 (insulin-dependent) diabetic patients: influence of glycaemic level B. Thorsteinsson, S. Fugleberg and C. Binder. Steno Memorial Hospital and Hagedorn Research Laboratory, Gentofte, Denmark Insulin is cleared from plasma at a lower rate in Type 1 (insulin-dependent) diabetic patients than in normal subjects. This difference might be related to the difference in glycaemic levels between the two populations. We therefore studied whether the insulin clearance rate depends on the actual glycaemic level in 16 Type 1 diabetic patients (age 33years (median, range: 20-50), diabetes duration 10 years (3-25) and haemoglobin Ale 7.9% (6.5-11.2)). Constant intravenous infusion of insulin (1-5 m U - k g - l . m i n -1) was used to achieve different levels of steady state plasma free insulin concentrations, while the blood glucose level was clamped at euglycaemia (4.0 mmol/1, 3.9-4.7) and hyperglycaemia (8.0 mmol/1, 7.6-9.0) on different days. The experimentally determined data were compared using a previously validated model of first order kinetics for insulin disappearance from plasma in Type 1 diabetic patients. The median clearance rate of insulin was 23 m l . k g - l - m i n -1 (16-26) at euglycaemia versus 23 ml.kg-a.min -1 (19-35) at hyperglycaemia (NS). In addition, no correlation was observed between the insulin clearance rate and the duration of diabetes or the haemoglobin Ale level (both Spearman's rho <0.1). In conclusion, the insulin clearance rate from plasma is independent of the actual glycaemic level in Type I diabetic patients. 547. Increased bloodflow in Type I (insulin-dependent) diabetic patients developing microvascular complications L.Thuesen, J.S. Christiansen, C.E. Mogensen and P. Henningsen. University Dept. of Cardiology and II University Clinic of Internal Medicine, Kommunehospitalet, Aarhus, Denmark Cardiac function was investigated by echocardiography in 80 Type 1 (insulin-dependent) diabetic patients with no signs of ischaemic heart disease and in 40 healthy control subjects. Echocardiographic findings were related to the urinary albumin excretion rate (UAE). In the diabetes group fractional shortening of the left ventricle (FS%) was 37.3% versus 34.3% (p< 0.01) in the control group, while indices of preload and afterload were at the same levels as in control subjects. In diabetic patients with preclinical nephropathy (UAE 20-200 p.g/min) (n=15) fractional shortening was 41.1% as compared to 37.0% (p< 0.002) in patients with no signs of nephropathy (UAE < 20 gg/min) (n= 48) and as compared to 34.8% (p< 0.001) in

Abstracts patients with clinical nephropathy (UAE <200 gg/min) (n=17). Further, in patients with preclinical nephropathy afterload was significantly decreased, while preload was at the same level as in the other two groups of UAE. In conclusion, a condition of cardiay hyperfunction has been found in diabetic patients with no signs of ischaemic heart disease and without severe microvascular complications, and seems pronounced in diabetic patients developing microvascular disease (patients with preclinical nephropathy) probably secondarily to a condition of hyperperfusion in these patients. 548. Renal 125I-BSA clearance and urinary albumin excretion are increased in mildly diabetic rats R. G. Tilton, G. Pugliese, K. Chang, C. Kilo and J. R. Williamson. Department of Pathology - Washington University School of Medicine, St. Louis, Missouri, USA To assess chronic effects of very mild diabetes on renal haemodynamics and filtration function, we compared 3 groups of male Sprague-Dawley rats: controls (n=9), diabetic (n=9; 60mg/kg body weight streptozotocin), and mildly diabetic ( n = 9 ; nicotinamide; 250 rag/ks body weight was measured 15 rain before injecting streptozotocin). Eight months later, renal blood flow (ml/min per g kidney) was measured with 8SSr-microspheres (15 ~tm), GFR and albumin clearance were assessed by measuring 57Co-EDTA and 125IBSA plasma clearances (g plasma/g kidney per rain), and 24 h urinary albumin excretion (mg/24h per 100g body weight) was quantified by radial immunodiffusion assay. In diabetic and mildly diabetic rats, nonfasting plasma glucose levels were increased (539 +_31 (SD) rag% and 156 +_14, respectively, versus 136 + 11 for controls; p<0.001 and p<0.005 respectively), as were renal clearance of 12SI-BSA (0.0064_+0.0032 and 0.0045-+0.0017, versus 0.0017-+0.0005; p<0.001) and urinary albumin excretion rates (3.09_+2.65 and 1.43_+1.29, versus 0.27_+0.14; p<0.005 and p < 0.025, respectively). Renal blood flow was decreased (/9<0.001) in diabetic but not in mildly diabetic rats (4.0_+ 0.4 and 5.0 +0.4, respectively, versus 4.9+__0.2 for controls) while GFR did not differ from controls (0.93 -+0.17 and 0.88 -+ 0.13, versus 0.87 -+0.07). These results suggest that glomerular capillary filtration function is impaired by very mild diabetes in the absence of significant haemodynamic changes. 549. Insulin stimulates the autophosphorylation of six tyrosines in three domains of the insulin receptor fl subunit both in vitro and in vivo H. E. Tornqvist, J. R. Gunsalus, R.A. Nemenoff and J. Avruch. Diabetes Unit, Massachusetts General Hospital, Boston, Mass, USA Tyrosine kinase activation and autophosphorylation of the insulin receptor (IR) fi subunit are obligatory steps in the chain of events in insulin action. We have shown that in vitro maximally autophosphorylated human IR contains at least six P-tyr residues, of which five have been identified directly by microsequencing: tyr 1146, 1150 and 1151 in the tyrosine kinase domain and tyr 1316 and 1322 in the Cterminal domain. The remaining site is most probably located close to the transmembrane region (tyr 953 and/or 960). IR was autophosphorylated with and without insulin, and the phosphorylation of the different sites with time was monitored and compared to tyrosine kinase activation. The data suggest that insulin causes a simultaneous phosphorylation of these three domains in the fl subunit paralleling the extent of tyrosine kinase activation. Studies of the insulin-induced autophosphorylation of IR in intact H4 hepatoma cells revealed that insulin triggers the autophosphorylation of the same six tyrosine residues that undergo insulin-stimulated autophosphorylation in vitro. 550. Factors associated with the 10-year survival of a cohort of diabetic patients Ch.Tountas, A. Kofinis, Th. Kardoyiannis, B. Pavlopoulos and B.Karamanos. Diabetic Center, Hippokration Hospital, Athens, Greece In order to evaluate factors related to the survival of diabetic patients we studied 500 unselected diabetic patients aged 61-80 initially examined between 1974-77. In 1987 information was obtained for 459 of them; 302 were alive (Group A) and 157 dead (Group B). We compared the two groups on the basis of the data of their initial examination. Group A were younger, 68.8 -+ 0.7 vs 74.5 + 0.7, p < 0.001, but longer diabetes duration 13.9 -+0.3 vs 11.4 __+_0.5, p < 0.001, lower systolic and diastolic blood pressure, 150.9 + 1.5 vs 191.2-+ 1.9, p < 0.001 and 87.4_+0.68 vs 94.5-+1.01, p<0.001, respectively; lower fasting blood glucose (FBG) 9.9-+0.3 vs 11.0-+0.01, p<0.05 but

589 A higher cholesterol 7.1 + 0.18 vs 6.35 + 0.2, p < 0.05 while there was no difference in % ideal body weight 112.9+0.1 vs 115.6-+ 1.6 mmol/1, p > 0.05 and triglycerides 1.26 + 0.07 vs 1.31 _ 0.1 mmol/1, p > 0.05. In Group A the prevalence of ECG abnormalities (Minnesota Code) was lower (12.4 vs 25.2%, p < 0.001) while that of retinopathy was not different (32.1 vs 37.9%, p>0.05). Causes of death were: cardiac events 29.2%, stroke 24.2%, neoplasms 16.5%, diabetes 5.1%, chronic renal failure 3.8%, other 21.2%. Diabetes appeared on the death cetificates only in 23.5%. Conclusions: Survival is positively influenced by lower age, blood pressure, FBG and prevalence of ECG abnormalities but not by diabetes duration, body weight, blood lipids and retinopathy. Cardiac events and stroke are the main causes of death. Death certificates are very inaccurate in relation to diabetes. 551. Reciprocal changes of glucose-induced insulin secretion and metabolism in cultured rat insulinoma cells M. E. Trautmann, B. Blondel, A. Gjinovci and C. B. Wollheim. Institut de Biochimie Clinique, University of Geneva, Switzerland Glucose-induced insulin secretion is partially preserved during perfusions of kidneys bearing the transplantable rat insulinoma. Cell isolation yielded approximately 20 x 1 0 6 cells per tumor. After overnight culture these cells responded to glucose (2.8-16.7 mmol/1) with a doubling of insulin secretion. Glucose utilisation was lower compared to RINm5F-cells and had a Km of about 5 mmol/l glucose. From a primary monolayer a new cell line was established with a population doubling time of 40 h. The insulin content stabilised at 350 ng/106 cells which is 1~ of the starting material. Initially this cell line secreted insulin in response to glucose (2.8-16.7 mmol/l). During subsequent culture the cells exhibited a greater insulin response to D-glyceraldehyde than RINm5F-cells but lost the glucose response. Other secretagogues were leucin, alanine and phorbol 12-myristate 13-acetate (PMA). Glucose utilisation was similar as in RINm5F-cells. Long-term culture in glucose-free medium did not alter insulin content or secretory responses. Addition of sodium butyrate to the culture medium yielded a reversible increase of insulin content up to 8-fold without affecting stimulated release. In conclusion, we showed that cells isolated from a glucose responsive insulinoma recognise glucose as secretagogue in vitro. After establishment of cell lines the glucose response disappeared while glucose metabolism increased. 552. Renal haemodynamics following oral protein load in Type 1 (insulin-dependen0 diabetic patients. Effects of indomethacin and glucagon R. Trevisan, R. Nosadini, P. Fioretto, A. Valerio, A. Doria, M.Velussi, A. Tiengo and G. Crepaldi. Cattedra di Patologia Medica 1 e di Malattie del Ricambio, Policlinico Universitario, Padova, Italy Glomerular filtration rate (GFR) is elevated following a meat meal in normals. Conflicting results are reported in Type 1 (insulin-dependent) diabetic patients. Our aim was to investigate, during an euglycaemic-insulin clamp, GFR (ml/min per 1.73 m :~) (mean+SD) at basal level and after a meat meal in 8 normal subjects, 9 diabetic patients with less than 5 years of diabetes (Group 1) and 9 diabetic patients with more than 5 years of diabetes (Group 2). GFR was 101 + 14 before and 136+14 (p<0.001) after protein load in normal subjects. Basal GFR was 122+18 in Group l and 125+13 in Group 2. Following the meat meal, GFR was 146_+20 in Group 1 (p<0.01) and 128 + 14 (NS) in Group 2. No differences were found between normal subjects and diabetic patients with regard to amino acid levels. The plasma glucagon increase was lower in Group 2 (p< 0.05) than in Group I and in normal subjects. Indomethacin (100 mg per oral) inhibited the protein stimulated GFR increase in all groups. The GFR increase after the meat meal was positively related to glucagon increase (r=0.56, p < 0.01). Conclusions: l) Protein-induced GFR increase is absent in long term diabetes without clinical nephropathy, 2) the lack of GFR increase following protein load with indomethacin suggests that prostaglandins mediate aminoacid stimulation of GFR, even if the correlation with glucagon patterns suggests a role for this hormone. 553. Amniotic fluid insulin and C-peptide in early pregnancy F.Troili, N.di Biase, G.di Rollo, A. Fortino, S. Balducci, E.Sciullo, A. Maldonato, E.Maggi 1, A. Pachi ~ and F. Fallucca. Diabetes Unit, C1. Medica 2 and 10. Ostetrica 3, "La Sapienza" University, Rome, Italy The widespread use of amniocentesis has led to an increased knowledge of the biochemical composition of the amniotic fluid (AF). Data about (AF) insulin (IRI) and C-peptide (CPR) in late gestation

590 A have been reported by several groups, but little information on these two peptides during early pregnancy is available. We have measured glucose (G), IRI and CPR and the C P R / I R I molar ratio in the AF collected during weeks 15 to 22 in 81 women undergoing amniocentesis for the following clinical indications: age ( > 35 years), fl-thalassemia, previous neonatal or foetal malformations. The results were not correlated with the clinical indication for amniocentesis, foetal sex, or family history of diabetes. A correlation was found between the week of gestation ( < 19 vs > 19) and both AF G (3.70 + 0.28 vs 2.65 + 0.44 mmol/1) and CPR (35 _ 2 vs 51 + 11 pmol/1) concentrations as well as between maternal glycaemia (<5.52 vs > 5.52 mmol/1) and AF-CPR (34 + 2 vs 42 _+3 pmol/1). In comparison to a previous study of ours performed in late gestation, all variables but IRI were markedly changed (early vs late gestation: G 3.31 + 0.22 vs 0.72_+0.11 mmol/1; IRI 6.2+0.3 vs 6.6_+0.7 pU/ml; CPR 37 + 2 vs 165 -+ 38 pmol/l; C P R / I R I 0.97 + 0.06 vs 3.8 -+ 1.6) suggesting that the foetal clearance and/or degradation of IRI and CPR may greatly change along gestation.

554. Diet efficacy on diabetic pregnant women in optimised metabolic balance G.F.Trossarelli 1, E.Meriggi 1, Q.Carta 2, G.Breccia 1, R.Bordon 1, A. Defilippi 1 and A. Bocci1. qnstitute of Obstetrics and Gynecology, University of Turin, Italy, 2Antidiabetic Center, San Giovanni Hospital of Turin, Italy In diabetic pregnancy dietetic restriction must consider foetus growth requirements (carbohydrates 150 g/day, protides 1.5 g/kg, at least). In 55 diabetic pregnant women (Type 1 (insulin-dependent): 21; Type 2 (non-insulin-dependent): 9; gestational diabetes: 19 diet (DGD), 6 insulin (IGD); pre-pregnancy BMI: 29 < 120%, 26 ~>120%) with optimised metabolic control (glycaemia: fasting 3.87_+ 0.55 mmol/1; 2-h post-prandium 6.63 + 0.55 mmol/1) we studied two diets randomly assigned: 1) kcal/day >i 1600, < 2000 (protides = 1.8 g/kg; carbohydrates =180 g/day lipids =65 g/day); 2) kcal/ day <1600, >11300 (protides 1.5 g/kg; carbohydrates 150g/day; lipids -~65 g/day). All women delivered at term. In Type 1 and DGD, diet ~>1600kcal induced a weekly higher weight gain (mean _+SD) (p< 0.05) (408 + 66 versus 198 g 417 + 109 versus 298 + 64 g). The weekly weight gain (diet I> 1600 kcal) in IGD was lower than in D G D (p<0.05) and Type1 (p<0.01). Except Type l (3199 _+288 versus 2630 g, p < 0.05; 4_+ 2 versus 1.3 + 0.5 ng/ml, p < 0.05), newborn weight and cord C-peptide (both diets) got similar results in all groups. Type 1, Type 2 Apgar score (diet < 1600 kcal) was lower (/7<0.05). As for all groups newborn weight, we had 6% large, 85% appropriate, 9% small gestational age (diet >~1600 kcal) and 9%, 73%, 18% respectively (diet <1600 kcal). These data suggest that a dietetic over-restriction can give a reduced maternal weight gain, a worse Apgar score and a higher rate of small babies.

555. Insulin in vivo reduces platelet sensitivity to aggregating agents M. Trovati, G. Anfossi, F. Cavalot, P. Massucco, E. Mulbaroni and G.Emanuelli. Cattedra di Clinica Medica Generale III, San Luigi Gonzaga Hospital, University of Turin, Italy The role of insulin in diabetic angiopathy is very complex. On the one hand, insulin promotes proliferation of arterial smooth muscle cells; on the other hand, insulin in vitro reduces platelet aggregation. We aimed at clarifying whether insulin at physiological concentrations reduces platelet function also in vivo. Five healthy male subjects, 26_+ 1 years, BMI 21 _+1, were submitted, after fasting overnight, to a 150 min study: 0-90rain: euglycaemic (4.97 mmol/1) hyperinsulinaemic (40 IxU/ml) clamp; 90-150 min: variable dextrose infusion to keep plasma glucose steady after the end of insulin infusion. Throughout the study, we measured plasma glucose, insulin and platelet sensitivity to ADP, Platelet Activating Factor (PAF) and Epinephrine, by means of Born's method, measuring ED50, the dose of each agent able to induce a maximal aggregation of 50%. Data (mean_+ SEM) are expressed as % of basal values. Insulin infusion caused an increase of ED50, that means a decrease of platelet aggregation, reversible after the end of infusion. ADP ED50 was 191 + 22% at 90 min (/7<0.01) and 110_+15 at 150 min (NS); PAF ED50 was 170_+35% at 90 min 09<0.05) and 109_+6% at 150 min (NS); Epinephrine ED50 was 139 _+18% at 90 rain (p< 0.05) and 103 _+3% at 150 rain (NS). In conclusion, insulin in vivo, at physiological concentrations, reduces platelet aggregation. A physiological modulating effect of insulin on platelet function is suggested.

Abstracts 556. Sulfonylurea-induced insulin release from isolated pancreatic islets correlates with the inhibition of ATP-dependent K§ G.Trube, K. M~inner, U. Panten and B.J. Ziinkler. Max-Planck-Institut for biophysikalische Chemie and Institut ftir Pharmakologie und Toxikologie der Universitat, G6ttingen, FRG The effects of several sulfonylureas and meglitinide on ATP-dependent K§ and insulin release in routine pancreatic B cells were compared. Currents were recorded from cultured single cells by using the whole-cell configuration patch-clamp technique. ATP-dependent K+-currents were activated by a low (0.3 retool/l) concentration of ATP in the solution dialysing the intracellular space of the cell. Addition of the above drugs to the extracellular solution caused a dose-dependent reduction of this current. Half-maximal inhibition was reached at tolbutamide 9 limol/1, meglitinide 2 lxmol/l, glipizide 6 nmol/1 or glibenclamide 4nmol/1 (media containing 2 mg/ml BSA). The effects of tolbutamide and meglitinide developed rapidly and were quickly reversible ( < 1 rain) whereas the slow ( > 15 rain) inhibition by nanomolar concentrations of glipizide or glibenclamide could not be reversed. Insulin secretion was stimulated by the hypoglycaemic compounds in the continuous presence of 10 mmol/1 glucose. Amounts released during 1-4 rain periods from approximately 50 freshly isolated islets were determined by radioimmunoassay. A half-maximal increase of steady-state (60 rain application) insulin release was obtained with tolbutamide 12 ~tmol/1, meglitinide 4 p~mol/ 1, glipizide 10 nmol/l or glibenclamide 6 nmol/1. Thus, concentrations at which these compounds stimulate insulin release correlate well with their potency as inhibitors of ATP-dependent K§ nels.

557. Radioisotopic assessment of A-V shunts in the diabetic foot. Correlation with cardiovascular tests for autonomic neuropathy L. Uccioli, G.Ghirlanda, L.Mancini, P. Cotroneo, P. Magnani, A. Manto, A. Solini and A.V. Greco. Istituto di Clinica Medica, Universitfi Cattolica del Sacro Cuore, Roma, Italy The aim of our study was to investigate the amount of A-V shunts in diabetics' feet and to correlate these with the autonomic neuropathy, as assessed by the cardiovascular tests (CVT). Fifty-four diabetic patients including 8 with neuropathic foot (NF), were analysed by CVT; pathological results were recorded in 75% of the patients with NF and in 37% of the remaining. A radioisotopic technique was used to study the A-V shunts in the 8 patients with N F and 8 control subjects, 4 of which showed pathological CVT. To measure A-V shunting we injected into the femoral artery labelled albumin microspheres which lodged in the limb capillaries, but passed through peripheral A-V shunts. The ratio between the pulmonary radioactivity detected after an arterial and after a venous injection of an equivalent amount of microspheres expressed the shunts value (physiologically less than 5%). While all the 8 patients with N F showed pathological shunts, only 25% of the control subjects did. Pathological shunts were present in 80% of the subjects wkh abnormal CVT and only in 33% of the others. In conclusion, pathological A-V shunts are always recorded in NF. A positive correlation between the abnormal CVT and the pathological A-V shunts cannot be ruled out.

558. Islet cell antibodies and ECHO-4 virus infection A.Uriarte, E.Cabrera, R.Ventura and J.Vargas. Department of Immunology, National Institute of Endocrinology, Cuba Environmental factors are known to trigger the development of Type 1 (insulin-dependent) diabetes. Among these factors, it is also known that some viruses can infect and damage pancreatic B cells in man and experimental animals. Circulating autoantibodies against pancreatic islet cells (ICA) have been reported in non-diabetic subjects infected by some types of viruses, but to our knowledge, not in patients suffering from ECHO-virus infection. We have found in the course of an outbreak of meningoencephalitis caused by an ECHO-4 virus, that 36% of infected children, at diagnosis, possess circulating ICA; 1-4 months after recovery from the disease, 24% still have autoantibodies and 21% of them were migration inhibitory factor (MIF)-positive in response to pancreatic antigens. Four children, at first negative for ICA, became positive in two months. ICA were detected by the indirect immunofluorescence technique on fixed blood group 0 human pancreas. No signs of thyroid autoimmunity were detected. These findings should alert us to the possible role of ECHO-viruses in the pathogenesis of Type 1 diabetes.

Abstracts

559. Glucosidase inhibition and viscous fibre: greater respective effects on postprandial glucose and fasting cholesterol L. O. Uttenthal, F. Requejo and S.R. Bloom. Department of Medicine, Royal Postgraduate Medical School, London, U K Inhibitors of intestinal a-glucosidases and purified viscous fibres, such as guar, are alternative or complementary means of reducing the postprandial blood glucose rise in diabetic patients. Absorbable a-glucosidase inhibitor BAYm1099 (50 mg thrice daily) and guar granules (5 g thrice daily) were tested separately and together in a randomised double-blind placebo-controlled crossover study on 12 Type 2 (non-insulin-dependent) diabetic patients on sulphonylureas. Each treatment period of 2 weeks followed a 2 week wash-out period, and was concluded with a 2.2 Megajoules mixed test breakfast. BAYm1099 and guar reduced the mean fasting blood glucose by 1.2 retool/l, p < 0.05 and 1.7 mmol/1, p < 0.01, respectively, with a similar but non-significant effect on serum fructosamine. Both BAYmI099 and guar lowered home-monitored postprandial blood glucose, the effect being highly significant for BAY m 1099 (p < 0.001, post-breakfast). Guar, but not BAYm1099, significantly lowered fasting serum cholesterol (,0<0.05). BAYm1099 reduced the test breakfast plasma responses of glucose (p<0.001) and gastric inhibitory polypeptide (GIP, p<0.001), and increased those of motilin (p<0.01) and peptide tyrosine-tyrosine (PYY, p<0.001). Guar had less effect on glucose (p< 0.05) and no significant effect on GIP, motitin or PYY, but increased the late release of neurotensin (p< 0.05). Combining treatments did not potentiate the desired effects of BAY m 1099, but retained the effect of guar on cholesterol. 560. Cardiovascular morbidity in middle-aged Type 2 (non-insulin-dependent) diabetic patients and control subjects during 5-year follow-up. The effect of risk factors on morbidity in diabetic patients M. Uusitupa, L. Niskanen, O. Siitonen and K. Py6r~l~. Department of Medicine, University of Kuopio, Finland During the years 1979-1981 a representative group of patients with newly diagnosed Type 2 (non-insulin-dependent) diabetes (70 men, 63 women) and non-diabetic control subjects (62 men, 82 women), aged 45-64 years were examined. Both groups were re-examined after 5 years to evaluate cardiovascular morbidity and to find out the relationship of risk factors to the incidence of myocardial infarction (MI) and claudication (CL). The incidence of MI (new major Q-QSchanges or hospital verified MI) was higher in diabetic men (11/57; 19%, p=0.02) and women (7/52; 14%, p=0.04) than in control men (2/57; 4%) and women (2/78; 3%). The incidence of CL was also higher in diabetic patients (20% in men; p = 0.07 and 22% in women, p<0.01) than in control subjects (7% in men and 5% in women). The presence of ischaemic ECG abnormalities (Minnesota code 4.1-3, 5.1-3, 7.1) at base-line predicted MI in diabetic men (p< 0.05). High systolic blood pressure was associated with CL in diabetic men. Diabetic patients with CL tended also to have higher total cholesterol (7.06+0.48 vs 6.22_+0.18 mmol/1, p=0.05 for men, 7.05+0.41 vs 6.46 _+0.22 retool/l, p = 0.20 for women), VLDL-cholesterol (1.67 + 0.43 vs 1.24_+0.14 mmol/1, p=0.26 for men, 1.83_+0.38 vs 0.91+ 0.08 retool/l, p = 0.001 for women) and triglyceride levels at base-line (3.10_+0.59 vs 2.38_+0.24mmol/1, p=0.18 for men, 3.68_+0.67 vs 1.95 + 0.19 mmol/1, p = 0.001 for women) than those without CL. In conclusion, diabetic patients showed 3- to 4-fold higher incidence of MI and CL than control subjects. The disturbed VLDL-metabolism may promote atherogenesis of lower limbs in diabetes.

561. Peripheral neuropathy and capillary permeability abnormality in diabetic patients P. Valensi, J. R. Attali, A. Behar and J. Sebaoun. Unit~ de Diab6tologie, H6pital Avicenne, Bobigny, Service de M6decine Nucl~aire, H6pital Broussais, Paris, France An increase in the capillary permeability to albumin (CPA) has been found in diabetic patients. We observed this frequently with a noninvasive isotopic test derived from the Landis method, using 99roTealbumin and measuring residual radioactivity externally after removal of forearm venous compression. The test was considered abnormal if albumin retention (AR) was >~8% at the 10th min or if AR was < 8% but the amplitude ratio of the low and high frequency peaks (LF/HF) obtained by analysis of the radioactivity disappearance curve using the Fourier fast function transfer was >~1%, which reveals a change in the lymphatic wash-out of albumin. Among the 25 nonnotensive diabetic patients with no retinopathy or signs of glomerulopathy, 11 [7 Type 1 (insulin-dependent), 4 Type 2 (non-insulin-dependent)] diabetic patients, with a 4-month to a 17-year du-

591 A ration of the disease, showed abnormal results. Eight of these 11 patients showed objective clinical signs of peripheral neuropathy and AR was either/> 8% (3) or < 8% with L F / H F /> 1% (5). None of the other 14 patients showed objective clinical signs of peripheral neuropathy. In conclusion, an increase in CPA can occur early, prior to retinopathy, glomerulopathy and hypertension and can then be significantly associated with peripheral neuropathy. The mechanism behind the relationship between CPA increase and peripheral neuropathy has yet to be elucidated.

562. Stimulation by D-glucose protein biosynthesis in tumoural insulinproducing cells I. Valverde, M. Barreto and W.J. Malaisse. Fundation Jimenez Diaz, Madrid, Spain and Brussels Free University, Brussels, Belgium Tumoural insulin-producing cells (RINm5F line) display a poor secretory response to D-glucose. Relative to basal insulin output, which corresponds to a fractional release of stored hormone close to 25 percent/90 min, the glucose-induced increment in insulin release does not exceed 28 + 7%. The low efficiency of D-glucose as an insulin secretagogue contrasts with a marked stimulant action of the hexose upon peptide biosynthesis. Thus, D-glucose increases in a rapid, sustained and dose-related manner the incorporation of (3H)leucine or (3H)phenylalanine into TCA-precipitable material (maximal stimulation: 7-fold as compared to basal). Although the low (pro)insulin content of the tumoural cells, relative to their total protein content, hampers the quantification of newly synthetised hormonal products, the ratio of immunoreactive proinsulin/insulin was found to exceed the value recorded in normal islet cells, this difference being more marked in secreted than stored material. It is concluded that, despite their poor secretory response to D-glucose, the tumoural cells are in fact quite sensitive to this hexose, as documented by its effect upon biosynthetic activity. The apparent discrepancy between the biosynthetic and secretory responses to D-glucose may be accounted, in part, by a severe perturbation in the capacity of the tumoural cells to store (pro)insulin. 563. Preservatives in insulin preparations impair phagocytosis: an in vitro study I. van Faassen, A. M. M. J. Verweij-van Vught, M.Z. Lomecky-Janousek, P.P.A.Razenberg, J.van der Meer and E.A.van der Veen. Departments of Endocrinology, Internal Medicine and Medical Microbiology, Free University Hospital, Amsterdam, The Netherlands Meta-cresol (m-cresol) and methylparahydroxy-benzoate (mph-benzoate) are used as preservatives in insulin preparations. In pump therapy the concentration of preservative of the infused insulin varies with the type of catheter: Polyvinylchloride (PVC) tubes retain preservative whereas Polyethyleen tubes do not. Among our patients infections at infusion sites occurred more often when m-cresol containing insulin was used. In 8 experiments the influence of insulin with and without preservative on human phagocytic function was studied. Insulin solution, polymorfonuclear leukocytes, serum and bacteria were incubated at 37 ~ After 60 min the number of viable bacteria was determined. Unperfused and peffused insulin (insulin pump and PVC catheter; 1 day and 4 days) with m-cresol and mphbenzoate, and insulin without preservative were tested. Phagocytic function was expressed as the percentage of bacteria killed. The killing-percentage (mean + SEM) was reduced by insulin with m-cresol (74.8+0.7) and mph-benzoate (85.0+0.9) as compared to insulin without preservative (90.4+1.9) and control solution (95.8+0.8) (p<0.001). Of the unperfused and 1 day perfused insulin the solutions with m-cresol caused lower killing-percentages than the mphbenzoate solutions (p< 0.05). The 1 day and 4 days perfused m-cresol solutions (remaining m-cresol concentration: 10% versus 30%) had the'same influence as the unperfused insulin. These data suggest an impairment of phagocytic function caused by preservative in insulin preparations. 564. Preclinical diabetic retinopathy yields normal values of vitreous fluorophotometry and static perimetry J.M.A.van Gerven, L.Vrij, H.H.P.J.Lemkes, J.A.van Best and J.A.Oosterhuis. Depts. of Endocrinology and Metabol. Dis. and Ophthalmology, The Netherlands Aims of this study were to investigate 1 : during which stage of diabetic retinopathy permeability of the blood-retinal barrier (BRB) is increased, and 2: whether increased BRB-permeability parallels abnormal retinal light sensitivity. In 21 diabetic patients (39 eyes) and 58 normal subjects, vitreous fluorophotometry (VF) was performed

592 A with a Fluorotron Master (Coherent Instruments, Palo Alto, Calif., USA) correcting for lens transmission and free plasma fluorescein. In 25 diabetic eyes, macular light sensitivity was recorded on a photopic and mesopic background, by Ttibinger static meridian perimetry. Level of retinopathy (assessed by fundus photography and fluorescein angiography) was divided into groups: I: no retinopathy (7eyes); II: minimal background (7eyes); III: background (16 eyes); IV: (pre)proliferative (9 eyes). Groups were similar in age, diabetes duration or HbA1. Static perimetry was normal in all patients. BRB-permeability was abnormal only in some eyes of groups III and IV: Normal (mean _+SD): 5.8 + 1.8; I (Geographical mean (range)): 4.20 (2.74-8.12) (NS vs normal); II: 5.58 (3.79-9.66) (NS); III: 8.26 (1.42-21.70) (NS); IV: 14.25 (7.29-44.91) (p<0.01 vs normal, p<0.05 vs III). BRB-permeability increased with diabetes duration, within groups: II (r=0.86, n=4, NS), III (r=0.82, n=10, p<0.01) and IV (r=0.83, n=7, p<0.05). In conclusion, clinical diabetic retinopathy can occur without BRB-disruption. In our patients, increased BRB-permeability occurred only if retinopathy was apparent, and macular light sensitivity was not reduced. These results suggest that VF is unsuitable for preclinical detection of diabetic retinopathy. 565. Effect of hyperglycaemia on arginine induced insulin secretion T.W.van Haeften, I.van Faassen and E.A.van der Veen. Free University Hospital, Amsterdam, The Netherlands Although it is well-known that glucose can potentiate the maximal responsiveness of the B cell to arginine, this requires unphysiological doses and is probably less important than B-cell sensitivity to arginine. As it is unknown whether hyperglycaemia per se changes B-cell sensitivity to arginine, dose-response curves of arginine-induced insulin secretion during euglycaemia and hyperglycaemia were compared. Seven healthy subjects were given five infusions with arginine (doses 0.1, 0.2, 0.3, 0.5, 0.7 g/kg, 30 min) both at euglycaemia and during hyperglycaemic clamps (plasma glucose level 17 mmol/1). Mean A plasma insulin levels were higher during hyperglycaemia at all arginine doses (t7< 0.01) reaching a maximum (during last 10 min of the infusions) of 425 _+77 mU/1 (mean _+SEM) compared to 31 _+ 5 mU/1 at euglycaemia (p< 0.002). Half-maximally stimulating arginine-dose (Kin) was not significantly altered by hyperglycaemia (0.27 _+0.02 vs 0.267 _+0.02). Conclusion: Hyperglycaemia potentiates arginine-induced insulin secretion without significantly affecting Bcell sensitivity to arginine. 566. Vitamin E status in normocholesterolemic and hypercholesterolemic diabetic patients M.G.Vandewoude and J.F.Vertommen. Dept. of Endocrinology and Metabolism, University of Antwerp, Belgium The close relationship between vitamin E, lipid and prostaglandin metabolism stresses the need for an accurate definition of the vitamin E status in a diabetic population. After an overnight fast, plasma vitamin E and plasma lipids were determined in 34 Type I (insulin-dependent) and 21 Type 2 (non-insulin-dependent)normocholesterolemic and in 7 hypercholesterolemic diabetic patients. They were also measured in 62 age- and sex-matched control subjects, 55 normocholesterolemic and 7 hypercholesterolemic individuals. Plasma vitamin E levels were significantly increased (p< 0.001) in hypercholesterolemic individuals, both in diabetic patients and in non-diabetic patients. The vitamin E/cholesterol ratio in these subjects was, however, not different from normolipemics. Plasma vitamin E was correlated with plasma lipids, especially with total cholesterol (p< 0.001). Since vitamin E is mainly transported by plasma lipoproteins, these strong correlations suggest that changes in plasma vitamin E in hypercholesterolemic diabetic patients should be considered as an epiphenomenon of increased plasma transport capacity in these individuals. 567. Islet secretion of thyrotropin releasing hormone and its paracrine effects E.Vara and J.Tamarit-Rodriguez. Departamento de Bioquimica, Facultad de Medicina, Universidad Complutense, Madrid, Spain Islets contain thyrotropin releasing hormone (TRH) of unknown physiological significance. Therefore, we have investigated its secretory pattern and its ability to modify the release of other islet hormones in incubated (60 min), collagenase-isolated, rat islets. Insulin, glucagon, somatostatin and TRH concentrations were measured in the incubation medium with specific radioimmunoassays. Within a range of glucose concentrations (0, 3, 10, and 20 mmol/1), insulin

Abstracts and somatostatin secretions were dose-dependently stimulated whereas those of TRH and glucagon were inhibited. Maximum increments of insulin (7-fold) and somatostatin (2.5-fold) occurred at 20 mmol/l glucose. Glucagon and TRH were similarly and almost maximally reduced (within 30%) after raising the glucose concentration to 20 mmol/1 and 3 mmol/1 respectively. Starved islets (48 h) showed considerably lower rates of all islet hormones except glucagon that was not reduced by 20 mmol/1 glucose. Addition of exogenous TRH (10 -8 mol/l) altered drastically the secretory pattern at 20 mmol/1 glucose: The insulin response was reduced (-66%) and that of somatostatin increased ( + 75%) whereas the inhibition of basal glucagon was completely blocked. These results are compatible with a paracrine, inhibitory role of TRH on glucose-stimulation and -inhibition of insulin and glucagon secretion, respectively, that is apparently mediated through an increase of somatostatin release. 568. Dietbylaminoethyl-dextran in the treatment of Type 2 (non-insulin-dependent) diabetes mellitus: a new therapeutic frontier? M. Vasta1, E. Sciullo, M. Guidi~, E. Pugliesi, S. Balducci, O. Falla, M.Pitaro, A.Monti, A.Maldonato and F.Fallucca. Diabetes Unit, C1. Medica 2, "La Sapienza" University, Rome, 1Ospedale Civile, Urbino, Italy Diethylaminoethyl-dextran (DEAED) (Pulsar) is a drug used in the treatment of hyperlipidaemias which has recently been reported to influence carbohydrate metabolism in non-diabetic subjects. To study its effects in Type 2 (non-insulin-dependent) diabetes, we added 1.5 g/24 h DEAED to the previous diet-alone treatment in ten patients (Group 1), or to the previous OHA treatment in 15 patients (Group 2). In 15 more patients we replaced the previous OHA treatment with 3 g/24 h DEAED (Group 3). Each subject performed 4 standard breakfasts (BTT, 200 g mild+50 g bread) at times 0, 7 and 30 days of treatment and 30 days after DEAED suspension. After 7 and 30 days of DEAED association the blood glucose levels during BTT were significantly reduced and a parallel significant reduction of HbAlc was observed (Group 1, 6.83 +0.23 vs 7.81 ___ 0.25%; Group2, 7.95+0.37 vs 8.54+0.39%); the suspension of DEAED worsened the metabolic control in both groups. The glycaemic control of Group 3 with DEAED alone was similar to that obtained with OHA; a significant increase of sensitivity to OHA was observed after DEAED suspension. In all three groups, DEAED induced a significant reduction of plasma insulin levels, as well as a reduction of plasma cholesterol and triglycerides. This study suggests that DEAED (alone or in association with OHA) might be very helpful in the treatment of Type 2 diabetes since it combines positive effects on both lipid and carbohydrate metabolism. 569. In vivo calibration of a needle-type glucose sensor to determine the kinetics of subcutaneous glucose concentration G. Velho, Ph. Froguel, G. Reach and D. Thevenot1. Service de Diab~tologie, Paris, and 1LABAM, Cr6teil, France The aim of this work was to investigate the changes in the signal generated by a needle type glucose sensor implanted in the subcutaneous tissue of anesthetized rats. 1) In 18 animals, glycaemia was decreased from 5.74 + 0.22 to a 3.04+ 0.27 mmol/1 plateau following the intravenous injection of I unit of insulin. This drop was followed by a decrease in the signal amplitude with a 5 min lagtime, the correlation coefficient (signal vs blood glucose level) ranging from 0.96 to 0.99. The current decreased from 14.33+1.32 to 11.31_+1.22 nA (p<0.02). 2)These two steady states allowed to calibration of the electrode and calculating of the apparent subcutaneous glucose level during subsequent glucose administration: a) Following a continuous glucose infusion (n=6) that increased glycaemia within 35 min to a 5.58_+ 8.38 mmol/1 plateau, the subcutaneous glucose level increased to a 5.03 _+0.38 mmol/1 plateau (NS); when glycaemia was increased to 10.66_+0.27 mmol/1 (n= 3), subcutaneous glucose concentration rose to 10.16 _+0.11 mmol/1 (NS). b)By contrast, after a glucose bolus (n=7), the glycaemic peak at 2min was 12.37+ 0.94 mmol/1, while the subcutaneous glucose peak, observed at 2 rain, was only 6.13 _+0.55 mmol/1 (p< 0.01). We conclude therefore that under steady state conditions, the subcutaneous glucose concentration reflects blood glucose level. This might not be true during an acute and transient change in blood glucose concentration. 570. Effects of protein and ketone body challenge on kidney haemodynamics. Is protein load adequate to evaluate renal reserve in Type I (insulin-dependent) diabetic patients? M.Velussi, P. Fioretto, R.Nosadini, R.Trevisan, A.Cernigoi, A. Doria, A. Valerio, A. Semplicini, P. Angeli and G. Crepaldi. Patologia

Abstracts Medica I, Istituto di Medicina Interna, Policlinico Universitario, Padova, Centro Antidiabetico di Monfalcone Gorizia, Padova, Italy The kidney capacity to increase glomerular filtration rate (GFR) following oral protein load has been defined as renal reserve in normal subjects. It has also been suggested that aminoacids stimulate proximal tubular Na + reabsorption (PRNa) thus decreasing distal tubular Na + reabsorption (DRNa) and elevating GFR by a macula densa feedback mechanism. We studied in 5 normal subjects and 5 Type 1 (insulin-dependent) diabetic patients GFR (107 +_10 vs 108 + 5 ml. rain -1.1.73 m 2) PRNa (11.0+0.0 vs 11.8+0.0 mEq/min, p<0.05) and DRNa (4.11 _+0.0 vs 3.29 + 0.09, p < 0.01) at basal level during euglycaemic insulin clamp with similar Na plasma concentrations. Following protein load (1 g/kg) GFR increased by 32 + 8% in normal and by 16 + 5% in diabetic patients (p< 0.01). Following ketone body intravenous infusion (25 ~tmol- kg-1 9min -1) GFR increased by 31+8% in normal subjects and by 34+5% in diabetic patients. PRNa increased by 20_+9% and 0 + 8% (t7< 0.01) in normal subjects and diabetic patients, respectively, following protein load whereas DRNa increased by 23 _+3% and 7 + 6% (/7< 0.05). Following ketone body infusion PRNa increased by 25 + 6% and 27_+5% in normal subjects and diabetic patients whereas DRNa increased by 7 + 6% and by 8 + 3%. Conclusions: 1) the kidney capacity to increase GFR is impaired in diabetic patients following protein but not following ketone body load, suggesting that protein oral load is not useful to evaluate renal reserve in diabetic patients; 2) either an increase or a decrease in PRNa following both protein and ketone body loads was not associated with concomitant opposite changes in DRNa, suggesting that a macula densa feedback mechanism does not mediate the effects of protein and ketone bodies on kidney haemodynamics. 571. Fibre intake does not affect insulin requirement and metabolic control in near-normoglycaemic pump-treated Type 1 (insulin-dependent) diabetic patients A.Venhaus, E.Chantelau and M.Berger. Medical Department of Nutrition and Metabolic Diseases, Dtisseldorf University, FRG Two females and eight males with Type 1 (insulin-dependent) diabetes mellitus (age 27 _+9 years, duration of diabetes 13 _+8 years, duration of insulin-pump therapy 22 _+5 months; mean _+SD) participated in a randomised cross-over trial which (after a 4-week run-in period) consisted of two 6-week study periods with a high-fibre-diet (HFD) and a low-fibre-diet (LFD), designed according to Thornton et al. Body weight, blood lipids, HbAlc (normal range 4.2-5.6% of total haemoglobin), insulin therapy, and the results of blood glucose self-monitoring were evaluated every fortnight. Food intake, including dietary fibres (McCance/Widdowson) was determined during one week of the run-in period and each of the study periods respectively. Results: Energy intake ranged between 2878+658 (run-in), 2372 + 669 (HFD), and 2757 _+654 (LFD) kcal/day (p< 0.05), and fibre intake varied between 22 _+10 (run-in), 35 _+13 (HFD), and 18 _+6 (LFD) g/day (p< 0.05). Body weight (69.3_+0.6 kg), HbAlc (5.99 + 0.74%), daily mean blood glucose (7.2 _+0.6 mmol/1), total cholesterol (4.8 + 0.8 mmol/l), HDL-cholesterol (1.45 _+0.4 mmol/1), and triglycerides (1.45 _+1.1 mmol/1) remained virtually unchanged during the entire study. Insulin requirement varied insignificantly between 40.1-+7.9 units/day (HFD) and 42.5-+10.1 units/day (LFD). Conclusion: neither increasing the habitual fibre intake by about 50% (HFD), nor decreasing it by about 20% (LFD) affected insulin therapy and metabolic control in these near-normoglycaemic, normolipaemic, normal-weight pump-treated Type 1 diabetic patients during study periods of six weeks. 572. Pituitary cell surface antibodies in Type I (insulin-dependent) diabetes M. Vercammen, F. Gorus, A. Foriers, O. Segers, G. Somers, M. Van De Winkel and D. Pipeleers. Dept. Metabolism & Endocrinology, Vrije Universiteit Brussels, Belgium Immunoglobulins from patients with Type 1 (insulin-dependent) diabetes often interact with cytoplasmic antigens of endocrine cells, in most cases islet cells. Binding has also been described with surface antigens of pancreatic B cells but not yet of other endocrine cell types. The present study examines the occurrence of anterior pituitary cell surface antibodies (APSA) in Type 1 diabetic patients younger than 20 years. Circulating immunoglobulins were fractionated into IgM and IgG and their binding to rat pituitary cells quantified by FACS-analysis. While none of 33 healthy age-matched control subjects presented APSA, 8/30 recent-onset Type 1 diabetic patients were positive in IgM and 3/30 in IgG. When tested for islet cell-surface antibodies (ICSA), 0/30 were positive in IgM and 7/30

593 A in IgG - the latter interacting selectively with islet B cells. Three years after diagnosis IgM-APSA were found in 4/31 patients and IgG-APSA in 7/31. That the presence of IgG-APSA was highly correlated to that of IgG-ICSA may indicate a reactivity to common antigens. Besides their possible use in screening tests, the IgM and IgG-APSA should also be examined for their possible pathophysiologic implications. 573. Disturbed white blood cell rheology in diabetic patients with microvascular complications I.Vermes, E.T.Steinmetz, L.J.J.M.Zeyen and E.A.van der Veen. Departments of Clinical Chemistry and Ophthalmology, H.d.K. Hospital, Oldenzaal & Department of Endocrinology, Free University Hospital, Amsterdam, The Netherlands Haemorheological alterations may act as accelerating factors in diabetic microangiopathy. Blood cell deformability is reduced in diabetic patients, but published results do not clarify which cell population might account for this abnormality. To investigate this question, isolated blood cell suspensions were filtered in the novel "St. George's Filtrometer" which is capable of discriminating between the two major filtration's factors: initial filtration rate as red cell and filter clogging as white cell component of filterability. Cell suspensions of 22 Type 1 (insulin-dependent) and 37 Type 2 (non-insulin-dependent) diabetes mellitus patients and 57 control subjects were studied. Patients were classified as no retinopathy (35), background retinopathy (16) and proliferative retinopathy (8). Initial filtration rate and red cell transit time as red cell components did not show significant differences between patients and control subjects. However, the number of clogging particles, as white cell parameter, was significantly higher in diabetic patients (146 _+5 ~1-1) than control subjects (98 -F _ 3 ul- I , p < 0.001). In addition, a significantly increased clogging was observed in patients with retinopathy (167+7 g1-1) compared with the retinopathy free diabetic patients (126 _+6 p~l-1,p < 0.01). In view of these observations, we surmise that decreased deformability and/or increased adhesiveness of leucocytes can play an additional role in the development of microangiopathy in diabetes mellitus patients. 574. Effect of proglumide (gastrin and cholecystokinin receptor antagonist, Milid) on glucose tolerance and plasma insulin levels in rats E.J.Verspohl, G.Wunderle and H.P.T.Ammon. Department of Pharmacology, Institute of Pharmaceutical Sciences, University of Tuebingen, FRG Cholecystokinin (CCK) receptors have been demonstrated in rat pancreatic islets. Proglumide (Milid, West Germany) was shown to possess a low affinity to these receptors and to be able to inhibit the CCK-modulated insulin release in vitro. The effect of proglumide on glucose tolerance and plasma insulin levels was tested in rats by using either CCK8 bolus injections or release by camostate (FOY-305; a trypsin inhibitor) of endogenous CCKs. In an intravenous glucose tolerance test (250 mg/kg) bolus injections of CCK8 between 0.1 and 5 nmol/kg dose-dependently increased plasma insulin levels (incremental increase up to 155 ixU/ml) with only a slight decrease of glucose levels. Proglumide (400 mg/kg intravenously 10 rain prior to the combined CCK8 and glucose dosage) reversed this CCK8 effect (0.5 nmol/kg). Proglumide had no effect on plasma insulin and glucose levels when glucose (250 mg/kg intravenous) bolus alone was used. Camostate (400 mg/kg per oral) via endogenous CCK increased plasma insulin levels by 10 ~tU/ml in an oral glucose (500 mg/kg) tolerance test. Pretreatment with proglumide (400 mg/ kg intraperitoneally) antagonised this camostate-mediated CCK effect. The data indicate: 1)an antagonistic effect of proglumide on CCK-mediated insulin release in vivo and 2) that therapy with CCK receptor antagonists should be carefully monitored for a diabetogenic action. 575. A proposal for a computerised system to evaluate personal dietary habits G. Vespasiani, M. Bruni, L. Clementi, N. Giostra, C. Cinciripini and A. Guido. Centro Antidiabetico - Ospedale S. Benedetto del Tronto, Italy Diet is the basis for treatment of diabetes mellitus. Nevertheless, compliance to dietary prescriptions is often rather poor. Correct education is one of the means to improve the motivation necessary for correct nutrition. However, the real amount of food intake of the individual patient is difficult to estimate in current clinical practice. The aim of our study was to select and develop a system for the pre-

594 A cise evaluation of individual eating habits. Three hundred foods, each comprising 22 nutritional components, have been codified by a bar code system. These codes have been printed in a handbook in which both the absolute and standard (cup, glass, etc.) amounts of the foods are indicated. The codes are interpreted with the use of a portable, pen-like reader which memorizes them according to the time of food intake. The data can be collected by the patient before each meal or easily at the end of the day. These data are periodically transmitted to a computer which carries out a quantitative and qualitative analysis, so presenting a nutritional profile of the patient. This system is suggested for use as an auxiliary aid parallel to self-monitoring of blood glucose for diabetic patients and as an educational means for anyone who needs to follow a diet.

576. Study of HLA phenotypes in 124 Type I (insulin-dependent) diabetes mellitus patients of the Cyclosporine-Diab~te-France trial P. Vexiau 1, V. Bochu 2, Ph. Passa 1, A. Marcelli2, P. Mercier4, J.D. Bignon 5, J. Seignalet6, G. Feutren 3, J. F. Bach 3 and J. Hors 2. ~Diabetology Department H6p. St. Louis, Paris, 2INSERM U 93 Paris, 3 Immunology Department Htp. Necker, Paris, 4CTS Marseille, 5CTS Nantes, 6CTS Montpellier, France One hundred twenty four insulin-dependent diabetic patients from the Cyclosporine-Diabtte-France (CDF) trial, 65 treated with Cyclosporine A (Cy A), 59 with placebo, all typed for H L A A , B, C, DR have been analysed at 6, 9 and 12 months follow-up. Partial or total remissions have been compared with failures in both therapeutic groups, according to the HLA phenotypes. Among the Cy A group more remissions were observed at 6 months in the case of patients bearing DR3 (DR3,X or DR3,4 subjects) than in case of non DR3, non DR4 patients (25/42=59% versus 2/9=22%, p<0.05); this difference remained significant at 12 months. In the placebo group, there was no significant difference according to HLA-types. The patients bearing DR3 with Cy A treatment had more remissions than DR3 subjects under placebo at 6 months (25/42= 59% versus 9/36=25%, p<0.001), 9months (p<0.0001) and 12months (17/42=40% versus 3/36=8%, p<0.0001). Moreover no significantly different result could be observed among the group of DR4,X and nonDR3, non DR4 patients comparing both treatments (5/23 =22% versus 5/22=23%). The existence of more frequent remissions within the group of HLA DR3 diabetic patients under Cy A suggests a heterogeneity of Type 1 diabetes mellitus, including a role of an HLA-associated autoimmune component. The HLA DR3 phenotype could then constitute a selective indication for immunosuppressive therapy.

577. Anti-Coxsackie B IgG and IgM antibodies in first degree relatives of Type I (insulin-dependent) diabetic patients. Lack of association with islet cell antibodies B.Vialettes, Ph. Demicco, C. Badier, J. Niccolino and V. LassmannVague. Department of Diabetology, University of Marseille, France To test whether Coxsackie B may initiate antipancreatic autoimmune reaction in Type 1 (insulin-dependent) diabetes, 158 first degree relatives of Type 1 diabetic patients (20.1 +_0.9 years, 8 I C A + , 150 I C A - ) were investigated for IgG and IgM antibodies against Coxsackie B1-B6 virus by ELISA and compared to 35 control subjects (22.3 +_1.3 years) and 29 recently diagnosed Type I diabetic patients (24.2 +_1.5 years). In comparison to control subjects, relatives were characterised by a high frequency of anti-Coxsackle B IgG positive response (84.2 vs 60%, p<0.01) but not of IgM (38 vs 28%) and diabetic patients by a high frequency of both IgG 100%, (p<0.01) and IgM (66.5%, p < 0.01) positive reactions. To overcome cross reaction between subtypes, specificity was allocated to the highest reaction: an excess of anti B 4 IgM antibodies was found in diabetic patients (41.4%, p<0.01) against 0% in control subjects, 8.3% in relatives. High frequency of positive IgG response in relatives did not account for an infection shared with the propositus as 20/22 relatives born after the onset of diabetes of propositus were positive. Among 8 positive ICA relatives (2 developed diabetes later) frequency of positive reactions was: IgG: 7/8, IgM: 1/8 (0/2 prediabetics). Conclusion: Coxsackie B recent infection of which serological trace is frequently found in recently diagnosed Type 1 diabetes does not initiate humoral antipancreatic reaction during preclinical phase of the disease. It is likely to play a precipitating role.

Abstracts 578. Effect of sulfonylurea plus insulin therapy on glucose control and insulin secretion in Type 2 (non-insulin-dependent) diabetic patients S. Vigili de Kreutzenberg, G. Lisato, A. Riccio, A.Tiengo and S. Del Prato. Cattedra di Malattie del Ricambio, University of Padova, Italy 6Type2 (non-insulin-dependent) diabetic patients (age=59_+ 2 years; BMI = 28.6 _+2.1 kg/m 2) with secondary failure to sulfonylurea, we evaluated the effect of 2 months treatment with a fixed dose of insulin (Ultratard Novo = 0.4 U / k g per day, suppertime injection) added to usual sulfonylurea (glybenclamide=15 mg/day) on glucose control and insulin secretion. Glucose control was assessed by 24 h plasma glucose and FFA profile, HbAac, basal hepatic glucose production (HGP) and disposal during +6.9 mmol/1 hyperglycaemic clamp. Insulin secretion was determined by 24 h plasma insulin and C-peptide profile, intravenous glucagon, hyperglycaemic clamp. Insulin was withdrawn 3 days before repeating studies. After 2 months sulfonylurea plus insulin, body weight was unchanged (74 vs 73 kg). HbAlc (11.8 vs 8.9%), fasting glucose (12.3 _+0.5 vs 8.9+_ 1.3 mmol/1), 24 h glucose (14.4 + 1.2 vs 11.5 _+1.7 mmol/1) and FFA (556 vs 391 lxmol/1), and HGP (14.4+_1.1 vs 11.1+_1.3 ~tmol/1. kg -1. rain -1) improved (p< 0.05). Glucose clearance was unchanged. After 2 months sulfonylurea plus insulin, fasting insulin and C-peptide were similar. 24 h profile increased slightly (insulin =14 +_4 vs 17 +_2 mU/1; C-peptide = 0.86 _+0.11 vs 0.99 +_0.16 pmol/1). Following acute hyperglycaemia, mean C-peptide increment was higher after sulfonylurea plus insulin (+0.20 vs +0.36 pmol/1), there was no difference after glucagon. Glucose disposal during hyperglycaemic clarfip was not affected by 2 months sulfonylurea plus insulin (M = 16.1 vs 18.3 lxmol/1, kg- 1 9rain- 1), the index of insulin sensitivity (M/I) slightly increased (24 vs 34%). Decrements in fasting and 24 h glucose correlated (r=0.70). Conclusion: in secondary failure Type 2 diabetic patients, addition of insulin at night reduces fasting glucose and improves overall glucose control. This is primarily mediated through increased insulin liver sensitivity. 579. The measurement of the pulse reappearance time in the diagnosis of peripheral vascular disease in diabetes mellitus K. H. Vogelberg, G. Helbig and W. Stork. Diabetes-Forschungsinstitut an der Universit~t Dtisseldorf, FRG The measurement of the ankle pressure index (API) is not reliable in diabetes mellitus, because of the hardening and poor compressibility of the arterial wall. We examined 40 patients, 12 with diabetes (10 M, 2 F, 61 + 9 years, body weight (BW 106+15%) and 28 without diabetes (20 M, 8 F, 62 + 11 years, BW 105 _+18%) with angiographically confirmed peripheral vascular disease (PVD) in order to discover whether the accuracy, sclerotic severity and localisation of PVD can be better determined by the pulse reappearance time (PRT) during reactive hyperaemia than by the measurement of the API using Doppler ultrasound methods for both. It was shown that the PRT and API were accurate for the frequency-diagnosis of PVD in patients with and without diabetes; however, with reference to the sclerotic severity and localisation of PVD there were significant differences: the sclerotic severity of PVD in diabetes was only correlated to the results of PRT (/,<0.0005); the API was not correlated in contrast to patients without diabetes (p<0.005). The localisation of occlusions could only be distinguished by measurements of the PRT in both diabetic and nondiabetic subjects. Therefore the measurements of the PRT by Doppler ultrasound seems to be more useful for the diagnostic assessment of PVD in diabetic patients than measurement of the API. 580. Muscle energy metabolism during buformin treatment K. Vondra, A. Bass, Z. Slabochov~ and M. And~l. Institute for Clinical and Experimental Medicine, Prague, Czechoslovakia The effect of buformin treatment on activities of selected energy supply enzymes in Type 2 (non-insulin-dependent) diabetic patients was studied using needle biopsy samples. Decreased activities of enzymes associated with glycolysis, cytoplasmic NAD regeneration and tricarboxylic acid cycle were found previously, no change in fatty acid catabolism. Results of this study, performed in 10 patients show that 4-week buformin administration led to a significant increase in activities of triosephosphate dehydrogenase by 21%, glycerol phosphate dehydrogenase (GPDH) by 18% and lactate dehydrogenase (LDH) by 28% (glycolysis, aerobic and anaerobic cytoplasmic NAD regeneration). This indicates increased muscle capacity to catabolise carbohydrates. This was reflected in improved carbohydrate tolerance. In view of unchanged activities of citrate

Abstracts synthase and malate dehydrogenase this also means that aerobic oxidation fails to keep pace with normalising glycolysis intensity and becomes relatively insufficient. Analysis of results showed that LDH activity and the L D H : G P D H ratio correlated positively with blood lactate. No ketonuria developed. Unaltered activity of 3-hydroxyacyl-CoA dehydrogenase did not suggest increased fatty acid breakdown either. Results confirm important role of skeletal muscle in pathogenesis of metabolic disturbances in diabetic patients and expand the body of knowledge regarding mechanism of biguanide action. 581. Peripheral diabetic neuropathy in patients treated by pancreas and kidney transplantation H. Vondrovfi, V. Bartog, I. Van~k and K. Vondra. Clinic of Neurology, Postgraduate Medical and Pharmaceutical Institute, Prague, Czechoslovakia Seven diabetic patients were followed by neurologic examination and electromyography over 4-22 months after pancreas and kidney transplantation. Full pancreatic graft function developed in four patients (no insulin, normoglycaemia - Group 1); partial function was maintained in three patients (low insulin dose, C-peptide secretion maintained - Group 2). Before transplantation, all patients had moderate to severe neuropathy demonstrable by neurologic examination and electromyography with motor nerve conduction velocity (MNCV) slowed by 30-50%. In all patients transplantation resulted in subjective improvement, e.g., steadier gait and regression of muscle spasm. Objective neurologic finding improved in three patients with moderate neuropathy resulting in, e.g., the reappearance of lower limb tendon reflexes. Neurologic finding was unchanged in the four remaining patients with extremely severe neuropathy. In one patient of Group 2 MNCV of all the studied nerves improved. In two Group 1 patients, MNCV of the n. tibialis and ulnaris improved as did that of the n. peroneus and medianus in one patient. We conclude that successful transplantation resulted in 1)regression of moderate neuropathy, 2) improved MNCV of the n. tibialis and ulnaris, observed more often than that of the n. peroneus and medianus, 3) no deterioration of peripheral neuropathy. 582. Cholinergic muscarinic receptor blockade suppresses arginineand exercise-induced growth hormone secretion in Type i (insulin-dependent) diabetic patients M. L. Wagner, J. Arends and B. H. L. Willms. Diabetesldinik Bad Lauterberg, FRG Cholinergic receptor antagonists may inhibit growth hormone secretion in normal man. To investigate whether growth hormone release may be suppressed similarly in diabetic patients, we studied the effects of cholinergic blockade on stimulated growth hormone levels in 18 Type 1 (insulin-dependent) diabetic patients. Paired tests were performed using one of two different stimuli (30 g arginine intravenous or physical exercise at 75 W for 30 min) with or without prior application of the selective cholinergic muscarinic receptor antagonist pirenzepine (30 mg intravenous at - 10 min). Arginine elicited a growth hormone secretory peak of 9.0 + 1.9 ng/ml (mean_+ SEM), which was completely suppressed by pirenzepine (1.5 _+0.4 ng/ml). Blood glucose rose during arginine infusion but was not affected by pirenzepine. Growth hormone levels increased during physical exercise in 8 of 10tested subjects to a mean peak of 9.0_+l.4ng/ml, which was abolished by pirenzepine (1.4_+ 0.4 ng/ml). Blood glucose was not influenced by pirenzepine. We conclude that growth hormone secretion in Type 1 diabetes mellitus is modulated by cholinergic pathways and is responsive to pharmacologic suppression by muscarinic receptor blockade. Pirenzepine should be tested for its long term effect on growth hormone release in patients with Type 1 diabetes. 583. Immune modulation by methimazole in Type I (insulin-dependent) diabetes? W.Waldh~iusl, P. Bratusch-Marrain, H.Vierhapper and K. Pirich. 1. Med. Univ. Klinik, Division of Clinical Endocrinology & Diabetology, Wien, Austria Methimazole (M) reduces synthesis of thyroid hormones, antibody titre vs thyroglobulin and microsomal thyroid antigen and possibly the number of helper T-lymphocytes. This study was consequently undertaken to assess the effect of M (100 mg/day) with thyroxine replacement (0.1 mg/day) sufficient to maintain normal T4 values (8.3 _+1.4 gg/dl) on the duration of primary remission and insulin requirement in Type 1 (insulin-dependent) diabetic patients (n=12;

595 A age, 24+ 11 years; ICA, 54% positive; onset of disease <2 months. Blood glucose (BG) 1 h postprandially 15.3+3.7 mmol/1; HbAIc, 8.5 _+1.6%; AUC, 0-120 rain, of arginine stimulated plasma C-peptide, 139 + 101 nmol. 1-1). Mean duration of methimazole administration was 8.3 +4.8 (SD) months. Five total remissions were seen at 1, 2, 4, 4 and 11 months of M administration. Periods without insulin requirement (HbAlc, < 5.8%) lasted for 4 months in one patient and still continue in four patients after 2, 7, 7 and 12 months respectively. Mean insulin requirement fell from 26 + 13 U/day to 15 _+13 U/day (2 p < 0.005), but only by 17% in patients not in remission. Associated metabolic variables were: BG l h postprandially, 8.6+ 3.6 mmol/1; HbAlc, 5.9 + 0.6%; AUC (0-120 rain) of arginine stimulated plasma C-peptide, 112_+47 mmol/1-1. No change was observed in peripheral blood of the helper/suppressor T-lymphocyte ratio (1.8 + 0.6). It remains to be seen whether the 41% of M-associated total remissions in Type 1 diabetic patients just reflect the natural course of the disease or a similar effect as that described for cyclosporin (Stiller, 1984:16/41 = 39% remissions). 584. Need for adequate community chiropody to help prevent diabetic foot disease J.K.Wales, P.G.Wiles, A.J.Birtwell, J.Wentworth, D.E. Price, P. J. Grant and P. Boyes. University Department of Medicine and Department of Chiropody, General Infirmary, Leeds, UK We have recently introduced a foot screening clinic for our diabetic patients. Medical and chiropody staff assess patients' risk of foot ulceration and need for regular chiropody. Patients are educated in appropriate foot care. Many variables are recorded and computerised including simple clinical assessments of neurological and vascular states using tuning fork, biothesiometry, palpability of pulses and ankle-brachial systolic ratio. Findings in the first 650 patients (age 60.6, range 19-83 years, duration of diabetes 12.4 (1-60) years) show that 158 (24%) have absent pulses in one foot, 90 (14%) being absent in both feet, and 147 (23%) have absent vibration sensation in one foot, 96 (15%) being absent in both feet. 279 patients (43%) required chiropody treatment, predominantly for callosities and nail problems, but only 130 (20% of the total) were already receiving appropriate chiropody care. Therefore, we have found a large proportion of diabetic patients with signs of neuropathy or ischaemia rendering them at risk of foot ulceration and an even larger proportion requiring regular, preventative chiropody. Greater resources need to be directed towards expanding community chiropody services and, thus, provide regular, frequent and convenient foot care for diabetic patients to avoid future morbidity. 585. Insulin is not required for reversal of the decreased glucose transport capacity in diabetic muscle H. Wallberg-Henriksson, N. Zetan and J. Henriksson. Karolinska Institute Department of Clinical Physiology at Huddinge University Hospital, Stockholm, Sweden In the untreated diabetic state, there is a marked insulin resistance for glucose transport. In the present study the reversibility of this decreased glucose transport capacity was investigated. Epitrochlearis muscles from control and diabetic rats (diabetes induced by streptozotocin, 125 mg/kg body wt) were incubated up to 14 h in media containing 6 mmol/1 glucose and 0 or 100 .uU/ml of insulin. As evidenced by determinations of tissue concentrations of ATP, phosphocreatine and lactate, the muscles maintained energy stores and adequate oxygenation during these long incubations. Insulin-induced (20 mU/ml) 3-methylglucose transport was markedly decreased in epitrochlearis muscles from 3-days-diabetic rats compared to control rats (3.77 _+0.38 vs 10.22 _+1.27 ~tmol- ml-1. h-l, p < 0.001). In vitro incubation gradually normalised insulin responsiveness within 12-14 h. The reversal of the insulin responsiveness took place even in the absence of insulin. In fact, insulin responsiveness was higher after incubation (14 h) with no insulin than with 100 uU/ml of insulin (9.85_+0.59 vs 8.06_+0.59 gmol.m1-1 -h -a, p < 0.05'). Glucose at 30 mmol/1 did not affect the normalisation of the insulinstimulated glucose transport capacity. The results indicate that the insulin resistance for glucose transport found in insulin-deficient diabetic muscle is not a direct consequence of the lack of insulin or of high glucose concentrations. 586. Response of subcutaneously implanted glucose sensor to intravenous glucose load in normal and diabetic rats R.V.Warhadpande, M. Dolderer, C.Zawadil, H.Zier, W. Kernel, G. Steinbach and E.F.Pfeiffer. Department of Internal Medicine, University of Ulm, FRG

596 A An implantable miniaturised enzymatic glucose sensor was used to study subcutaneous tissue glucose changes in normal (n=11) and streptozotocin induced diabetic (n= 8) rats. After implanting the sensor in subcutaneous tissue in flanks, glucose was injected intravenously (1 g/kg). Serial blood glucose values were recorded and compared with that of current output from the implanted sensor, which was already tested and calibrated in vitro. Increase in sensor current, after intravenous injection of glucose, was seen after 4.1 (2-6) min and it reached maximum after 23.0 (10-50) min in normal rats. The corresponding values in diabetic rats were 3.8 (2-9) min and 34.4 (15-70) min. The peak excursion in current output from the basal value was 234.6% and 233.6% in normal and diabetic rats, respectively, whereas the increase in blood glucose was 322.6% and 326.7% in normal and diabetic rats. The time taken by blood glucose for return to original level was 101.2 min in normal and 125 min in diabetic rats. The similar changes in sensor output took 118.1 min and 114.2 min respectively. These results indicate that tissue glucose measured indirectly by a glucose sensor showed close relationship with blood glucose and was similar in normal and diabetic rats.

587. Iatrogenic hyp~rinsulinaemia in Type 2 (non-insulin-dependent) diabetic patients as cause for excessive hyperlipoproteinaemia M. Week, M. Hanefeld, S. Gehrisch, K. Schollberg, W. Leonhardt and W.Jaross. Department of Internal Medicine, Medical Academy of Dresden, G D R We observed 6 diabetic patients treated with high dosages of insulin ( 2 = 48 1.U.), which were trnasferred to our metabolic ward because of massive therapy refractory dyslipoproteinaemia ( 2 triglycerides (TG)=21.6mmol/1, total cholesterol (TC)=13.4mmol/1, HDLC = 0.52 mmol/1, LDL-C = 7.9 mmol/1, Apo C-II = 92 rag/l, Apo CIII=734 rag/l) and insufficient glucose control ( 2 of glycaemic index= 13.8 mmol/1). Anamnesis, medical data and C-peptide determination ( 2 before glucagon stimulation = 1.15 nmol/1, ~ after glucagon stimulation=l.5 nmol/1) before insulin treatment clearly revealed them to be Type 2 diabetic patients with presumably primary hyperlipoproteinaemia. Our working hypothesis was that nonindicated insulin treatment induced a vicious cycle via hyperinsulinaemia, insulin resistance, hyperglycaemia, excessive hyperlipoproteinaemia. Semi-starvation should counteract this circulus vitiosus. Therefore we stopped insulin injections and put the patients for 4 weeks on protein-supplemented VLCD (330 kcal/d). After 6 weeks with refeeding, the following metabolic parameters were observed: TG = 2.9 mmol/1, TC = 5.2 mmol/1, HDL-C = 0.79 mmol/1, LDLC=3.0 mmol/1, Apo C-II=55 mg/1, Apo C-III=177 mg/1, glycaemic index = 7.1 mmol/l. In 2 patients glucose control was possible with diet alone, 4 needed additionally 1-5 mg glibenclamide per day.

588. Glucose does not block ATP-regulated K+-channel activity in fetal B cells M.Welsh, P.-O.Berggren and P.Rorsman. Dept. of Medical Cell Biology, Uppsala University, Uppsala and Dept. of Med. Physics, G6teborgs University, G6teborg, Sweden Fetal B cells do not respond properly with increased secretion of insulin when stimulated with glucose, and therefore may represent an interesting model for studies of Type 2 (non-insulin-dependent) diabetes. We have addressed the question whether this secretory defect is correlated with electrophysiological disturbances of the cells. Islets were prepared from fetuses of 21-day pregnant rats and cultured in 11 mmol/l glucose 3 days before use. Single-channel currents were measured on cell clusters of 25-50 ~tm in diameter using standard patch-clamp techniques. When recording channel activity from cellattached patches (intact cells) in the absence of glucose, openings of a 60 pS K+-channel were observed. After increasing glucose to 20 mmol/1, channel activity persisted for more than 10 min, and in some cells even increased. Exposure of the cells to 0.2-0.4 mmol/1 of the sulphonylurea tolbutamid resulted in a rapid and complete closure of the channel, and the appearance of action potentials. The abnormal response to glucose is not a consequence of an altered ATPsensitivity of the channel since recordings of inside-out patches demonstrated a 50% inhibition of channel activity with 10 p~mol/1 ATP, i.e. similar to that of adult B cells. The data therefore suggest that the incomplete blockage of the ATP-regulated K+-channel in fetal B cells results from immature substrate metabolism.

Abstracts 589. Erecaid system: an effective, non-invasive device for managing erectile impotence in diabetic men P. G. Wiles. University Department of Medicine, General Infirmary, Leeds, U K The Erecaid system uses a suction device to induce penile engorgement, the erection is then maintained by a constrictive band for up to 30 min. Ten male diabetic patients median age 55 years (range 34- 64), duration of diabetes 18 (5 - 31 ) years, together with their partners, were instructed in the use of the device. They had suffered erectile impotence for 3 (2-15) years having first sought medical help 2 (1-15) years previously. After 3 months the effectiveness of the device was assessed. Two patients were not regularly using the system. One had separated from his partner, the other was experiencing marital disharmony. Both patients had been satisfied with the device. Results of patients' assessments using a visual analogue scale (0-10) were: for ease of use: 9.0+0.5 (mean+SEM); effectiveness of device: 8.0+1.0; satisfaction with erection achieved: 8.2+0.5; partner's satisfaction: 8.3 + 0.5. Using a scale - 5 to + 5 they also graded for: change in overall relationship with partner: +3.2+0.8; and change in general self esteem: +3.4+0.8. Patients were having intercourse 4 + 2 times/month. All recommended the system for use by others. There were no unpleasant side effects. The Erecaid system is a simple effective means of overcoming erectile impotence and is safe for use in diabetic patients.

590. Islet-cell autoimmunity and cystic fibrosis T. Wilkin 1, D. Heaf ~, M. Dalzell2, C. Smith 2, A. Tuck a and P. Stutchfield2. aDepartment of Medicine II, Southampton General Hospital; 2Respiratory Unit, Alder Hey Children's Hospital, Liverpool, U K Such terms as autoimmune attack and autoaggression are commonly applied to autoimmunity in the belief that damage results from dysregulated immunity. Our alternative hypothesis - the primary lesion theory - argues that autoimmunity represents a secondary, protective and above all appropriate response to antigen presented by tissues undergoing primary disruption. In order to establish whether, in vivo, the primary disruption of cells provokes a sustained humoral response analogous to that of classic autoimmunity, we used an ELISA to look for insulin autoantibodies (IAA) in the sera of 29 insulinnaive children with cystic fibrosis of the pancreas, 58 homozygous twins discordant for Type I (insulin-dependent) diabetes and 60 childhood control subjects. IAA were present in 6.7% control subjects (range 10-18 binding units), 47% twins (range 10-32 binding units) and sustained in 21% cystic patients (range 10-38 binding units) although none had an autoimmune diathesis. It is conventionally argued that sustained autoantibodies are markers for a unique process called autoimmunity. The present data suggest, to the contrary, that autoantibodies are a physiological response to non-specific cell damage. As the intensity of immune response is HLA-determined specifically for each antigen, it may be argued that the IAA titre recorded in each case was a function of insulin exposure (obligate) and immune response genotype (facultative).

591. First international workshop on insulin autoantibodies T.Wilkin 1, 2J.Palmer and participants of First International Workshop on IAA. aDepartment of Medicine II, Southampton General Hospital; 2Diabetes Research Centre, Seattle, USA The first international workshop on insulin autoantibodies (IAA) was held in Perth, Australia, during January 1987. Twenty-one centres worldwide participated in two studies, the first designed to reflect the degree of consensus in IAA measurement and the second to compare particular parameters of assay behaviour. Viewed uncritically, the variance in IAA results from the 49 coded samples in Study I was considerable, but could be largely attributed to two independent factors: assay type and assay ligand. Thus, fluid phase radiobinding assays appeared to respond more strongly to insulin antibodies from insulin treated diabetic patients and solid phase ELISA's to insulin autoantibodies from insulin-naive individuals. A number of IAA sera were clearly human insulin specific in both assay types so that laboratories using a porcine insulin ligand were not in a position to detect them. Study 2 confirmed Study 1 but also showed that many assays were troubled with non-specific binding from irrelevant serum proteins. The data indicate the need in phase 2 for a standard calibration curve, reference serum and ligand in order to reduce variance and improve consensus.

Abstracts 592. Statistical analysis of a large computerised clinical database containing diabetic outpatients records: retinopathy C. D. Williams, S. Till, P. Calder, S. Pack and P. H. S6nksen. Department of Medicine, United Medical and Dental Schools of Guy's & St.Thomas' Hospitals, St.Thomas' Hospital, London, UK

Information on all newly referred diabetic patients (45% newly diagnosed, 55% established) has been collected prospectively since 1974 using a structured questionnaire containing 190 data items, and entered into a microcomputer-based clinic management system. Crosssectional studies using bivariate analyses revealed highly significant associations (p<0.005) between retinopathy (based on records of microaneurysms, haemorrhages, exudates and new vessels) with proteinuria, peripheral neuropathy, and diabetic foot problems ( n = 1216). Potential predictor variables for retinopathy were identified using principal components and correspondence analyses, and examined further with several other multivariate techniques. Retinopathy was associated significantly with duration of diabetes and systolic blood pressure but not with smoking (n= 879) nor glycosylated haemoglobin at first visit (n=584) or follow-up (n=81). In Type 2 (non-insulin-dependent) patients retinopathy was found in 9.2% at diagnosis and associated with random blood glucose ( n = 227). Additional indicants for retinopathy in Type 1 (insulin-dependent) patients included total daily insulin dosage and ethnic origin (n=180). Despite significantly higher HbA1 levels, Afro-Cart]bean patients had a lower incidence of retinal involvement. These data suggest that in contrast to duration of diabetes, systolic blood pressure and ethnic origin, smoking and HbA"1 are poor predictors for development of retinopathy. 593. Hypothalamic regulatory peptide disturbances in animal diabetes (3. Williams, H.M. Cardoso, M.A. (3hatei, A.R. Diani, (3. C. (3erritsen, J. M. Burrin, J. M. Polak and S.R. Bloom. Departments of Medicine and Histochemistry, Royal Postgraduate Medical School, London, UK and Diabetes Research Division, Upjohn Co., Kalamazoo, Mich, USA Several hypothalamic peptides may affect feeding and glucoregulatory hormone secretion. We examined hypothalamic peptides in 3 models of animal diabetes_+ obesity. KKAy mice spontaneously develop hyperinsulinaemia, hyperphagia and obesity ( = prediabetic phase: 30 days) and finally Type 2 diabetes (90 days). Susceptible Chinese hamster sublines develop B-cell failure and insulin-deficient diabetes. Wistar rats were rendered diabetic by intravenous streptozotocin (STZ: 55 mg/kg). Peptides were extracted from hypothalamic blocks by boiling in 0.5 tool/1 acetic acid and bombesin, galanin, neuropeptide Y (NPY), neurotensin, somatostatin and VIP were measured by radioimmunoassay. KKAy mice showed significantlyreduced (by 30-50%, p < 0.02) hypothalamic NPY, galanin and VIP in both pre-diabetic (n="12) and diabetic (n=12), compared with matched C57BL/6 mice. In diabetic hamsters (n=12), NPY and somatostatin were significantly lower (by 30%, p<0.05) than in matched non-diabetic hamsters. STZ-diabetic rats (n= 12 per group) showed significantly-increased NPY (by 30-50%, p<0.01) after 4 and 6 weeks of diabetes, compared with non-diabetic rats. Hypothalamic peptide changes may be associated with central metabolic adjustment and/or abnormal pituitary function in diabetes, and perhaps with the primary hypothalamic defect postulated in KKAy mice. Centrally-administered NPY stimulates feeding and insulin secretion: NPY disturbances in all 3 models suggest a key role in hypothalamic metabolic regulation. 594. Effects of a single nocturnal administration of a long acting somatostatin analogue, SMS 201-995 on blood glucose profile during the night, human growth hormone and cortisol secretion in Type ~1(insulindependent) diabetic patients B. Willms, A. Harris and B. Mehmke. Diabetesklinik Bad Lauterberg, FR(3 We studied the effect of a long acting somatostatin analogue, SMS 20"1-995, on nocturnal blood glucose profles, (3H and cortisol levels. Methods: In the first study, 5 Type "1 (insulin-dependent) diabetic patients were given 100 ~g SMS 201-995. In the second study, "10Type 1 diabetic patients were given 25 ~tg SMS 201-995. SMS was injected for three nights subcutaneously at 22.00 hours. Results: 100 ~xg SMS suppressed (3H levels to values below 1 ng/ml up to 01.00 hours, then GH began to increase to a maximum value at 05.00 hours. 25 ~g SMS suppressed (3H to a smaller extent and for a shorter period; the rise began at midnight up to a maximum at 03.00 hours. Blood glucose (BG) concentration: With 100 ~g SMS,

597 A blood glucose was lowered, beginning two hours after injection, reaching its nadir at 04.00 hours (between 2.81 and 2.98 mmol/1) and rising again in a rebound to fasting BG levels of 5.08 mmol/l (night 2), 6.46 mmol/l (night3) and 8.23 mmol/l (night 4). With 25~g SMS mean BG was lower ( n i g h t 2 : 4 . 1 4 m m o l / 1 at 02.00 hours; night 3: 4.70 mmol/1 at 02.00 hours; night 4: 4.64 mmol/l at 03.00 hours). Compared with 100 gg SMS the hypoglycaemia was less with 25 l-tg SMS, no rebound was seen. Fasting BG was 7.96mmol/1 (night2), 11.77mmol/1 (night3) and 9.10 mmol/1 (night 4). Cortisol levels were not significantly different. Summary and Conclusions: A single nocturnal administration of SMS 201-995 depresses GH secretion for a short time, induces hypoglycaemia after midnight and does not prevent the early morning increase of blood glucose concentration.

595. Pharmacokinetics and bioavailability of human proinsulin/ insulin admixtures given in one syringe subcutaneously or injected separately G.Willms, H.Reichert and M.Berger. Department of Nutrition and Metabolic Diseases, University Duesseldorf, Duesseldorf, FRG Human proinsulin (HPI) may be an alternative to NPH long-acting insulin preparations. In this study, we have investigated the pharmacokinetics of mixtures of HPI and regular human insulin (Eli Lilly, NI) injected subcutaneously. Sixteen healthy male volunteers received 4 different ratios of HPI (strength "10 m g / m l = 4 0 U/ml) and NI (1.45 m g / m l = 4 0 IU/ml). Every protocol consisted of injections of 0.5 ml H P I / N I (20 IU) in the following ratios: 4:1, 2 : 1, 1 : 1, and ] : 2. The preparations were injected either mixed in one syringe or in separate syringes. Each volunteer was subjected to all eight different experiments in randomised order, single blinded. Venous blood was sampled at - 1 5 rain, at injection, and thereafter in regular intervals until 13 h after the subcutaneous injection of HPI/NI, in order to determine plasma levels of glucose, free insulin and proinsulin. The area under the plasma glucose time curves were different between the various H P I / N I ratios (p<0.01) and also, for a given HPI/NI ratio, between the injections of HPI-NI mixtures in one syringe and separate injections of HPI and NI in two syringes (p<0.01). The plasma sugar was still 33% below baseline 10 h after the injections of 4:1 and 2 : 1 H P I / N I ratios; and 8 h after the injection of 1 : 1 and 1 : 2 ratios when mixed in one syringe. Plasma glucose was 33% below baseline 6 h after injection of the ratio 1:2 in separate syringes. Conclusion: The admixture of NI with HPI in one syringe before administration alters the bioavailability of regular insulin on subcutaneous injection.

596. The effects of 3-hydroxybutyrate, in the presence and absence of insulin, on fatty acid mobilisation in isolated human adipocytes L.Wilson and D.G.Johnston. Department of Medicine, University of Newcastle upon Tyne, The Medical School, Framlington Place, Newcastle upon Tyne, UK In rat adipocytes ketone bodies are antilipolytic and increase the sensitivity of adipocyte metabolism to insulin. The effects of ketone bodies on fatty acid (NEFA) mobilisation in human adipocytes are unknown. This study has investigated the effects of D-3-hydroxybutyrate (3-OHB), with and without insulin, on lipolysis and NEFA esterification in isolated human adipocytes. Lipolysis was assessed by glycerol release, and esterification by the non-isotopic technique (NEFA: glycerol ratio). Adipocytes were prepared by collagenase digestion on abdominal wall adipose tissue biopsies obtained during elective surgery. Statistical analyses were performed using analysis of variance. 3-OHB alone (1.25 to 10 retool/l) had no effect on basal lipolysis but inhibited noradrenaline (10 -6 mmol/1) stimulated lipolysis in a dose-dependent manner (at 1.25mmol/1:0.129 vs 0.118 lxmol glycerol/10s cells/4h with least significant difference (LSD)0.05= 0.010). Noradrenaline stimulated NEFA release was similarly inhibited (at 1.25 mmol/1:0.389 vs 0.332 Ixmol NEFA/105 cells/ 4 h, with LSD005= 0.053). No effects on esterification were observed. Insulin a l o n e (8 x 10 -1~ mmol/1) inhibited noradrenaline stimulated lipolysis (0.180 vs 0.1"17Ixmol glycerol/105cells/4h, with LSDa05=0.045) and the inhibition observed in the presence of both insulin and 3-OHB (5 and 10 mmol/1) was additive. These studies suggest that 3-OHB directly inhibits noradrenaline stimulated lipolysis in isolated human adipocytes and does not increase the sensitivity of lipolysis to insulin.

598 A

Abstracts

597. Lymphocyte transformation and neutrophil phagocytic activity in elderly Type 2 (non-insulin-dependent) diabetic patients P.H.Windocour 1, J.Lenton 2, J.A.H.Puxty 2 and D.C.Anderson 1. University Departments of Medicine I and Geriatrics 2, Hope Hospital, Salford, Lancashire, UK

measure max. 0.72 mm x 0.57 mm. Between the proliferating ductules insulin reactive single cells and also compact B cell conglomerations were located. Mitoses were not visible. The findings suggest that the hyperplasia of B cells in the graft is originated by the induction of streptozotocin diabetes in the host animal.

Although hyperglycaemia and the ageing process are presumed to increase the risk of infection, definitive evidence for this is sparse. The early Type 2 (non-insulin-dependent) diabetic patient, however, might be predisposed to bacterial infection if ageing and diabetes are cumulative in altering host defence mechanisms. We have examined lymphocyte transformation to phytohaemagglutinin (PHA), concavalin A (Con A) and pokeweed nitrogen (PWM), and neutrophil microbicidal activity by measuring nitroblue tetrazolium (NBT) reduction in 11 healthy elderly control subjects (3 M, 8 F), aged 70_+ 4.5 years (mean+ SD), and 23 poorly controlled elderly Type 2 diabetic patients (11 M, 12 F), aged 68_+5.1 years. HbA1 in the diabetic patients was 11.2+2.3% and mean blood glucose (MBG) 11.0_+ 3.2 mmol/1. Lymphocyte transformation to 5 t.tg and 10 ~g of PHA, to 10 ~tg and 20 p~g of Con A and to 5 I.tg or 10 ,ug of PWM did not differ between the healthy control subjects and diabetic patients. NBT reduction likewise was no different between the 2 groups. When those diabetic patients with the worst glycaemic control (number= 14) were compared to those with only moderately poor metabolic control (number= 9), HbA1 12.8 +_1.3 vs 8.8 + 0.8% (p< 0.001); MBG 12.3 _+3.2 vs 9.0 _+2.0 mmol/l (p< 0.005), lymphocyte transformation and NBT reduction were equivalent. These data suggest that elderly Type 2 diabetic patients have equivalent host defence responses to a healthy elderly population irrespective of the degree of glycaemic control and are not therefore more susceptible to infection secondary to disturbed lymphocyte transformation or phagocytic killing.

600. The effect of a new oral antidiabetic drug (etomoxir) on glucose turnover and recycling with [1-14C1, [3-3HI-glucose tracer in pigs H. P. O.Wolf and K.V. Brenner. Research Laboratories, Byk Gulden Lomberg, Konstanz, FRG Etomoxir (INN) is a new antidiabetic agent from the class of oxirane carboxylic acids. The mechanism of action of this class of compounds is specific inactivation of carnitine palmitoyltransferase I (CPT I, EC 2.3.1.21). Infusion of etomoxir to 4 fasted pigs caused significant (48%) falls in blood glucose concentrations. To assess whether inhibition of hepatic glucose production or increase of peripheral glucose utilisation is causally associated, a primed infusion of [3-3H]-glucose and [1-14C]-glucose was used, and glucose turnover rates, recycling and metabolic clearance rate of glucose were determined. No effects of infusion of etomoxir on glucose turnover rates could be found. Recycling of glucose carbon was affected to a relatively small extent. The metabolic clearance rate, however, increased by 126% from 5.0_+0.7ml.kg-l.min -1 in the control group to 11.3 _+3.5 ml. kg -1- min -1 in the treated group (mean_+ SEM, p < 0.05). We conclude that under fasting conditions this increase in glucose utilisation plays a major part in the blood glucose lowering effect of etomoxir.

598. High yield production of murine monoclonal islet cell surface antibodies in ascites of irradiated recipients S. Witt, B. Ziegler, N. Nadrowitz I and M. Ziegler. Central Institute of Diabetes "Gerhardt Katsch", Karlsburg, aDepartment of Radiology, University of Greifswald, G D R Islet cell surface antibodies (ICSA) have been detected in sera from newly-diagnosed Type 1 (insulin-dependent) diabetic patients. A prerequisite to study the pathogenic role of ICSA is the generation of monoclonal ICSA against different antigenic determinants of B cells as occurring in the polyclonal sera from Type 1 diabetic patients. A large amount of islet cell reactive monoclonal antibodies can be produced in ascites fluid by injecting hybridoma cells into a mouse. However, quite often only a few hybridomas induce formation of ascites tumor. In the present study the average ascites tumor formation rate of 10 different hybridomas could be increased from 32% (n = 338 mice) to 78% (n= 112 mice) by only one whole body irradiation (500 rad) of paraffin-pretreated Balb/c mice. The volume of ascites fluid was significantly (p<0.01) higher in male recipients (10.3 ml vs 5.6 ml). One monoclonal ICSA, M3aG9, produced by this procedure still showed a striking surface immunofluorescence on islet cells at an ascites fluid dilution of 1:10000. From the increased tumor formation rate in irradiated mice it is suggested that in non-irradiated recipients the tumor growth rate was lowered by immunological reactions against hybridoma cells provoked by cell surface neoantigens revealed by cell fusion or tumor-associated antigens of the myeloma parent cells. 599. Proliferation of B cells in pancreatic islet syugeneic transplantation into the spleen of streptozotocin diabetic Lewis rats F. Wohlrab, S. Schmidt, B.Wilke and L. Cossel. Institute of Pathology, Karl-Marx-University, Leipzig, GDR; Central Institute for Diabetes Research "Gerhardt Katsch", Karlsburg, G D R An islet transplantation model was established with the two eongeneic Lewis rat strains LEW l W and LEW 1A. Female rats (weighing 150-180 g) were made diabetic by a single injection of 50 mg/kg BW streptozotocin. Syngeneic transplantation of 800 to 1000 islets of neonates into the spleen resulted in permanent graft survival and in normoglycaemia of diabetic rats of the two strains. Two hundred days after transplantation, morphologically and functionally intact islet tissue were demonstrable in the spleen by histological, histochemical and electron microscopical investigations. The remaining pancreatic islets in diabetic rats contained only few B cells. In the graft tissue of the spleen compact B cell complexes (max. length 1,86 mm; max. breadth 1.14 mm) were observed, which derive from transplanted pancreatic ductules. The areas of proliferated ductules

601. A randomised cross-over trial of oral versus insulin therapy in patients with Type 2 (non-insulin-dependent) diabetes mellitus (preliminary results) B. H. R. Wolffenbuttel, R.F.A. Weber, P.M.v. Koetsveld and L. Verschoor. Dept. of Internal Medicine III, University Hospital Rotterdam - Dijkzigt, The Netherlands The therapeutic approach to patients with Type 2 (non-insulin-dependent) diabetes, who are hyperglycaemic despite dietary treatment, remains a debate. We investigated the effects of six months' treatment with oral (O) and insulin (I) therapy in non-obese patients with fasting blood glucose above 7 retool/1 on metabolic control and B-cell function in a randomised cross-over fashion. Twelve patients (age 60 _+3 years, known duration of diabetes 6 + 2 years) were treated with both glibenclamide (18 _+1 rag/daily), and insulin administered twice daily (35 + 3 U daily). In 3 patients, receiving insulin initially, subjective and objective signs of hyperglycaemia occurred after switching to oral therapy, necessitating reinstitution of insulin treatment. The complete cross-over results could be obtained in 9 patients. Weight increase was more pronounced during I (2.8 _+0.9 vs 1.3 _+0.7 kg, p < 0.05). Fasting blood glucose (12.6 + 0.8 retool/l) was significantly (p<0.05) lower during I (8.0+0.6 retool/l) than during O (10.8_+1.1 mmol/1). Glycosylated haemoglobin showed comparable changes. Fasting C-peptide increased during both O and I (from 508 _+73 to 617 _+89 and 564 _+97 pmol/1 respectively), as did C-peptide release after glucagon (from 2449 + 420 to 3964_+ 592 and 3476+628pmol/15min). HDL-cholesterol was higher (p<0.05) during I (1.19 + 0.17 retool/l) compared to O (0.97_+0.10 mmol/1). Five patients experienced improved well-being during I. We conclude that in non-obese Type 2 diabetic patients treatment with insulin causes: 1)clinical improvement; 2)better metabolic control; 3) higher HDL-cholesterol levels. 602. Glucose-induced changes of the sodium content of isolated pancreatic islets G. H. J. Wolters and M.W.A. Vonk. Laboratory Experimental Endocrinology, Dept. of Surgery, University of Groningen, The Netherlands The effect of glucose on islet Na + content and its role in the insulin secretory process is not clear. We developed a method to rapidly remove extracellular Na + and to measure intracellular Na + by atomic emission spectrometry. At 2.5 mmol/1 glucose the islets contain about 7 mmol Na + per ~tg DNA, which rapidly decreases by 20% at 15 mmol/1 glucose. Tetrodotoxin did not alter the Na+content nor the effect of glucose. However, activation of the Na + channel by veratridine strongly increased islet Na + content and even enhanced the effect of glucose. Inhibition of the Na+-H + antiporter by amiloride did not alter the Na+content nor abolished the effect of glucose. Acceleration of Na+-H + exchange by monensin increases the Na + content by 60%, however, the effect of 15 mmol/1 glucose was even

Abstracts larger. Inhibition of the Na+-K + pump by ouabain enhanced the Na + content by 75% and abolished the effect of glucose. This might indicate that glucose decreases the Na + content of islets by increasing the activity of the pump. However, manipulation of the activity of the pump by changing K0+ concentration or islet ATP content suggests that the effects of glucose are probably due to another mechanism, presumably due to inhibition of the Na+-Ca 2+ countertransport. 603. Confounding effects of short-term metabolic control and C-peptide status on insulin tolerance, TRH, GnRH and exercise overload tests in Type I (insulin-dependent) diabetes M. I. Wih-zburger, G. M. Prelevi6, P. H. S~nksen, L. A. Peri6, S. Till and R. Morris. Medical Centre "Zvezdara", Department of Endocrinology, University of Belgrade, Yugoslavia and Department of Medicine, St Thomas's Hospital, University of London, U K

In order to evaluate different aspects of pituitary function in Type 1 (insulin-dependent) diabetes and influence of metabolic control on it, exercise overload, ITY, TRH and GnRH tests were performed in a group of 25 patients during poor (Study I) and after two weeks of significantly improved metabolic control (Study II). According to serum C-peptide levels patients were divided into the following groups - CpN - 10 patients with undetectable C-peptide and CpP - 15 patients with residual B-cell activity. The results show: a) The same exercise overload induces significantly higher growth hormone response in patients without, than in those with, residual pancreatic B cell function independent of the level of metabolic control; b) TRH induces higher growth hormone secretory response in CpN patients and this response seems to be under the influence of metabolic control; c) Growth hormone and cortisol secretion in response to insulin-induced hypoglycaemia are independent of the presence of residual B-cell function as well as metabolic control; d) TSH and PRL secretion in response to TRH are independent of metabolic control as well as the presence of endogenous pancreatic reserve; e) LH response to GnRH in CpN patients is significantly lower than in CpP patients during both study periods. 604. Determinants of mortality from myocardial infarction in diabetes J. S. Yudkin and G. A. Oswald. Academic Diabetic Unit, Whittington Hospital, London, UK

We have investigated the outcome of admission with myocardial infarction in 83 diabetic patients and 380 non-diabetic subjects whose levels of glycohaemoglobin excluded prior diabetes. Of the diabetic patients 42.2% died compared with 24.7% non-diabetic subjects (p<0.005). There was no difference in age or sex ratio but peak levels of aspartate transaminase were significantly lower in the diabetic patients (p<0.01). Twenty-seven of 35 diabetic deaths were caused by left ventricular failure or cardiogenic shock (pump failure), and another 8 patients who developed pump failure survived. Among the diabetic patients there was no relationship between age, diabetes duration or glycohaemoglobin level and either pump failure or death. These outcomes were also unrelated to prior treatment, gender or infarct site, but levels of plasma glucose and peak aspartate transaminase were higher in those who developed pump failure or died. The 83 diabetic patients were compared with 249 age- and sex-matched diabetic clinic attenders without myocardial infarction to assess whether diabetes duration, prior treatment or control influence the risk of incidence of myocardial infarction. Levels of glycohaemoglobin were significantly lower and duration of diabetes shorter in infarct patients than matched control subjects, but there was no difference in prior treatment. Cardiovascular disease and hospital mortality are thus unrelated to poor diabetic control or long duration. 605. Regulation of plasma free fatty acid concentration by glucose and insulin in diabetic versus nondiabetic obese subjects C.Zancanaro, M. Cigolini, E. Bonora, P. Moghetti, M. Querena and M. Muggeo. Institute of Clinical Medicine and Chair of Metabolic Diseases, University of Verona, Italy This study evaluated the in vivo suppressive effect of glucose and insulin on plasma free fatty acid (FFA) concentrations in diabetic vs nondiabetic obese subjects. For this purpose plasma FFA were measured during a 4 h hyperglycaemic glucose clamp (11.05 mmol/1) in 6 obese nondiabetic and 6 weight-matched obese newly diagnosed Type 2 (non-insulin-dependent) diabetic subjects. Somatostatin was also infused (250 meg/h) during the third hour of clamp to inhibit glucose-stimulated insulin secretion. Plasma insulin was similar in the two groups at fasting and all throughout the study. The amount

599 A of glucose metabolised during the clamp was significantly lower in diabetic subjects all throughout the study. Plasma FFA were similar in the two groups at fasting, and in both groups decreased during hyperglycaemia plus glucose-induced hyperinsulinaemia (0-120 min), rose during hyperglycaemia and inhibition of insulin secretion by somatostatin (120 180 min), decreased again during hyperglycaemia and recovering hyperinsulinaemia (180-240 min). However, plasma FFA were significantly higher in diabetic than in nondiabetic subjects throughout the three periods of the study (0-120 min, F = 29.10, p<0.0001; 120-180 min, F=21.09, p<0.0001; 180-240 min, F = 4.94, p < 0.05). These data suggest that plasma FFA suppressibility by glucose and insulin is lower in obese Type 2 diabetic patients as compared to obese nondiabetic subjects. 606. The importance of cardiovascular autonomic neuropathy in Type 1 (insulin-dependent) diabetic patients with different stages of diabetic nephropathy E.Zander, E. Grimmberger, G. Zander and B. Schulz. Central Institute of Diabetes "Gerhardt Katsch", Karlsburg, G D R

Diabetic nephropathy (dNP) is known to be associated with increased cardiovascular mortality. This can possibly be also due to cardiovascular autonomic neuropathy (CAN). The aim of our study was to investigate the impact of CAN in Type 1 (insulin-dependent) diabetic patients with different stages of dNP. We studied patients with incipient dNP (I, n= 8), with overt dNP (II, n= 17), with renal insufficiency (III, n=20), with end-stage-renal-failure (IV, n=12) compared with 37 control subjects (V) without clinical signs of dNP, but of similar diabetes duration (17_+ 8.1 years). We investigated heart rate variation during deep breathing (A) (computer-aided), RR-interval variation during standing (B), R-R-interval during Valsalva-manoeuvre (C), blood-pressure response to standing (D), as well as clinical features of autonomic and peripheral neuropathy. By testA we found in I 10.5+6.48; II 7.35_+5.0; III 4.85_+3.67; IV 5.33 _+4.58 as compared with V 14.5 _+8.87 beats/rain (p< 0.01). Similar abnormal findings in other autonomic tests were also in close correlation with degrees of dNP. Summing up we conclude: cardiovascular autonomic neuropathy is very common in patients with dNP and might be an important risk factor for increased cardiovascular mortality; CAN has been shown to be the more frequent the more dNP has progressed; patients with incipient dNP should be followed up also in CAN function tests. 607. Influence of insulin on blood pressure in non-obese and obese nondiabetic subjects I.Zavaroni, E. Bonora, E. Dall'Aglio, A. Pezzarossa and M. Pagliara. Institute of Clinica Medica Generale, Parma University, Italy

Hypertensive patients have plasma insulin levels higher than normotensive subjects. In addition, a significant relationship between plasma insulin and blood pressure in various populations has been reported. To further clarify the association between these variables, we have measured blood pressure and plasma insulin at fasting and following an oral glucose load in 367 (247 non-obese, 120 obese) normotensive to mildly hypertensive untreated subjects in good health. After controlling for age and body weight, we observed a significant relationship between diastolic blood pressure and either 1 h or 2 h plasma insulin (r=0.14, p<0.01, and r=0.12, p<0.005 respectively). When non-obese and obese subjects were examined separately significant relationships were found between 1 h plasma insulin and either systolic (r= 0.16, p < 0.01) or diastolic (r= 0.21, p< 0.001) blood pressure and between 2 h plasma insulin and diastolic blood pressure (r=0A2, p<0.05) in non-obese but not in obese subjects. Accordingly a comparison of sex-, age-, and weight-matched hyperinsulinaemic vs normoinsulinaemic subjects showed that the former had significantly higher values of blood pressure only if not obese. These results demonstrate that plasma insulin is independently correlated with blood pressure and support the hypothesis that insulin is a factor regulating blood pressure in man. 608. The use of cuprophan H D F in immnnoisolated transplantation of allogeneic rat islets T. Zekorn, L. Filip, U. Komp, K. Maurer, R. (3. Bretzel and K. Federlin. Medizinische Klinik III der Universit~t Giessen, FRG

Immunoisolated transplantation (Tx) of islets inside hollow fibres (HF) may avoid rejection. Rat islets were cultured inside HF for 2 weeks. Glucose-stimulation (16.5 mmol/1) was done on day 0 to 14. Following 4 day culture HF were filled with fresh islets and stimulated. Seven hundred BDII-islets were HF-transplanted into

600 A peritoneum or liver of Lewis rats (controls: HF without islets). On day 0 insulin secretion after 90 min was 67% of the same quantity of free islets. After day 2 and 4 in culture the secretion after 90 min was 6% and 4%, respectively, of free floating culture. On day 8 and 14 no insulin secretion could be observed. Inside HF there remained only very few islets. The insulin secretion of fresh islets enclosed in precultured HF showed no significant difference to non-precultured HF. On day 4 both Tx groups were normoglycaemic, controls remained hyperglycaemic. On day 8 and 12 blood glucose levels of liver-Tx were normoglycaemic significantly lower than in peritoneal Tx or controls. Afterwards no differences were observed. Histologically on day 28 only very few islets were demonstrated inside liver-Tx-HF. By use of Cuprophan-HF in vitro and in vivo survival rates of islets were not satisfying. 609. Assessment of D-glucose transport in isolated human adipocytes: which method is most appropriate? S.Zeuzem, C.Rosak, M.Podda, E.Jungmann and K.Sch6ffiing. Centre for Internal Medicine, University Hospital, Frankfurt a.M., FRG Glucose transport into human adipocytes has usually been assessed using non-metabolisable sugar-analogues. Two different methods using the physiological substrate D-glucose have been described. Method A uses U-14C-D-glucose at a concentration of 20 llmol/1 and an incubation time of 20 s. Method B uses a concentration of 0.3 ~tmol/l with an incubation period of 60 rain and is based on the premise that glucose uptake provides a measurement of glucose transport when studies are carried out at very low glucose concentrations. The time course of D-glucose uptake in method A was linear over the initial 30s (0.055pmol/s per 10cm 2 basally and 0.093 pmol/s per 10 cm 2 with maximal insulin stimulation). The basal glucose uptake in method B was linear over the initial 50 rain (0.025 pmol/min per 10 era2), the maximally insulin stimulated uptake only up to 40 rain (0.045 pmol/min per 10 cm2). Taken into account the differences in extracellular glucose concentration and incubation time, the data for the calculated membrane permeability of both methods were equivalent. Half-maximal insulin stimulation occurred at 84.6_+17.6pmol/1 and 85.7+14.3 pmol/1 for method A and B respectively (n= 6). Correcting the incubation time from 60 to 40 rain, method B is suitable for the assessment of insulin sensitivity of the D-glucose uptake in human adipocytes. The method is reliable, requires less cells and 14C-labelled D-glucose. 610. A discrepancy between anti-islet cytotoxicity of spleen cells measured in vitro and survival of islet isografts B.Ziegler, E. K0hler, K.-D. Kohnert, S.Tietz, B. Hehmke and M. Ziegler. Central Institute of Diabetes "Gerhardt Katsch', Karlsburg, G D R Abnormalities in both humoral and cellular immunity have been demonstrated in Type 1 (insulin-dependent) diabetes, but the significance of these phenomena is not dear. However, previous analyses of autoantibodies and cytotoxicity against islet cells detected in vitro have shown conflicting results. In the present study the in vivo relevance of spleen cell anti-islet cytntoxicity measured in vitro was examined by transplantation of syngeneic islets using the recently reported experimental diabetes model. Lewis rats RTI a developed severe hyperglycaemia after three combined treatments in weekly intervals with 0.5 ml complete Freund's adjuvant (CFA), 24 h before 25 mg/kg body weight streptozotocin (SZ) was given. Control rats received CFA or SZ alone remained normoglycaemic. After the first combined treatment with CFA and SZ the pancreatic insulin content was reduced by 76.5% and splenocytes showed a significant cytotoxicity (p< 0.01) against syngeneic islet cells measured y specific 51Cr release of 11.4 + 1.5% (n = 8). Surprisingly, syngeneic islets survived when transplanted at the same time into CFA/SZ-treated rats (n= 10). There was no difference between the insulin content of islet isografts in CFA/SZ-treated and control rats. This finding reveals a possible discrepancy between organ-specific immune reactions measured in vitro and those affecting the B cells in vivo. 611. Detection of ICSA titre in descendants of diabetic patients by indirect immunofluorescence using live rat insulinoma cells M. Ziegler, B. Ziegler, P. Heinke and I. Rjasanowski. Central Institute of Diabetes "Gerhardt Katsch", Karlsburg, G D R

Abstracts Islet cell surface antibodies (ICSA) have been detected with a high prevalence in newly diagnosed patients with Type 1 (insulin-dependent) diabetes using normal rat islet cells or rat insulinoma cells, The prognostic value of ICSA and their pathogenic role is not yet clarified. In the present study the indirect immunofluorescence assay was substantially modified to develop a sensitive assay by detection of ICSA titres. Patients' sera diluted 1 : 20, 1 : 40 to 1 : 320 were incubated with 105 RIN cells overnight at 4~ in 0.2 ml followed by incubation with fluorescent antibodies against human immunoglobulins. The cells were evaluated by fluorescence microscopy by two independent readers. The titre of ICSA was calculated by linear regression analysis. A serum is considered as ICSA-positive when the titre was higher than mean + 3 SD of control group. The interassay variation coefficient amounts to 19.2% (n=20 days). One out of 61 control sera and 21 out of 86 non-diabetic children of diabetic patients were ICSA-positive, the latter with an average ICSA titre of 1: 423 + 97 (1:180 to 1:1700). Using this more sensitive and objective assay for detecting ICSA, studies are now in progress to test the hypothesis that the appearance of ICSA is a prerequisite to develop Type 1 diabetes. 612. Pregnancy and diabetic retinopathy Chr.Zoupas, Ch. Kalaitzidou-Kollia and K.S. Karaiskos. Diabetes Center "Hygeia" General Hospital and "Mitera" Maternity Hospital, Athens, Greece Diabetic retinopathy (DR) is of paramount importance for planning or continuing pregnancy. The aim of our study was to present the effect of pregnancy on DR. A total of 71 Type 1 (insulin-dependent) pregnant women, mean age 28.5+11 years and diabetes duration 11.6+13years (mean_+SEM) under strict metabolic control (32 treated with pumps), were followed throughout pregnancy and two months after delivery. Thirty-two patients (45%) had DR. Nineteen had minimal or background retinopathy (BR) and 13 proliferative retinopathy (PR). Eleven patients had been treated before pregnancy with photocoagulation and 2 with vitrectomy. During gestation 4 of 19 patients (21%) with minimal or BR and 3 of 13 (23%) with treated PR, deteriorated their eye disease. In one patient with PR progression of retinal disease was arrested with photocoagulation during pregnancy. Two patients with BR who deteriorated during pregnancy, regressed after delivery. A positive correlation was found between progressive PR and diabetes duration, but not with age and blood glucose control. Our results demonstrate that photocoagulation before pregnancy may protect against rapidly progressive PR. Deterioration is observed in about 20% of women with BR but regression is common after delivery. Therefore with appropriate management PR or BR are not contraindications to pregnancy. 613. Content, distribution and biosynthesis of fl-endorphin in islets of Langerhans of Wistar rats H. Ziihlke, G. Jess, T. Bauch, T. Rosolski and H. Hahn von Dorsche. Department of Biochemistry, University of Greifswald, G D R In vivo experiments show that fl-endorphin--like peptides (E) play a role in carbohydrate metabolism. Moreover, E are located and synthesised in islets of Langerhans. The aim of our investigations was to establish what kind of islet cells contain E, 'what is the content of E in islets, and whether the E biosynthesis is regulated by glucose. For the localisation of E in islets the immunohistochemical double labelling technique for pancreas slices was used. Islets were isolated from the pancreas of Wistar rats by collagenase digestion. The content of E in the islets was measured by a radioimmunoassay. Two hundred islets were incubated for 5 h KRB buffer containing 3HLeuzin (spec. activity: 1.8 TBq/mmol) and different concentrations of glucose (1.5, 6.0, 15.0 mmol/1 respectively). After homogenisation of the islets E were bound to endorphin antiserum from rabbits and identified by gel chromatography. E are localised in A as well as in 0 D cells. The content is 9.1-15pg/islet, VC=30V0, (adult) and 0.5-5 pg/islet (neonatal). By gel chromatography three 3H-labelled peaks (high MW proteins, fl-lipotropin and fl-endorphin) were obtained. A rise in glucose concentration diminishes the E. In conclusion, fl-endorphin is synthesised via high molecular weight proteins reacting with endorphin antibodies; glucose diminishes the rate of synthesis.