Diabetologia 1 1 , 3 2 9 - 3 8 5 (1975) 9 by Springer-Verlag 1975

Eleventh Annual Meeting of the European Association for the Study of Diabetes Munich, F. R. G., September 4 - 6, 1975

Abstracts 1. Diabetogenic and Insulin-Like Actions of Growth Hormone in Man. U. Adamson, E. Cerasi and J. Wahren. Department of Endocrinology, Karolinska Hospital, S-104 01 Stockholm, and Department of Clinical Physiology, Serafimer Hospital, S-112 83 Stockholm, Sweden. The acute effects of human growth hormone (GH) on glucose homeostasis were investigated in normal man. GH was given i.v. after an overnight fast (40 ~tg/kg/30 min). One hour after GH, the insulin responses to i.v. glucose, glucagon and tolbutamide, respectively, were measured. In separate experiments, the effect of GH infusion on splanchnic glucose output (GO) was investigated by the liver vein catheterization technique. - GH reduced the basal insulin level by 10-20%. The insulin response to glucose, glucagon and tolbutamide were all inhibited by about 40%. When high doses of glucose were used, GH-induced inhibition of insulin response was partly overcome. - Basal blood glucose levels were slightly but significantly decreased by GH, while glucose tolerance was diminished. The basal GO was reduced by 40% after GH. The effect of a small glucose infusion (2 mg/kg/min) on GO was not modified by GH. - It is suggested that the initial effect of GH consists of a) a diabetogenic one, mainly mediated by suppression of insulin secretion, and b) an insulin-like, mainly due to suppression of GO. 2. Glucose Phosphorylation and Insulin Release of the Pancreatic islets from Newborn Rats. A. ~gren, S.E. Brolin and C. Hellerstr6m. Histological Department, University of Uppsala, Uppsala, Sweden. The fetal B-cell is characterized by a deficient insulin release in response to glucose. Little is, however, known about the molecular mechanism involved in the transition from a glucose refractive to a glucose sensitive state. In the present study the significance of the glucose phosphorylation for the maturation of the B-cell has been evaluated by measurements of the glucose phosphorylation rate and the stimulation of the insulin release by D-glyceraldehyde, which enters the glycolytic pathway well below the glucose phosphorylation step. - The rate of glucose phosphorylation in islet specimens of 1-day-old rats remained unchanged at glucose concentrations of 0.5 mM and 100 raM, whereas a significant increase was observed at the higher glucose concentration in 6 day-old and in adult rats. These findings suggest that phosphorylating enzymes with different K m values for glucose might be present in the glucose sensitive islets of the two older animal groups. - Incubation of isolated islets from 1-day-old and adult rats with D-glucose at 3.3 mM and 16.7 mM confirmed previous observations that islets isolated from newborn animals do not increase their insulin release in response to a raised extracellular glucose concentration. By contrast, in corresponding experiments 10 mM D-glyceraldehyde was found to significantly stimulate insulin release. This stimulating action of D-glyceraldehyde was potentiated by addition of 10 mM theophylline to the incubation medium. All together the results are compatible with the idea that the glucose phosphorylation of the B-cell is of significance for its functional maturation. 3. Ultrastructural Cytochemistry of Islet Cells. Amherdt M., Rufener C., and F. Malaisse-Lagae. Institute of Histology and Embryology, University of Geneva Medical School, Geneva, Switzerland. Ultrastructural localization of different phosphatases was

performed in rat isolated islets and monolayer culture (ML) of neonatal rat pancreas. Concanavalin A (ConA) binding sites were also investigated using a peroxidase labelling technique. - In all islet cell types, perinuclear and rough endoplasmic reticulum cisternae contained reaction product revealing the presence of glucose-6-phosphatase. No labelling, however, occurred in ML cultures. Inosine-diphosphatase and thiamine pyrophosphatase reaction product was present in the cisternae and associated vesicles of the concave (mature) face of the Golgi complexin every cell type of both ML cultures and isolated islets. Acid ph0sphatase was also found in the Golgi complex of all cells, and in secretorygranules of B-cells only. Using Ernst's technique, adenosine triphosphatase was detected on the plasma membraneof pancreatic endocrine cells. Plasma membrane of all endocrine cells contained ConA binding sites. Intracellular ConA-binding sites were restricted to the secretory granules in ML cultures, but included all above-mentioned membrane-bound compartments in the isolated islets. These findings suggest: 1) an association of each type of phosphatase to a specific membrane compartment; 2) a broad distribution of a-methyl-glucoside and a-mannoside groups revealed by ConA binding. 4. HL-A Factors in Insulin Dependent Diabetes Meliitus: Clinical Significance. O. Andersen, L. Vejtorp, M. Christy, P. Platz, A. Svejgaard, M. Thomsen, P. Reersted and J. Nerup. Med. Dpt. E. Frederiksberg Hospital, Med. Dpt. T. Bispebjerg Hospital, Steno Memorial Hospital, Tissue Typing Lab. Rigshospitalet, Dpt. of Ophthalmology and Med. Dpt. F. Gentofte Hospital, Copenhagen, Denmark. Recently we described the association between HL-A 8 and W15 and insulin-dependent diabetes mellitus (Lancet 2: 864, 1974). The clinical significance of this association is unknown so far, but preliminary studies have shown: 1. The relative risk (RR) of insulindependent diabetes for carriers of HL-A 8 and W15 is 2.3 and 2.2 respectively. RR of maturity (non-insulin-dependent) diabetes was not increased. 2. RRs for HL-A 8 and W15 heterozygotes and homozygotes were identical, i. e. no "gene dose effect" exists. 3. RR of insulin-dependent diabetes for HL-A 8 + W15 carriers was the sum of the individual RRs of HL-A 8 and W 15.4. The prevalence of diabetes mellitus among siblings of diabetics with HL-A 8, W15 is 10, 11 and 20 per cent, respectively, corresponding to the calculated RRs. 5. Early insulin response (IVGTT) in first degree relatives (siblings) of HL-A 8 and W15 positive diabetics was decreased in non-diabetic HL-A 8 and W15 carriers. 6. Proliferative retinopathy was predominantly found in HL-A 8 and W15 positive diabetics. Thus the HL-A factors seem to be of some significance for the development and course of the disease. 5. Tissue Culture of Isolated Pancreatic Islets: Long-Term Effects of L-leucine on the Specific Functions of the Pancreatic B-Cells. A. Andersson, J. H6iriis-Nielsen and H. Borg. Dept of Histology, University of Uppsala, Uppsala, Sweden and the Research Laboratory, Steno Memorial Hospital, Gentofte, Denmark. Previous studies have shown that isolated pancreatic islets maintained in tissue culture for one week at a non-stimulating glucose concentration are characterized by an impaired insulin release and biosynthesis as well as glucose metabolism. In the present study we investigated whether a high concentration of L-leucine in the culture medium could maintain the specific functions of the B-cell despite a low extracellular glucose concentration. - Pancreatic

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islets isolated from normal mice were kept in tissue culture for one week at a glucose concentration of 0.6 mg/ml in the presence or absence of 15 mM L-leucine. The functional characteristics of the cultured islets were tested in short-term experiments at the end of the culture period. - Electron micrographs of leucine stimulated B-cells revealed morphological signs of a high biosynthetic and secretory activity. Furthermore the accumulation of insulin in the culture medium was increased and in acute experiments the rate of insulin secretion in response to glucose and leucine was enhanced. It was also observed that insulin biosynthesis as well as glucose metabolism proceeded at rates similar to those for islets cultured at a stimulating glucose concentration. - The present results suggest that L-leucine may substitute for glucose as a long-term stimulus of the B-cells in order to preserve their specific functions. 6. Effects of N-acetyl-D-glucosamine (NAG) and Related Compounds on the Biosynthesis and Secretion of Insulin. S.J.H. Ashcroft, J.R. Crossley, P.C. Crossley. Department of Biochemistry, University of Bristol, U.K. The aim of the study was to investigate the mechanism of action of NAG on the rat 13-cellglucoreceptor. - Insulin releasestudieswere performed in vitro using batch incubation and perifusion of islets, and in vivo by intravenous administration to conscious rats. Pro)insulin biosynthesis was determined by affinity chromatography of extracts of islets incubated with [3H]-leucine. Islet metabolism of NAG was studied using [14C] NAG and by assay of islet NAG kinase. - Insulin release in vitro: stimulation by NAG .and related sugars required the presence of a substimulatory concentration of glucose, and was abolished by mannoheptulose. - Insulin release in vivo: NAG was a potent stimulator; N-dichloracetylglucosamine was even more effective. Glucosamine derivatives with a longer acyl chain were less effective. NAG-stimulated release was abolished by mannoheptulose. - Insulin biosynthesis." NAG was a more effective stimulator than equimolar glucose. This response did not require the presence of glucose and was not abolished by mannoheptulose. - N A G metabolism: islets oxidized [14C] NAG and possessed a specific NAG kinase. - NAG 'stimulates insulin biosynthesis but insulin release does not occur unless glucose is also present. Mannoheptulose abolishes the effect of NAG on release but not on biosynthesis. These results indicate differences in signal reception and/or transduction for insulin biosynthesis and release. 7. Arginine-lnduced Variations of Hormones and Fuels in Diabetics; Effect of Somatostatin. R. Assan*, A. Basdevant*, Ch. Heuclin*, A. Selmi*, J. R. Girard**. * Dept. of Diabetology, H6tel-Dieu, Paris, France. ** Faculty of Science, Paris, France. A synthetic linear somatostatin (SRIF) efficient on rat glucagon and growth hormone (GH) secretions in vitro, was infused for 75 min at increasing dosage (7.5, 20, 50 ~xg/min) to diabetic patients deprived of insulin for 15 hours. Arginine (25 g) was infused alone ("controls") and in presence of SRIF. - The arginine-induced GH release (controls: 6.5 + 2.0 ng/ml) was blunted by SRIF 7.5 ~tg/min and higher doses. Glucagon (IRG) release (controls: 1150 + 400 pg/ml) was reduced by 60%, 80% and 95 % respectively in presence of SRIF 7.5, 20, 50 txg/min. Cortisol response was unaffected. Blood alanine decreased in controls ( - 90 + 36 ~tM/60 min) and this fall was reduced by 32%, 73% and 90% respectively in presence of SRIF at 3 doses. The arginine-induced blood glucose (BG) rise (+ 130 + 19 mg/100 ml/60 min) was not significantly inhibited (+ 94 + 56 mg/100 ml/60 min) with SRIF 50 ltg/min. In depancreatectomized patients, arginine induced a rise in BG (+ 200 rag/100 ml/60 min) and a fall in alanine ( - 500 gM/60 rain); IRG (K30 antiserum) was present in plasma and weakly increased by arginine (+ 25 pg/ml at 60 min). SRIF inhibited the IRG rise and the alanine fall and reduced by 75 % the BG rise. - Conclusion: 1/ The arginine-induced decrease in alanine may reflect a glucagonstimulated enhancement of gluconeogenesis, since it disappeared when IRG release was blocked. 2 / I n the absence of exogenous insulin, SRIF infusion did not decrease BG level in diabetics. 3/ Blockade of IRG release required higher doses of SRIF than GH inhibition.

8. In Vitro Effect of Insulin upon Hepatic Lipogenesis of Normal (C57BL/6J) Mice. F. Assimacopoulos-Jeannet, B. Jeanrenaud. Laboratoires de Recherches M6dicales Avenue de la Roseraie 64 1211 Geneva, 4, Switzerland. The purpose of this study was to investigate whether insulin added in vitro influences lipid metabolism of perfused livers. - Livers from normal mice were perfused in situ with a Krebs-Ringer bicarbonate buffer containing bovine albumin, serum and erythrocytes. - Insulin (500 ~tU/ml) added to perfusate failed to modify hepatic lipogenesis. It was hypothesized that normal in vivo insulin levels might suffice to stimulate lipogenesis of subsequently perfused livers. Mice were therefore treated with anti-insulin (AIS) serum 2 hrs prior to perfusion. Such treatment increased blood glucose and FFA, without altering hepatic glycogen or cyclic AMP. It reduced basal lipogenesis of subsequently perfused livers. Moreover, the addition of insulin (500 ~tU/ml) to perfused livers from AIS-treated mice resulted in a two-fold stimulation of lipogenesis but only in the presence of substrates such as glucose or acetate. Stimulatory effect of insulin upon lipogenesis was already observed at 50 ~tU/ml. The decrease in lipogenesis brought about by addition of oleate was not alleviated by superimposed addition of insulin. It is concluded that insulin stimulates substrate dependent hepatic lipogenesis in vitro, a stimulation that does not appear to be related to changes in fatty acyl-CoA but to some effect at or beyond the acetyl CoA-carboxylase step. 9. The Influence of 1-Butylbignanide Hydrochloride (Silubin retard) and 1-Phenylethylbiguanide Hydrochloride (Phenformin) on the Influenza Epidemic in 1971 among Diabetics Treated with Oral Antidiabetic Agents. S.A. Babinski, M.D. Department of Internal Medicine, Clinical Teaching Hospital, al. Kogciuszki 52/9 90-428 07 Ldd~, Poland. Observation of the degree of morbidity of influenza among diabetics treated with derivatives of bignanide. - During the 1971 epidemic of influenza 110 patients with diabetes treated with derivatives of biguanide (group A) and 79 diabetics treated with insulin or sulfonylurea derivatives (group B) were observed. - The incidence of influenza was significantly lower in group A than in group B. - The reports of antiviral activity of bignanide derivatives are also confirmed by a lower number of complications after influenza in group A compared with group B. Biguanides seem to play a significant role in the treatment of viral infections among diabetics. 10. D-glucose Uptake by Rat Liver Plasma Membranes. W. Bachmann, D. Challoner. Indiana University, Indianapolis, USA and Diabetes Research Unit, Munich, FRG. The exact mechanism of glucose transport across the plasma membrane in liver is not established. The uptake of glucose by the plasma membrane can be regarded as part of this mechanism. Rat liver plasma membranes were isolated by a modification of the method of Neville. Purity of membranes was checked by marker enzymes and electron microscopy. Uptake studies were performed by a Millipore filtration technique in order to separate the membrane bound from the unbound 3H-D-glucose. - The uptake process has the following characteristics: 1. Rapid, temperature dependent uptake. 2. Linearity of uptake up to a glucose concentration of 500 mM as well as linear dependency on membrane protein. 3. Rapid release of membrane bound glucose, indicating reversibility. 4. Uptake is blocked by phloretin, HgC12 and N-ethylmaleimide. 5. Evidence for a counterflow phenomenon. 6. Sonication of membranes diminishes drastically the uptake-capacity and leads to a release of membrane protein with a higher specific uptake as compared to the intact membrane. - Liver plasma membranes exhibit a combination of transport characteristics typical for simple diffusion and facilitated transport. 11. lmmunoreactive Insulin in the Pancreatic Juice and Bile of Rats. C.J. Bailey, P.R. Flatt, T.W. Atkins, L.C. Best and A.J. Matty. Department of Biological Sciences, University of Aston in Birmingham, Birmingham B4 7ET, U.K.

Abstracts Immunoreactive insulin (IRI) was estimated in pancreatic juice and hepatic bile collected from fasted anaesthetised and conscious rats. Insulin recovery studies indicated no distortion of IRI determinations in these fluids at 4 ~ C over the range of concentrations encountered. - In anaesthetised animals IRI levels in pancreatic juice and bile were slightly higher than in arterial blood and rose significantly during an i.v. infusion of either glucose or insulin, but did not exceed the levels in hepatic portal blood adjacent to the liver. - In conscious animals IRI levels in pancreatic juice and bile were marginally higher than in anaesthetised animals at the same time of day. However, the flow rate and therefore the quantity of IRI in pancreatic juice were significantly raised in the conscious animals, whereas the flow rate and quantity of IRI in bile were not significantly altered. - The IRI levels in pancreatic juice and bile showed a circadian pattern similar to the IRI levels in peripheral blood, with maximum levels during the early dark phase (21.00 to 24.00). The quantities of IRI entering the duodenum in the pancreatic juice and bile during 24 hrs in fasting conscious rats were 1614 + 337 ~tU/Kg body weight respectively.

12. Evidence for the Existence of an Entero-Hypothalamo-lnsular Axis. Bajaj, J.S., Chhina, G.S., Mohankumar, V., Garg, S.K., Baldev Singh. All-India Institute of Medical Sciences, New Delhi, India. Maintenance of glucose homeostasis is under the influence of medially located satiety (VMN) and laterally situated feeding areas (LH) of the hypothalamus. The present study aimed at elucidating the mechanisms which regulate the activity of VMN and LH. - In acute experiments, using chloralose anaesthetised cats, evoked responses (ER) from VMN and LH were recorded on stimulation of mesenteric nerves. Insulin administration produced alterations in E R from VMN and LH; initial increase in amplitude followed by a decrease was elicited from VMN while an increase in amplitude, preceded by an initial short lasting inhibition, was recorded from LH. - In chronic experiments in conscious male rhesus monkeys, electrical stimulation of VMN and LH through stereotactically implanted electrodes resulted in significant alterations in peripheral immunoreactive insulin (IRI) content; a significant decrease in IRI at 30, 150 and 240 min followed VMN stimulation while a marked increase in IRI occurred 30 min following LH stimulation. - The data suggest the existence of an enterohypothalamo-insular axis wherein insulin modifies the activity of VMN and LH as evidenced by alterations in ER on stimulation of mesenteric nerves, while changes in serum IRI follow stimulation of VMN and LH. This axis may have a key role in the regulation of food intake. 13. Isotopic Studies in the Ketosis of Fasting and Diabetes in Man. E.O. Balasse, M.A. Neef. Metabolic Unit and Laboratory of Experimental Medicine, University of Brussels, Brussels, Belgium. Earlier studies from our group have shown that in dogs, diabetic ketosis is due to a combination of overproduction and underutilization of ketones. In order to test whether this applies to human ketosis, the rate of transport of total ketone bodies (TKB) was measured in 20 obese subjects fasted 1-23 days and in 8 insulindependent diabetic subjects deprived of insulin for at least 3 days. Overnight fasted normal subjects infused with variable amounts of exogenous ketones served as controls. The tracer used w a s 3 - 1 4 C acetoacetate given as a primed constant i.v. infusion. - After the onset of the fast, ketonemia and ketogenesis rose progressively with time, attaining a steady level of respectively 7.2 + 0.4 ~tmol/ml and 1.87 + 0.08 mmol/min after 3 days. Corresponding rates of uptake of ketones by tissues (production minus urinary loss) averaged 1.72 + 0.08 mmol/min. Ketone concentration was exponentially related to the rate of production and to the rate of uptake. In the 8 diabetic subjects tested, ketonemia ranged between 1.7 and 17.7 ~tmol/ml and ketogenesis between 0.8 and 3.8 mmol/min. The relationships between ketone concentration on one hand and ketone production or utilization on the other hand, were similar to those observed in starved subjects. The maximal capacity of tissues to utilize ketones was around 2.5 mmol/min for both groups, whereas control subjects could utilize as much as 3.5 to 4.0 mmoles/min. - In conclusion, kinetics of TKB appear to follow a similar pattern in fasting and in

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diabetic ketosis. In severe diabetic ketosis, ketogenesis largely exceeds maximal utilization rate of ketones. A decreased ability of peripheral tissues to utilize ketones can be demonstrated in both fasting and diabetic ketosis.

14. Glucose Metabolism of Isolated Human Fat Cells. I. Bal~si, M. Vars~inyi-Nagy and D. Szy. Third Department of Medicine and Department of Radiology, Semmelweis University of Medicine, Budapest, Hungary. The insulin insensitivity of obese patients should be connected with augmented body fat tissue mass. Experiments were done to determine the effect of insulin on the glucose metabolism of isolated human fat cells. - Subcutaneous fat tissue of 30 patients was obtained by surgery. Fat cells were isolated with collagenase by the method of Rodbell. Fat cell suspension was incubated 2 hrs at 37 ~ C with 100 ~tU/ml porcine insulin. Incorporation of glucose-U-14C was measured into CO2, triglycerides and fatty acids of triglycerides. - The diameter and trigiyceride content of fat cells from patients yeith different nutritional status gave positive correlations with ponderal index and body fat mass. No correlation could be demonstrated with age. - The basal and insulin stimulated incorporation of the glucose-14C-activity into CO 2 and triglycerides gave a negative correlation with the degree of obesity. - The obesity of adults is related to the enlargement of fat cells containing more depot-fat. The glucose metabolic activity of adipocytes from obese persons was decreased and insulin resulted in less augmentation of glucose assimilation. This phenomenon is connected with the limited capacity of fat cells to store triglycerides. 15. The Prognostic Importance of Fnndus Angiography in Diabetic Children. L. Barta, G. Brooser and M. Molnfir. First Department of Paediatrics, Semmelweis University Medical School; Sections of Ophthalmology, Postgraduate Medical School, Budapest, Hungary. The aim of the study was to establish the importance of the fluorescein angiography findings for the development and prognosis of angiopathy in ophthalmoscopically negative cases. - Fluorescein fundus angiography has been carried out yearly and ophthalmoscopy every 6 months throughout 4 years in 140 children, 10-16 years of age. - In chemical diabetes and at the onset of manifest diabetes the fluorescein angiographic finding was sometimes positive. In the first 5 years after the manifestation of diabetes the fluorescein angiographic finding was positive in 50% of those cases where ophthalmoscopy was negative, while 6-9 years after the onset of diabetes ophthalmoscopy proved to be positive in 6 cases out of 20 patients, who displayed microaneurysms at the first fluorescein angiography. In contrast, out of 35 fluorescein anglographically negative patients there was not a single ophthalmoscopically positive one. - 29 patients, 14-20 years of age, developed an ophthalmoscopically positive retinopathy. Thus, the prognosis proved to be significantly better in those patients whose fluorescein angiographic finding was negative when their disease had become manifest. - Fluorescein angiography thus offers a possibility of defining the risk of retinopathy and thus of organizing the careful control of such patients at risk. 16. Partial Purification and Characterization of Active "Enteroglucagon" from Porcine Jejuno-l]eum. Dominique Bataille, G. Rosselin, P. Freychet, and V. Mutt. Unit6 INSERM U.55, H6pital Saint-Antoine, 75571 Paris-Cedex 12, France and GIH laboratory, Karolinska Institutet, Stockholm 60, Sweden. The low molecular weight glucagon-like activity, or small "enteroglucagon" (EG), extracted from porcine jejuno-ileum, was partially purified on the basis of 3 criteria: 1) ability to react in radioreceptorassay (RRA) for pancreatic glucagon (PG) in liver membranes (LM); 2) ability to stimulate adenylate cyclase (SAC) in LM; and 3) glucagon-like immunoreactivity (GLI). The concentration of E G in these extracts as measured by R R A was found to be 36 times higher than in similar extracts from duodenum. On gel filtration, E G was eluted as a single peak later than PG and vasoactive

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Abstracts

intestinal peptide (VIP). Running this peak on cation exchangers resulted, very reproducibly, in two sharp peaks of activity as measured by RRA, SAC or GLI. The two peaks behave similarly in their immunological features: the activity of both peaks on a nonspecific anti-PG antiserum is 100 times higher than on the PG-specific Unger's 30K. However, on the basis of its GLI content, referred to as 100%, the second peak was much more effective on RRA (25%) and SAC (20%) than the first one (1% and 0.5% respectively) and then must be considered as the active form of EG. The pH i of this second peak was found to be over 9.5 by isoelectrofocussing. - We conclude that the bioactive form of EG is a peptide which is smaller and more basic than PG, is mainly present in the lower part of the small intestine and is capable of activating the LM-AC probably through the receptor for PG.

17. Characterization of the Residual Beta-Cell Function in Diabetics by a New C-Peptide Radioimmunoassay. W. Beischer, S. Raptis, L. Keller, E. Heinze, K.E. Schr6der, E.F. Pfeiffer. Department of Endocrinology and Metabolism, Center of Internal Medicine and Pediatrics, University of Ulm, FRG. The suitability of C-peptide in serum as an indicator for beta-cell function was examined. In regard to a possible oral treatment of diabetics receiving insulin we were specially interested in serum C-peptide levels following intravenous administration of glibenclamide-glucose (25 ~g-0.33 g/kg). - A radioimmunoassay for human C-peptide was developed. Its sensitivity is 0.3 ng C-peptide/ml serum; human proinsulin is the only substance known to cross react. - In fasting serum of normal subjects molar C-peptide concentration was about fivefold higher than insulin. Insulin and C-peptide showed similar patterns on stimulation. - In serum of 30 diabetic children C-peptide was not measurable or subnormal. However, in 6 diabetic children with clinical signs of remission normal to raised levels were found. Arginine was a preferential stimulus to glucose during the remission period. Fasting serum C-peptide values from 30 diabetics of the adult onset type were grouped into subnormal, normal and raised levels. According to the extent of C-peptide stimulation following glibenclamide-glucose the groups were divided further. These data were considered together with the corresponding blood sugar patterns and with the clinical responses to oral treatment with glibenclamide. - In addition to insulin, C-peptide proves to be a reliable indicator for beta-cell function, being of special interest in diabetes. C-peptide determinations were shown to be superior to any other method for predicting the success of oral therapy in previously insulin-treated diabetics. 18. Effect of Diabetes, Obesity and Ageing on Glycolytic and Lipogenic Enzymes of Adipose Tissue in Man. F. Belfiore, V. Borzi, A.M. Rabuazzo and E. Napoli. I Clinica Medica, University of Catania Medical School, Ospedale Garibaldi, 95123 Catania, Italy. The activity of several glycolytic and lipogenie enzymes was assayed, by spectrophotometric methods, in homogenates of adipose tissue from normal, diabetic, obese and aged subjects. - In the diabetics there was a decreased activity, both on a protein and on a wet-weight basis, of hexokinase (especially type II) and pentose cycle dehydrogenases, as well as of pyruvate kinase. This could be connected with the defective glucose utilization by adipose tissue in diabetes. Besides the above-mentioned dehydrogenases, NADP-malate dehydrogenase was also diminished. The reduction of these NADPH-forming enzymes would suggest a depressed lipogenesis. Obese diabetics, however, had a glucose-6-phosphate dehydrogenase activity 4.55-fold higher than nonobese diabetics. - In obese subjects with moderately enlarged fat cells, the activity of hexokinase and 6-phosphofructokinase was increased on a protein basis, thus suggesting augmented capacity to metabolize glucose and then to generate alpha-glycerophosphate. The key lipogenic enzyme ATP citrate-lyase was also elevated, which is consistent with the occurrence of enhanced fatty acid synthesis. In subjects over sixty there was a decrease in the activity of hexokinase (type II), 6-phosphofruetokinase and glucose-6-

phosphate dehydrogenase. These changes, that resemble those found in diabetics, would imply that in the aged there is a reduced capacity to utilize glucose.

19. Cheiroarthropathy of Juvenile Diabetes. A. Benedetti, G. Cattano, S. Macor, C. Noacco. Diabetes Unit, Udine General Hospital, Udine, Italy. A peculiar involvement of interphalangeal joints of both hands with palmar flexion of the fingers has been observed in 9 insulin-treated, nonrheumatoid, juvenile diabetic patients (5 males, 4 females) aged 13 to 35 (mean age 20yrs). - The onset of diabetes occurred between 1 and 12 years of age. Painless deformities of the fingers with progressive stiffness and impaired extension started 4 to 10 years after the beginning of insulin therapy. Only one patient complained of pain and periarticular swelling. X-ray and circulatory changes were absent or minimal. - Prepubertal patients showed delayed puberty and stunted growth (4 below the 3rd percentile, 1 below the 10th percentile. Adult patients had normal sexual development and a short stature. - Electromyography showed minor abnormalities of the motor units, normal or subnormal motor nerve conduction velocity, increased median terminal latency, in the absence of muscular atrophy or thickening of palmar tendons. The sense of vibration was impaired in one subject. - The articular changes, reported also by Rosenbloom and Frias (Clin Res 22:92A, 1974), are distinct from previously described forms of "diabetic hand" as atrophic neuropathic osteoarthropathy, Dupuytren's contracture, carpal tunnel syndrome. Juvenile cheiroarthropathy is associated with a) early onset, b) poor control of diabetes, c) stunted growth, d) hepatomegaly, e) delayed puberty, and f) longstanding administration of insulin. 20. Automatic Continuous Recording of Metabolic Parameters in the Treatment of Diabetic Coma. G. Berg, D. Sailer, R. Kellner. Forschungsabteilung f/Jr Ern~ihrung und Stoffwechselkrankheiten an der Medizinischen Klinik der Universitgt Erlangen-Nfirnberg, Erlangen, FRG. 1 ml/h of extracorporeally heparinised whole blood was taken by means of a cannula and passed across ion selective electrodes to obtain the pCO 2 and pH values. Subsequently, glucose was determined by means of an autoanalyzer. - The voltage on the electrodes and the photometer was measured by a digital voltmeter and delivered to a computer. With a special programme, the data were plotted continuously. - The purpose of recording these data was to provide additional guidelines for infusion therapy. The system was applied, for instance, in the control and therapy of acidotic metabolic situations in diabetic coma. In this way the critical metabolic situation could be equilibrated within a few hours. The data will be completed by the determination of serum electrolytes, especially potassium. We are planning further the complete automatic control of infusion therapy based on the obtained values and trend analyses. Electrolytes will be infused according to the electrolyte balance which is generally calculated for four hours, taking into account the infusion and the amount excreted in the urine. 21. Suppressibility of Serum Insulin in Patients with lnsulinomaby Somatostatin (S. S.), Diazoxide, and Diphenylhydantoin (DPH). M. Berger, P. Berchtold, W. Wiegelmann, H. Drost, H. Zimmermann. 2nd Medical Department and Diabetes Research Institute, University of Dfisseldoff, FRG. In 3 patients insulinomas were diagnosed measuring blood glucose and serum insulin during fasting tests (blood sugar mg/di) / serum insulin ~tU/ml ratio < 2.5). The suppressibility of serum insulin was investigated using 3 pharmacological inhibitors of insulin secretion: 1) S.S. given as an i.v. bolus (250 ~g) followed by an infusion of 750 ~tg over 3 hours; 2) diazoxide, infusion of 600 mg over 1 hour; 3) DPt-I peroral 600 mg/day for 5 days, comparison of insulin secretion after an oral 100 g glucose load before and after DPH treatment (DPH-test). - Blood glucose and serum insulin were determined; in fasting and S. S.-tests glucagon, gastrin, and growth hormone were assayed additionally. The diagnosis of insulinoma was histologically confirmed after operation. - Results:

Abstracts

Pat.

Suppression of serum insulin by Histology of insulinoma S.S. diazoxide DPH

A B C

total 0 0

total 0 0

total partial 0

adenoma, isomorph, cells adenoma, polymorph, cells carcinoma

Basal glucagon levels (measured using K 30 antibody) in all 3 patients were above 200 pg/ml; S.S. suppressed glucagon in all cases. - The results demonstrate differences in suppressibility of insulin secretion in patients with insulinomas which reflect differences in the histology of the tumors. This preliminary study suggests the possible use of various insulin suppression tests in the differentiation of insulinomas. 22. Hypoglycemic Effect of Proguanil (N1-Parachlorophenyl , N sIsopropylbignanide). W. Berger, S. Aner, H. Gfschke. Abteilung fiir Diabetologie, Medizinische Universit~itsklinik, Kantonsspital Basel, Switzerland. Previous studies from this laboratory and from others have suggested that inhibition of intestinal glucose absorption is a contributory factor in the hypoglycemic effect of biguanides. This study was designed to investigate whether Proguanil, a biguanide used for Malaria treatment, has a hypoglycemic effect and an inhibitory action on glucose absorption. - Two hours prior to oral glucose tolerance tests 8 nondiabetics and 7 diabetics received a single dose of Proguanil (300 mg orally) or Placebo, each subject serving as his own control. Furthermore, 7 patients in poor diabetic control on Sulfonylureas (SU) alone were given a combination of SU + Proguanil for 3 weeks. - Proguanil administration did not affect blood glucose and plasma insulin responses to oral glucose in diabetics and nondiabetics. However, a significant improvement of diabetic control was achieved in patients previously on SU alone when Proguanil was added: on SU alone mean blood glucose levels at 8, 10, 11, 12, 14, 15 and 16 h were: 205,290, 273,239,261,236,222 rag/100 ml. On SU + Proguanil corresponding values were: 161, 230, 210, 176, 195, 179, 163 mg/100 ml. When Proguanil was stopped, blood glucose increased to pretreatment (SU alone) levels. It is concluded that Proguanil is a potent antidiabetic drug and that inhibition of glucose absorption is not a prerequisite of the hypoglycemic action of biguanides. 23. The Effect of D-Glucose on Phospholipid Biosynthesis in the Pancreatic B-Cells. C. Berne. Department of Histology, University of Uppsala, Uppsala, Sweden. Phospholipids are important constituents of all cellular membranes. The turnover of membranes in the pancreatic B-cells is closely linked to the synthesis, storage and release of insulin. To study the regulation of B-cell membrane turnover, the biosynthesis of phospholipids was examined in islets exposed to a glucose stimulus. - In the presence of phospholipid precursors (~4C-labelled D-glucose and palmitate; 3H-labelled L-serine, choline, ethanolamine and myo-inositol) islets collagenase-isolated from obob-mice were incubated at low (3.5 mM) and high (17.0 mM) glucose. After extraction of the lipids and separation by TLC the incorporation of labelled precursors into different phospholipids was analyzed. - Sixty to 80% of the radioactivity from glucose and palmitate was recovered in the phospholipid fraction and 10-30% in the triglyeerides. While high glucose caused a two-fold increase in the triglyceride incorporation a five-fold increase was found in the phospholipids. Hydrolysis of the islet lipid extracts revealed that 95% of the radioactivity was confined to the glycerol moiety. A profound effect of glucose on phospholipid biosynthesis was also found in all phospholipids irrespective of whether serine, choline, ethapolamine or inositol was the precursor. Generally, omission of . . . . Ca 2 " 7 " failed to .mhlNt the stimulant effect of glucose on this process. It is concluded that the effects of glucose on the B-celts include a stimulant action on the biosynthesis of membrane phospholipids. 24. Withdrawn

333

25. Studies on the Mechanism of Action of Streptozotocin. L. C. Best, T. W. Atkins, C.J. Bailey and A. J. Matty. Department of Biological Sciences, University of Aston in Birmingham, Gosta Green, Birmingham B4 7ET, U.K. The diabetogenic effect of streptozotocin has been attributed to a cytotoxic action on the pancreatic [3cell. To further investigate this action, the influence of streptozotocin on basal, glucose-induced and hormone-induced insulin secretion was examined using perifused mouse islets. - Mice were treated with streptozotocin (200 mg/kg i. p) and hyperglycaemia was confirmed four days later. - Perifused islets exhibited reduced basal insulin secretion in the presence of 3 mM glucose and failed to respond to 25 mM glucose. However, secretin (0.2 clinical units/ml) and pancreozymin (1.5 Ivy dog units/ml) produced a small transient rise in insulin secretion and ghicagon (5.0 gg/ml) produced a protracted stimulatory effect. Islets isolated from untreated mice and perifused with streptozotocin (1.0 mg/ml) also exhibited reduced basal and glucoseinduced insulin release, but the stimulatory action of glucagon was still observed. - Pretreatment of the islets with streptozotocin (1.0 mg/ml) for five minutes inhibited the effects of a subsequent glucose challenge. - These data confirm a direct action of streptozotocin on the pancreatic flcelland implicate an influence at the cell membrane level. 26. Diurnal Variations in Plasma Prolactin, Growth Hormone, and Blood Glucose in Labile Diabetes Mellitus. C. Binder, J. Drejer, C. Hagen, C. Henriksen, H. Kehlet and L. M. Nielsen. Hvidore Hospital, DK-2930 Klampenborg, Denmark, and Department of Reproductive Physiology, St. Bartholomews Hospital, London ECI, U.K. We have studied prolactin levels in labile diabetes without ketonuria, and the possible relationships between prolactin, growth hormone, cortisol, and blood glucose during 24-hour periods. The hormones were determined by competitive protein binding techniques and blood glucose by a glucose oxidase method. Six male, insulin dependent patients were studied. They all showed fluctuations of blood glucose levels, but none had ketonuria at the time of investigation. Their ages were: 16-38 (mean: 28) years, the duration of disease: 4-13 (mean: 7) years, and the insulin doses: 24-56 (mean: 39) units. - Prolactin levels varied within the normal range, with a slighter than usual increase at night. Growth hormone pattern showed, in three cases, the known abnormalities, with great fluctuations, uncorrelated with changes in blood glucose concentration. Cortisol showed a normal rhythm. Blood glucose concentrations fluctuated greatly, but the variations could not be related to changes in the plasma hormone levels, neither could a relationship be demonstrated between the concentrations of prolactin, growth hormone or cortisol. - Thus changes in blood glucose concentration were not a direct determinant of changes in prolactin, growth hormone or cortisol concentrations in plasma. 27. Register of Newly Diagnosed Diabetic Children in Great Britain and Ireland. A. Bloom, D.R. Gamble, T.M. Hayes. British Diabetic Association. School of Medicine, Heath Park, Cardiff GF4 4XN, U.K. The study was designed to furnish epidemiological data and to provide a cohort fo r prospective studies. - We regularly circularised all physicians and paediatricians in Great Britain and Ireland. Notification forms were supplied and a fee paid on completion. - Over 2500 newly diagnosed children under the age of 16 were notified in the first two completed years (November 1972 to end of October, 1974). Incidence was 7.67 per 100,000 children. 11% gave history of a first degree relative with diabetes. - Geographical incidence varied in urban and country areas, and in each year, probably partly dependent on degree of ascertainment. Age incidence was bimodal, with peaks at about 5 and 11 years of age, falling off sharply after age 12. - Marked seasonal variation occurred in older children with more than twice as many cases occurring in the winter as in the summer, replicated in each completed year. In children under the age of 5, no seasonal variation was apparent in either year. - In four instances, diabetes appeared in more than one sibling within a few weeks of each other.

334

Abstracts

28. GIP in Diabetes. S.R. Bloom. The Royal Postgraduate Medical School, Hammersmith Hospital, London and Radcliffe Infirmary, Oxford, U.K. Gastric inhibitory peptide (GIP) is an intestinal hormone with potent insulin releasing properties and may be the main component of the entero-insular axis. It has been reported as raised in diabetics. - Plasma levels of GIP have therefore been measured by a specific radioimmunoassay in 11 diet treated maturity onset diabetics and 18 normal controls after stimulation by oral glucose and also by a standard meal. The basal plasma GIP level was 59 + 12 (SEM) pg/ml in the diabetics and 65 + 11 in controls and the peak after oral glucose was 156 + 28 and 150 + 20, and after the meal 173 + 27 and 206 + 21 pg/ml, respectively, which was not significantly different. - There was a very good correlation between the integrated GIP release after oral glucose and the standard meal (r = .82, n = 29) but there was no correlation between GIP and insulin release (r = .23, n = 49). Integrated GIP release was significantly greater in 5 obese diabetics than in 6 normal weight diabetics (oral glucose 1341 + 148 v 800 + 147, meal 1581 + 107v962 + 174), but no difference was seen between obese and normal weight nondiabetic controls. Thus GIP abnormalities can be demonstrated in diabetics only on analysis of separate subgroups. The significance of this and the correlation with other gut hormones will be discussed. 29. Further Characterization of the Insulin Secretory Defect in Genetically Diabetic Mice. L. Boquist, B. Hellman, J. Sehlin, I.-B. Tfiljedal. Departments of Pathology and Histology, University of Ume~, S-901 87 Ume~, Sweden. The aim was to elucidate further the insulin secretory defect which occurs in the C57BL/KsJ strain of mice carrying the mutated gene db. For comparative purposes, the effects of the diabetes mutation were also studied in mice with the genetic background C57BL/6J. - Structurally the B-cells of diabetic KsJ-mice showed degenerative and atrophic alterations, whereas those of the diabetic 6J-mice exhibited hypertrophy and hyperplasia. - The islets of the diabetic Ks J-mice showed an impaired release of insulin in response to glucose, iodoacetamide, chloromercuribenzene-p-sulphonic acid, methylxanthine, and the ionophore X-537A. The content of insulin in these islets was decreased, but the remaining amount was larger than that released from stimulated islets of normal controls. The diabetic 6J-mice were hyperinsulinemic and moderately hyperglycemic and possessed islets which responded to glucose, iodoacetamide, chloromercuribenzene-p-sulphonic acid and methylxanthine. In the presence of 0 or 20 mM glucose, the islets of diabetic KsJ-mice and 6j-mice exhibited the same concentration of cAMP. However, in the presence of 20 mM glucose, 1 mM 3-isobutyl-1-methylxanthine provoked a smaller increase of cAMP in the islets of diabetic KsJ-mice than in those of diabetic 6J-mice. At the same glucose concentration, the lanthanum method for measuring intracellular uptake of 45CaZ+ revealed a slower uptake of the isotope in the islets of diabetic KsJ-mice than in those of normal KsJ controls (P < 0.05). Neither in the islets of KsJ-mice nor in those of 6J-mice had the diabetes mutation any demonstrable effect on the rate of 3H20formation from (5-3H) glucose. 30. Investigations on Glucose Turnover by 3H-2-C-Glucose during Treatment with Prednisolone and Biguanides. P. Bottermann, U. Schweigart, R. Ermler. II. Medizinische Klinik der Technischen Universit/it Munich, FRG. The purpose of our investigations was to determine whether the decreased glucose tolerance during treatment with prednisolone could be prevented by biguanides. In 7 healthy volunteers the k-value decreased after treatment with three times daily prednisolone (25 mg i.v.) from 1.17 + 0.09 (I) to 0.98 + 0.08 (II). After two weeks with daily treatment of buformin 300 mg the k-value improved to 1.74 + 0.21 (III). After combination with prednisolone the k-value became 1.08 + 0.14 (IV). This shows that prednisolone decreases the glucose-tolerance significantly (I : II = p < 0.01) and biguanides improve it (I:III = p w 0.001). The glucose tolerance test impaired by prednisolone was improved but

not normalised (II:IV n.s.) by biguanides. In each i.v. glucose tolerance test (0.33 g/kg) a tracer dose of 3H-2-C-glucose was injected simultaneously. From the slope of the concentration of 3H-2-C--Klucose the k*-value was calculated. Since there is no recyclingof 3H-2-C-glucose it is a very suitable tracer. The k*-value is caused by glucose assimilation alone; the k-value is the result of assimilation and gluconeogenesis. - Therefore k*-value was higher than the corresponding k-value. The difference between kii and k* H was higher than between kHi and k'Hi; this means that gluconeogenesis is increased and glucose utilisation decreased by prednisolone. On the other hand glucose utilisation is improved and gluconeogenesis is decreased by biguanides. Similar results were found in 7 volunteers under basal metabolic conditions. The glucose pool and glucose turnover-rate was calculated. During treatment with prednisolone blood glucose, serum-insulin, glucose pool and glucose turn-over-rate increased significantly. Biguanides caused only an increase in glucose turnover-rate; blood glucose and serum insulin remained constant. 31. Effects of Fasting on Insulin Release, Cyclic AMP and Glucose Oxidation in Isolated Islets of Langerhans. P.R. Bouman, W. Konijnendijk and G.H.J. Wolters. Endocrine Research Unit, Department of Pharmacology, University of Groningen, The Netherlands. Fasting is known to inhibit the insulinogenic effect of glucose. The possible role of islet cAMP and glucose oxidation was investigated in this study. - Pancreatic islets of fed, 24 h fasted and 72 h fasted rats were incubated for varying periods of time at different levels of glucose. Islet cAMP and insulin release were measured by radioimmunoassay. Overall glucose oxidation was estimated by measuring the cumulative 14-CO 2 production from D[U-14C]glucose during the initial 30 or 60 rain of glucose stimulation. - In fed rat islets glucose 15 mM induced a 3-fold rise of cAMP at 15 min. Both this effect and the effect on insulin release were inhibited by over 50% after 24 h of fasting, but glucose oxidation was not affected. The inhibition of insulin secretion was most evident during the initial 30 min of incubation. Fasting for 72 h further increased the inhibitory effect on insulin release and islet cAMP in addition to causing a 20-25% inhibition of glucose oxidation at glucose 7.5 and 15 mM. Fasting did not affect the initial cAMP content nor basal insulin release at glucose 2.5 raM. - These results indicate that the reduced insulinogenic effect of glucose during short-term fasting is due to inhibition of a glucose-induced rise of islet cAMP, additional depression of glucose oxidation occurring at a later stage. 32. Effect of Prednisolone on Glucagon Secretion "in Vitro". C. Calle, J. A. Hedo, M. L. ViUanueva, and J. Marco. Clinica Puerta de Hierro, Universidad Aut6noma de Madrid, Madrid, Spain. Previous work from our laboratory has shown that glucocorticoid (g-c) treatment enhances basal and arginine-induced glucagon secretion in man. To gain further insight into the influence of g-c on alpha-ceil function, in the present study we have examined the effect of prednisolone (Prd) on glucagon release in a preparation of mouse pancreatic islets. - The islets were isolated by the collagenase technique, and batches of 10 were incubated for 60 min in 2 ml of Krebs-Henseleit buffer containing glucose (60 mg%), albumin (0.2%) and Trasylol (2000 U) (basal medium). Arginine (7.5 mM) was used as glucagon secretagogue. - Pretreatment of the donors with Prd (0.2-0.3 mg daily, for four days, dissolved in normally-consumed drinking water) induced an increase in both basal (from 796 pg + 46, SEM, to 1005 pg + 75; P = 0.019) and arginine-provoked (from 1236 pg + 61 to 1500 pg + 119; P = 0.05) glucagon release (N = 80). The glucagon content of the islets, estimated after their disruption by sonication, was not modified by the treatment (28.06 + 1.19 vs. 28.68 + 1.13 ng/50 islets (N = 58). The addition of Prd (5 • 10-5 mM) to the medium failed to affect basal glucagon secretion significantly. - In good agreement with previous "in vivo" studies, our data indicate that chronic g-c administration augments the secretory activity of the alpha-cell. This does not seem to be a result of increased glucagon synthesis nor a direct effect of g-c on the glucagon-releasing mechanism. Rather, environmental changes induced by these hormones could be responsible for alpha-cell hyper~unction.

Abstracts 33. Nerve Terminal Norepinephrine uptake in Collagenase Isolated, Perifused Rat Islets. L. A. Campfield, L. Girardier. Institut de Biochimie Clinique, Geneva, Switzerland. Ddpartement de Physiologic, Geneva, Switzerland. The role of norepinephrine (NE) uptake by nerve terminals within collagenase isolated rat islets in determining the local B-cell NE concentration was investigated. - Percent decrease of insulin secretion (IS) caused by 2 x 10-8 M norepinephrine (NE) was estimated in the presence and absence of a specific blocker of norepinephrine uptake, 10-6 M desipramine (D). While perifused with KRB containing 0.3% BSA and ascorbic acid (1 mM), islets were exposed first to 5 mM glucose during 45 min, glucose being then increased to 20 mM (G). After 10 min, 4 successive 30 minute experimental periods followed; with exposure to G, G + NE, G + NE + D, G + D. - The percent decrease in IS for NE alone was 27 + 5% (N = 7); for NE + D it was 28 + 4%, using G and G + D as the respective control periods. The mean of the individual differences was 0.9 + 4.8%. - Block of neuronal uptake of NE by D does notresult in a longer inhibition of IS by 2 x 10-8 M NE. Either the uptake process is "non-functional" due to enzyme exposure or the rate of uptake is not quantitatively important. Therefore, nerve terminal NE uptake is not a major determinant of B-cell NE concentration in collagenase isolated, perifused rat islets. 34. The Effects of Physiological Concentrations of Norepinephrine and Epinephrine on Insulin Secretion. L. Arthur Campfield, Albert E. Renold. Institut de Biochimie Clinique, Geneva, Switzerland. The hypothesis that the sympathetic nervous system (SNS) modifies glucose induced insulin secretion (IS) in both stressed and unstressed states was indirectly assessed by defining the effects of plasma concentrations of norepinephrine (NE) and epinephrine (E) reported in resting man (2 • 10-9 M NE and 4 x 10-r~ M E). - Rat islets were perifused with KRB containing 0.3% BSA and ascorbic acid (1 mM). NE or E was added after 40 minutes or when glucose was increased from 5 raM. 2• 1 0 -9 M NE and 4• 10-1~ M E decreased IS at all times with 10, 20, and 40 mM, but not 5 mM G. Mean percent decrease in IS was 21 (NE) and 18 (E) (p < .05). Both shifted the input-output (IS vs [6]) relationship (threshold: 5 mM G; half-maximal: 9 mM; maximal: 20 mM) downward without translation along the horizontal glucose axis. A particular catecholamine concentration caused a constant percent decrease that was independent of [glucose]. Percent IS decrease vs [NE] revealed the following: threshold: 2 x 10-9 M NE; half-maximal: 1.5 x 10-7 M NE; maximal: 2 x 104 M NE. - Physiological concentrations of NE or E decreased IS in a dose-dependent manner that was independent of [glucose]. Higher concentrations, possibly corresponding to increased SNS activity, caused more effective and sensitive decrease in IS. In conclusion, the SNS could indeed modify IS in stressed and unstressed states. 35. Influence of Epinephrine and Insulin on White Adipose Cell Membranes: a Freeze-Fracture Study. J.-L Carpentier, L. Orci. Institute of Histology and Embryology, University of Geneva Medical School, Geneva, Switzerland. Data concerning the influence of hormones on the morphology of the white adipose cell plasma membranes are contradictory. Usin~ the freeze-fracture technique, the invaginations ("pinocytosis") and the protein-containing particles of plasma membranes were quantified morphometricaIly. Epididymal adipose fragments from 6 rats were incubated 45 minutes with or without epinephrine (1 gg/ml) or insulin (1000 ~tU/ml). Results were expressed per ~tm2 of membrane. Epinephrine raised membrane invaglnations from 47.3 _+ 1.0 (control with ascorbic acid) to 52.8 -+ 1.4, (p < 0.02). Insulin also increased membrane invaginations from 50.0 _+ 1.7 (control) to 56.6 _+ 2.5 (p < 0.05). The number of protein-containing particles (A + B faces) rose from 761.7 _+ 52.0 (control) to 914.7 _+ 36.0 (p < 0.05) under the influence of insulin, whereas epinephrine decreased the number of protein-containing particles from 862.2 + 38.5 (control) to 780.8 +- 55.5; this latter difference, however, did not reach statistical significance. Preliminary results, obtained on

335

adipocyte plasma membranes of 3 day fasted rats also showed an augmentation in membrane invaginations. - It is concluded: a) that both epinephrine and insulin increase the membrane invaginations; b) that it is not possible to establish whether this surface activity results from increased endocytosis, exocytosis, or both; c) that insulin increases the protein-containing particles of the plasma membrane. 36. In Vivo Regulation of Adipose Tissue Protein Kinase by Cyclic AMP Mediated Glucagon Stimulation. R.E. Catalan, M.P. Castillon, V.G. Corces and C. Avila. Hospital Clfnico, Departamento de Medicina Nuclear (JEN) and Departamento de Bioquimica, Universidad de Madrid, Madrid-3, Spain. The regulation of protein kinases by hormones has been reported for some tissues but most of the results have been obtained by in vitro experiments. It was the aim of the present work to examine the in vivo regulation of adipose tissue protein kinase by glucagon. Male rats were injected with glucagon with a dose of 0.2 rag/100 g body weight. Protein kinase was assayed by measuring the incorporation of ATP- 32 into histone (Gorin and Goodman, Horm. Metab. Res., 1974, 6, 146) and cyclic AMP by radioimmunoassay. Glucagon produced an increase in cyclic AMP which reached a plateau between 5 and 15 min and a decrease at 30 min. These significant changes were concomitant with similar increments in the protein kinase ratio (Corbin et al., J. Biol. Chem., 1973, 248, 1813). Total protein kinase (measured in presence of cyclic AMP) showed a decrease in specific activity at 5 and 15 rain. - In conclusion, in vivo administration of glucagon causes an increase in the dissociation of protein kinase subunits which is accompanied by elevated cyclic AMP concentrations in the rat adipose tissue. 37. HL - A and Juvenile Diabetes. G. Cathelineau, L. Cathelineau, J. Hors, M. Schmid, J. Dausset. Hopital Saint Louis, 2 Place Alfred Fournier Paris Cedex 10, France. 75 cases (all Caucasian) of insulin-dependent diabetes were tested for 28 HL-A antigens using the microlymphotoxicity method. The increase in frequency of the antigen HL-A 8 was confirmed, and found statistically significant in the patients (55) in whom the onset of the disease occurred before the age of 35 years: 35% vs 11% ina control population of 152 healthy individuals (p: < 0.001; Pc: corrected by multiplying by the number of comparisons. On the other hand, in the 20 cases where diabetes appeared after the age of 35 years, the frequency of HL-A 8 (20%) was not significantly increased. The antigen W15 however, was not found to be increased in comparison with the controls (11% and 12 % respectively). The variation in the results from one population to another are probably due to variations in the linkage disequilibrium (A) between the gene possibly governing susceptibility to diabetes and those at the second HL-A locus and the MLC (mixed lymphocyte culture) locus. 38. The Prevalence of Diabetes Mellitus (DM) and Non-Diabetic Glycosuria (NDG) in a Rural Population in Greece. P.D. Christacopoulos, B.G. Karamanos, C.D. Tountas, D.N. Chaniotis and G. E. Merikas. Diabetic Center, Second Department of Medicine, University of Athens, Ippokration Hospital, Greece. This study aimed to ascertain the prevalence of DM and NDG in a rural Greek population in which genetics and diet differ from those in the rest of Europe. - From 12357 inhabitants (males (m) 5826, females (f) 6531), 11749 (95%) were tested for glycosuria 2 hours after a carbohydrate rich meal. An oral glucose tolerance, test (OGTT) was done in glycosurics without known DM. - There were 153 (89m, 64f) glycosurics, 74 of which (42m, 32f) had abnormal OGTT and 126 (52m, 74f) known diabetics. The number of nondiabetics with glycosuria was 79 (47m, 39f). Since DM prevalence varied with age it is best expressed by age. DM and NDG Prevalence % Age DM Males Females NDG Males Females

0-19 .06 .05 .34 .82

20-39 .46 .39 .50 .39

40-59 1.71 1.28 .94 p <.05 .26

> 60 4.71 5.10 1.65 .55

p <.01

Total 1.71 1.70 .85 p < . 0 5 .53

336

Abstracts

Conclusions: 1) DM prevalence in the total population approximates that repported from other countries. 2) No difference was found between sexes. 3) NDG prevalence in males was higher; it increased with age but only in males.

39. An Improved affinity Chromatographic System for the Purification of Glucagon-Like Immunoreactivity (G.L.I.). J.M. Conlon, R.W.J. Flanagan and R.F. Murphy. Departments of Biochemistry and Medicine, The Queen's University of Belfast, U.K. G.L.I. exists in multiple forms distributed throughout the gastrointestinal tract. Antibodies to glucagon of different specificities, YY89 and YY92, binding C-terminal and N-terminal fragments of glucagon respectively, were coupled to Sepharose-4B and used to purify immunoreactive polypeptides from porcine pancreas, stomach and gut. - Using the YY89 conjugate, material immunometrically and electrophoretically indistinguishable from glucagon was isolated from the pancreas and stomach fundus. Using the YY92 conjugate, polypeptides were isolated from the pancreas, stomach body, jejunum and ileum which showed less than 10% cross-reactivity with YY89 under conditions of radioimmunoassay. The pylorus and duodenum contained only trace amounts of immunoreactive material which was bound by the YY92 but not the YY89 conjugate. Colonic G.L.I. was bound by both conjugates and could be separated by gel-filtration on Sephadex-G50 into a major fraction, M.W. > 30,000 and a minor fraction, M.W. 1500. - An immunoaffinity chromatography system, using immobilised antibodies of known specificities, has been developed which is capable of simultaneously purifying and distinguishing between various forms of G.L.I. in tissue extracts. 40. The Effect of Hydrocortisone Phosphate, Methylprednisolone and Phenytoin on Pancreatic Insulin Release and Hepatic Gintathione - Insulin Transhydrogenase Activity in the Rat. A.G. Cudworth, M. D., M. R. C. P., Harold E. Barber, B. Sc., Ph. D. University of Liverpool, Department of Medicine, U.K. Following intravenous injection of glucose to rats, blood was collected from the carotid artery and the portal vein, and insulin was determined by radioimmunoassay. Pancreatic insulin release and hepatic insulin extraction were increased following the administration of glucocorticoid drugs and reduced following phenytoin. Hepatic glutathione-insulin transhydrogenase activity (GITA) was measured in each animal and found to be increased after glucocorticoid therapy, but unaffected by phenytoin. In alloxandiabetic rats, GITA was significantly increased following treatment with both methylprednisolone and Phenytoin compared with control alloxan-diabetic rats. This is suggestive evidence that both these drugs can initiate an ihcrease in GITA. - It is concluded that the markedly raised GITA in steroid treated non-diabetic rats is the combined result of a drug induced effect plus the major inducing effect of an increased hepatic uptake of insulin on insulin degradation, whereas the main effect of phenytoin is a reduction of pancreatic insulin release. 41. Evidence for HL-A Linked Genes in 'Juvenile' Diabetes Mellitus. A.G. Cudworth, M.D., M.R.C.P., J.C. Woodrow, M.D., F.R.C.P. University of Liverpool, Department of Medicine, U.K. Any enquiry into the basic mechanisms underlying 'juvenile' onset diabetes must take into account the role of genetic determination. There is recent evidence that one of the loci involved is in the HL-A chromosomal'region. In order to throw further light on this, a larger study of 218 patients with diabetes mellitus (150 with an age of onset of 30 years or less and 68 with an age of onset of 31 to 80 years) has been carried out, together with a study of seventeen families containing two or more 'juvenile' diabetic siblings. By HL-A typing the parents it was possible to establish whether identical haplotypes shared by the affected siblings were present on identical chromosomes inherited from one or other parent. - A positive association of HL-A8 and W15 with juvenile onset diabetes is confirmed. The relative risk for juvenile diabetes in HL-A8 positive individuals is 2.17 (p = 0.023) and in W15 positive individuals the relative risk is 2.35 (p = 0.0037). - This study also

demonstrates a striking and significant departure of the pattern of zygotic assortment in respect of the HL-A chromosome in diabetic sibships. The number of those siblings possessing identical haplotypes is markedly increased. - These data are consistent with the presence of a locus, closely linked to the HL-A loci, at which there is a gene or genes having an important effect in producing susceptibility to juvenile onset diabetes.

42. Evidence that the Glucose-Fatty Acid Cycle is Operative in Isolated Skeletal (Soleus) Muscle. G.S. Cuendet, Ernest G. Loten, Albert E. Renold. Institut de Biochimie Clinique and Laboratoire de recherches mrdicales, University of Geneva, 121l Genrve 4, Switzerland. The effect of palmitate, acetoacetate or [5-hydroxybutyrate (/3OH) on glucose metabolism and transport were studied in isolated soleus muscle of fed C 5 7 B 1 - + / + mice. - The muscles were dissected, fixed on holders and incubated (KRB-buffer, 5 mM glucose, 2% defatted albumin). The presence of /3OG resulted in decreased release of lactate and pyruvate (14 and 40% respectively), decreased oxidation of 1~C-glucose (51%), decreased glycolysis (~H incorporation from 5- 3H-glucoseinto 3HzO) from 7.7 to 6.1, while glycogen deposition was increased from 0.67 to 1.60 nmoles/mg ww/hour. Total glucose uptake, i.e. the sum of glycolysis + glycogenesis, remained unaltered. Insulin increased glucose uptake, glyeolysis and glycogenesis by 160, 140 and 420% respectively. In the presence of /3OH, insulin-stimulated glucose uptake and glycolysis were decreased by 35 and 57% respectively, whereas glycogenesis doubled. In the presence of 10 mM glucose, flOH similarly inhibited basal glucose uptake. - 2-deoxyglucose uptake was not inhibited by/3OH. 3-O-methylglucose uptake was inhibited only when glucose was also present in the incubation medium. The effects of fatty acids and of aeetoacetate were comparable to those of /3OH. - These results suggest that ketones and fatty acids decrease (1) the utilisation and (2) the uptake of glucose. (3) this latter inhibition does not result from a direct effect on transport. 43. Infection and Immnnoreactivity to Insulin. A. Czy~yk, H. Rogala, J. Lawecki and A. Symonides-Lawecka. Department III of Internal Diseases, Medical Academy, Warsaw, Poland. The aim of this communication is to present the clinical data indicating that besides the degree of purification of insulin the production of insulin-binding antibodies in man may also depend on other factors. - In two groups of diabetics (12 adults and 10 children) monocomponent insulin (MC) was administered from the discovery of the disease for 1-2 years. Serum insulin-binding antibodies were determined by the method of Christiansen employing immunoelectrophoresis in agar gel, based on binding 1125 ox-insulin by serum IgG. - In most adult diabetics no significant rise in serum levels of insulin-binding IgG was observed. However, a moderate increase was found in three patients with virus hepatitis developing during or just before the onset of treatment with MC-insulin, and in one patient after influenza. The effect of infection was more marked in children: in five of them a rise of insulin-binding antibody level coincident with infection, vaccination or liver disease was noted. These observations suggest that both viral and bacterial infection may induce a change in immunogenicity with respect to pure insulin. However, other factors must also be involved in this phenomenon since in observed children infection was not always followed by an increase in the serum level of insulin-binding IgG. 44. An in Vitro System for Elucidating Mechanisms Responsible for Plasma Membrane: ~ Granule Interaction. Brian Davis, Norman R. Lazarus. Diabetes Research Laboratory, The Wellcome Foundation Limited, Temple Hill, Dartford, Kent, DA1 5AH, U.K. The aim was to develop an in vitro insulin "secreting" system to study t-granule: plasma membrane interactions. - Plasma membranes from cod islets were purified by density gradient centrifugation, fi-granules were isolated from mouse islets. Granules and membranes were incubated in 4 mM tris pH 6.5, 15~ The following were present, MgSO4 (15 mM), KCI

Abstracts (5.5 mM), papaverine (4 mM). Insulin release took place when granules were incubated with membranes in the presence of added Ca 2+ (0.05 ~tM), ATP (5 pM) and glucose. Plasma membranes from pituitary (rat) and liver (mouse and cod) were without effect. L-glucose, galactose, frnctose-6-phosphate, fructose 1,6-diphosphate, glyceraldehyde-3-phosphate, ribose-5-phosphate could not substitute for glucose; UTP, GTP could not replace ATP. Mannose was almost as potent as glucose. Tolbutamide with Ca 2+ and ATP caused insulin release; diazoxide inhibited. Granules without membranes were stable to all stimulators alone or in combination. The in vitro insulin "releasing" system described shows specificity that is compatible with in vivo insulin secretion. Glucose, without having to undergo metabolism, is capable of causing insulin release; metabolites of glucose, specifically glucose-6-phosphate and phosphoenol pyruvate are also capable of inducing release. The above system should be a useful tool in dissecting not only t-granule: plasma membrane interactions, but also those system in which exocytosis is necessary for secretion. 45. Prognosis in Juvenile Diabetes Meilitus. T. Deckert, J.E. Poulsen and M. Larsen. Steno Memorial Hospital, Gentofte, Denmark. In order to study the prognosis of juvenile diabetes all diabetic patients hospitalized at the Steno Memorial Hospital between 1933 and 1973 were examined. 307 patients fulfilled the following criteria: diagnosis made before the age of 30 and diabetes onset before 1933. 5.8% of the patients could not be followed. 88% of the patients were treated with insulin two times a day. - Up to the 1st January 1973 duration of life, duration of diabetes, cause of death and "quality of life" were investigated. Furthermore the influence of frequency of examination, the degree of control, weight, blood pressure and insulin dose/kg bodyweight were examined. - 40% of the patients were still alive, 47% of these were able to work. 16 % of all patients developed blindness, 21% coronary infarction, 10% cerebro-vascular accident. 12% had undergone an amputation, 38% developed proteinuria and 22% uremia. Frequent examination, good degree of control, normal bodyweight and low insulin dose/kg were found to give a highly significant better prognosis than sporadic examinations, bad degree of control over- or underweight and an insulin dose higher than 0.50 U/kg/day. 46. Plasma Levels of Placental Hormones in Diabetic Pregnant Women. R. de Hertogh, K. Thomas, J.J. Hoet and I. Vanderheyden. Unit6 d'Endocrinologie et Nutrition. Cliniques Universitaires Saint-Pierre - Louvain, Belgium. In diabetics functional alterations of the hormonal secretory activity of the foeto-placental unit have been reported, although published results diverge in their conclusions. In the present study, unconjugated estrogens and HCS were measured by radioimmunology in the same plasma samples of twenty well controlled, insulin treated, diabetic pregnant women throughout gestation. A group of 20 normal pregnant women was studied in a similar way and provided the reference values. Plasma levels of unconjugated estrogens and of HCS increased throughout pregnancy in both normals and diabetics, and the general shape of the curves were similar. The main difference was a significantly higher plasma level of estradiol in the diabetics in the second half of gestation. - Estriol levels were not significantly different. - In conclusion, no placental hormonal deficiency could be demonstrated in this group of well controlled insulin treated diabetic pregnant women. Contrarywise, higher estradiol levels could correspond to higher estrogen production by the diabetic placenta. However, the normal increase of estriol suggests a qualitative modification in the estrogen precursors delivered to the placenta in favor of the 16-non-hydroxylated compound. The latter could be of maternal origin (by increased adrenal activity) or of foetal origin (by decreased hepatic 16-hydroxylating activity with or without increased adrenal secretion). Further study is required to explore this alternative. A third possibility would be a change in the peripheral metabolism of estrogens in the diabetic pregnant women.

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47. Bone Mass in Diabetics. I.H de Leeuw, R. Abs. Rucaplein 155 B 2610 Wilrijk Belgium, Middelheimhospital, University of Antwerp, Belgium. Previous radiological studies suggested that the cortical thickness in the second metacarpal bone was significantly different in some age-and-sex related groups of diabetic patients as compared to matched controls in the non-diabetic population. - In order to verify these results, the Norland-Cameron "bone mineral analyzer" was used. This instrument measures the trabecular and cortical bone mass at the distal end of the radius. - Diabetics were examined after careful subdivision into sex- and age groups. Cortical and particularly trabecular bone mass was found to be lower, as compared to the non-diabetic population, in insulin dependent diabetics in the 20-29 year age group. In contrast, in maturity-onset diabetics the cortical and trabecular bone mass was significantly greater in the 60-69 year age group, in both sexes. These findings confirm the conclusions of the radiological studies. - The reduction in bone mass in juvenile diabetics is most probably the result of chronic keto-acidosis. The higher bone mass in the older, maturity-onset group is not easy to explain. Vascular changes and multiple endocrine influences, must play a role in the genesis of this phenomenon, which seems to protect the diabetic temporarily against the physiological age-related bone loss. 48. Copper and Glucose Metabolism in the Aged. P. de Nicola, G. Casale, E. Ferrero, M.R. Lissandrin. Istituto di Gerontologia e Geriatria, Universith, Pavia, Italy. Copper levels in the blood are supposed to be related to aging on one hand and to lipid and glucose metabolism on the other. - In 30 aged subjects, 18 of whom were diabetic, blood glucose, serum insulin, FFA and copper were studied after a glucose load (0.75 g/kg of body weight). In diabetic patients the insulin levels slowly increased until the 180th minute, FFA remained unaffected and the concentration of serum copper decreased significantly. - In nondiabetic subjects the insulin levels rapidly increased and reached their peak at the 60th and 90th minute, the FFA fell to 50 per cent of basal value at the 2nd and 3rd hour, while the serum copper concentration decreased until the 180th minute. - On the basis of these results it might be assumed that the FFA level in the blood was more closely related to the insulin level than to glucose blood levels. Interestingly enough, copper levels were not elevated in fasting diabetic subjects but were rather high in fasting non-diabetic subjects, while the trend was opposite after loading. 49. Retrospective Study of the Cardiovascular Fate of 190 Diabetics Treated for Five Years or More with Biguanides Alone. M. Derot, M. Lavieuville, F. Isnard. (Statistical analysis by E. Eschwege and L. Papoz of the Inserm statistical Research Unit.) Paris, France. This retrospective survey covers a sample of 190 patients whose histories were secured from 28 French diabetologists. Patients have been treated with Mefformin, as either the sustained release or normal preparation, or Phenformin sustained release, and had not received any other antidiabetic therapy. The mean duration of treatment for the entire sample was 73 months and the range was 5 to 12 years. - Cardiovascular assessment was based on the development of cardiovascular complications specific for diabetes (diabetic retinopathy, glomerulopathy) or alternatively specific vascular change (coronary insufficiency, infarction, cerebral, arteriopathy or vascular disease of the lower limbs). Weight variations and diabetic control were also analysed. The mean loss of weight at the end of the study period was 6 kg and at the most recent check-up, after 5 to 12 years treatment, control of the diabetic condition remained good (blood sugar under 140 mg/I00 ml in 59%) The small number of deaths known to be of cardiovascular origin was under 3%. Analysis of the patients'cardiovascular complications when admitted to the survey showed that during an identical surveillance period those initially affected showed a greater progression than those without disease at the outset (p less than 0,05). The authors will discuss the results found in this retrospective survey,

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50. Competition and Synergism between Calcium and Barium upon Insulin Release. G. Devis, G. Somers, E. van Obberghen and W.J. Malaisse. Laboratories of Experimental Medicine, Brussels Universities, Brussels, Belgium. In order to document the determinant role of divalent cations in insulin release, we have characterized the insulinotropic modality of Ba 2 t in the isolated perfused rat pancreas. The stimulant effect of Ba: + was antagonized by extracellular Ca 2+, so that the i0amediate but rapidly degreasing release of insulin evoked by Ba 2 r in the presence of Ca 2-r became a much higher and more sustained response in Ca2+-depleted media e0riched with EDTA. This paradoxical finding suggests that Ba 2-~ and Ca 2n- might compete for the ~ame carrie(, as also suggested by both the inhibitory effect o f Ca 2"r o n 133Ba2-1- net uptake by isolated islets and the reduction of Ba 2+-induced insulin release by verapamil, an inhibitor of Ca 2+ inw~d transport in the B-cell. - Nevertheless, the process of Ba 2-r-induced secretion remained apparently influenced by intracellular Ca 2-1- since agents thought to provoke Ca 2+ accumulation in a critical site within the B-cell, e. g. glugose and theophylline, poterltiated the insulinotropic effect of Ba 21-. _ The aptitude of Ba 2-1- to stimulate insulin secretion with, under appropriate conditions, a biphasic pattern strongly supports the view that the dynamics of the secretory process are governed by events triggered by divalent cations. Moreover, the present work reveals some properties of the carrier system responsible for the inward transport of these cations in the B-cell. 51. Glucose Production from Working Human Forearm Muscle in Diabetics and in Obese during Complete Starvation. P. Dieterle, I. Bachl, G. Bachl, J. Henner, P. Minkus, D. Frommeld, C. Dietetic. III. Med. Dept., St~idt. Krankenhaus MiinchenNeuperlach D-8 Miinchen 83, Oskar-Maria-Graf-Ring 51, FRG. Muscle Glucose uptake is stimulated by insulin and exercise. It is not known if exercise stimulates glucose uptake in human muscle when insulin is absent. Therefore diabetics and starved obese subjects, known to have an altered insulin secretion, were studied. Studies were undertaken in 3 groups: 15 subjects with various degrees of diabetes (treatment was withheld 24 hours before the experiment), 6 obese subjects after 14 to 21 days of starvation, and 9 controls. - The forearm technique was used in all experiments. The forearm exercise of 20 minutes' duration was performed on a hand ergometer. Before, during and after work blood samples were drawn for analyzing substrates. - In the controls glucose uptake increased 8-fold during exercise. No increase of the mean glucose uptake was observed in the diabetics and in the starved obese subjects. Moreover a net release of glucose during physical work was seen in a high percentage in these two groups. Though work intensity was the same in the various groups studied, the lactate production was statistically significantly lower in the diabetics and the starved obese subjects. - It is suggested that glucose release and diminished lactate production from working muscle might be the result of elevated catecholamines in non treated diabetics and starved subjects. 52. Glucose Production of Skeletal Muscle in Acute Insulin Deficiency Stimulated by lsoproterenul. G. Dietze, M. Wicklmayr and H. Mehnert. 3rd Medical Department, Schwabing City Hospital, Munich, FRG. In spite of the absence of a glucose-6-phosphatase activity in skeletal muscle, production of free glucose could be demonstrated in acute insulin deficiency. This phenomenon is thought to be due to enhanced glycogenolysis. In order to test this hypothesis, metabolic balances of human forearm were calculated from arterio-deep venous substrate differences and muscle perfusion rates during intrabrachial isoproterenol infusion (0.4 ~g/min.) in 9 diabetic and 7 healthy subjects. In acute insulin deficiency, during catecholamine infusion glucose production of the forearm increased from - 0.84 + 0.10 to - 2.35 _ 0.11 ~moles/100 g x min. (p < 0.05), but lactate output did not rise significantly ( - 0.51 + 0.13 to - 0.73 + 0.17, p < 0.1). Under normal conditions glucose utilization diminished from 0.95 + 0.04 to 0.71 + 0.1 Itmoles/100 g • min. (p

< 0.05) and lactate production showed a 3-fold rise ( - 0.71 + 0.11 to 2.1 + 0.2 lxmoles/100 g x rain., p < 0.05). These data suggest that, in acute insulin deficiency, glucose production of skeletal muscle is not due exclusively to enhanced glycogenolysis, but also to limited activity of the glycolytic pathway.

53. Inhibitory Effect of Plasma Triglycerides on Oxygen Release from Blood in Patients with Hyperlipoproteinaemias. J. Ditzel, M.D. Section of Endocrinologyl Department of Medicine and Clinical Chemistry, Aalborg Regional Hospital, Aalborg, Denmark. In a study of oxygen affinity of whole blood, a newly diagnosed diabetic with lactescent plasma was found to exhibit an oxyhaemoglobin dissociation curve (ODC) markedly shifted to the left (P-50 act.pH 21 mm Hg vs. normal 26 mm Hg) despite normal concentrations of haemoglobin and red cell 2,3-diphosphoglycerate. With insulin the plasma cleared with gradual normalization of the position of the ODC. For this reason the ODC and related arameters were determined in 36 cases of hyperlipoproteinaemia HLP). A significant inverse correlation was found between the concentration of plasma triglycerides and the P-50 act.pH. Type I and familiar and acquired type V HLP showed the lowest P-50 (18-22 mm Hg). This suggests that particularly the chylom~cron fraction interferes with normal oxygen transport. It is likely that in hyperlipoproteinaemias, lipoproteins form a diffusion barrier for tissue oxygen delivery at the microcirculatory level. This mechanism may explain the clinical observation that postabsorptive lipidaemia can adversely affect patients with coronary heart disease with angina pectoris.

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54. Glucagon in Relationship to the Type of Insulin Secretion in Diabetics Using the Successive Glucose-Tolbutamide-Test. H. Drost, D. Grtineklee, F.A. Gries. Diabetes Research Institute, University of Diisseldorf, FRG. Weinges (1972) described different types of insulin secretion using the successive glucose tolbutamide test. We investigated the role of glucagon secretion in this test system. Subjects were divided in four groups based on their type of insulin secretion: juvenile type diabetics (group 1), normal weight maturity onset diabetics with delayed insulin release after glucose (group I1), obese maturity onset diabetics with hyperinsulinemia (group II1) and healthy controls (group IV). The fasting subjects were given glucose 100 g p. o. and after 180 minutes tolbutamide 1 g i.v. Blood glucose, serum insulin and plasma glucagon (using antiserum K30) were determined. - Results." Fasting levels of glucagon were in group I (128 + 29 pg/ml), group I1 (173 _ 34 pg/ml), group 11I (237 _+ 51 pg/ml) and in the controls (115 + 19 pg/ml). The glucagon levels in group 1I and IlI were significantly different from those in group 1V (p 0,005). After glucose load glucagon significantly decreased in group II (108 + 30 pg/ml) and group IV (59 + 10 pg/ml) whereas glucagon levels in group 1 and III remained unchanged. After tolbutamide there was a slight rise in glucagon levels in group I and II1 whereas in group 11 (201 + 45 pg/ml) and group IV (132 + 3l pg/ml) the increase was statistically significant. Glucagon response to tolbutamide seems to be not a specific effect of sulfonylureas but merely due to the induced hypoglycemia. - Conclusions: In juvenile type diabetics and obese maturity onset diabetics fasting glucagon levels were significantly higher than in normal subjects. After stimulation with glucose and tolbutamide glucagon levels did not change in these types of diabetics. Maturity onset diabetics with normal weight, delayed insulin response to glucose and immediate, insulin response to tolbutamide exhibited glucagon levels" as in healthy subjects. 55. Steady-State Plasma Levels of Glibornuride during Longterm Oral Administration in Man. U.C. Dubach, A. Korn, J. Raaflaub. Medizinische Universit~itsPoliklinik, Basel, 1. Medizinische Universitiitsklinik, Wien, F. Hoffmann-La Roche & Co., Limited Company, Basel, Department of Experimental Medicine, Switzerland. The multiple dose pharmacokinetics of the sulfonylurea derivative glibornuride (GLUTRIL) were examined in 7 diabetic subjects

Abstracts treated with the drug over a period of 4 weeks (25 mg every twelve hours). - Glibornuride in plasma was determined by differential pulse polarography. - The steady-state levels of glibornuride (maximum and minimum concentrations) were determined experimentally and by calculation (using the pharmacokinetic parameters obtained following oral administration of 50 mg GLUTRIL on the 1st day of application). There was good agreement between experimental and calculated values. - The results show a regular pharmacokinetic behaviour of glibornuride. There was neither unexpected accumulation of the drug nor diminution of the steady-state levels as might occur e. g. as consequence of induction of microsomal enzymes. 56. Ultrastructural Changes in the Renal Tubule of the Diabetic Mouse (KK Strain). R. du Boistesselin, J. Duhault and O. Racadot. Laboratory of Histology, CHU Piti6-Salp6tri~re, 105 boulevard de l'H6pital 75634 Paris Cedex 13, France. Nodular and diffuse types of advanced glomerulosclerosis have been previously reported by ourselves and others. However, changes in tubules have been considered as not significant. - The present study points out the ultrastructural changes occurring in the proximal convoluted tubule of the diabetic KK mouse. - M e t h o d s : Light microscopy. Trichrome stain and P.A.S. - Electron microscopy:glutaraldehyde OSO4 fixation, Epon embedding. 10 KK mice and 10 CD mice as controls. Both groups were 14 months old. - Results: We observed: - An increase of the thickness of the basal lamina in the glomerulus and in the proximal convoluted tubules. - Several ultrastructural lesions: a) lysosomes increase in size and in number, filling up the tubular cells. These lysosomes present a myelin-like aspect and sometimes crystals were observed. b) Reduplication of the mitochondrial membrane appears. Some mitochondria contain dense granules, c) Expelling of tubular cells into the lumen. - Conclusions: These observations suggest that diabetic nephropathy depends on the development of several individual lesions. Each of them may be influenced by various factors such as a metabolic disturbance. However, these different lesions are closely interrelated. Some relationships are proposed with ultrastructural alterations in the pituitary of diabetic subjects. 57. Egfect of Somatostatin on Insulin Biosynthesis. S. D. Garcia, G. Rosselin. Unit6 INSERM U.55, H6pital SaintAntoine, 184 Rue, du Faubourg Saint-Antoine, Paris 12 ~ France. Somatostatin is known as an inhibitor of growth hormone, TSH, glucagon and insulin release. However no data are reported for its effect on hormonal biosynthesis. The effect of somatostatin on glucose induced insulin biosynthesis was measured by the incorporation of 3H-leucine into immunoreactive insulin (IRI) of newborn rat isolated pancreas. The incorporation of tritiated leucine increases linearly with time at the rate of 3.8 (r: 0.99, p < 0.001) femtomoles of 3H-leucine per minute in 10 ~tgof IRI, that is in one explant. Optimal doses of somatostatin (5 ~tg/ml) reduce the rate of tritiated leucine incorporation by half in presence of the same concentration of glucose (11 mM). This inhibitory effect of somatostatin on insulin biosynthesis appears at 5.5 mM glucose and is not modified by further increase in glucose concentrations up to 27.5 mM. Theophylline or dibutyryl cyclic AMP (db cyclic AMP) which, in this system, at 10 mM concentration, strongly increase insulin biosynthesis, did not reverse the somatostatin induced inhibition of insulin biosynthesis. A 10 ~g/ml concentration of somatostatin gives the maximum blockade, 50% of the fallin insulin biosynthesis being obtained with dose below 0.5 ~tg/ml; similar dose responses are observed for the blockade by somatostatin of the insulin biosynthesis induced by theophylline or db cyclic AMP. Somatostatin also inhibits insulin biosynthesis in isolated islets from newborn rat pancreas; this finding suggests that inhibition of insulin biosynthesis is not mediated by exocrine pancreas but is related to a direct interaction of the pepfide with the endocrine tissue. The impairment by somatostatin of the insulin biosynthesis induced by glucose as well as by cyclic AMP suggests that this peptide inhibits the biosynthesis of insulin and insulin release in the same way.

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58. lmmunohistochemical Evidence for the Presence of Somatostatin-Like lmmunoreactivity in the A1-Cells of Rat Pancreas. S. Efendi6, T. H6kfelt, C. Hellerstr6m, O. Johansson, R. Luft and A. Arimura. Department of Endocrinology and Metabolism, Karolinska Hospital, S-104 01 Stockholm 60, Sweden. In the present study the occurrence of somatostatin or a somatostatin-like peptide in the islets of the rat was investigated. Cryostat sections of formalin fixed pancreas were incubated with antibodies to somatostafin and glucagon followed by FITC conjugated antibodies, according to the indirect technique of Coons et al. Silver staining with the method of Hellman & Hellerstr6m was performed on the same sections. - Somatostafin positive cells were found in the periphery of islets. These cells were all argyrophil and constitute the so called Al-cells. The glucagon positive cells were localized mostly directly beneath the somatostatin containing cells, but rarely exhibited argyrophilia. - The present results strongly indicate the presence of somatostatin or a somatostatin-like peptide in the argyrophilic Al-cells. 59. Multifactorial in Vivo Control of NSILA-S in the Rat and in the Dog. J. E. Eigenmann, B. Kammermann, M. Becker and W. Leemann. Metabolic Unit, Department of Medicine, Kantonsspital Ziirich, Departments of Veterinary Medicine and Veterinary Surgery, School of Veterinary Medicine Ziirich, Switzerland. Non suppressible insulin-like activity (NSILA-S) is increased in acromegalics and decreased in pituitary dwarfs. NSILA-S exerts not only insulin-like activity but also growth promoting and sulfation activity. Therefore, we studied NSILA-S levels in different endocrine and metabolic situations. - Dogs were studied after hypophysectomy (hypox) or pancreatectomy, rats during fasting, after induction of streptozotocin-diabetes or after hypox. Sera from these animals were passed over Sephadex G-200 in 1 M acetic acid. This method separates NSILA-S (average MW 7000) from its binding protein and other high molecular weight proteins. After chromatography the fractions between 75 and 120% of total bed volume were pooled, lyophilized and their NSILA-S content was determined in the rat fat pad assay. - In pancreatectomized and in hypophysectomized dogs NSILA-S dropped to about 20-30% of the initial levels. Hypox rats exhibited similarly low levels of NSILA-S. In pancreatectomized dogs NSILA-S rose slowly back to initial levels under insulin therapy. In fasted and in streptozotocindiabetic rats NSILA-S levels fell to about 50% of those of control animals. With the exception of hypox animals NSILA-S did not seem to correlate with growth hormone levels. - From these experiments it is concluded that NSILA-S is subject to multifactorial endocrine and metabolic control. Insulin is likely to play a key role in the regulation of NSILA-S. 60. The Effect of Thyroid Status and Insulin Administration on Plasma Cyclic AMP Response to Glucagon. R.S. Elkeles, K. Siddle, J.K. Shetty and J.H. Lazarus. Division of Clinical Investigation, Clinical Research Centre and Northwick Park Hospital, Harrow and Departments of Medicine and Medical Biochemistry, Welsh National School of Medicine, Cardiff, U.K. Intravenous glucagon causes a rise in plasma cyclic AMP (cAMP), which has been shown to be hepatic in origin. In vitro, thyroid hormone increases glucagon stimulated rise in intrahepatic cAMP, while insulin inhibits hepatic glucagon stimulated cAMP production. Studies have been undertaken to ascertain whether thyroid status or insulin administration can modify plasma cAMP response to glucagon in vivo. - Plasma cAMP was measured by radioimmunoassay. Both plasma cAMP and serum insulin response to glucagon 1000 ~tg i.v. were exaggerated in 9 hyperthyroid patients and reduced in 5 hypothyroid patients compared with 10 eutbyroid controls. - In 4 normal subjects glucagon 10, 100 and 1,000 ~tg produced a dose related rise in plasma cAMP. Insulin 6 U i. v. failed to modify the cAMP response to 10 and 100 ~tg glucagon given simultaneously. In normal subjects insulin 6 U i.v., either alone or with glucose, produced no significant reduction in basal plasma cAMP compared with a control saline injection. - Plasma cAMP response to glucagon 1,000 ~tg i.v. was unchanged in 8 insulin

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Abstracts

requiring diabetics before and 1 week after starting treatment with insulin. - Plasma cAMP response to glucagon is considerably modified by thyroid status and may provide a sensitive index of tissue exposure to thyroid hormone. No significant effect of insulin either acutely or in the longer term was demonstrated. 61. Interactions between Insulin and Chemically Modified Insulins at the Cellular Level. M.J. Ellis, P.H. Sfnksen, R. Corkhill. Department of Medicine, St. Thomas's Hospital Medical School, London SE1 7EH, U.K. The interaction between native and chemically modified insulins was investigated to define structural analogues which would potentiate or antagonise the metabolic effect of the hormone. Insulin analogues substituted at the A A 1, B 1 or B29 positions or including a synthetic A1-B29 or A1-B 1 cross-link were examined in an isolated fat cell bioassay system. Potencies relative to insulin varied between 0.2 and 100%. The interactions of insulin with A1, B29 diacetyl insulin (relative potency 30.2%) and with A1-B29 suberoyl insulin (relative potency 5.6%) were studied over a wide range of molar ratios. The results were analysed by two methods. Firstly by construction isobols to demonstrate whether the action of insulin was potentiated or antagonised by the presence of the analogue. Secondly, by fitting the data to two mathematical models; one assuming a constant potency relative to insulin and the other a varying potency according to the relative proportions of insulin and analogue. - The fat cell response to insulin was antagonised bythe presence of A1-B29 suberoyl insulin whereas there was a slight potentiating effect of A1, B29 diacetyl insulin. - Minor alterations to the insulin molecule can result in materials capable of modifying the effect of the native hormone. 62. The Biological Activity on Rat Fat Cells of Insulin from the Atlantic Hagfish (Myxine glutinosa). S.O. Emdin, S. Gammeltoft and J. Gliemann. Institute of Medical Physiology C, University of Copenhagen, Rfidmandsgade 71, 2200 Copenhagen N, Denmark. We have studied insulin from the atlantic Hagfish, which represents the phylogenetically oldest insulin characterized in detail so far, in order to correlate its highly substituted amino-acid sequence with its biological activity on isolated rat fat cells relative to pig insulin. We determined the binding affinity from the competition with ~25I-labelled pig insulin for receptors, the potency from the stimulation of lipogenesis from 3-3Hglucose and the rate of inactivation from the decrease in potency after incubation with fat ceils in a high concentration. The binding affinity was 25% the potency 5% and the rate of inactivation 10% relative to pig insulin. Hagfish and pig insulin exerted additive effects in submaximal doses and identical time-courses of onset and cessation of activation of lipogenesis. The discrepancy between the relative binding affinity and potency is so far unique for hagfish insulin, since chemically modified insulins and insulins from other vertebrates have shown parallel changes of these two parameters. The fact that hagfish insulin is not antagonistic to pig insulin is in agreement with the concept that fat ceils possess an excess of insulin receptors, with respect to activation of lipogenesis. The amino-acid sequence of hagfish insulin gives little basis for the explanation of its altered biological properties as compared with pig insulin. 63. Insulin Secretion in Insulin-Requiring Diabetics before and during Insulin Treatment. B. Enk, T. Deckert. Steno Memorial Hospital, Gentofte, Denmark. Since relatively non-immunogenic insulin preparations became available, measurements of endogenous insulin secretion in insulin requiring diabetics, after stimulation with different secretagogues became possible. - Four groups of non-obese diabetics were investigated. Group I: 14 patients with mild diabetes, not yet insulin requiring, diagnosed before the age of 30. Group II: 19 ketonuric patients just before insulin treatment. Group III: 18 patients during the remission period after average 16.5 months of insulin treatment. Group IV: 12 patients with no remission period or relapse after an average 19.5 months of insulin treatment. Blood-glucose and im-

munoreactive insulin was measured fasting and after i.v. secretin, during i.v. GTT, oral GTT and tolbutamide/glucagon stimulation. - Considerable insulin secretion could be demonstrated in group I, whereas in group II only a very small insulin peak was obtained after Secretin and the combined injection of Glucagon and Tolbutamide. During the remission phase (group III) considerable insulin secretion was demonstrated, whereas in group IV only a very small insulin peak was obtained. - Significant correlation was found between the quality of diabetic regulation and endogenous insulin secretion. 64. Effects of Chlorpromazine (CPZ) on Blood Glucose and Plasma Insulin Levels in Healthy Subjects and in Diabetic Patients. G. Erle, M. Basso, C. Zaccaria, C. De Palo, E. Zago. Divisione di Malattie Metaboliche. Ospedale Civile di Vicenza, Istituto di Semeiotica, Universit/i di Padova, Italy. CPZ inhibits insulin release from isolated pancreatic islets of rats; moreover, in hypoglycemic patients this drug diminished hypoglycemia and decreased plasma insulin levels. In this study the effect of CPZ has been investigated in non-diabetic subjects and in patients with latent diabetes. In normal subjects we have shown that: a) CPZ treatment (75 mg/day for 1 week) is ineffective in decreasing basal insulin levels in plasma and glucose-stimulated insulin response, b) CPZ (50 mg/day for 1 week) did not modify glucose assimilation rate and insulin response to i. v. glucose, c) CPZ infusion (50 mg in 60 rain) slightly increased blood glucose, but did not decrease plasma insulin levels significantly. At termination of CPZ infusion, insulin/glucose ratio rose significantly, d) in patients with latent diabetes IVGTT was performed during CPZ infusion and the results compared with another IVGTT performed previously, Statistical analysis of the data demonstrates that glucosestimulated insulin response is reduced by CPZ perfusion. - These results suggest that oral administration of CPZ at doses shown to be effective in patients with insulinoma, have little or no effect on insulin secretion in normal man. Infusion of CPZ produces slight hyperglycemia which is probably due, at least in part, to inhibition of insulin secretion. - In subjects with latent diabetes CPZ infusion inhibits pancreatic response to i.v. glucose. 65. Plasma C-peptide in Insulin Requiring Diabetes Mellitus: Diurnal Variation and Response to Oral Glucose Load during the First 9 Months of Treatment. O. K. Faber, C. Binder, J. Markussen, K. Naithani & L. G. Heding. Hvidore Hospital, Emiliekildevej 1, DK-2930 Klampenborg, Denmark. Plasma C-peptide concentration during a normal day and in response to an oral glucose load was used as a measure of endogenous insulin secretion. - C-peptide was measured by a radioimmunoassay on unextracted plasma. - The material comprised 17 patients who, at the time of diagnosis fulfilled the following criteria: < 50 years of age, bodyweight < + 10% of ideal weight, blood-glucose concentration _-_. 12,0 mmol/1 and significant ketonuria (> 50 mg/100 ml). - Investigations were carried out after initial metabolic stabilization on insulin, 1 month later, and then every third month. - After initial metabolic stabilization fasting C-peptide concentration averaged 0,32 with a range from 0,21 to 0,50 pmol/ml plasma (normal range 0,26-0,63, mean 0,40). All except one (n = 8) showed a significant increase after food intake, but compared to normal subjects the rise was smaller and less rapid. The individual responses underwent only small changes during the following investigations in the three patients followed so far. - The results from 17 initial studies will be presented together with follow-up studies in 11, 7, and 3 patients after 3, 6, and 9 months duration of disease respectively. - One out of seven patients followed for 6 months showed a complete remission. This could be predicted from the C-peptide response to meals and especially to the oral glucose load. 66. Effects of Glibenclamide on Glucagon and Growth Hormone Secretion in Insulin Treated Diabetic Patients. F. Fallucca, FP. Tinelli, C. Mirabella, G. Menzinger, D. Andreani. Cattedra di Endocrinologia e Medicina Costituzionale. Policlinico Umberto I. Universit~ di Roma, Italy.

Abstracts The absence of definite increments of plasma insulin during long term treatment of diabetes with sulphonylureas indicates that non betacytotropic mechanisms may be involved in the action of these drugs. The authors had observed previously a reduction of arginineinduced secretion of growth-hormone (GH) and glucagon (IRG) after glibenclamide (GB) treatment in maturity-onset diabetes. In order to exclude that these effects were mediated by insulin the authors have studied the effect of GB in a group of insulindependent diabetics (IDD). - Methods: 9 IDD were submitted to an arginine test (ATT) before and after treatment for one month with GB (5 mg/day) Diet and insulin dosage were not changed during this period. - Results: After GB blood glucose values did not change significantly but GH response to arginine was clearly reduced with a significant difference (p < 0.05) between peak values (28,4 + 7,7 --~ 13, ! + 2,4 ng/ml), and arginine-induced IRG secretion was also reduced with significant differences at 45' (271 _+ 23 ~ 173 _+ 23 pg/ml) (p < 0,05) and at 60' (185 + 1 6 ~ 108 + 18 pg/ml) (p < 0,005). A control study two months after suspension of the treatment is in progress. - Conclusion: The reduction in arginine-induced increments of plasma GH and IRG in these patients, who presumably are not capable of secreting insulin, indicates that the GB-induced suppression of GH and IRG secretion is not mediated through a betacytotropic effect. At the moment it is not clear whether the suppression of GH, or of IRG secretion, is primary or if both are independent effects of the drug. 67. The Role of GH and ACTH in Exercise-Induced Fatty Acid Mobilization in Rats. G. Federspil, C. de Palo, N. Sicolo, G. Udeschini, G. Erie, C. Scandellari. Istituto di Semeiotica Medica, Universiffi di Padova. II Cattedra Istituto di Farmacologia, Universitg di Milano, Italy. Prolonged muscular exercise increases plasma FFA and glycerol in rats. In previous researches we have found that, in hypophysectomized rats, plasma FFA did not rise; the present research has been carried out to investigate the role of two pituitary hormones - GH and ACTH - on exercise-induced lipid mobilization. - The experiments performed showed that: a) plasma R-GH decreases sharply in rats submitted to prolonged muscular exercise (forced swim in water at 32-33 ~ C for 60 rain), b) administration of Dexamethasone (400 gg/100 g b.w.) in resting rats did not modify plasma FFA. c) administration of Dexamethasone in doses shown capable of blocking ACTH release (40 and 400 ~tg/100 g b.w.), does not inhibit lipid mobilization, d) Morphine Sulphate, a drug shown to release endogenous ACTH in rats, did not increase plasma FFA levels in resting rats. - These findings suggest that, since muscular exercise has an inhibitory effect on R-GH secretion in rats, this hormone cannot stimulate lipid mobilization during exercise. Moreover our results suggest that, in vivo, release of endogenous ACTH has no appreciable lipolytic effect in rats, and, moreover, that this hormone alone does not play a fundamental role in exercise-stimulated lipid mobilization in rats. - These results support the hypothesis that the pituitary plays a permissive rather than stimulatory role in exercise-induced lipid mobilization. 68. Fetal Blood Glucose and Insulin (IRI) - Response during lv.-Glucose-lnfusion to the Mother During Delivery. A. Feige, W. Kiinzel, H.J. Mitzkat. Universit~its-Frauenklinik Wiirzburg, 87 Wiirzbnrg, Josef-Scbneider-Str. 4, Arbeitsgruppe Diabetologie, Department Inhere Medizin, Medizinische Hochschule Hannover, Pasteurallee 5, FRG. Newborn infants of mothers with diabetes mellitus often demonstrate islet-cell-hypertrophy with hyperinsulinism. These findings are possibly correlated with hyperglycaemia of the mother. Until now data are available for humans only postpartum, whereas during pregnancy animals were chosen. We were interested in a possible stimulation of fetal IRI by intravenous glucose infusion to healthy subjects during delivery. - According to Cerasi and Luft iv.-glucose-infusion was given to 11 subjects during the first stage of labour. Blood samples were taken for determination of fetal glucose, IRI, pH and base excess according to Saling and simultaneously maternal blood samples for glucose and hormone measurements. - During glucose-infusion fetal glucose increased in

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parallel with maternal glucose levels. An increase in maternal IRI occurred whereas fetal IRI concentration was unchanged. During stimulation there was a significant correlation between the fetal insulinogenic index and increasing birth weight. There was no change in maternal HPL-levels during infusion; also pH-and baseexcess were unchanged. - Our results show no alteration of fetal insulin secretion in response to glucose infusion in the mother during delivery. A correlation existed between fetal insulinogenic index and birth weight. 69. Effect of Glucagon on Renin Secretion in Man. A. Fernandez-Cruz (Jr)., R.H. Noth and R. Hendler. Yale University and Complutense University of Madrid, Spain. Glucagon (G) increases renin secretion in the isolated rat kidney, possibly via cyclic-AMP. - (Res. 32: 290, 1973). We have studied the effect of low (0,01 mg) and high (0,2 mg) dose intravenous infusions of (G) in normal supine male subjects. Simultaneous measurements of (G), blood glucose (BG), pulse (PR), blood pressure (BP), dopamine-B-hydroxylase (DBH), cortisol (F), and plasma renin activity (PRA) were obtained over 180 rain. Although low dose (G) produced a rise in BG no response occurred in PRA. After high dose infusion mean PRA varied from a baseline of 0.44 s 0.2 (mean + SEM) ng/ml/hr to 0.37 s 0.1 at 3 min, 0.40 + 0.1 at 5 min, 0.76 + 0.3 at 10 min, 0,67 + 0,3 at 15 min, 0,55 + 0,2 at 30 min, 0,50 _+ 0,2 at 60 min, 0.35 + 0,1 at 120 min. This increase was largely attributable to the response in two subjects and was not statistically significant. After sodium deprivation, reinfusion of 0,2 mg of (G) into two previously unresponsive subjects increased PRA by 66 and 68%. 70. Liver Glycogen in Normal and Diabetic Subjects of Different Weight on Fasting and during Carbohydrate Intake. W. Fill, A. Beringer, S. Afschar. ENT Service I, University of Vienna Medical School, Austria. Determination of assimilation and release of liver glycogen in normal and diabetic subjects. - Quantitative glycogen test by Beringer and Thaler in 700 needle biopsies of the liver. - Substantial differences in glycogen metabolism of normal and diabetic subjects were found - 1. In overweight non-diabetics glycogen was metabolized at a lower rate than in low-weight normal subjects. 2. This was also demonstrable in diabetics on insulin or biguanide, diet and SH therapy. 3. In overweight subjects and decompensated diabetics on different therapeutic regimes, glycogen accumulation after variable glucose doses was reduced. On continued food ingestion, glycogen concentrations did not differ in diabetics and nondiabetics. 4. Endogenous de novo glucose synthesis, measured as excess glycogen storage after small glucose doses in normal-weight healthy fasting subjects, amounted to 135 mg/kg body weight/hour. Only part of it was released into the periphery. There was, however, considerable variation during the day. 5. De novo glycogen synthesis in normal subjects was only insignificantly suppressed by carbohydrate intake. In decompensated diabetics it was less markedly increased than is generally assumed. 71. The Insulin Response in Chemical Gestational Diabetes. P.M. Fisher, H. W. Sutherland, P.D. Bewsher, A. Campbell. Department of Obstetrics and Gynaecology, University of Aberdeen, U.K. Our aim was to examine the hypothesis that chemical gestational diabetes occurs in women with impaired pancreatic beta-cell function in the non-pregnant state. The primary defect in diabetes mellitus is believed to be a selective impairment of B cell glucoreceptor sensitivity and may be identified by the 'low' insulin response to glucose infusion. - 12 untreated chemical gestational diabetic (IVGTF increment index < 2 997 in late pregnancy with > 2 997 after pregnancy) women had 90 minute glucose infusion tests (plasma glucose maintained by infusion at 14 94 - 1 5 . 5 mMOL/L with plasma immunoreactive insulin levels measured at 1 minute intervals for 5, and 5 minute intervals for 85 minutes. A summated plasma insulin response was calculated from the observed biphasic insulin response. Low insulin responders were defined as those in the lowest 15th centile found for control groups of non-diabetic

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pregnant (n-20) and non-pregnant (n-20) women. - Three insulin response groups were defined: 1. 'Low' during pregnancy and postpartum (n-6). 2. 'Low! during pregnancy, 'high' postpartum (n-3). 3. 'High' during pregnancy and postpartum (n-3). - 1. Chemical gestational diabetes is associated with a low insulin response in pregnancy. - 2. Diminished beta-cell gluco-receptor sensitivity was found in 50% of the women in the non-pregnant state. - 3. Chemical gestational diabetes is associated with inadequate insulinogenic compensation for pregnancy.

72. Purification and Characterisation of Plasma Glucagon-Like lmmunoreactivity by Immunoaffinity Chromatography. R.W.J. Flanagan, E.R. Trimble, J.M. Conlon and R.F. Murphy. Departments of Medicine and Biochemistry, The Queen's University of Belfast, and The Metabolic Unit Royal Victoria Hospital, Belfast, U.K. Circulating glucagon and glucagon-like immunoreactivity (GLI) from the pancreas and/or gut is difficult to measure by direct radioimmunoassay (RIA) of plasma. Discrepancies may be due to different specificities of assay antibodies or to artifacts resulting from plasma proteases. In this study immunoaffinity chromatography was used to purify circulating GLI prior to characterisation. Antibodies specific for either the C- and Nterminal regions of glucagon were immobilised on Sepharose-4B and columns of each conjugate linked in series. Following application of plasma from fasting human subjects, and washings to remove non-specifically bound materials, the columns were separated and eluted: fractions containing GLI were then gel-filtered on Sephadex G-50. Species of molecular weights > 30,000, 12,000, 7,000, 3,500 and 2,000 were recovered after purification on the C-terminal reactive affinity column, and all these species reacted with both N-terminal and C-terminal antibodies in the RIA. Species of similar molecular weights were also recovered from the N-terminal reactive affinity column, but these species only reacted with Nterminal antibody in the RIA. In different subjects different profiles were noted in that in some, large molecular species predominated whereas, in others small molecular material was most abundant. Hence, the combination of affinity chromatography and gel-filtration allows the opportunity to study the variety of molecular species of GLI in plasma. 73. Osteoporosis and Hyperostosis in Diabetes Mellitus. S. Forg~ics, L. V~rtes, Radiological Clinic of Med. University and J~inos Hospital Budapest, (1082 Budapest. 1Slll6iut 78), Hungary. The aim of our work was to study the osteoporotic and hyperostotic changes of the bony system in diabetics. - Determination of the metacarpal index was carried out in 428 diabetics and in 646 healthy persons. Roentgenograms of the skull, the spine and the pelvis were evaluated in 500 non-selected diabetic patients above the age 40. According to former observations diabetes is considered predisposing to osteoporosis. In our material comparative regression of the metacarpal indices on age showed that the difference between diabetics and non-diabetics is not significant either in males or in females. Spontaneous vertebral compression has a lower frequency of occurrence in diabetics than an average population of the same age. Thus osteoporosis is not increased in degree or incidence in our diabetic material. This can be explained by the modern therapy of diabetes, as metabolic disturbances leading to demonstrable bone loss do not develop. - Hyperostotic changes were observed in about 25% of diabetics. Its manifestations are: spondylosis hyperostotica, hyperostosis frontalis interna, calcification of the ligaments and hyperostosis of the bones of the pelvis. These hyperostotic changes and diabetes mellitus form a characteristic entity. It is rather rare in juvenile diabetes, but it is frequent in mild maturity onset diabetes. The overproduction of growth hormone might be responsible for the development of hyperostosis. 74. Adenosinetriphosphatase Activities in Homogenates and Subcellular Fractions of Mouse Pancreatic Islets. B. Formby, K. Capito. University of Copenhagen, Dept. of Biochemistry A, Panum Institute, Copenhagen, Denmark.

Membrane bound (Na, K)ATPase and CaATPase are considered to participate in the active transport of Na plus K and Ca, respectively. Mitochondrial MgATPase is supposed to be involved in active transport of protons. Studies on these activities are reported. Attempts to measure (Na, K)ATPase activity in pancreatic 13-cells have hitherto been in vain, but from studies on 2:Na net uptake in microdissected islets (Sehlin and T~iljedal, FEBS letters 39, 209, 1974), the specific activity of a ouabain-sensitive active Na-pump can be calculated to be about 0.06 ~tmoles ATP hydrolysed per hr per mg membrane protein, corresponding to a negligible percentage of the total ATPase activity. Using ('r with high specific activity (20-30 Ci/mmole) as substrate, we have measured (Na, K)ATPase activity, defined as the activity in a medium containing Mg, Na, K minus the activity in a medium with the same ions plus 1.0 mM ouabain, in a microsomal fraction. Pretreatment of the microsomes with 2.5 M urea removed more than 50% MgATPase activity. Specific (Na, K)ATPase activity was 0.11 + 0.05 ~tmoles ATP hydrolysed/mg protein/hr and found to be inhibited by chloromercuribenzene-p-sulfonate, but not by cyclicAMP, Diphenylhydantoin, Ca or Caffeine. - CaATPase activity, as previously reported by us, is found especially in secretory granules and mitochondria. A different inhibition pattern of CaATPase in these two subfractions by La and Gd has been found. Furthermore, preliminary experiments have shown an increased CaATPase activity in homogenates during starvation (48 hr). - Using Naazide and Oligomycin as inhibitors, different inhibition patterns of CaATPase and MgATPase were found, thus suggesting these activities to originate from two different enzymic entities.

75. The Effect of Diabetic Control on Motor Nerve Conduction Velocity. C. Fox, C. Lowy and V. Wright. Department of Medicine, St. Thomas' Hospital Medical School, London SE1, U.K. A colony of diabetic rats has been set up to investigate the factors responsible for long-term diabetic complications. - Eighty Streptozotocin-treated rats have been randomly allocated to one of the following groups: A. Untreated. Normal diet. B. Low carbohydrate diet. C. Insulin plus normal diet. D. Insulin (same dose) plus low CHO diet. - Representative animals from each group have been studied individually in metabolic cages and motor nerve conduction velocity (MNCV) has been measured in the tail nerves of the same animals. - Urinary glucose excretion/rat/24 hrs (mean + SEM) was as follows: Before treatment: 89 mM + 3.3. After 3 month's treatment: A. 94 mM + 6.7. B. 43 m M + 3.9. C. 32 mM + 5.5. D. 5 mM + 1.1. - MNCV after 3 month's treatment: Non-diabetic 44.1 M/see + 1.3. A. 37.6 M/see + 1.3. B. 41.3 M/see + 0.7. C. 42.7 M/see + 1.2. D. 43.1 M/see ___ 1.2. - MNCV was significantly reduced in untreated diabetes as compared with normal (p < .005) and with the three groups of treated diabetes (p < .05). There was a correlation between individual glucose excretion and MNCV (p < .01). - Thus we have shown that a low CHO diet alone causes a marked reduction in glycosuria and results after 3 months suggest that MNCV is related to the degree of diabetic control irrespective of the presence of insulin. 76. Maintenance of Pancreatic t-Cell Function by 1-5 Week Storage in the Cold. B. Frankel. The University of Umeh, Department of Histology, S-901 87 Ume~ 6, Sweden. Improved techniques for storage of isolated pancreatic islets will greatly facilitate accumulation and transportation of islets for use in transplantation and will aid metabolic studies. - Therefore a method was developed for cold storage of hand microdissected ob/ob mouse islets in HEPES-buffered Tissue Culture Medium 199 containing no serum. - Maintenance of normal glucose-stimulated insulin release for 1 week depended on: a low storage temperature (8 ~ or 22 ~ C), a high glucose concentration in the medium (18 mM), and a 4-hr period at 37 ~prior to preincubation and measurement of insulin release during incubation. Loss of islet insulin occurred at 37 ~ but was prevented by storage at 8 ~or 22 ~ However, islet insulin content after storage did not correlate with insulin responses to glucose. Islets stored at 8 ~ with 18 mM glucose for 3 or 5 weeks, or

Abstracts with 3 mM glucose for 1 week, contained the same amounts of insulin as fresh islets but lost the insulin response to glucose. Both insulin content and glucose-stimulated insulin release were largely maintained if the islets were transferred weekly to a 37 ~medium for 7 hr. - Glucose-stimulated insulin release can be well maintained for at least 5 weeks if islets are stored in a high concentration of glucose at 8 ~ with a short interval at 37 ~ every 7 days. 77. Purified Insulins. A Clinical Study on the Occurrence of Insulin Binding Antibodies. I-I. Frerichs, U. Deuticke, H. Drost, G.J. Kremer, O. Jacobi, P. Schmidt, Aa. Volund. Depts. of Internal Medicine, Univ. of G6ttingen and Mainz. Med. Depts. City Hospital MiinchenSchwabing and Wupperta], FRG. NOVO Research Institute, Copenhagen, Denmark. Between April 1970 and September 1973 a total of 61 insulindependent diabetic patients, none of whom had received insulin before, were treated in alternating order either with MC-insulin NOVO (c-component prepared from crystalline porcine insulin by gel chromatography) or with MS-insulin NOVO (severalfold recrystallized porcine insulin). A comparable (control) group comprised 96 diabetic patients treated for periods of up to 36 months with non-purified commercial insulin preparations available in the FRG. - Titres of insulin-binding antibodies were determined in serum by radioimmunoelectrophoresis, anti human-IgG (containing low titres of anti-IgA/anti-IgM) raised in rabbits serving as antibody and porcine 125-I insulin as the antigen. Detection level was 0.05 mU/ml. - Patients of the MC-group (19 female, 14 male; age 17-73 yrs; mean insulin dose 25 U/day) developed insulinbinding AB 3-6 m. after starting insulin. At 3, 6, 9, 12, 18, 24 and 30 m. the mean values were 0.05, 0.15, 0.19, 0.29, 0.43 resp 0.20 mU/ml. (n of patients 33, 31, 31, 25, 19, 15 and 8 resp). - In patients of the MS-group (10 female, 18 male; age 10-73 yrs; mean insulin dose 29 U/day) AB were found even before 3 m. after start of treatment. The mean values at the times indicated above were 0.17, 0.43 +, 0.62 +, 0.45, 0.61, 0.68 resp 0.59 mU/ml. (n of patients 28, 26, 24, 19, 11, 7 or4). (+p < 0.05 compared to MC-group in the Wilcoxon rank test). - Of the 96 patients on commercial insulin (49 female, 47 male; age 5-73 yrs; mean insulin dose 35 U/day) 30 had been on insulin from 1-6 m., 21 from 7-12 m., 27 from 13-24 m. and 18 from 25-36 m. The mean level of AB at 3, 6, 9, 12, 18, 24 or 30 m. was 1.84, 3.22, 5.11, 5.07, 4.22, 2.86 resp 3.30 mU/ml. Porcine monocomponent-insulin is immunogenic in patients never before treated with insulin. However, insulin-binding AB are lower than in patients treated with porcine insulin purified by multiple recrystallization or with insulin purified by usual procedures A. 78. Blood Sugar as a Predictor for Coronary Heart Disease. J.H. Fuller, P. McCartney, L.M. Colwell. Unit for Metabolic Medicine, Guy's Hospital, London, S.E. 1. Dept. of Epidemiology, London School of Hygiene & Tropical Medicine, London, W.C. 1, U.K. Most earlier work on the relationship between glucose intolerance and coronary heart disease (CHD) has been based on prevalence studies. We report a prospective study of the predictive value for CHD death of glucose tolerance measured under standard conditions in 19,000 male London Civil Servants, aged 40 or more, examined in 1967-69 and centrally monitored for mortality ever since. At baseline, capillary blood sugar concentration was recorded two hours after a 50 g oral glucose load as was weight, blood pressure, blood cholesterol, smoking status and electrocardiogram. During subsequent follow-up there have been 780 deaths of which 325 (42%) were attributed to CHD. - The 2 hr blood sugar concentration was significantly correlated with the risk of CHD death, a relationship which is shown to be independent of the other baseline variables measured, by the application of a multiple regression statistical model. The strength of the predictive power of blood sugar has been estimated and the influence upon it of other measured variables has been calculated. It is concluded that measures of glucose tolerance add valuable additional information for the assessment of the future risk of CHD.

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79. The Effect of Nicotinic Acid and of Propranolol on Glucagon and Plasma Cateeholamine Responses to Prolonged Exercise in Man. H, Galbo, J.J. Holst, N.J. Christensen and J. Hilsted. Institute of Medical Physiology B, University of Copenhagen; Department of Gastroenterology A, Bispebjerg Hospital Copenhagen; and Second Clinic of Internal Medicine, Kommunehospitalet, /~rhus, Denmark. The present study evaluates the importance of adrenergic receptor stimulation and substrate metabolism for the exercise induced glucagon secretion in man. Seven healthy subjects ran at 60% of individual maximal aerobic power to exhaustion during ~adrenergic blockade with propranolol (P), during lipolytic blockade with nicotinic acid (N) and without drugs (C). Venous blood was drawn for analysis every fifteen minutes. The total work times differed significantly 83 + 9 (P), 122 + 8 (N), 166 _+ 10 (C) rain (mean and SE). After an initial decrease glucagon concentrations increased progressively and became, at exhaustion, identical and three times the preexercise values. During exercise epinephrine concentrations rose throughout and were always above preexercise levels (0,06 _+ 0,01 ng/ml). At exhaustion concentrations 2,15 + 0,41 (P) were larger than 1,08 _+ 0,31 (N) and 0,72 __ 0,28 (C). Norepinephrine and insulin levels uniformly increased and decreased, respectively. The carbohydrate combustion rate was smaller and FFA and glycerol values larger during C-experiments. Identical glucagon concentrations and identical, decreased plasma glucose concentrations at exhaustion after different work times, irrespective of larger a-adrenergic stimulation during r-blockade, indicate that change in glucose concentration and not stimulation of adrenergic receptors is the major determinant for exercise induced glucagon secretion. Circulating epinephrine possibly significantly influences substrate mobilisation during prolonged exercise. 80. Hypertriglyceridaemia and the Esterification of Fatty Acids by Adipose Tissue in Diabetes Mellitus. D.J. Galton, P. Clifton-Bligh. Diabetes and Lipid Research Laboratory, St Bartholomew's Hospital, London E.C. 1, U.K. Patients with adult diabetes commonly develop hypertrigiyceridaemia associated with raised preB-lipoproteins. This may be due to defects of lipoprotein lipase or of the uptake and esterification of fatty acids to adipocyte lipid. To see if esterification defects in adipose tissue are primarily related to hypertriglyceridaemia we have measured palmitate conversion to glycerides in groups of adult diabetics with or without hypertriglyceridaemia. The patients were: 7 non-diabetic and normotriglyceridaemic (age 27 + 2.7 years, per cent obese 42 + 2.7, plasma triglycerides 101 _+ 11 mg%, blood sugar 77 _+ 3 mg%); 6 diabetic and normotriglyceridaemic (age 56 -+ 3 years, per cent obese 37 + 2, plasma triglycerides 108 + 16 rag%, blood sugar 169 + 22 mg%); 8 diabetic and hypertriglyceridaemic (age 48 + 3 years, per cent obese 35 + 2, plasma triglycerides 1515 + 867 rag%, blood sugar 135 + 17 rag%). Esterification of palmitate to cell lipid correlated with the triglyceride content of adipocytes (r = 0.76, n = 21, p < 0.001). In diabetics palmitate esterification to triglycerides was 1.93 + 0.32 (6) for normotriglyceridaemics versus 4.18 + 0.96 (6) n. mole/105cells/hr for hypertrigiyceridaemics (p < 0.05); esterification to diglycerides was 1.58 + 0.08 (6) for normotriglyceridaemic versus 2.33 + 0.4 (6) n. mole/105 cells/hr for hypertriglyceridaemics (p N. S.). Esterification was decreased in diabetic compared to non-diabetics (3.06 + 0.6 (12) versus 5.72 _+ 1.1 (7) n. mole/105cells/hr, p < 0.05). The reduction in palmitate esterification in adult diabetes does not therefore relate to the presence of hypertriglyceridaemia. Also in diabetics, with or without hypertriglyceridaemia, esterification of palmitate is reduced compared to non-diabetics. 81. The Significance of Insulin Receptors in Isolated Rat Fat Cells. S. Gammeltoft. Institute of Medical Physiology C, University of Copenhagen, R~dmandsgade 71, 2200 Copenhagen N, Denmark. We have studied the specific binding of 125I-labelled insulin to receptor sites in isolated rat fat cells attempting to solve some of the controversy about insulin receptors which has appeared in reports

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from several laboratories. The following was found: 1) Monoiodoinsulin and native insulin were inactivated at the same rate, whereas the relative biological potency and binding affinity was about 70%. 2) The receptor binding of 125I-labelled insulin was homogeneous at concentrations from 1 pM to 1 ~tM, but occasionally heterogeneity was observed, i.e. a second class of binding appeared at insulin concentrations below 100 pM. 3) The activation of lipogenesis by insulin, when measured simultaneously on the same fat ceils, was present also when the insulin receptor binding was homogeneous. 4) Equilibrium of receptor binding was obtained after 180 min at 20 ~C and 45 min at 37 ~ C. The receptor binding of a2SI-labelled insulin was increased by a factor of two. The rate of dissociation followed first-order kinetics at 20 and 37 ~C with half-times of 40 and 10 min. The rate was not changed by addition of insulin 0.7 ~M. It is concluded that the effect of insulin on lipogenesis, which was half- 9 maximally stimulated by an insulin concentration of 60 pM is mediated through binding of insulin to receptors with a K d of 3 nM. The variable "high affinity binding" could either be linked with other insulin effects e.g. antilipolysis, express negative cooperativity or result from heterogeneity of the ~:SI-labelled insulin.

82. Relationship between Serum Calcium and Carbohydrate Metabolism. L. Ger6, M.D., I. Hollo, F. Szalay, L. Kor~nyi, K. Steczek, Gy. Tamfis, jr. First Department of Medicine, Semmelweis University, Budapest, Hungary. Our aim was to evaluate. 1. the role of ca!cium in insulin secretion. 2. the role of calcium in insulin action. - Methods: 1. intravenous calcium load. 2. oGTT on normocalcemie and hypocalcemic patients. 3. determination of serum calcium following single injection of insulin. - Results: 1. Calcium load reduced significantly blood glucose level without altering IRI. 2. During oGTT on hypocalcemic patients no difference was found in the IRI responses and the time-course of blood glucose with or without simultaneous calcium injection. 3. Serum calcium level decreased considerably following a single injection of insulin. - Conclusions: 1. Calcium may enhance glucose utilization without influencing IRI. 2. Insulin action is accompanied - rather preceded - by a decrease of serum calcium level.

antiserum). In order to obtain information on the factors which can modify fetal IRG secretion, pancreas from full term rat fetuses and 8 to 27 week old human fetuses were studied in vitro. 100 mg of pancreatic pieces (0.5/0.5 mm) were perifused with KrebsHenseleit medium containing glucose (G) and stimulatory agents. IRG was estimated by radioimmunoassay. Infullterm ratfetus, IRG release was not affected by G level 1 to 6 mM but was decreased by G 30 mM. Arginine (Arg) 30 mM in presence of G 4 mM induced a biphasic IRG release: this response was blunted by G 30 mM and potentiated by G 1 raM. Norepinephrine (NE) (10 ~tg/ml) stimulated IRG release in the presence of G 4 mM. In human fetus, IRG release was not modified by G 1 mM to 4 mM. Arg 30 mM in presence of G 4 mM induced a biphasie IRG release in all the fetuses studied (n = 14). NE (10 ~tg/ml) in presence of G 4 mM stimulated IRG release in 5 fetuses studied. - Conclusions: 1/Fetal A cells of pancreas are relatively insensitive to glucose levels in the physiological range. 2/Arg and NE are strong stimulators of fetal A ceils.

85. Investigations Concerning Glucose and Lipid Metabolism in Subjects Born with Fetal Macrosomy. V. Gligore, M. Motocu, V. Duma, D. Petcu, S. Blaga, R. Trandafoiu. Ilnd Medical Clinic, Gluj-Napoca, Rumania. The aim of the present paper was to assess the possible diabetogenic risk of subjects born with fetal macrosomy, in order to reduce this risk or to delay the clinical manifestation of diabetes. - A clinical and biological investigation has been carried out in 54 subjects born as "big-babies" and in a control group of 30 persons, by the oral glucose tolerance test and measurement of FFA during the test. The results showed a flat OGTF. There was a slow, reduced, and delayed depression of FFA levels following the glucose load. A lack of diabetes-type GT was observed, together with a lack of FFA dynamics as described in prediabetes. - The evaluation of the diabetogenic risk of former big babies is dependent on their young age. The risk seems to depend on the possible later intervention of other diabetogenic risk factors (obesity, psychological stress, pancreatotropic infections, climacteric, etc.)

83. The Influence of ACTH, STH, and TSH on the Dynamics of Insulin Secretion by Perfused Rat Pancreas.

86. Variation of GH, Prolactin, Blood Sugar and Insulin in Subjects with Insulin Dependent Diabetes after OGTT and Pretreatment with 2-a-Br-ergocryptine.

D. Ghiea, Z. Mirodon, M. Oprescu. International Atomic Energy Agency Research Contract 1215/R1R B. The Institute of Endocrinology of the Academy of Medical Sciences, Bucharest, Rumania. In order to find evidence of the direct action of some hypophyseal hormones on pancreatic ~-cells, a study was carried out concerning the effect of these hormones on the dynamics of insulin secretion. Using the method of perfusion of the rat pancreas "in vitro" the authors measured the discharge of insulin under the influence of a constant glucose infusion (controls) and under different experimental conditions. - Constant glucose infusion (60 min./8.3 or 16.7 raM) and ACTH (10 I.U. or 20 I.U./100 ml) significantly increased insulin secretion. - Constant glucose infusion (60 min./8.3 or 16.7 mM) and TSH (5 I.U. or 10 I.U./100 ml) likewise brought about a significant increase in insulin secretion. - When ACTH or TSH infusion was interrupted 30 minutes after beginning the experiment a return to normal insulin secretion values occurred. - Constant glucose infusion (60 min/8.3 or 16.7 raM) and STH did not significantly modify insulin secretion. - The data obtained suggest the possible existence of a hypophyseal control of insulin secretion by direct intervention of ACTH or TSH at the level of pancreatic if-cells.

A. Gnudi, R. Lugari, M.G. Cavazzini. Istituto Patologia Medica. Endocrinologia e Pat. Costituzionale. Universit~ di Parma, Italy. In insulin dependent diabetics high GH values are usually observed. The OGTT is unable to modify this level. It was observed that CB154(2-a-Br-ergocryptine) seems capable of lowing G H in acromegalic subjects. We thought it pertinent to check whether or not CB154 can reduce GH also in diabetics. - The OGTT was applied in insulin dependent diabetics before and after 5 days of treatment with 2.5 mg of CB154. IRI, GH, hPRL and Blood Sugar were measured during the test. - Significant GH and hPRL variations were not observed after OGTT in the diabetic subjects, IRI was always absent and glycemia followed the model of the diabetic curve. On the contrary, after pretreatment with CB154, we observed 1) hPRL reduction 2) G H increases, sometimes as early as 30' from the oral glucose, 3) IRI was never present and Blood Sugar values were lower than with glucose alone. - Contrary to obversations in acromegalics, CB154 is unable to lower the increased level of GH, normally present in the insulin dependent diabetic. After pre-treatment with CB 154, oral glucose seems instead to release GH. - Such behaviour can be explained only if we postulate an antagonism at the hypothalamic level between the adrenergic and serotoninergic pathways of G H stimulation.

84. Giucagon Release from Perifused Fetal Rat and Human Pancreas.

87. Metabolites and Insulin Release in Pregnancy.

J.R. Girard*, P.F. Plouin**, Ch. Heuclin**, N. Bourdillat**, R. Assan**. * Faculty of Sciences, Paris, France. ** Department of Diabetology, H6tel-Dieu, Paris, France. Glucagon (IRG) has been found in the plasma of rat fetus and also in human fetuses, 15 to 22 weeks old, (mean 250 pg/ml, 30 K

I.C. Green, K.W. Taylor. Biochemistry Group, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K. Islets from pregnant rats are found to be more sensitive to stimulation by glucose than are those from normal rats. However, they do not appear to be similarly sensitive to stimulation by leucine o r

Abstracts glucagon. We have further investigated the secretory sensitivity by comparing the response to various metabolites of glucose of islets from pregnant and non-pregnant rats. - Glyceraldehyde, citrate, succinate and pyruvate stimulate insulin release to varying degrees in islets from pregnant rats, and all four compounds markedly potentiate the effect of increasing the cyclic AMP levels in these islets. However, only glyceraldehyde and citrate stimulate insulin release from islets of normal rats, and only glyceraldehyde is able to potentiate significantly the effect of raising the cyclic AMP levels in these islets. Furthermore, the metabolism of 14C-glucose is increased in islets from pregnant rats. - Thus, islets from pregnant rats are particularly sensitive to stimulation by a number of glucose metabolites as well as glucose itself. This sensitivity may be related to facilitated metabolism of these compounds in islets in pregnancy, or to production of higher cyclic AMP levels, leading to higher rates of insulin release. 88. Decreased Cyclic AMP and Insulin Response to Glucose from Isolated Islets of Neonatal Rats. V. Grill, K. Asplund. Department of Endocrinology, Karolinska Hospital, Stockholm, and the Department of Histology, University of Uppsala, Sweden. Neonatal beta cells are insensitive to the insulin-releasing action of glucose. To investigate a possible role for cAMP in this condition (3H) cAMP accumulation and insulin release were measured after prelabelling with (3H)-adenine in incubates of islets from 1, 6, and 35 day old rats exposed to 3.3 mM or 16.7 mM glucose, with or without 0.1 mM 3-isobutyM-methylxanthine (IBMX). In 35 day old rats (3H) cAMP accumulation was enhanced after 60 min incubations with glucose 16.7 mM and further increased by IBMX. These changes were paralleled by stimulated insulin release. In 1 day old rats no effect of glucose, with or without IBMX, was seen on (3H) cAMP, while minor insulin release due to high glucose alone was potentiated by IBMX. The stimulatory pattern of glucoseinduced insulin release in 6 day old animals was intermediate between the other two age-groups. At this age, stimulation of (3H) cAMP due to glucose was observed only in the presence of IBMX. Measurements of (3H) cAMP after 3 rain incubations confirmed these different stimulatory patterns of glucose in the age-groups studied. - It is suggested that inefficiency of glucose in stimulating the adenyl cyclase - cAMP system of neonatal islets may be important in determining insensitivity to the insulin-releasing action of glucose in neonates. 89. Cyclic AMP and Insulin Response to Different Hexoses and Mannoheptulose in Islets of Langerhans from the Rat. V. Grill, E. Cerasi. Department of Endocrinology, Karolinska Hospital, S-104 01 Stockholm 60, Sweden. Earlier work demonstrated a correlation between glucose-induced cAMP and IRI response in rat islets. The specificity of these correlations was investigated by measuring the effects of D-mannose, D-fructose, D-galactose, L-glucose and 3-0methylglucose on (3H) cAMP (3 and 60 rain incubations) and simultaneous insulin release (60 min). (3H) cAMP was determined after prelabelling the islets with (3H)-adenine for 60 min. - Only D-glucose and D-mannose directly stimulated insulin release and (3H) cAMP. The threshold of stimulation was higher for D-mannose than for D-glucose. D-fructose elicited insulin and (3H) cAMP response at 38.8 mM only when 3.3 mM glucose was present. With 8.3 mM glucose, D-galactose stimulated insulin secretion and (3H) cAMP. L-glucose or 3-0-methylglucose were ineffective at 60 min in the presence of 8.3 mM D-glucose, while at 3 min small effects were noted on (3H) cAMP together with 8.3 mM D-glucose. D-mannoheptulose and D-glucosamine inhibited insulin release and accumulation of (3H ) cAMP (27.7 mM D-glucose), inhibition by mannoheptulose being complete within 90 sec. - The mechanism of glucose action was further investigated by using D-glucose anomers. In 3 min incubations, (3H) cAMP was preferentially stimulated by a-glucose (8.3 mM). At higher concentrations no differences were seen. - It is concluded that hexoseinduced cAMP accumulation participates in the initiation of insulin release, possibly through actions of the sugars on cell-membrane receptors.

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90. Intrarenal Localization of Gluconeogenesis. W.G. Guder, U. Schmidt. Klinisch-Chemisches Institut, Stfidt. Krankenhaus Mfinchen-Schwabing, 8 Munich 40, K61ner Platz 1, FRG. Renal gluconeogenesis increases in starvation and diabetic acidosis by hitherto unknown mechanisms. Since renal glucose production contributes little to blood glucose levels, its significance seems more related to renal function. In order to get more insight into this relationship, we investigated the intrarenal localization of gluconeogenesis by radiochemical determination of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) in microdissected defined structures of rat kidney. PEPCK was found to be localized exclusively in structures of the proximal tubule, decreasing from the convoluted to the more distal straight portion. Juxtamedullary nephrons contained less activity than superficial nephrons. Starvation and metabolic acidosis led to a 3-4 fold increase in activity in the proximal tubule without any detectable activity in glomeruli, thick ascending limbs of Henle's loop and collecting ducts. The localization of renal PEPCK correlates well with renal lactate and amino acid reabsorption. Since isolated tubules from starved rats convert glutamine and lactate to glucose, renal gluconeogenesis can be interpreted as a mechanism that preserves filtered organic acid carbon. This function seems to be part of renal acid base regulation. 91. Adaptive and Insulin Regulation of Amino Acid Transport in Mesenchymal Cells. G.G. Guidotti, A.F. Borghetti, R. Franchi-Gazzola and G.C. Gazzola. Istituto di Patologia Generale, Universitg di Parma, 43100 Parma, Italy. The transport of amino acids across the cell membrane is regulated by adaptive and hormonal control mechanisms which continuously modulate its efficiency to meet the actual requirements of the intracellular machinery. Purpose of this work was to verify the occurrence of adaptive and insulin regulations for amino acid transport in a variety of mesenchymal cells. - The activity of the transport systems (and changes as a function of time in the presence and absence of insulin) have been evaluated from measurements of initial entl~ rates with representative amino acids. - A time-dependent adaptive regulation of transport system A (for neutral amino acids) is operative in such mesenchymal cells as fibroblasts, chondroblasts, osteoblasts and myoblasts. The same A mediation is responsive to insulin in these cells. Adaptive control in thymic lymphocytes and peritoneal macrophages is restricted to some amino acids (c~-aminoisobutyric acid, glycine and L-proline) among those considered typical substrates of the A mediation. All substrates of transport system A (including L-alanine and L-serine) respond to insulin with increased uptake in thymocytes, whereas no hormonal effect has been observed in macrophages. - These results suggest that the A mediation is heterogeneous and that adaptive and insulin control mechanisms act independently on amino acid transport. 92. The Pupil in Long-Term Diabetes, Nervous and Muscular Function. H.J.G. Gundersen. 2nd University Clinic of Internal Medicine, University of Aarhus, Denmark. The incidence and severity of autonomic nervous abnormalities versus abnormal muscular dynamics in the pupil of tong-term diabetics was studied. - Employing infra-red TV-videopupillography the area of the pupil was measured in complete darkness and after a short pulse of light in eight normals and 29 juvenile diabetics (5-28 years duration of diabetes), aged 30-40 years. The degree of retinopathy ranged from no lesion to proliferative retinopathy, but the area selected for light-stimulation was in all cases without visible changes. - A significant correlation (Kendall's thau = - 0.36, 2p = 0.018) was found between the area of the pupil in darkness and the duration of diabetes. The area was 35.2 + 7.2 m 2 in normals and 32.3 + 6.6 in patients with 5-10 years of diabetes, but it was reduced to 21.1 + 7.2 after _-< 20 years of

346

Abstracts

diabetes (t --- 4.22, 2p < 0.001). The dynamics of the light response were normal in the diabetics: 1) latency time was 274 + 16 msec in the normals and 293 _+ 39 in patients with -> 20 years of diabetes. There was no correlation to the duration of diabetes, 2p = 0.40.2) Maximal constriction during light-response was 35.6 + 8.8% and 42.3 + 12.4 and 3) redilatation after 5 see was 88.1 + 5.6% and 93.5 + 6.1 in the same two groups. 4) Maximal velocities during constriction and redilatation were - 349 + 28 sec-~ and 119 + 19 in normals and - 3 6 0 _+ 16 and + 108 -+ 17 in long-term diabetics. The small pupil in diabetics indicates that the sympathetic activity in the pupil is gradually reduced in the course of diabetes, allowing dominance of the parasympathetic autonomic innervation. - The muscular dynamics of the pupil appear normal even in long-term diabetics.

93. Deficient Insulin Biosynthesis and RNA Metabolism in Pancreatic B-Cells of Mice with Spontaneous Diabetes. R. Gunnarsson. Department of Histology, University of Uppsala, Uppsala, Sweden. Diabetic mice homozygous for the mutations "diabetes" (db) and "misty" (m) were studied with regard to body weight, serum glucose and insulin concentrations and islet morphology at 5-75 weeks of age. In addition, the pancreatic islet metabolism was studied during the development of hyperglycaemia by using islets, isolated with a collagenase technique, from animals 5, 10 and 20 weeks of age. The unaffected littermates were used as controls. - The diabetic mice showed marked obesity associated with hyperglycaemia and hyperinsulinaemia, which was most pronounced in the 20 week old animals. The isolated islets were studied with regard to glucose oxidation rate, insulin content, insulin synthesis and release and RNA metabolism. In the 5 week old diabetic mice the only significant difference from the controls was an exaggerated insulin response to glucose. At 10 weeks there were no striking differences between diabetic and nondiabetic animals. In the 20 week old diabetic animals there was a decreased insulin content and a lowered insulin biosynthesis. There was also a block of the glucose stimulated RNA synthesis at this age. These functional disturbances of the islet metabolism were, however, not associated with signs of morphological degeneration. Such changes were not observed until about 40 weeks of age.

94. A Prospective Study of Intensive Dietary Management of Maturity-Onset Diabetes Mellltus. D.R. Hadden, E. A. Wilson, J.A. Weaver and D.A.D. Montgomery. Diabetic Clinic, Royal Victoria Hospital, Belfast BT12 6BA, U.K. As a logical sequence to retrospective and prospective data associating myocardial infarction with the use of oral hypoglycaemic agents in the management of maturity-onset diabetes mellitus, we have initiated a prospective study of intensive dietary management of a similar group of diabetic patients, without any other hypoglycaemic agent. - Between September 1972 and December 1974, 216 newly diagnosed diabetics aged 40-70 have been referred to the diabetic clinic: of these 129 have been acceptable for the defined purpose of the study: during subsequent intensive dietary management only 7 of these patients have shown persistent fasting blood glucose above 200 mg% and have been treated with an oral hypoglycaemic agent. A system of grading dietary adherence has been devised. - 43 patients have completed a 6-month initial study: their mean weight at diagnosis was 178 Ibs (standard 151 Ibs), mean age 57 years. After 6 months, mean weight was 163 lbs. Mean fasting glucose at onset was 177 mg%, failing to 114 mg% at 6 mmmtts. There was a considerable reduction after 6 months in blood glucose values during an oral GTT (mean 2 hour value fell from 260 to 160 mg%) and a commensurate rise in mean plasma insulin response. The "shape" of the GTT expressed as an harmonic function remained abnormal. - The practicality of intensive dietary management of the maturityonset diabetic will be discussed and the relation of this type of study to possible multicentre trials assessed.

95. The Responsiveness to Insulin, the Rate and the Pattern of Glucose Metabolism in Isolated Fat Cells From Rats of Different Weight. F.M. Hansen. Steno Memorial Hospital, Gentofte, Denmark. The investigation was carried out to elucidate further the decreased insulin effect on glucose uptake in fat cells from heavy old rats. Isolated epididymal fat ceils from ad lib fed male Wistar rats were incubated for two hours at 37 ~ C without and with insulin (10 mU/ml) in HEPES buffer containing albumin (10 mg/ml) and glucose (4.7 mmol). Both [U-!4C] -, [1-14C]- and [6J7C] glucose were used in separate incubations. The animals were divided in group A (100-300 g) and group B (> 300 g). - The insulin effect (per cent increase over the basal rate) on triglyceride synthesis and total CO2 formation was significantly lower in group B. The insulin effect on fatty acid - (360%), glycerol synthesis (30%), the pentose - (176%) and the citric acid cycle (0%) was the same in both groups. - The rate of the fatty acid synthesis and the pentose cycle was significantly lower and the glycerol synthesis and the citric acid cycle was significantly higher in group B. There was a significant positive correlation between the fatty acid synthesis and the pentose cycle, insulin caused increase in glucose uptake. - It is concluded that the insulin effect on different pathways of glucose metabolism is constant from animal to animal. - In group B with low insulin effect on total glucose uptake, fatty acid synthesis and associated pathways with high insulin effect is decreased and the pathways with low or no insulin effect increased. 96. Amines and Amino Acids in Mouse Pancreatic Islets. Effects of Starvation. S.E. Hansen, C.J. Hedeskov. University of Copenhagen, Department of Biochemistry A, Panum Institutet, Blegdamsvej 3, DK 2200 Copenhagen N, Denmark. It has been suggested that the decreased sensitivity of the insulin secretory mechanism during starvation may be associated with an increased concentration of certain amines in the pancreatic islets. We have tried to test this hypothesis by direct measurements of the content of adrenalin, noradrenalin, DOPA and serotonin in the islets. - The method of Brown and Perham, in which a doubleisotope-labelling technique using dansyl chloride is utilized, was further developed for this purpose. The method enabled us to measure accurately as little as 10--20 pmol of amines or amino acids respectively. - The following values for amino acid content in islets from normal fed mice have been found (expressed as mmoles/kg dry weight): gly 21, leu 8.3, lys 11.1, phe 3.0, pro 5.7, val 10.7. These values are of the same order of magnitude as those found by Gylfe (Acta Endocrinologica, 75, 105-118, 1974) for islets from obese, hyperglycemic mice. Like the islets from obese, hyperglycemic mice, the islets from normal mice contain GABA and large amounts of taurin. - Preliminary experiments have shown that adrenalin, noradrenalin, DOPA and serotonin can be measured in an amount of tissue corresponding to 15-20 ~tg dry weight (30-40 islets) and further that islets from starved mice contain increased amounts of serotonin. 97. Increased Serum Prolactin in Diabetic Ketoacidosis; Relationship to Serum Osmolality and Serum Sodium Concentration. K.F. Hanssen, P. Torjesen. Medical Department B & Hormone and Isotope Laboratory, Aker sykehus, Oslo, Norway. We studied 8 patients with diabetic ketoacidosis (initial pH 6.99-7.30, serum glucose 1127-473 rag%). All patients were treated with continous i. v. infusion of insulin (16 I. U./hour) and all responded satisfactorily to this treatment. Blood samples for determination of blood glucose, acid/base status, serum prolactin, serum osmolality and serum Na, K, C1 were taken on arrival and 0, 1, 2, 3, 4, 6 and 8 hours after the start of the insulin infusion. Serum prolactin was determined by a homologous radioimmunoassay. The pr01actin preparation used for iodination and for the production of antiserum was prepared in our own laboratory from frozen pituitary glands according to Hwang et al. (1973). Human prolactin obtained from NIH (HPr V - - L - S 1) was used as standard. In the assay system, our prolactin preparation was immunologically identical to

Abstracts the NIH preparation. The serum prolactin concentrations in normal fasting subjects were; men: 4.9 _+ 0.8 ng/ml (n = 9); women: 5.0 + 1.6 ng/ml (n --- 10) (mean -+ SUM). In diabetic ketoacidosis, before starting treatment, serum prolactin concentration was 23.5 + 8.3 ng/ml. During treatment serum prolactin decreased to 14.4 + 5.1 ng/ml (Mean +SEM). We studied the correlation between serum prolactin and serum osmolality/serum sodium concentration. The correlation coefficient between serum prolactin and serum osmolality did not differ significantly from zero, whereas there was a highly significant inverse correlation between serum prolactin and serum sodium concentration (r = - 0 . 5 7 ; p < 0.01; n = 46). In conclusion, the serum prolactin concentration is considerably elevated in untreated diabetic ketoacidosis and decreases following treatment. There is a highly significant inverse correlation between serum prolactin and serum sodium concentration before and during insulin treatment, supporting the role of prolactin as a sodium regulating hormone, as observed in animal experiments. 98. A Mathematical Approach to the Dose-Response Relation of Blood Glucose and Serum Insulin in Man. Haslbeck, M , Pr/51s, H. Diabetes Research Unit and 3rd Med. Dept., Schwabing City Hospital, 8 Munich 40, Krlner Platz 1, FRG. A sigmoidal relationship between glucose concentration and insulin release is well established in vitro and during glucose infusions in man. On the other hand, little is known about dose-response relations after oral glucose, especially after high doses. - Blood glucose and IRI were measured after 9 different oral doses of glucose (30 - 300 g, 40 g/m 2, 1.75 g/kg) in 111 normal subjects. The data of one curve were transformed into a single term with a new mathematical method. This allows the investigation of changes of the measured parameters in equidistant time intervals related to the varying doses of glucose. The results confirm the linear relation between IRI-increment and oral glucose over a broad range of 13 ~tU/ml x h (after 30 g glucose), 38 (after 1.75 g/kg glucose), up to 77 (after 300 g glucose) over a period of 3 hours. However, blood glucose is regulated within a relatively narrow range (venous: 86 - 101 rag/100 ml X h, capillary or arterial: 94 - 135 mg/ml x h, capillary-venous difference (CVD): 4 - 3 2 rag/100 ml x h). The relation of "mean blood glucose concentration x h" as well as CVD x h and IRI response x h follows a sigmoidal curve. This shows the range in which blood glucose, serum insulin and peripheral glucose uptake is regulated under physiological conditions. The mathematical index allows a better characterization than the absolute measurements at a given time point. 99. Influence of Insulin and Glucose on the Hydroxylation of Lysine in the Glomerular Basement Membrane of the Rat. Ch. Hasslacher, P. Wahl, K. Beier, K. H. Munderloh. Medical University Clinic, Heidelberg, FRG. In order to investigate the influence of insulin and glucose on the hydroxylation of lysine in the glomerular basement membrane, glomeruli of normal and diabetic rats were isolated and incubated with ~4C-lysine. After isolation, glomerular basement membranes were hydrolyzed and lysine and hydroxylysine were separated by thinlayer chromatography. After two hours of incubation 3 - 5 % of the incorporated radioactivity of lysine was hydroxylated in normal glomeruli. The degree of hydroxylation decreased with weight (age) of rats. Insulin (0.1 E/ml) and high glucose concentrations did not influence incorporation of lysine and its hydroxylation. There were no differences in the hydroxylation rates of diabetic and normal rats. These results did not confirm the observations of Cohen, who found a higher hydroxylation rate of lysine in diabetic rats. 100. Basal and Post-Glucose Proinsulin and Insulin Secretion in Overweight Women before and after Administration of Mefformin. L. Hausmann, R. Schubotz, H. Kaffarnik. Medizinische Universit~its-Poliklinik Marburg/Lahn, FRG. Serum proinsulin and insulin levels were measured in 35 normal or overweight women before and after the administration of Metformin. Proinsulin like components were determined by means

347

of an "Insulin Degrading Protease" from rat liver which degraded insulin into non-radioimmunoassayable fission products. The proinsulin fraction of the basal total insulin was 68 per cent in normal women. With increasing obesity the relation shifted in favour of insulin. After stimulation with I00 g oral glucose proinsulin levels rose in parallel with total insulin, but were less marked. The increased total insulin secretion in obesity was, therefore, largely due insulin and less to proinsulin. The greater the obesity the later maximal insulin levels were reached after oral glucose administration: proinsulin peaks occurred later than insulin peaks. - After 4 weeks administration of 1.7 g Metformin daily the weight reduction was on average 1.4 kg, after 16 weeks 5.2 kg. In relation to the weight reduction lower proinsulin and insulin levels were found before and after stimulation with glucose. Even those test persons who did not lose weight on Metformin showed lower proinsulin and insulin levels. It might be that Metformin leads to a slowing in glucose absorption and consequently to a decrease of insulin secretion. 101. Diurnal Variations in Plasma Glucose and Insulin Levels after I.V. Gincagon and Arglnine Infusion in Men and Women. M. Hautecouverture, A. Basdevant, G. Slama and G. Tchobroutsky. Hrtel-Dieu, Service de Diabrtologie, place du Parvis Notre-Dame, 75181 Paris Cedex 04, France. A diurnal pattern of insulin secretion has been shown in man using several stimuli including meals, oral and intravenous glucose loads and tolbutamide. Up to now, this has not received a clear-cut explanation. We have used glucogon and arginine because of their different insulinogenic mechanisms. - 6 men and 6 women were tested. All were healthy lean young non-diabetic volunteers. The test procedure consisted in the I.V. administration of 0.1 mg/kg of glucagon, followed 150 minutes later by a thirty minute arginine infusion (15 g/m2). - The insulin secretion induced by glucagon and by arginine was significantly lower in the afternoon than in the morning in both sexes. Because of higher morning plasma insulin levels in women than in men, the diurnal pattern was more pronounced in women than in men. No difference was observed in a. m. versus p.m. plasma glucose values. - Diurnal changes in insulin secretion were observed after glucagon and also arginine administrations. The fact that such a pattern was observed whatever the mechanism involved in insulin secretion supports the concept of an endogenous rhythm of B cell function. Sex modulates this pattern as previously shown in this laboratory. (Diabetologia 10, 725-730, 1974). 102. Enteric Hormones and Insulin Secretion in Addison's Disease. Effect of Hydrocortisone. F.G. Hawkins Carranza, L. Larrodera, M. Fldrez Tasc6n, A. Martinez and A. Schiiller. Ciudad Sanitaria 1 de Octubre, Madrid, Spain. Insulin secretion after OGTT in Addison's disease has been shown to increase with hydrocortisone treatment. To investigate the possibility that glucocorticoids potentiate insulin release by an indirect effect mediated by the enteroinsular axis, the following studies were made: 1) Six non obese patients with Addison's disease (Normal OGTF) (167 cm, aged 3 4 - 4 5 y) were subjected to 2 different tests (before treatment): a) Secretin iv. 2U/kg. b. wght., ( J - M ) ; b) C C K - P Z iv. 2 U/kg. b. wght., (impure preparation, J - M ) . 2) Patients were again studied after treatment (hydrocortisone, 20 mg daily/10 days, and 1i/2hrs before pertinent test. 3) Control group of 5 non obese healthy subjects (164 cm. aged 1 8 - 2 9 y). Plasma glucose (glucose-oxidase) and insulin (I. R. I., method of Heding) were assayed at fixed intervals during each test. Results: 1) Insulin release after Secretin and C C K - P Z was not significantly different in patients from that in the control group; 2) After hydrocortisone I.R.I. release induced by Secretin was not significantly different; 3) I. R. I. release induced by C C K - P Z was significantly different (p 0.01) after hydrocortisone treatment, in the first minute postinjection. Conclusion: 1) Insulin release by enteric hormones in Addison's disease is not different from normal subjects. 2) Hydrocortisone increases I.R.I. release induced by C C K - P Z in Addisons' patients.

348

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103. Inosine-Stimulated Insulin Release and the Metabolism of Inosine in Isolated Mouse Pancreatic Islets. C.J. Hedeskov, K. Capito. Department of Biochemistry A, University of Cogenhagen Panum Institute, Blegdamsvej 3, Copenhagen, Denmark. 10 mM inosine was demonstrated to be a potent primary stimulus of insulin secretion in isolated pancreatic islets from normal fed mice. A markedly diminished response was seen during starvation. Caffeine and mannoheptulose potentiated the effect of inosine, a phenomenon also observed when glyceraldehyde acts as secretagogne. - The possible mechanism behind the inosine stimulation was investigated. A nucleosidphosphorylase, capable of catalyzing the conversion of. inosine to hypoxanthine and ribose-l-phosphate was shown to be present in the islets. A suggestion that this conversion actually takes place and that ribose-l-phosphate is further metabolized to glyceraldehyde-3phosphate through the pentose-phosphate shunt, was given by the demonstration of an inosine-induced increase in the concentration of ATP and fructose-l.6-diphosphate and in the production of lactate in islets from fed mice. Also the content of cAMP in islets from fed mice was found to be increased after incubation with inosine, whereas no change in cAMP concentration could be observed in islets from starved mice. - The observed changes in islet metabolism mimic what is seen after glucose stimulation and it is suggested that inosine may act as a functional glucose analogue. As hypoxanthine has only an insignificant effect on secretion and the ribose part of inosine does not in itself stimulate insulin release, the results support the substrate-sitehypothesis for glucose-induced insulin secretion. 104. Human Plasma C-Peptide Assay, Sources of Error and Examples of Its Use. L.G. Heding, U. Dahl Larsen, J. Markussen and V.K. Naithani. Novo Research Institute, Novo Allee, DK-2880 Copenhagen, Denmark. A sensitive radioimmunoassay to human C-peptide has been established. As this assay is being used to study pathological conditions such as diabetes and insulinomas, it was considered a prerequisite that the routine assay involved a removal of proinsulin to ensure specificity. Antibodies against human C-peptide were raised in guinea pigs immunized with crude human b-component, and 9 out of 12 animals developed useful antibodies. The detection limit was approx. 0.02 pmol/ml standard (100 ~1volumes), and from 0 to 0.5 pmol/ml in 100 ~1 volumes the binding of (I-125)-human Tyr-C-peptide fell from 48 to 16%. - Removal of proinsulin and intermediates was performed by adding a suspension of SepharoseAIS (50 ~1) to each serum sample (500 ~tl)followed by centrifugation. C-peptide is then determined in the supernatant. - Three main sources of error have been encountered: (i) non-identity between the immunoreactivities of synthetic standards and natural C-peptide (ii) sera without human C-peptide may give values significantly different from zero and, consequently, sera with high C-peptide concentration (> i pmol/ml) may show a dilution effect. By selection of antisera these 3 sources of error were eliminated. The mean fasting level (_+ SEM) of C-peptide and IRIin 15 normal persons was: 0.39 _+0.02 and 0.048 _+0.009 pmol/ml; 1 h after 1.75 g glucose/kg orally the values increased to: 2.24 _+ 0.18 and 0.52 + 0.08 pmol/ml. In ten newly diagnosed insulin-requiring diabetics the corresponding values in the fasting state were: 0.21 _+ 0.04 and 0.06 + 0.009 pmol/ml and I h after glucose 0.42 _ 0.08 and 0.11 + 0.014 pmol/ml. Nine maturity onset diabetics, never treated with insulin, had fasting values of 0.74 _+ 0.18 and 0.11 _+0.029 pmol/ml and post glucose values of 2.34 -+ 0.42 and 0.49 + 0.11 pmol/ml. 105. Potentiation of Glucagon Secretion by Serotonin (5-HT) Antagonists in Man. J. A. Hedo, J. Martinell, C. Calle, M. L. Villanueva, and Jose Marco. Cllnica Puerta de Hierro, Universidad Aut6noma de Madrid, Madrid, Spain. This work was undertaken to study the possible implication of endogenous 5-HT in the control of glucagon secretion in man. Young, healthy volunteers were treated with two 5-HT antagonists - either cyproheptadine (CPHT) (16 mg daily, for two days) or

methysergide (MTS) (9 mg daily, for two days) - or with an inhibitor of 5-HT synthesis, p-chlorophenylalanine (PCPA) (2 gr daily, for four days). Alpha cell stimulation was obtained by administration of i.v. arginine (468 mg/kg) and by insulin-induced hypoglycemia (0.1 U/kg). Each subject served as his own control. Plasma glucagon was assayed radioimmunologically with Unger 30 K antiserum. - Pretreatment with any of these drugs did not significantly affect basal glucagon levels. However, after CPHT administration, the glucagon responses to arginine (N = 12) andto insulin-induced hypoglycemia (N = 9) were greater than in the control experiments (+ 105 pg/ml, p = 0.01, and + 197 pg/ml, p = 0.01, respectively). A potentiation of arginine-provoked glucagon secretion was also observed after MTS treatment (+ 260 pg/ml, p = 0.01; N = 7). Similarly, after PCPA administration the alpha-cell responsiveness to both aminogenic (N = 1 2 ) a n d hypoglycemic (N = 7) stimuli was enhanced (+ 108 pg/ml, p--0.04, and + 164 pg/ml, p = 0.03, respectively). - In conclusion, glucagon secretion is potentiated by treatment with drugs which either antagonize 5-HT action or inhibit its synthesis, thereby suggesting that a serotoninergic mechanism may modulate alpha-cell function in man. 106. Structure and Metabolism at Human Pancreatic Islets Maintained in Tissue Culture. C. Hellerstr6m, A. Andersson, C.G. Groth, R. Gunnarsson, G. Lundgren, J. Westman, J. Ostman. Departments of Histology and Human Anatomy, The University of Uppsala and Departments of Surgery and Internal Medicine, The Huddinge Hospital, Huddinge, Sweden. Transplantation of human pancreatic islets to diabetic patients may require that donor islets are kept viable in vitro for hours or days before they can be transferred to the recipient. We have therefore studied the survival in vitro of adult human pancreatic islets, which were isolated by means of collagenase digestion and identified in a microscope with dark field illumination. Immediately after isolation the islets were placed in tissue culture and maintained for 7 days at a glucose cone. of 6.1 mM. Light microscopic and ultrastructural analyses of the cultured islets showed a satisfactory preservation of the islet cells and the occurrence of both B-cells and A2-cells. During culture there was a continuous release of insulin to the medium and short-term incubations at the end of the culture period showed that the rate of insulin release was strongly stimulated by 16.7 mM glucose plus 10 mM theophylline. Incubation of cultured islets with 3H-leucine for 3 h in 16.7 mM glucose demonstrated distinct peaks of radioactivity in both the proinsulin and insulin regions of the gel chromatographs. Measurements of the islet oxygen uptake showed linear rates of respiration for several hours but little respiratory stimulation by glucose. It is concluded that the isolation procedure itself is a highly critical step in studies in vitro of human pancreatic islets. After isolation, however, the islets can survive in tissue culture and may be suitable for transplantation purposes. 107. The Interaction of Ca + § and cAMP in Glucose Stimulation of Insulin Secretion. B. Hellman. The University of Ume~, Department of Histology, S-90187 Ume~ 6, Sweden. An attempt wao rpade to delineate the interrelationship between cAMP and Ca n- n- during glucose-stimulated insulin release with islets microdissected from ob/ob-mice. After addition of 20 mM glucose to medium containing a phosphodiesterase inhibitor, cAMP levels were increased in islets frprq fed mice but not from mice starved for 18 h. Omission of Ca - ~ - 7 - from the incubation medium abolished the stimulatory effects of glucose on both insulin release and islet cAMP without affecting the basal level of cAMP. Increasing the extracellular Ca r 7- above the concentration necessary for the maximum secretory action of glucose inhibited the glucosestimulated release but did not affect basal secretion and islet cAMP. The maximum secretory + + effect of glucose was reached at lower concentrations of Ca when the islets contained high concentrations of cAMP. Exposure of th,e islets to ionophores which facilitate intracellular transport of Can- n- (X-537A and A-23187) resulted in stimulatory as welI as inhibitory effects on insulin re-

Abstracts lease. At least part of the ionophore inhibition of glucosestimulated insulin release was caused by reduced adenylate cyclase activity. The data suggest that both decreases anc~i~creases of the functionally important intracellular pool(s) of Ca n- n- can diminish insulin resnonses to glucose. The interrelationship between cAMP and Ca-t- + in the process of insulin secretion is complex: apparently Ca -t-+ alters adenylate cyclase activity and cAMP has a permissive effect on the mechanism by which Ca t- + regulates the insulin dicharge from the t-cell.

108. Regulation of Rat Skeletal Muscle Pyruvate Dehydrogenase Activity by Fasting, Diabetes and Work. G. Hennig, G. L6ffler and O.H. Wieland. Diabetes Research Unit and Inst. Clin. Chem., Schwabing City Hospital, 8 Munich 40, K61ner Platz 1, FRG. To get more insight into the regulation of glucose metabolism in skeletal muscle, the activity of the pyruvate dehydrogenase (PDH) complex was determined, which is the rate limiting step in the glucose breakdown to CO2. The enzyme exists in an active (PDHa) and inactive (PDHb) form, which are interconvertible by enzyme catalyzed phosphorylation and dephosphorylation respectively. Therefore total PDH activities and the proportion of PDH a were determined in the gastrocnemius muscle of normally fed, fasted and diabetic rats at rest and during electric stimulation (10 impulses/ sec., 2 Volt). 21,7% of total PDH (2.2 mU/mg protein) were present as PDH a in normally fed animals. This proportion decreased to 6.8 % and 4.9 % in streptozotocin diabetes and fasting respectively, whereas the total activity did not change. Electric stimulation led to an immediate increase of PDH,, which could be detected after 20 sec. of stimulation. After stimulation for 5 rain. PDHa amounted to 49.7% in the fed, and 7.8% in the fasted state. These results clearly demonstrate the importance of PDH interconversion for glucose breakdown energy production in skeletal muscle. 109. Bicarbonate Requirement for Normal Insulin (IRI) Release by Isolated Rat Islets. J.-C. Henquin and Andr6 E. Lambert. Unit6 de Daib~te et Croissance, University of Louvain School of Medicine, St Pierre Hospital, 3000 Louvain, Belgium. This study investigated the effects of changes in extracellular HCO 3 concentration upon the secretory and metabolic functions of the/3 cells. Experiments were carried out in a Krebs-Ringer medium buffered with 10 mM Hepes (pH 7.4) and containing 25 mM NaHCO 3 or not (Bic-O). - Glucose ( 1 5 0 - 5 0 0 rag%) stimulated IRI release was inhibited by 55 % in Bic-O, but unchanged between 3 and 25 mM NaHCO 3. Bicarbonate omission for only 5 min before glucose stimulation (300 rag%) abolished the early secretory response of perifused islets, whereas the late phase was the more depressed as HCO 3 absence was longer. This inhibition was completely reversible upon HCO 3 reintroduction, immediately for the first phase and after 20 min for the second one. Theophylline, dibutyryl-cAMP and cytochalasin B partially corrected the late response to glucose in Bic-O but failed to restore a rapid response. Stimulations by high K or parachloromercuribenzoic acid were unaffected in Bic-O, but those by D-glyceraldehyde, leucine and glucagon were suppressed. - Glucose oxidation and utilisation as well as glucose transport in islet cells were normalin HCO 3 absence. In contrast, Ca influx and uptake were reduced by about 25% in Bic-O, but fully restored by only 5 mM HCO 3. - It is concluded that extracellular H C O 3 is a prerequisite for a normal insulin secretion and that its absence affects differently both phases of glucoseinduced release, through a mechanism that might involve an abnormal Ca handling by the beta cells. 110. Pancreatic and Gut Hormones in Starvation Diabetes. R.W. Henry, J. Ardill and K.D. Buchanan. Department of Medicine, The Queen's University of Belfast, U.K. The phenomenon of starvation diabetes is ill-understood, but it may serve as an experimental model in the understanding of idiopathic diabetes mellitus. Accordingly, we induced the state in nine volunteers using a 72-hour acute starvation. This period was initiated and terminated by a mixed meal (first and second meals

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respectively). Blood samples were taken before and during the meal at 0, 15, 30, 45, 60, 90, 120 and 180 minutes and the following substances were measured: - immunoreactive insulin (IRI), immunoreactive gastrin (IRGa), immunoreactive secretin (IRS), glucagon-like immunoreactivity (GLI) measured both by Nterminal (N-GLI) and C-terminal (C-GLI) antibodies, and plasma sugar. - IRI concentrations displayed a delayed and exaggerated response following the second meal and the plasma sugar values for this meal showed glucose intolerance. Basal concentrations of IRGa and N-GLI were similar before and after starvation and the responses of these hormones to each meal showed no change after starvation. C-GLI however, rose during starvation and the meal responses varied; the first meal stimulating C-GLI release and the second meal depressing the raised values following starvation. The initial meal had little effect on IRS concentrations, but the second meal rapidly suppressed markedly raised levels attained during the fast. - It is possible that the elevated C-GLI and IRS levels following starvation may be responsible for the impaired IRI secretion and glucose intolerance of starvation diabetes.

111. Intravenous Insulin Therapy under Conditions Imitating Physiological Profiles. K.D. Hepp, R. Renner, H.J. von Funcke, H. Mehnert, R. Haerten and H. Kresse. 3rd Med. Dept. Schwabing City Hospital and Diabetes Research Unit, Munich, and Siemens AG Medical Engineering Group, Erlangen, FRG. A key problem in the development of an 'artificial beta cell' is a glucose sensor suitable for implantation. In spite of great progress with sensors based on the principle of "the biofuel cell", it still remains open as to whether all technical and medical problems can be overcome in the near future. - An alternative concept is the control of an insulin pump by a fixed profile individually adjusted to the patient's needs, based on physiological secretion patterns. In order to test this possibility, a commercial infusion pump has been connected with a special controlling device which allows for programming of an infusion profile over 24 hrs, with delivery rates from 0 . 3 - 6 units/hr. 4 stable and 3 labile diabetics were observed in the hospital under such intravenous therapy for 2 - 3 days and the following results were obtained: (1) Better control as compared to that of conventional therapy with one or more insulin injections. (2) A small reduction in insulin requirement. Although our final goal is a sensor-controlled device, the system described seems feasible and could be beneficial in the treatment of the unstable diabetic. For further development more flexible controlling devices are taken into consideration, comprising the adjustment to fluctuating insulin requirements (exercise, infection). 112. Studies on the Participation of Duodenal Insulin Releasing Activity in the/3-Cytotropic Action of Glibenclamide. W. Heptner, H.-M. Kellner, I. Hilwig, O. Christ, A. Zermatten, J.-P. Felber. HOECHST AG, Medical Department, Frankfurt/Germany and Division de Biochimie Clinique, D6partment de M6decine, H6pital Cantonal Universitaire, 1011 Lausanne, Switzerland. In 1969 it was suggested that the highly effective antidiabetic drug glibenclamide causes the release of gut hormones effective in stimulating insulin release. In animal experiments it was shown that the labelled drug does not penetrate from blood into the pancreatic tissue. - In recent experiments maximal serum IRI-levels were observed between 1 to 3 rain after intragastric administration of 0.2 mg/kg (threshold dose) glibenclamide to fasted rats. At the beginning of elevated IRI levels only 1.4 _+0.3 ng/ml of the drug - that is less than 0.5% of the amount administered - could be detected in peripheral blood. In the duodenal mucosa, however, levels of fixed Glibenclamide are well correlated to serum IRI-concentrations. Furthermore we could measure the liberation of insulin releasing activity by glibenclamide from duodenal mucosa of both rat and man. The well confirmed in vitro effect of the agent on insulin release from pancreas could be confirmed in a protein free medium and in the presence of albumin, but not in the presence of complete serum. - Because of our findings one has to consider the possibility that in maturity onset diabetes the typical pattern in serum insulin

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following the intake of food components may be primarily due to a certain defect in the mucosa of the gastrointestinal tract rather than in the t-cells of the pancreas itself. 113. Insulin Release: the Permissive Role of Calcium Inward Transport in the B-Cell. A. Herchuelz, J. Levy*, M. Mahy, J. Schoonheydt, A. Sener. Laboratory of Experimental Medicine (Head: Dr. W.J. Malaisse), Brussels University, Brussels, Belgium. Glucose provokes immediate and sustained changes in Ca 2+ handling l?y pancreatic islets. It is said to inhibit the outward transport of c a zT across the B-cell membrane and, subseauently, to increase the net uptake of Ca 2+ by the B-cell, causing Ca~+ accumulation in the cytosol and secretory granules. Under steady-state conditions, this sequence of events would be quantitatively dependent on the integrity of calcium inward transport in the B-cell. The present work aims at documenting such a dependency. - At increasing Ca 2lconcentratiorts (0.1 to 4.0 mEq/l), the changes in both glucoseinduced Ca 2~ net uptake and insulin release were suggestive of a first-order relationsllip. A high Mg 2-r concentration inhibited glucose-induced Ca 2-r net uptake and subsequent insulin release, without causing any facilitation of 45calcium effiux. Verapamil (1 to 10 ~ M) also inhibited Ca 2• net uptake under basal conditions or on stir0ulation by glucose, sulfonylureas and Ba 2+ , without altering 45Ca2t efflux. Incidentally verapamil, like omission of extracellular Ca 2+ , caused little alteration of glucose metabolism and proinsulin synthesis. - These findings suggest that the inward transport of Ca 2-v in the B-cell is mediated by a carrier systeln characterized by first-order ~ype regulation by extracellular Ca 2-~-, competition between Ca 2"V and Mg 2"ff , and sensitivity to verapamil. Our data also document the permissive role of a sufficient inward calcium transort for stimulation of insulin release by different secretagogues. * Recipient of a 1975 Pfizer-Europe Travel Fellowship of the E.A.S.D.).

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114. Digitoxose and the Existence of a Glucoreceptor in the Beta Cells of the Islets of Langerhans of the Rat. O.G.Hermida, J. Gomez-Acebo. Laboratorio de Morfologla. Instituto G. Marafi6n C. S. I. C. VeNzquez 144, Madrid (6), Spain. Digitoxin is the only drug reported that stimulates insulin release but partially inhibits glucose stimulated insulin release. In this work the idea that the competition glucose-digitoxin may be at the level of a hypothetical glucoreceptor in the beta cells (binding of the glycoside sugar molecule) has been investigated. Our findings indicate that the integrity of the chemical structure of digltoxin is needed to exert its pharmacological action on insulin release. We also demonstrate that digitoxose inhibits glucose stimulated insulin release without inhibiting the glucose oxidation rates of the islets. The discovery of a sugar (digitoxose) that inhibits the insulin secretory response to glucose without inhibiting glucose metabolism is strong evidence that the action of glucose itself is blocked at the level of glucoreceptors located in the cell membrane, which appear to be the regulator sites of insulin release. The influence of digitoxose on leucine, tolbutamide and other sugars distinct from glucose will also be presented. 115. Influence of L and D-Fucose on Glucose Stimulated Insulin Release. O.G. Hermida, J. G6mez-Acebo. Laboratorio de Morfologia. Instituto G. Marafi6n Centro de Investigaciones Biol6gicas. C. S. 1. C. Vel~zquez 144, Madrid (6), Spain. On the basis that 2-deoxyglucose and digitoxose (2-6-deoxy-Dribohexose) inhibit glucose stimulated insulin release, we thought it of interest to investigate the influence of other deoxy-sugars on glucose stimulated insulin release. One of the most common deoxysugars is fucose (6-deoxy galactose) which occurs in human milk oligosaccharides, blood group polysaccharides and numerous glycoproteins. - In these experiments the effect of L- and D-fucose on glucose stimulated insulin release were investigated using the perifusion system of the islets of Langerhans of the rat. - Our results indicate that L-fucose inhibited glucose stimulated insulin

release at concentrations between 16 mM and 0.25 mM, showing a maximum inhibition of 67% and a inhibition constant of 0.083. D-fucose inhibited glucose stimulated insulin release at concentrations between 16 and 0.25 mM showing a maximum inhibition of 14% and a inhibition constant of 0.043. - The rates of glucose oxidation measured by the formation of 14CO2 from D-(U14C) glucose showed an inhibition in respect to the controls, L-fucose (49%) and D-fucose (45%). 116. Different Plasma Lipid Responses of Diabetics According to Dietary Advice During One Year's Treatment. T. D. R. Hockaday, J. M. Hockaday, J. I. Mann. Radcliffe Infirmary, Oxford, U.K. In a prospective study of newly diagnosed diabetics in which metabolic features are collated with clinical events, patients received one of two types of randomly allocated dietary advice. This report examines the effect on plasma lipids over the first year. The calorie intake was determined by weight and height. One diet (LC) was classical, with carbohydrate weight (gms) restricted to one-tenth of the daily calories, while in the other (LF) there were advised limitations of (a) calories from fat to 30% of the total, (b) daily cholesterol to 300 mg and (c) ratio of polyunsaturated to other fatty acids above 0.9. - 56 diabetics under 66 years old at diagnosis were allocated between 2 groups, which did not differ in age or initial fasting plasma cholesterol or triglyceride. Patients were studied again after one month and one year. The decrease over the year in weight (mean = 4.8 on 76.6 kg) and in fasting blood glucose (68 on 208 mg/100 ml) was also similar in the two groups (much of it occurred in the first month), nor did fasting insulin values differ. However, a significant decrease in cholesterol occurred only in the LF group (195 to 177 rag/100 ml at one year) and on subdivision by sex, only in LF males (196 to 167 mg/100 ml, Wilcoxon ranking p < 0.01). Triglyceride decreased after a month on LC (132 to 106 mg/100ml) but not LF, and more in males than in females, but this effect was lost after a year. - Advice on a LF diet led to a fall in plasma cholesterol not seen after LC advice. Changes in cholesterol or triglyceride could not be predicted from change in weight, fasting glucose or insulin concentrations. 117. Sexual Differences in Metabolic Features of Diabetics before and after the First Month's Treatment. T. D. R. Hockaday, J. M. Hockaday, J. I. Mann. Radcliffe Infirmary, Oxford, U.K. Data from a prospective study of newly diagnosed diabetics under 66 years old (in which metabolic features are collated with clinical events) were analysed for difference by sex. - Metabolic investigation was made after an over-night fast and after intravenous injection of glucose. Treatment was by dietary advice, randomly allocated between two types, emphasising either carbohydrate or lipid restriction. - Mean fasting blood glucose was higher among the 37 women than the 42 men (230 v 188 rag/100 ml, p < 0.02), as was blood glycerol (0.13 v 0.10 raM, p < 0.02). After intravenous glucose, women showed a lower rate factor (Ko, 0.58 v 0.72) and a trend towards a smaller 30 rain lactate increment (0.09 v 0.16 raM). After a month, with a mean loss of 3 Kg by each sex, glucose and glycerol were still higher in women (185 v 146 rag/100 ml; 0.13 v 0.09 raM) and K o lower (0.66 v 0.76). - On both tests, plasma growth hormone was lower in women, especially after glucose (e. g. 50 rain value, initial, women 1.3, men 4.3 mU/L: one month, women 0.7, men 2.1 mU/L). There was no lowering of fasting insulin in the female. - The groups were of similar age but the women were plumper (ponderal index 0.30 v 0.26). However, the index did not correlate with the values reported. - The results suggest a metabolic difference between sexes among diabetics. 118. Reversal of Phenformin-lnduced Hyperlactataemia by Dichloroacetate in Normal and Diabetic Rats. P.A.H. Holloway, K.G.M.M. Alberti. Faculty of Medicine, Chemical Pathology and Human Metabolism, General Hospital, Southampton S09 4XY, U.K. Hyperlactataemia and lactic acidosis are major complications of phenformin (PH) therapy. Dichloroacetate (DCA), an activator of

Abstracts pyruvate dehydragenase, lowers blood lactate concentration in normal and diabetic rats. The present study was designed to investigate whether DCA could reverse phenformin-induced hyperlactataemia in the rat. - Starved, conscious normal and streptozotocin diabetic rats were infused for 21/2 hr with saline, phenformin (0.21 mM/kg/hr) or phenformin plus DCA (2.35 mM/kg/h). Arterial blood samples were drawn from the femoral artery at frequent intervals. - In normal rats, phenformin caused a 5% fall in (glucose), an eight-fold (+ 2.79 mM) increase in (lactate), a 56% (0.053 raM) increase in (pyruvate) and a 3.5-fold increase in (alanine) after 150 minutes. In contrast DCA plus p henformin caused a 50% fall in (glucose) by 150(minutes; lactate). increased by only 0.32 mM, (pyruvate) and (alamne) decreased and ketone body concentration increased by 85 %. In severely diabetic animals phenformin caused significant increases in blood glucose (+ 4 mM), lactate (+ 1.2 raM), pyruvate ( + 0.02 mM) and alanine + 0.09 raM). When DCA was added to phenformin (glucose) - 2.2 mM), (pyruvate) ( - 0.06 raM) and (alanine) ( - 0.049 raM) all decreased, and (lactate) increased by only 0.4 mM compared with saline-infused controls. - Thus DCA reverses the hyperlactataemic, hyperpyruvataemic and hyperalaninaemic effects of phenformin in both normal and diabetic rats whilst enhancing hypoglycaemia.

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119. The Influence of Glucagon on Carbohydrate and Fat Metabolism during Prolonged Exercise in Rats. J.J. Hoist, H. Galbo, E. A. Richter and J. Henriksson. Department of Gastroenterology A, Bispebjerg Hospital, Copenhagen; and Institute of Medical Physiology B, University of Copenhagen, Denmark. The importance of glucagon for the alterations in carbohydrate and fat metabolism during swimming has been evaluated. Fed, male rats were used. Blood was drawn by cardiac puncture for lactate and glucose analyses and either rabbit-antiglucagon serum (A-rats) or normal rabbit serum (N-rats) injected. 30 rats were then forced to swim (S-rats) with a tail weight for 60 min, while 16 rats were resting controls (C-rats). Then blood was drawn again and samples of liver and muscle tissue collected. Glucagon concentrations increased from 151 _+ 18 (SE) pg/ml (CN-rats) tO 332 + 61 (SN-rats) (p < 0.05). All rats treated with antiserum had excess antibody in their plasma at sacrifice. Liver glycogen (p < 0.05) and plasma insulin (p < 0.02) decreased and blood glucose increased (p < 0.05) in SN-rats, while these parameters did not change significantly in SA-rats. FFA rose in SA-rats (p < 0.005) as well as in SN-rats (p < 0.05) as did lactate concentrations (p < 0.05). Glycerol concentrations, however, only increased in SA-rats (p < 0.05). Muscle glycogen diminished uniformly in exercised rats. These results indicate that increased glucagon secretion during prolonged exercise induces increased hepatic glycogenolysis. Possibly lipolysis was greater in antiglucagon-antibody-treated swimming animals indicating a compensatory stimulus allowing a shift in fuel combustion. 120. Calcium Pools in Rat Pancreatic B-Cells. S.L. Howell, W. Montague and M. Tyhurst. School of Biological Sciences, University of Sussex, Falmer, Brighton, Sussex, U.K. Changes in intracellular calcium distribution may be involved in the regulation of insulin secretion. We have therefore studied concentrations of calcium, and factors which may regulate calcium accumulation, in various B cell organelles. Calcium levels were determined directly by electron microscope X-ray microanalysis of frozen sections of unfixed islets; calcium-45 accumulation by particulate components of islet homogenates or subcellular fractions was estimated by incubation with calcium-45 followed by separation of organelles bound from free calcium by Millipore filtration. - Mitochondria and storage granules were found to contain high concentrations of calcium as determined by X-ray microanalysis; calcium was also present in endoplasmic reticulum and in other organelles. Calcium-45 accumulation by homogenates was rapid, temperature-dependent, stimulated by ATP and involved mitochondrial and microsomal fractions, but not storage granules, which seemed to provide a relatively inert calcium pool.

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Net accumulation of calcium-45 by homogenates or mitochondrial fractions was reduced by cyclic AMP (10 ~tM-1 raM), by dibutyryl cyclic AMP or by cyclic GMP. It seems possible that mitochondria may play an important role in the regulation of intracellular calcium levels in the B cell, and of insulin secretion.

121. Hepatic Triglyceride Secretion and Free Fatty Acid Flux in Vivo after Ventromedial Hypotbalamic (VMH) Lesions. B.-E. Hustvedt, A. Haug and A. Lovo. The Institute for Nutrition Research, School of Medicine, University of Oslo, Blindern, Norway. Ventromedial hypothalamic (VMH) lesions in adult rats result in hyperphagia, hyperinsulinemia and accumulation of body fat. Hyperinsulinemia is present already 2 days postoperatively even when hyperphagia is prevented. In this study the triglyceride (TG) secretion to the plasma and the flux of free fatty acids have been studied in vivo after 24 hrs without food 2 days post-operatively. Triton WR 1339 and 3H20 were given i.v. to VMH-lesioned and sham-operated controls for measurement of TG secretion from the liver and hepatic lipogenesis respectively. The flux of FFA through the plasma pool was measured after single i.v. injection of (1-14C)palmitic acid complexed with albumin and successive rapid sampling of arterial blood. - The VMH-lesioned animals exhibited a 50% increase in TG secretion. Hepatic lipogenesis was also increased, but not enough to account for the increased TG secretion. The plasma FFA concentration and flux was significantly decreased in the VMH-lesioned rats. As hepatic uptake of FFA is claimed to be proportional to the plasma concentration of FFA, it is unlikely that increased hepatic secretion of TG was due to an increased supply of FFA to the liver. - In conclusion; VMH-lesions, which resulted in hyperinsulinemia, caused decreased release of FFA from adipose tissue. The hepatic metabolism of fatty acids was also changed, leading to greater TG secretion at the expense of other processes. 122. Effect of Anoxia on the Glycolytic Flux in Pancreatic t-cells. L.-]k. Idahl. The University of Umefi, Department of Histology, S-90187 Ume~ 6, Sweden. The glycolytic rate of t-cell-rich islets of non-inbred ob/ob-mice, measured as the production of 3HzO from D-(5-3H)glucose, was inhibited by anoxia at glucose concentrations above 5 raM. Pieces of exocrine pancreas, which exhibited a slower aerobic 3H20-production than the islets, showed a clear enhancement of the process during anoxia. The islet transport of D-glucose appeared unaffected by anoxia. An activation of phosphofructokinase in anoxic islets was indicated by decreased concentrations of glucose-6-phosphate and increased concentrations of fructose-l.6-diphosphate plus triose phosphates. It is suggested that the concomitant inhibition of glycolytic flux is due to a low lactate dehydrogenase activity in the islets resulting in a slow reoxidation of NADH and a slow phosphoglyceraldehyde oxidation under anaerobic conditions. In contrast to the rate of 3H20-production, the concentration of fructose- 1.6-diphosphate plus triose phosphates in anoxic islets was already at its maximum with 1 mM glucose. This incongruence may indicate that glucose directly or indirectly activates some step distal to phosphofructokinase. 123. B-cell Function of Insulin-Dependent Young Onset Diabetics Assessed by C-Peptide Immunoreactivity. Ikeda, Y., N. Ando, N. Minami, and Y. Ide. 4th Dept. of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan. Human serum C-peptide immunoreactivity (CPR) of insulindependent diabetics whose onset was under 25 years was measured in order to detect pancreatic B-cell function. - Sixteen insulindependent diabetics were studied; 10 were controlled (FBS < 160 mg/dl), 6 were uncontrolled (FBS > 160 mg/dl). Eight insulinindependent cases (FBS < 160 mg/dl) were used as control. Blood sugar and CPR were measured every 30 minutes for 120 minutes during 50 g OGTT. Blood sugar was measured by Autoanalyser and CPR by a two-antibody method using synthetic human C-peptide. - CPR response of the insulin-dependent cases was lower than

352

Abstracts

control in both groups (P < 0.05): controlled group ZCPR = 11.84 + 2.28 ng/ml, uncontrolled group YCPR = 11.67 _+ 1.76 ng/ml; control ZCPR = 33.31 + 4.46 ng/ml. No significant correlations were found between CPR levels and the state of control, duration of the disease and dose of insulin. - The study indicated insufficiency of B-cell function of insulin-dependent diabetics compared with the insulin-independent group. The low response of CPR of insulindependent diabetics does not correlated with the state of control. And their control state seems to be influenced by other factors than endogenously secreted insulin, ,such as insulin sensitivity of peripheral tissues, hepatic metabolism or other hormones. 124. The Incidence of Hyperlipoproteinemia (HLP) in Different Forms of Diabetes Meilitus. C. Ionescu-Tirgovi~te, I. Mincu, J. St~nescu, E. Popa, E. Ghi~eBeer, D. Chela. Clinic of Nutrition and Metabolic Diseases, Bucharest, Rumania. The lipid profile was studied in 149 diabetics: 90 women and 59 men, aged 6 to 71 years, and the duration of diabetes 2 to 41 years. The distribution of the diabetes forms in terms of age was the following: juvenile diabetes (onset under 20 years) 20 cases; youthonset diabetes (onset between the ages of 2 1 - 4 0 ) 35 cases; maturity-onset diabetes (onset between 41 - 6 5 years) 94 cases. Of the 149 cases 77 were under insulin treatment and 72 under oral treatment. - HLP was encountered in 89 cases (59.6%), distributed as follows: type II-a in 24.7%, type II-b in 14.6%, type III in 5.6%, type IV in 49.4% and type V in 5.6% of the cases. It was more frequent in males (66.1%) as against 55.5% in women. The incidence of HLP was of 64.5% in maturity-onset diabetes, 51.4% in youth-onset diabetes and 55.0% in juvenile diabetes. The incidence of HLP was not significantly higher in the diabetics receiving an oral treatment (62.1%) than in those under insulin treatment (51.1%). - In conclusion, diabetes itself and not the age or treatment applied (oral or insulin) is the factor that determines the high incidence of HLP in this disease. 125. Further Characterization of the Inhibitory Effect of Somatostatin on Insulin and Gincagon Release from the Isolated, Pedused Canine Pancreas. J. Iversen. Second University Clinic of Internal Medicine, Kommunehospitalet, 8000 Arbus C, Denmark. Somatostatin is a powerful inhibitor of insulin and glucagon release from the isolated, peffused canine pancreas. To further characterize the inhibitory effect on islet cell hormone release the effect of somatostatin was investigated in the presence of potentiators of glucose induced insulin release, secretagogues other than glucose and catecholamines, and alpha- and beta-adrenergic blockade. During perfusion with a glucose cone. of 150 rag%, somatostatin at a concentration of 1 ng/ml abolishes both the first and the second phase of insulin release. When glucose-induced insulin release is augmented by arginine 1 mM or potentiators of the adenylcyclase system, such as glucagon 2 ng/ml, cyclic-AMP 1 rnM and theophylline 1 mM, somatostatin at a concentration of 1 ng/ml is able to overcome the stimulation and suppress insulin maximally. When insulin is stimulated by secretagogues which do not require the presence of glucose, such as leucine 1 mM and tolbutamide, somatostatin at a concentration of 1 ng/ml also inhibits insulin maximally. When acetyl choline 10 gM is perfused, glucose-induced insulin release is also inhibited. When epinephrine 2 ng/ml is perfused, somatostatin inhibits the inhibited insulin release further. Phentolamine 1 ~tM or propanolol 1 gM does not influence somatostatin inhibited insulin release. Somatostatin has no effect upon endogenously released norepinephrine from the pancreas. During perfusion with both low and high glucose, somatostatin totally abolishes glucagon release. When glucagon is stimulated by arginine l raM, leucine 1 raM, acetyl choline 10 pM or epinephrine 2 ng/ml, somatostatin overrules the stimulation and inhibits release. Alpha- and betaadrenergic blockade does not influence somatostatin inhibited glucagon release. - Somatostatin inhibits insulin and glucagon release in all test situations, irrespective that the stimulus of release is an initiator (glucose, leucine or tolbutamide), a potenti-

ator (arginine or stimulators of the adenylcyclase system), or an autonomic modulation, sympathetic or parasympathetic. An effect of somatostatin distally in the secretory process is suggested. 126. Kinetics of Insulin Secretion in Chronic Pancreatitis and Mild Maturity Onset Diabetes. W. P.U. Jackson, A.I. Vinik, J. Botha. Department of Medicine, Medical School, University of Cape Town, South Africa. We studied insulin responses to glucose with and without cholecystokinin-pancreozymin (CCK-PZ) and aminophylline infusions in 27 controls, 17 chronic pancreatitis patients who had normal oral glucose tolerance tests, and 17 mild diabetics. Glucose alone was given as 5 and 10 g boluses followed by infusion at 250 mg/min and 500 mg/min. The insulin responses of controls and pancreatitics were similar, but the diabetics had a raised Km and a reduced Vmax. - Crude CCK-PZ without additional glucose caused a negligible spike in insulin release, but a progressive increment at higher glucose concentrations. Pancreatitis patients and controls had similar responses, but in mild diabetics there was a marked increase in Vmax without a change in the Kin. - Infusions of aminophylline (12.5 mg/min) enhanced insulin responses to glucose injection in controls with an increase in Vmax and no change in Kin, but had no effect in mild diabetics. Pancreatitics who had normal responses to other stimuli had a lower than normal Vmax response to glucose plus aminophylline. - This suggests that impairment of CAMP generation is an early lesion in pancreatitis, that in both pancreatitis and genetic diabetes the gut hormone stimulation of insulin secretion is intact in the early stages, and that this hormone acts distally to the glucoreceptor and the CAMP amplification system. 127. Reduced Nerve Fibre Diameter in Acute Experimental Diabetes. J. Jakobsen. University Institute of Pathology and the Second University Clinic of Internal Medicine, Kommunehospitalet, Aarhus, Denmark. Newly diagnosed juvenile diabetes as well as experimental diabetes of short duration are associated with reduced nerve conduction velocity. The morphological changes underlying this functional abnormality have not been clarified. The present study is a light microscopic, morphometric analysis of the common peroneal nerve in acute experimental diabetes of 4 weeks duration. - Internodal length and fibre thickness were measured in 20 isolated nerve fibres from each of 10 streptozotocin-diabetic rats and 10 normal rats. Measurements were also performed on transverse sections. - A reduction in the calibre of the five thickest fibres from each animal was found in the diabetic group (12.10 ~t against 11.50 9, 2 p < 0.05), while the internodal length was the same in the two groups (1.26 mm against 1.25 ram). In the diabetic group as a whole the slope-coefficient of regression lines between internodal length and fibre thickness was 1.04 against 0.95 in normals (not statistically significant). No evidence of demyelination was observed. Neither 9 was there any evidence of remyelination except in one of the 200 fibres from the control group. - A reduction in myelinated fibre diameter without sign of demyelination and remyelination is suggestive of axonal dwindling. Axonal diameter is an important determinant of conduction velocity. The results obtained may therefore explain this functional abnormality occuring in acute diabetes. 128. Genetic Association of the Insulin-Antibody Production with Histocompatibility (HL-A)-Antigens in Diabetics. F.K. Jansen, J. Bertrams, F.K. Jansen, D. Griineklee, H. Drost, H.E. Reis, J. Bever, E. Kuwert, F.A. Gries, E. Altrock. Laboratories of St. Elisabeth-Hosp., Essen, Diabetes-Res.-Inst., Dfisseldorf, University Clinic, Mainz, Medical Clinic, Wuppertal, Inst. Med. Virol. Immunol., Essen and University Clinic G6ttingen, FRG. Since in inbred strains of mice insulin-antibody production is genetically linked to histocompatibility genes (Jansen and Kiese11975), the existence of Ir-genes for insulin in human diabetics could be expected. - 55 diabetics with a high immune response to insulin

Abstracts ("High responders"), 54 patients lacking antibody production to insulin ("Low responders") and 1000 healthy controls were HL-Atyped. - Micro-lymphocytotoxicity-technique according to Kissmeyer-Nielsen (1967) for HL-A antigen-determination and cellulose-adsorption-technique and radio immuno assay according to Christiansen (1970) for insulin antibody determination. - In all patients." significant increase of HL-A8 (33.6% vs. 20.5%, p ---0.0018), significant increase of W15 (24.1% vs. - 5 . 0 % , p = 0.016) and sign. decrease of HL-A7 (12.9% vs. 26.0%, p -0.0029). In high-responders: normal freq. of HL-A8 and increase of W15 (25.9% vs. 15.0%, p = 0.049). In low-responders: normal freq. of W15 and increase of HL-A8 (34.5% vs. 20.5%, p = 0.021) and decrease of HL-A7 (10.9% vs. 26.0%, p = 0.019). - Firstly, these results corroborate the association of HL-A8, W15 and possibly HL-A7 with insulin dependent diabetes. Secondly these data indicate the existence of insulin-It genes, which are linked to genes of the human histocompatibility complex, namely HL-A8, W15 and HL-A7. 129. Defining Maturity-Onset Diabetes. R.J. Jarrett, H. Keen. Guy's Hospital, London, U.K. There are numerous minimum criteria for diagnosing diabetes metlitus on the basis of glucose tolerance tests and none of these have any proven validity in terms of subsequent individual outcome. We have followed two groups of subjects with "borderline diabetes", i. e. glucose intolerance discovered in the course of a population survey: - 1) 248 individuals of both sexes having a two hour post glucose blood sugar between 120 and 200 mg/dl and followed for at least 10 years, derived from the population of Bedford, England; 2) 207 male Civil Servants identified by a screening two hour blood sugar exceeding 110 mg/dl and a subsequent abnormal oral GTT in which the two hour blood sugar was less than 200 mg/dl and followed for at least 5 years. In addition subjects from the Bedford Study with two hour blood sugar of 200 + mg/dl, called diabetic and referred for conventional treatment have been reviewed 5 years later. - The striking finding in the borderline diabetics is an absence of clinical retinopathy despite, in many individuals, persisting glucose intolerance. In contrast, in the Bedford diabetics, after five years, the prevalence of retinopathy was over 30%. This suggests that specific diabetic tissue damage only occurs above a higher level of glycaemia than that indicated by most diagnostic criteria. Many individuals now labelled as diabetic might more appropriately be called glucose intolerant. Such glucose intolerance might be regarded as indicating a risk of atherosclerotic disease rather more than that of specifically diabetic tissue damage. 130. Interactions of Fructose and Dichloroacetate (DCA) in the Isolated Perfused Rat Liver. G. Johnson, K.C. Man. Faculty of Medicine, Chemical Pathology and Human Metabolism, General Hospital, Southampton S09 4XY, U.K. Fructose infusion in man and animals causes hyperlactataemia due to rapid conversion of fructose to lactate by the liver. In contrast dichloroacetate, an activator of pyruvate dehydrogenase, decreases blood (lactate). The present study was designed to show whether dichloroacetate could prevent fructose-induced lactate production by liver. - Livers from 48 hr starved or fed rats were perfused with medium containing 10 mM fructose, 10 mM DCA or both together. At the end of perfusion livers were freeze-clamped for metabolite estimation. - Fructose caused a rise in medium (lactate) to 2.3 mM and 5 mM with livers from starved and fed rats respectively, but only 1.2 mM (p < 0.01) and 2.5 mM (p < 0.01) when DCA was present as well. DCA also decreased medium (pyruvate) by 88% in starved and 80% in fed rat liver perfusions. The decreased lactate production was accompanied by an increase in medium ketone body concentration of 88% in starved and 128% in fed experiments. Tissue metabolite analysis revealed decreased hepatic (lactate), (pyruvate) and (alanine) and increased (ketone bodies) compared with fructose alone. - The results are consistent with DCA activation of pyruvate dehydrogenase and suggest that DCA could be useful in preventing fructose-induced hyperlactataemia.

35 3

131. The Metabolism of Insulin, Proinsulin and Insulin Analogues. R.H. Jones, D.I. Dron, K.H. Quine. Department of Medicine, St. Thomas' Hospital Medical School, London SE1 7EH, U.K. Degradation mechanisms for insulin have been described, and the in vivo kinetics of insulin metabolism extensively studied. We have used structural analogues of insulin tonnexamine the specificity of these processes. - Insulin, proinsulin, A1, B29 diacetyl insulin and three analogues with a synthetic A1-B29 crosslink have been studied by a priming dose constant infusion technique in greyhounds. Metabolic clearance rate (MCR), half life (Tl/2), apparent distribution space, and urinary clearance have been derived for each material. - All the analogues had significantly lower MCRs and longer Tl/2s than insulin (p < .01 in every case). - There were no differences in distribution space. Insulin and proinsulin showed saturation of the degradation system at high physiological levels, whereas the analogues did not. Urinary clearance of A x B29 modified analogues was ten times that of insulin and proinsulin. - The presence of the C-peptide in proinsulin markedly reduces the rate of degradation of the molecule but does not prevent reabsorption from the nephron. The A1, B29 site on the insulin molecule is of key importance in its degradation and renal handling. 132. Inhibition of Insulin and Glucagon Release by Cyproheptadine in the Rat. H. G. Joost, S. Lenzen, J. Beckmann and A. Hasselblatt. Institut fiir Pharmakologie und Toxikologie der Universit/it G6ttingen, D-3400 G6ttingen, Geiststrage 9, FRG. It has been established recently that cyproheptadine inhibits insulin release in vivo and in vitro. To gain further insight into the specifity and the mechanism of this effect, insulin and glucagon release were studied in the perfused rat pancreas. In addition, cyclic AMP content and 45Ca accumulation of isolated islets were measured. Cyproheptadine significantly inhibited the insulin secretory response to glucose and tolbutamide. When the pancreas was stimulated by an arginine infusion, cyproheptadine reduced the secretion of insulin as well as the biphasic glucagon release. Isolated islets of obese mice released less insulin when incubated with cyproheptadine. Simultaneously their cyclic AMP content remained unchanged. Theophylline increased cyclic AMP content and insulin release of islets in the presence of cyproheptadine, but failed to completely restore the secretory response. Furthermore, incubated islets accumulated significantly less 45Ca when cyproheptadine was present. - The results suggest that cyproheptadine has a general inhibitory effect on hormone release. The inhibitory effect on the B-cell is probably not mediated by cyclic AMP, although increase of the nucleotide partly reversed the inhibition. Finally, the effects of cyproheptadine on Ca metabolism of the islet may provide an approach to the mechanism of the inhibitory action. 133. The Effects of Exercise, Catecholamines and the Blockade of Adrenergic Beta-Receptors on the Assimilation of Glucose. E. Jungmann, G. Bach, D. B6hmer, D. Eckardt and I. Hass. AbteiIung f[ir Endokrinologie des Zentrums der Inneren Medizin der Universit~itsklinik Frankfurt am Main und Sport~irztliche Hauptuntersuchungsstelle, FRG. Epinephrine, norepinephrine and blockade of the adrenergic fi-receptors inhibit insulin secretion. Exercise decreases the insulin requirement of diabetic patients. In this study we investigated the effects of exercise, catecholamines and two /3-receptor blocking agents on glucose metabolism. Glucose (0.33 g/kg BW) was injected i.v. in six young healthy volunteers at rest and during exercise on a bicycle ergometer (1 h, 600 kpm/min). For the inhibition of the fl-receptors, propanolol (PROP) (5 mg) or pindolol (PIND) (0.4 rag) were given i.v. The following parameters were determined: glucose, free glycerol, NEFA, IRI, lactate, potassium and epinephrine norepinephrine. At rest, fl-receptor blocking agents decrease glucose assimilation and inhibit the regular decrease of potassium. PIND stimulates insulin secretion, PROP decreases insulin and NEFA. Free glycerol and catecholamines do not change. Exercise stimulates catecholamine-release, especially after PROP

354

Abs~a~s

and PIND. In all cases, the glucose tolerance is improved and insulin secretion inhibited. Lipolysis, glycogenolysis and glycolysis are stimulated to various degrees. PROP inhibits lipolysis nearly completely and induces a significant hypoglycemia for about 30 minutes. The different effects of PROP and PIND during exercise can only be explained by a mechanism of glucose assimilation, which is partly independent of insulin, but regulated by catecholamines and the energy supply from glycolysis and free fatty acid oxidation.

134. The Pathology of the Islets of Langerhans in Early Juvenile Diabetes Mellitus. K. Junker, J. Egeberg, H. Kromann and J. Nerup. Med. Dpt. F., Gentofte Hospital, Med. Dpt. E., Frederiksberg Hospital & Anatomy Dpt. B, University of Copenhagen, Denmark. Lymphocytic inflammation of the islets of Langerhans - insulitis has been found in 6 0 - 70 per cent of patients with juvenile diabetes mellitus (JDM) of short duration. However, insulitis as a feature of JDM was recently questioned (Doniach & Morgan. Clin. Endocr. 2:233, 1973). - An attempt to elucidate the question was undertaken by studying i g pancreatic sections of autopsy specimens from 12 patients with JDM dying within a year of the onset of the disease and 24 cases of sudden deaths matched for age, sex and autopsy procedures. - The results were: 1) In the controls islets were demonstrable in all cases but one. B and A cells were present in all islets. Insulitis was absent. - 2) In the diabetics islets could be demonstrated in all cases but one. The number of islets was normal as was the number and the granulation of A-cells. Aldehydefuchsin staining made B-cells visible in only one case. Insulitis was present in varying degree in 6 of the patients (50 per cent). - Thus a selective B-cell destruction and a non-cicatricial lymphocytic inflammation was demonstrated in 50 per cent of JDM of short duration, suggesting that autoimmunity plays a part in the pathogenesis of this condition. 135. Levels of 2.3-Diphosphoglycerate and Other Phosphates in the Erythrocytes of Diabetics and Nondiabetics with Chronic Ulcers. Y. Kanter, A.N. Bessman, Diabetes Service, Rancho Los Amigos Hospital, Downey, California, and University of Southern California School of Medicine, Los Angeles, California, USA. The red blood cell (RBC) concentrations of total phosphate (Pt), inorganic phosphate (Pi), nueleotide phosphate (P~) and 2.3-diphosphoglycerate (2.3-DPG) were studied, utilizing an automated ashing method for measurement of ashed metabolic phosphates, in 20 hospitalized diabetics with chronic peripheral ulcers (UD), 20 hospitalized nondiabetics with chronic pressure sore ulcers (UN), and 20 normal nondiabetic subjects (N). - The following data (Mean + SEM) were obtained (in mM/LRBe): P,

Pi

P,,

2.3-DPG

N 17.85 + 0.65 2.11 _+ 0.11 4.67 + 0.19 4.69 + 0.12 UN 19.01 + 0.34 2.71 _+ 0.11 6.14 + 0.32 5.76 + 0.26 UD 22.90 + 0.60 3.18 _+ 0.14 6.39 + 0.28 6.81 + 0.15 Levels of Pt were higher CO< 0.005) in UD, compared to both UN and N. Pi levels were higher Co< 0.005) in UN and highest in UD (p < 0.0005) when compared to N. Pn levels were higher CO< 0.005) in both UN and UD, compared to N. 2.3-DPG levels were higher (p < 0.0025) in UN compared to N and highest CO < 0.0005) in UD compared to N. - This study demonstrates that the Pn and 2.3-DPG concentrations of the RBC are significantly higher in all patients with chronic tissue damage. In the presence of diabetes, further elevation of Pn and 2.3-DPG is found, possibly due to an attempt by the RBC to improve 02 delivery to the hypoxic extremity of the chronic diabetic patient.

136. Hyperinsulinemia Following Ventro-Medial Hypothalamus Lesions: Possible Consequences upon Liver Metabolism. C. Karakash, B. Hustvedt, Y. Le Marchand, B. Jeanrenaud. Laboratoires de Recherches M6dicales, Geneva and Institute of Nutrition Research, Oslo. Avenue de la Roseraie 64, 1211 Geneva 4, Switzerland.

We have studied the metabolism of perfused livers from normal rats and from rats made hyperinsulinemic via ventro-medial hypothalamus (VMH) lesions. - Bilateral lesions of VMH were made by applying anodal current through a stereotaxic instrument. Nonoperated rats served as controls. VMH lesions were verified by microscopy and by appearance of hyperphagia. To eliminate the possibility that observed metabolic differences could be related to hyperphagia, lesioned rats were placed on restricted food intake that matched that of controls. Seven days after operation, blood samples were withdrawn, and livers perfused in situ. - VMH-lesioned rats became hyperinsulinemic and their plasma glucose and FFA levels reached values that were lower than those of controls. Moreover, under all experimental conditions (no substrate or various substrates in perfusate) perfused livers of VMH-lesioned animals exhibited greater lipogenesis, greater acetyl-CoA carboxylase and fatty acid synthetase activities, lower glucose output and greater ureogenesis than control livers. Administration of anti-insulin serum to VMH-lesioned rats prior to perfusion restored these anomalies towards normal. - It is concluded that VMH lesions play a role in appearance of hyperinsulinemia, and that hyperinsulinemia is partly responsible for the metabolic disorders of livers from VMH-lesioned rats.

137. Oral Glucose Load (OGL) Acting as a Non-Glycemic Stimulus in Insulin Secretion. B.G. Karamanos, P.D. Christacopoulos, P. J. Papadimitriou, C. G. Kardatos and S.D. Korkolis. Diabetic Center, Second Department of Medicine, University of Athens, Ippokration Hospital, Greece. This study to investigated the possibility of a further stimulation of insulin secretion by an OGL without any corresponding increase of the already high blood glucose level (BGL). - Eight fasted male volunteers having normal glucose tolerance were given sufficient amounts of glucose intravenously to obtain a constantly high BGL. The infusion ceased 30' later 5' after a 50 g. OGL. Blood was taken at 5' intervals from 0' time for 60', and glucose and insulin were measured. - Before the OGL the constant BGL had a mean 376 _+ 27 SEM, which decreased during the first 15' to 351 + 30 CO> .05) and the last 15' to 275 + 24 (vs 376 + 27 as well as 351 + 30 p < .001). - In the same periods the respective mean insulin values were 49.4 + 6.6, 78.9 + 13.5 CO< .05) and 102 _+ 19.7 (vs 49.4 + 6.6 as well as 78.9 + 13.5, p < .05). - Therefore, further insulin secretion, in normal subjects with induced high BGL, can be stimulated by an OGL without a corresponding increase in the already high BGL; moreover insulin levels continue to increase while BGL decrease significantly. - Thus the observed significant insulin secretion by the already glucose-primed b-cell could be mediated by OGL via gut factors. 138. Membrane SH-Gr0ups and Glucose Uptake in Adipocytes. H. Kather, B. Simon. Klinisches Institut fiir Herzinfarktforschung an der Medizinischen Universit~itsklinik, Heidelberg, FRG. To study the participation of membrane SH-groups in glucose transport of isolated rat fat ceils we compared the effects of a small organic mercurial reagent pCMB with those of a large pCMB derivative-pCMB-Dextran, M W - 10.000. Intact fat cells and fat free homogenate were prepared according to Rodbell (J. Biol. Chem. 239 : 375, 1964, 241 : 130,1966). Intact fat cells were preloaded with pCMB and pCMB-Dextran (7 • 10-7 to 7 x 10~ - xM) for 30 min, washed and subsequently incubated for 1 h with and without insulin. In another series pCMB-Dextran (up to 7 x 10-~M) was present during the whole incubation procedure. The conversion of (U- 14C) glucose to 14CO 2 and 14C triglycerides was measured. - It could be shown that both compounds inhibited the metabolic activities of fat cell homogenates to about the same extent. When applied to intact fat cells uncoupled pCMB showed (1) an insulin like effect (2) inhibition of insulin stimulated glucose metabolism and (3) inhibition of basal glucose uptake dependent on the concentrations used. In contrast, pCMB-Dextran did not substantially alter basal glucose transport nor did prevent the insulin stimulatory effect. - Considering the identical reactivity of pCMB and pCMBDextran towards SH-groups, the lack of effect of the large derivative on glucose metabolism of intact adipocytes indicates that

Abstracts the fat cell membrane is impervious to this reagent. We conclude that superficial membrane SH-groups are not functionally important in basal glucose uptake and insulin mediated enhancement of glucose transport. The various effects of uncoupled pCMB may be caused by blocking of SH-groups deeper in the membrane and/or at the intracellular level. 139. Epidemioiogy of Diabetes in an Urban Area of Greece. N. Katsilambros, J. Steryotis, N. Moiras, E. Bezos, G.K. Daikos. First DPT of Propaedeutic Medicine, University of Athens (Director: Professor G.K. Daikos), Greece. No epidemiological study on diabetes has been previously carried out in urban areas of Greece (approximately 50% of the country's total population). - 21, 410 inhabitants of the town of Aegaleo (suburb of Athens) were included in the study. Initially, a postprandial urine specimen was examined for the presence of glycosuria by two different enzymatic methods (Testape and Clinistix). An oral GTT was performed on glycosurics (50 g Glucose; venous blood; autoanalyser). - B.D.A. criteria were applied. - 417 glycosurics were submitted to OGTT. 135 new cases of diabetes were discovered. Among them, obesity was common and it was more pronounced in females. The prevalence of both known as well as of previously unknown cases of diabetes was 2.41% (confidence limits 2.12 to 2.70) in males and 3.24% (2.92 to 3.56) in females. The higher prevalence rate in females was statistically significant only in the 60 to 69 year age group. - The results will be presented in relation to age and sex. In addition, some observations about etiology and frequency of nondiabetic glycosuria will be mentioned. - It appears that diabetes is not infrequent in the urban population of Greece. 140. Binding of NSILA-S by a Carrier Protein of Serum in Vivo. U. Kaufmann, E.R. Froesch. Metabolic Unit, Department of Medicine, Kantonsspital Zurich, Switzerland. Previous work from this laboratory has shown that NSILA-S is specifically bound to a serum protein in vitro. To study this phenomenon in vivo we injected rats intravenously with ~25I-labelled NSILA-S. After various time intervals, serum and organs were rernoved and the radioactivity was counted. - Results: 1. 10 minutes after injection of 125I NSILA, 25% of the tracer was found in serum, compared to 12% when either labelled insulin is given, or 125I NSILA-S is given with an excess of cold NSILA-S. 2. The half-life of the 25% of 125INSILA-S in serum is about 3 hours. 3. Chromatography on Sephadex G-200 at neutral pH reveals 4 radioactive peaks. The one corresponding to low molecular weight NSILA-S disappears after 20 minutes. Simultaneously 2 peaks in the high molecular weight range increase in size. A peak at 70% bed volume dominates during the first minutes. Then the radioactivity shifts to a peak at 50% bed volume. The fourth peak at 100% bed volume (mostly free 1250 begins to increase 1 hour after the injection. 4. The kidneys accumulate up to 20% of the label. The other organs (heart, liver, lungs, diaphragm, spleen, adrenals) contain only small amounts of 125I. - Conclusions: 1. Our findings confirm the existence of a specific carrier protein for NSILA-S. This may answer some of the questions related to its physiological role. 2. The appearance of NSILA-S in 2 different high molecular weight peaks may be due to aggregates of the NSILA-binding protein. 3. The accumulation of tracer in the kidneys suggests either degradation or increased binding of NSILA-S in this organ. 141. Controlled Trial of Phenformin in Borderline Diabetics: 5 Year Findings. H. Keen, R.J. Jarrett, J. H. Fuller, P. Zimmet and J. D. Ward. Unit for Metabolic Medicine, Guy's Hospital, London, S.E. 1, U.K. In the course of the Whitehall Health Survey of 20000 male Civil Servants aged 40 or more (Reid et. al., 1974), 203 newly detected borderline diabetics were recruited between 1967 and 1969 to a double-blind, randomfsed trial of phenformin (50 rag/day) versus placebo. Half the members of each treatment group were advised and taught to restrict their carbohydrate intake to 120 g/day, the rest merely being advised to reduce table sugar. - Standardised

355

serial observations have been made at regular visits to a follow-up clinic of cardiovascular, ocular, neurological, renal and metabolic parameters. Subjects failing to attend have been exhaustively investigated and deaths classified by certified cause. All subjects have now passed the five-year observation point chosen for this report. - Significant effects of treatment upon body weight and blood pressure have been demonstrated and the relative contributions of carbohydrate restriction and phenformin will be presented. Of 18 deaths, 9 had occurred in the phenformin and 9 in the placebo treatment groups. The proportions attributed to cardiovascular causes were also similar. Indices of cardiovascular morbidity are being analysed and will be presented. - The findings of other controlled clinical trials of phenformin will be reviewed in relation to our results. 142. Influence of Histocompatibility Antigens and of "Foreigness" of Insulin on Titre and Heterogeneity of Insulin Antibodies. U. Kiesel, F. K. Jansen. Diabetes Research Institute, 4 Diisseldorf, Hennekamp 65, FRG. The quantity (titre) and quality (specificity and heterogeneity) of antibodies is regulated by T- and B-lymphocytes. The T-cell-function is genetically associated with histocompatibility antigens (h. ag.). Therefore we studied the influence of h. ag. and of "foreigness" of heterologous insulins (i. e. the differences in aminoacidsequence from autologous insulin) on the procuction of antibodies in both cell-systems. Concerning the influence of h. ag. we immunized mouse strains with different h.ag. (congenic-resistant strains) with hihghly purified vobine and porcine insulin. The heterogeneity of antibodies was demonstrated by isoelectric focussing (i. e. f.) of antisera against insulin of sheep, sattle rabbits, guinea pigs and mice. Only H-2-d-mice were high responders; H-2-b-mice produced lower titres and H-2-k-mice did not show detectable antibodies. Bovine insulin was always more immunogenic than porcine insulin. The number of bands in the i. e.f. could not be correlated with the number of foreign aminoacids. Our results seem to indicate that T-cell-function against insulin is genetically influenced by h. ag. and needs foreign aminoacids to stimulate antibody production. B-cells seem not to be tolerant against autologous insulin and therefore be able to produce antibodies to autologous determinants after stimulation by T-cells. 143. Perifusion of Liver Fragments - a Convenient System for Metabolic Studies in Limited Tissue Availability. M. Kikuchi. Institut de Biochimie Clinique, Geneva, Switzerland. This study was undertaken to investigate kinetics of metabolic pathways in small liver tissue fragments. - Rat liver pieces were prepared using a tissue chopper (0.3 X 0.3 x 0.3 mm). Portions of 100 mg were placed in chambers of 0.7 ml volume, through which perifusion medium (KRB, i% gelatine, 20% red cells) were pumped at 1.0 ml/min. Both recirculation and open system were used. Metabolites were determined in the effluent and the tissue. Electron microscopic examination revealed only small layers of necrosis at the surface of the pieces. After 30 min preperifusion, lactic dehydrogenase efflux was negligible, ATP content > 85%, pH constant, lactate efflux constant, but glycogenolysis was substantial. However, glucagon 10-7 M stimulated glycogenolysis to 3 0 0 - 350% basal (p < 0.01), the threshold stimulatory concentration being 10-9 M (p < 0.05). Insulin 10-5 M reduced (p < 0.01) glucagon (10- 7 M) stimulated glycogenolysis. Basal gluconeogenesis from fructose was enhanced by glucagon 10-7 M. - This preparation is a useful tool for kinetic studies of liver metabolism when a) only small amounts of tissue are available such as liver biopsies and b) where perfusion techniques are not accessible. 144. Hemoglobin Ale in the Diabetic Mouse. R. J. Koenig, D. C. Araujo and A. Cerami. The Rockefeller University, New York, New York 10021, USA. The glycohemoglobin AIc (Hb Aic) is present in twice-normal concentrations in diabetic humans. Diabetic mice were examined for a similar hemoglobin and its synthesis studied. - C5 7BL/KsJ-db/db and streptozotocin diabetic mice were studied. Hemoglobins were

356

Abstracts

separated by column chromatography on Bio Rex 70, quantified with Drabkin's solution, and monomers isolated by CM-cellulose chromatography in 8M urea. To study Hb AIo synthesis, + / + and db/db mice were injected with radiolabelled blood from + / + and db/db donors injected 3 days prior with 59Fe. The cpm of Hb's A and A~cwere determined periodically for 47 days. - Hb Aic comprises 4.5 % of diabetic mouse hemoglobin, 1.8 % of wildtype (p < 0.001 ). Hyperglycemia precedes by 4 weeks the increase in Hb A~c. Beta chains of mouse (and human) Hb Aic contain a NaB3H4-reducible linkage. Mouse Hb AI~ is synthesized at a constant rate throughout each red cell's life. Its rate of synthesis in diabetic recipients is 2.7 times that in wild-type (p < 0.005) and is independent of the genotype of red cells donated to that recipient. - Mouse Hb A~c results from a post-synthetic modification of Hb A. The synthesis rate of Hb A~cis determined by an unknown plasma factor present in increased amount in diabetes. 145. The Influence of the Diabetic State on the Performance and Metabolism of the Rat Heart. M. Koepe, H. Reinauer. Biochemical Department of the DiabetesForschungsinstitut 4 D/isseldorf, Auf'm Hennekamp 65, FRG. Isolated hearts of normal and diabetic rats were perfused with Krebs-Henseleit buffer. The following were recorded: left ventricular pressure, diastolic pressure, dp/dt, the aortic pressure, coronary flow, oxygen consumption and glucose utilization. In the frozen heart tissue the energy rich compound glycogen, lactate, pyruvate, and the activity of the pyruvate hydrogenase complex were estimated. - Under low work conditions there is little difference between the heart function of normal or diabetic rats: In the latter, the heart contraction rate (dp/dt) and the content of creatine phosphate were reduced, and glycogen breakdown enhanced. Under high work conditions there is no difference in the glucose utilization of normal and diabetic rats with or without insulin addition to the perfusion medium. Insulin deficiency reduces the glycogen and creatine phosphate content to a higher degree. Lactate production is reduced in the hearts of diabetic rats with or without insulin. - The reduced glucose utilization in diabetic rats can be stimulated to a normal extent by high work. 146. The Natural History of Diabetic Maculopathy. ' E~MI Kohner,J. Grindle and H. Cheng. Hammersmith Hospital, Du Cane Road, London W12 OHS, U. K. Maculopathy is the commonest cause of visual loss in diabetic retinopathy. It is defined as visual loss due to macular oedema in the presence of hard exudates and/or micro-aneurysms and haemorrhages. This study reports 75 patients with this condition followed for at least 1 year and having at least one eye untreated. In most patients the visual acuity deteriorated. The mean initial visual acuity was significantly better than that at 1-2-3-4-5 years (p < 0.001 - < 0.05). - Features indicating poor visual prognosis were: extensive haemorrhages in comparison with hard exudates, the worst prognosis being in those who had hardly any hard exudates visible, but extensive oedema. - On fluorescein angiography those with good capillary perfusion and focal rather than generalised leakage had the best prognosis. Peffusion of the immediate peri-foveal capillaries was of less importance than large areas of peripheral non-perfusion. Eyes with this lesion were most likely to develop new vessels and vitreous haemorrhage. - It was not possible to predict patients who were likely to develop plaques at the macula. - It is concluded that if patients have deteriorating vision or large areas of non-perfusion treatment should not be delayed. 147. Studies on Insulin-Degrading Enzymes in Pancreatic Islets. K.-D. Kohnert, S. Ansorge, S. Schmidt, H. Jahr, H.-J. Hahn, H. Kirschke, H. Ziihlke and H. Fiedler. Central Institute for Diabetes, 2201 Karlsburg/Greifswald, GDR. The nature of insulin-degrading enzymes of pancreatic islets and their function in maintaining an intracellular insulin steady-state are as yet unknown. 125I-labelled insulin, glucagon and A-chain were used as tracers and the degradation of these substrates was followed by a precipitation technique with 5% trichloroacetic acid or by gel filtration. - Pancreatic islets of various rodents and man contain:

thiol-protein disulfide oxidoreductase (TPO) and lysosomal proteinase capable of degrading insulin in a stepwise manner. These enzymes were demonstrated using antibodies to TPO and lysosomal proteinases. Heavy metals, SH-reagents and antibodies to TPO abolished insulin-degrading activity of the islets. - In islets of fed, 12 h'-starved Wistar rats, albino mice and non-glycosuric spiny mice TPO-activity was found to be about 10 U/mg protein. Starving of Wistar rats for 72 h caused a decrease by 54%. TPO-activity in islets of glycosuric spiny mice and sand rats was 6.04 + 0.46 and 4.04 _+ 0.39 U/mg protein, respectively. 2 h after i.v. glucose loading (1 g/kg) TPO-activity was diminished to 2.09 + 0.39 U/mg protein in normal sand rats, whereas in the prediabetic group displaying weight gain and elevated blood glucose levels the activity increased from 3.37 + 1.08 to 13.92 + 1.75 U/mg protein. - Our results suggest that TPO and proteinases may play a role in the regulation of the intracellular insulin content.

148. Norepinephrine-lnduced Lipolysis in Diabetes: A New Aspect in Fuel Production at Rest and during Exercise. V. A. Koivisto, H. K./~kerblom. The Children's Hospital, University of Helsinki, and Department of Paediatrics, University of Oulu, Finland. The lipolytic effect of norepinephrine (NE) in adipose tissue in vitro and the lipolysis in vivo were studied before and after exercise in non-fasted rats with severe, untreated streptozotocin-diabetes. 1. Before exercise NE in increasing concentrations stimulated in vitro glycerol release to an equal extent from adipose tissue of nondiabetic and diabetic rats. However, the re-esterification of free fatty acids (FFA) in vitro was decreased by low concentrations of NE in diabetic but not in control adipose tissue. - 2. Exercise did not change the NE-induced glycerol release in vitro from diabetic adipose tissue. - 3. After exercise a high concentration of NE decreased the FFA re-esterification in diabetic but not in control adipose tissue. - 4. In diabetic animals the increase in plasma glycerol and FFA during exercise correlated inversely with the NE-induced release of glycerol (r = 0.94) and FFA (r = 0.85) from the adipose tissue of the same animals after exercise. - The lipolyric effect of NE in vitro is not increased in diabetic adipose tissue before or after exercise as compared to normal. However, by decreasing the FFA reesterification NE helps yield a better energy supply in insulin deficient animals.

149. Exercise-Induced Hormonal and Metabolic Changes in Diabetes. V. A. Koivisto, R.M. Nousiainen, E.A. Nikkil~i und J. Heikkil~i. The Children's Hospital and the Third and First Departments of Medicine, University of Helsinki, Helsinki, Finland. Exercise-induced metabolic and hormonal changes were studied in non-obese and obese untreated diabetic patients. The ergometric exercise load was 70% of the maximal working capacity and lasted for 30 rain. - 1. During exercise the serum immunoreactive insulin (IRI) level remained unchanged in control subjects and in obese diabetics, but decreased markedly in nonobese diabetics. The decrease of IRI correlated with the rise in heart rate in nonobese diabetics, who with similar work load showed a higher pulse rate than the subjects of the other groups. After exercise IRI rose to the pre-exercise level, and the magnitude of this rise correlated with the simultaneous increase of blood glucose (r = 0.91). - 2. In nonobese diabetics the rise of serum immunoreactive growth hormone (HGH) during exercise was higher than in controls, and it was inversely correlated with the pre-exercise level of IRI (r = -0.73). In obese diabetics, who all had increased basal IRI level, exercise induced no response in HGH. - 3. The plasma glycerol response to exercise was identical in obese and nonobese diabetic patients in spite of the lower IRI and higher HGH plasma levels in the latter group. - The exercise-induced fall of plasma IRI in nonobese diabetics might be accounted for by a high sympathetic stimulation induced by exercise. It is possible that in obese diabetics the high level of IRI prevents exercise-induced HGH secretion.

Abstracts

150. Neurological Findings in Juvenile Diabetics. A. D. Korczyn, M. Karp and Z. Laron. Department of Neurology and Counselling Centre for Juvenile Diabetics, Beilinson Hosp., Tel Aviv Univ. Med. School, Israel. Information is scarce concerning the prevalence of neurological abnormalities in patients with juvenile diabetes. It is a matter of controversy whether neuropathy in patients with diabetes is directly related to the degree and duration of the metabolic disturbances connected with this disease. - Over one hundred non-selected juvenile diabetics were subjected to a complete neurological examination. - Neurological abnormalities were present in about one third of this group. These were usually asymptomatic. In most cases abnormalities consisted of depressed tendon reflexes and distal sensory impairment. - Abnormal findings were less common in younger patients. The duration of the disease from the time of its diagnosis was not found to have influenced the neurological status. Males and females showed the same frequency of abnormal results. The relationship between the degree of control attained and the neurological deficits was complex and will be described and discussed in detail. 151. The Effect of Ethanol on Serum Insulin Concentrations in Portal, Hepatic and Peripheral Venous Blood. C. Ktihl, O. Andersen, S. Lindk~er Jensen and O. Vagn Nielsen. Department of Internal Medicine T, Bispebjerg Hospital and Department of Surgical Gastroenterology, Rigshospitalet, Copenhagen, Denmark. Recently, we found that infusion of ethanol induced elevated concentrations of insulin in peripheral venous blood during a subsequent IVGTT. The present study was initiated in order to investigate if the elevated insulin levels seen after ethanol infusion might, in part, be ascribed to a diminished hepatic insulin degradation rate. Anesthetized pigs were subjected to an IVGTT after infusion of ethanol (10 mM or 20 mM) or saline for 4 hrs. During the procedure venous blood drawn simultaneously from the portal-, hepatic- and jugular veins. Compared to saline, 10 mM of ethanol induced a threefold increment in the insulin secretory response to glucose in the portal blood (p < 0.01). However, 20 mM of ethanol did not elevate insulin levels further. Ethanol did not influence hepatic insulin degradation rate. The results suggest: 1. Ethanol elevates insulin concentration after glucose stimulation through an action directly on the beta-cell. 2. The potentiating effect of ethanol on glucose-induced insulin secretion is dose-dependent. 3. Ethanol does not affect hepatic insulin degradation. 152. Is Prolactin a Diabetogenic Hormone? R. Landgraf, A. Weissmann, R. H6rl and M. M. C. Landgraf-Leurs. II. Clinic of Internal Medicine, University of Munich, Munich, FRG. Prolactin (PRL) administration in animals and man can cause alterations in glucose metabolism. The aim of this study was to examine the long-term effects of PRL on glucose-induced insulin secretion in patients suffering from hyperprolactinemia and the short-term effects of PRL on glucose-induced insulin release in the perfused rat pancreas. - All patients (n = 17) with hyperprolactinemia (PRL-levels ranging from 1250 to 90000 ~tU/ml) had pituitary tumours, but had no signs of acromegaly nor were obese. There was no difference of basal blood glucose and insulin values between normal subjects and the hyperprolactinemic patients. After glucose loading (100 g) the patients with high PRL-levels showed a significant decrease in glucose tolerance and an increased insulin response. These alterations were not correlated with the height of the PRL-values. Bromocriptine (CB 154) which suppressed PRL-secretion improved glucose tolerance and significantly decreased glucose-induced insulin release in these patients. - Human PRL (1000 or 5000 ~tU/ml), which was purified from serum of hyperprolacfinemic patients by column chromatography, or CB 154 (20, 100 or 200 ~g%) had no significant influences on the biphasic insulin release due to glucose (20 mM) in the perfused pancreas. - These results suggest that PRL may decrease glucose utilisation by increasing the peripheral resistance to insulin and therefore may be added to the list of diabetogenic hormones.

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153. Insulin Response of Healthy and Diabetic Subjects to Oral Glucose Tolerance Test before and after the Administration o f Cinnarizine. I. L~ing jun., A. K~ldor, IV[. Stiitzel, B. Gach~ly, I. Bal~zsi, L. Littmann, Second and Third Departement of Medicine, Semmelweis University of Medicine, Budapest, Hungary. Recently experimental data have been published about the inhibition of glucose-stimulated insulin secretion by indole amines and about the potentiation of the same process by so called anti-amine drugs. We studied the effect of an often used anti-amine drug, cinnarizine (Stugeron) on glucose-stimulated insulin secretion in humans. Oral glucose tolerance tests with IRI estimation were performed in healthy and maturity-onset diabetic subjects before and after a four days course of 3 x 50 mg cinnarizine. Each patient served as his own control. - We could see a significant potentiation of glucose-stimulated insulin secretion in healthy individuals but no such effect could be demonstrated in the group of diabetic patients. - We suggest that these results also represent data concerning the possible role of monoamine activity influencing insulin secretion. We plan further investigations in search of an explanation of the different response of healthy and diabetic subjects to cinnarizine. 154. Physiological Action of Avian Pancreatic (Endocrine) Polypeptide (APP). D.R. Langslow, R.L. Hazelwood. Department of Biochemistry, University of Edinburgh Medical School, Edinburgh, U.K. APP is a small polypeptide found only in avian pancreas (Langslow et aL, Endocrinology, 1973) and possesses both gastric and metabolic effects (Hazelwood et al., Gen. Comp. Endocr., 1973). The objectives of the present study were to elucidate further any metabolic effect or an effect on plasma glucagon and insulin in response to APP. Adult male chickens were starved for 24 hours, anaesthetised, and the proventriculus cannulated for collection of gastric juice every 15 minutes. Blood samples were collected from a cannulated femoral artery over 60 minutes; injections were made into an alar vein. Control birds received 0.5 ml of saline (0.9%). Plasma glucose, FFA, uric acid, IRI and IRG concentrations were determined in response to 50 gg APP/kg body weight. Gastric volume was measured and acid, pepsin and total protein estimated. - The powerful gastric effect of APP was shown by an increase in volume flow (73.0%), pepsin (71.3%), acid (39.1%) and protein (37.2%). Plasma glucose, IRI and uric acid levels were unaffected by APP. FFA concentrations decreased immediately by 20% and continued to fall to 35% of initial values. These data are consistent with previous reports on plasma glycerol and glucose effects of APP. - It is concluded that, at this dose level, APP is a much more potent exocrine secretagogue than it is a metabolic/endocrine regulator. 155. Cells Storing "Pancreatic Polypeptide". Identification by Light and Electron Microscopy. L.-I. Larsson, F. Sundler and R. H~&anson. Depts. of Histology and Pharmacology, University of Lund, Lund, Sweden. While purifying chicken insulin Kimmel and coworkers (1968) isolated a 36 amino acid peptide which they named avian pancreatic polypeptide (APP). By radioimmunoassay this peptide was found to circulate in plasma where its levels varied with the prandial state. In chickens, APP stimulated gastric acid secretion and possibly also hepatic lipid synthesis. Analogous peptides have subsequently been isolated from the pancreas of mammals. Using antisera to human and bovine pancreatic polypeptide (HPP and BPP respectively) we have succeeded in identifying the cells storing pancreatic polypeptide in a number of mammals, including man. The cells share many features with known peptide hormone-producing cells including the content of characteristic cytoplasmic granules and the ability to take up and decarboxylate administered amine precursors. They are distinct from the A, B and D cells and often predominate in the duodenal lobe ("uncinate process"). In some mammals they occur also in the gastrointestinal tract. - Available data support the alleged hormonal role of the pancreatic polypeptide. Its physiological significance remains to be established.

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Abstracts

156. Insulin Biosynthesis and Secretion in "Sucrose-Diabetic" Rats. H. Laube, H. Schatz, Ch. Nierle, E.F. Pfeiffer. Department of Endocrinology and Metabolism, Center of Internal Medicine, University of Ulm, FRG. Following prolonged feeding of sucrose and other fructose containing diets glucose assimilation in rats has been shown to be markedly delayed. Despite a diabetic glucose tolerance, substrate induced insulin secretion was found to be enhanced in Vivo and in vitro as well. The following experiments were conducted to find out if the state of hyperinsulinemia was correlated to a corresponding increase in insulin biosynthesis. - In sucrose and starch fed rats, insulin secretion was examined in the perfused pancreas and in isolated islets of Langerhans, following perfusion and incubation containing various glucose concentrations. Incorporation of 3H-leucin into the proinsulin and insulin fraction of the islet protein was taken as an index of insulin synthesis. - Following sucrose feeding, insulin secretion was markedly enhanced, compared to a starch rich diet. As in other kinds of diabetes glucagon levels were elevated. The incorporation of 3H-leucin into proinsulin increased with rising glucose concentrations and was significantly higher in animals kept on a sucrose rich diet. Since fructose alone did not enhance insulinbiosynthesis, the present findings suggest that the effect of sucrose is an indirect one and caused by the immediate postprandial impact of glucose on the ~cells. 157. The Course of Diabetic Retinopathy: a Statistical Study on Its Development, Progression and Regression in 4.400 Diabetics. J. P. Lauvaux and J. Pirart, C. Eisendrath. H6pital Saint-Pierre and Clinique Cesar de Paepe-Brussels, Belgium. The aims of this study were the assessment of the natural history of retinopathy and of the possible role of glycemic control. Ophthalmoscopic examination was carried out at least yearly and methods and criteria standardized. Coded data were submitted to statistical analysis (C.D.C. Computer). - Preliminary results of this prospective study conducted from 1947 to 1973 have been present (I. D. F. Brussels 1973, EASD Jerusalem 1974): Prevalence of retinopathy is strongly related to known duration and to glycemic control, assessed each year and expressed as a cumulative value for the whole duration. - The yearly development rate (incidence), progression rate, and regression rate (proliferative retinopathy excluded) have now been studied. - The incidence of retinopathy increases with duration of diabetes and is strongly related to the cumulative and the yearly degree of control. - Increased progression rates and reduced regression rate are related to poor cumulative control or to poor yearly control. - It is concluded that chronic hyperglycemia is the etiological factor of diabetic retinopathy. - Long term and even short term (one year) alleviation of hyperglycemia has a benefical influence on the course of retinopathy. 158. Reversible Enhancement of Glucagon Release during Calcium Deprivation: Comparison with the Effect of Verapamil. V. Leclercq-Meyer, J. Marchand. Laboratory of Experimental Medicine (Head: Prof. W.J. Malaisse), Free University of Brussels, Brussels, Belgium. We recently observed that calcium deprivation causes an immediate increase in glucagon secretion by the isolated perfused rat pancreas, this enhancement being positively related to the glucose concentration (3.3, 5.5, 8.3 and 16.6 mM) of the perfusate. A further investigation of this paradoxical phenomenon revealed that the enhancement of glucagon release was, at all glucose levels, rapidly reversed on restoration of a normal calcium concentration. However, verapamil (10 gM), which is thought to inhibit inward calcium transport in many tissues and was found to inhibit insulin release, had little if any effect upon glucagon secretion at both 3.3 and 16.6 mM glucose levels. - These results suggest that the exquisite sensitivity of the a-cell to changes in calcium concentration is not the consequence of calcium depletion in the adjacent t-cell. Nevertheless, and although the possible influence of verapamil upon calcium handling by the a-cell remains to be assessed, our data would be compatible with a direct and localized

effect of extracellular rather than intracellular calcium in the determination of the observed phenomenon, pointing for instance towards a possible role for altered intercellular binding in the paradoxical behaviour of the a-cell during calcium deprivation.

159. Diabetic Macroangiopathy: in Vitro Study of the Growth of Aortic Smooth Muscle Cells Propagated in Human Serum from Young Juvenile Diabetics. T. Ledet. Department of Pathology and the Second Clinic of Internal Medicine, Kommunehospitalet, 8000-Aarhus C, Denmark. Earlier studies have shown a significant effect of diabetic rabbit serum on growth of aortic smooth muscle cells in vitro (Ledet et al. Am. J. Path. 74:50a, 1974). This paper describes the effect of human diabetic serum (dialysed and non'-dialysed) on growth of aortic medial cell cultures. - Primary explants of rabbit aortic smooth muscle cells were propagated in tissue cultures. Growth medium (BME) was supplemented with: a) serum from untreated and short-term treated (3 days) diabetics, b) dialysed and non-dialysed serum from treated diabetics, c) normal serum with and without supplementation with 50 ~tU/ml. 100 ~U/ml and 200 ~U/ml Insulin Leo Neutral | The glucose concentration in the fasting serum was 79 mg% (control), 120 mg% (treated) 257 mg% (untreated-diabetics) and 258 mg% (short-term treated). The lipid values were normal, and the serum-insulin in the diabetic serum 0 ~U/ml (untreated), 6 ~tU/ml (short-term treated diabetics) and 12 ~tU/ml in the normal serum. - Serum from untreated and treated diabetics stimulated significantly the outgrowth of aortic smooth muscle cells (2 p < 0.01). The growth was also enhanced significantly by dialysed diabetic serum (2 p < 0.01). The growth was the same in normal with and without insulin addition. - If this in vitro model has an in vivo counterpart, human diabetic macroangiopathy cannot be imputed to increased glucose, increased ketone-bodies, increased seruminsulin or increased lipids. 160. Glucagon Metabolism by the Isolated Pertused Dog Kidney. P.J. Lefebvre, A.S. Luyckx and A. H. Nizet. Institute of Medicine, University of Li6ge, 4000 Liege, Belgium. Previous studies from our laboratory using dog kidneys acutely transplanted to the neck vessels of a perfused anesthetized dog demonstrated a significant kidney glucagon uptake, averaging 89 + 14 pg/min/g of kidney under basal conditions. Within the limits of arterial plasma glucagon levels reached in these experiments, a correlation between arterial glucagon and kidney glucagon uptake was not found. We speculated that this might be due to the relatively narrow range of arterial glucagon concentrations ( 5 0 - 2 5 0 pg/ml) reached in these studies. We now report on the effect of increased plasma glucagon levels on kidney glucagon uptake using isolated perfused dog kidneys infused with exogenous glucagon at doses ranging from 5 to 30 ng/min. This resulted in arterial plasma glucagon concentrations ranging from less than 150 pg/ml in control experiments to 1200 pg/ml when the maximal glucagon dose was used. The results indicated that kidney glucagon uptake was highly and significantly correlated with arterial plasma glucagon (r = 0.925, 32 determinations) or the quantity of glucagon entering the kidneys per rain (r = 0.958, 32 determinations). The regression line for arterial plasma glucagon (y) on kidney glucagon uptake (x) corresponded to the equation y = 115 + 0.535 x, indicating that no significant kidney glucagon uptake was observed for arterial plasma glucagon values less than 115 pg/ml. - These studies demonstrate for the first time that increasing arterial plasma glucagon is accompanied by a proportional increase in renal glucagon uptake. 161. Visualization by an Immunocytochemical Method of Insulin Binding to Cultured Human HeLa Cells. F. Legros, P. Uytenhoef and V. Conrad. Laboratoire de Physiologie et de Physiopathologie 2, rue Evers, 1000 Brussels, Belgium. Human HeLa cells in culture have been used in order to study insulin binding at the membrane level. - HeLa cells are grown as monolayers on microscope cover slides in Ham's F12 medium enriched with calf serum. After 24 hours at 37 ~ C, cells are incubated for 30 minutes in the same medium without or with native

Abstracts porcine insulin at the concentration of 3.10 -7 M. After incubation, cells are fixed in 1% paraformaldehyde. Fixed cells are successively treated with guinea pig antiporcine insulin serum and sheep peroxidase labelled anti-rabbit IG antibody. Peroxidase catalyzing oxidation of 3-3'-diaminobenzidine in the presence of H202 visualizes insulin specific bindings. - Cellular localization of hormone fixation is observed for cells incubated in insulin 3.10 - 7 M as well as for cells cultured in Ham's F12 medium enriched with calf serum. Control slides which have not been submitted to anti-insulin serum after fixation are not stained. Immunocytochemical treatment is managed so that spontaneous oxidation of diaminobenzidine is avoided. When 3.10 -7 M insulin is added to the medium, the number of revealed binding sites is significantly more numerous than for cells cultured in Ham's F12 medium enriched with calf serum. - It may be concluded that HeLa ceils possess specific receptor sites for insulin. The enrichment of the medium with calf serum provides minute amounts of immunologically active hormone molecules. Variations in reaction intensity between ceils treated with exogenous insulin and cells incubated in the medium might be used for measurements of cell receptivity to insulin. 162. Selective and Reversible Inhibition of Glucose-Induced lnsulin Secretion Caused by Thyroxine Treatment. S. Lenzen, H.G. Joost, J. Beckmann, A. Hasselblatt. Institut fiir Pharmakologie und Toxikologie, Universit~it G6ttingen, G6ttingen, FRG. Thyroxine and triiodothyronine treatment inhibit the late phase of glucose-induced insulin release from the peffused rat pancreas whereas propylthiouracil treatment and thyroidectomy result in a stimulation. - Thyroxine (600 ~g/kg b. wt./day, for five days) treatment inhibits selectively glucose-induced insulin secretion. Tolbutamide (20 rag%), D-glyceraldehyde (10 mM), and D-mannose (16.7 mM) are glucose-independent insulin secretagogues and induce secretory patterns distinctly different from D-glucose (16.7 mM). Insulin release due to these secretagogues is not inhibited by thyroxine treatment. - Several substances were examined for their ability to reverse inhibition of glucose-induced insulin secretion caused by thyroxine treatment. Some are not calorigenic (tolbutamide 20 rag%, L-glucose 16.7 raM), others are calorigenic and provide energy via glycolysis (D-fructose 16.7 raM, DL-glyceric acid 10 raM, pyruvate 15 mM) or by some other mechanism (propionic acid 10 raM, a-ketobutyrate 10 mM). - Out of all these substances only c~-ketobutyrate and as we have shown before (Diabetologia 11, 4 9 - 5 5 , 1975) pyruvate are able to restore impaired glucose-induced insulin secretion in thyroxine treated rats. - The results indicate that the defect of glucoseinduced insulin secretion after thyroxine treatment may not solely be attributed to involvement of thyroxine with glycolysis. 163. Excessive Adipose Tissue Hypertrophy in Maturity Onset-Diabetics in Relation to Weight Reduction. W. Leonhardt, M. Hanefeld, H. Hailer, H. Schneider. Medical Clinic of Medical Academy, 8019 Dresden, Fetscherstr. 74, GDR. In relation to their degree of obesity, maturity onsetdiabetics (MOD) have larger adipocytes than controls. We tested whether weight reduction diminished the adipocyte volumes in both groups to a different degree. - 23 obese MOD (15 females and 8 males) and 25 obese controls (17 females and 8 males) were reduced in body weight (minimum 4 kg) during a stationary regime of 4 to 6 weeks. Before and after treatment, adipose tissue was biopsied from the subcutaneous adipose tissue of the abdomen. Volumes of isolated adipocytes were measured by pulse counting. Initial weight (85.8 kg MOD, 111.3 kg controls) and weight loss obtained (7.7 kg MOD, 12.5 kg controls) were smaller in MOD than in controls (p < 0.01). By contrast, initial adipocyte volume (0.64 nl MOD, 0.55 nl controls) and volume reduction (0.18 nl MOD, 0.08 nl controls) were highest in MOD (p < 0.05). Adipocyte volumes were also compared with normal values _+ 2sd based on ideal weight and sex, obtained in 142 controls. In MOD, volumes before weight reduction were in 67% above 2sd and after weight reduction in 91% within 2sd. A certain weight loss in MOD had a much stronger effect

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upon reduction of adipocyte volumes than in controls. Obese MOD exhibit excessive adipose tissue hypertrophy which reduces to the hypertrophy of obese controls following weight reduction. 164. Isolation of Rat Islet Plasma Membranes Using Specific Labelling of Cell Surface Receptors as a Marker. A. Lernmark (Ume~, Sweden), D. F. Steiner (Chicago, Illinois, USA), H. Ziihlke (Karlsburg, GDR). Department of Biochemistry, University of Chicago, Chicago, Illinois, USA. Pancreata from pilocarpine-injected rats were digested with collagenase and the islets separated on Ficoll gradients. Batches of 1000-2000 islets were incubated with 10-9 M highly radioactive 125I-labeled wheat germ agglutinin (125I-WGA). The islets were washed and lysed by mechanical shaking in a hypotonic buffer and the lysate was then subjected to two consecutive isopycnic centrifugations on continuous sucrose gradients. The radioactivity was found in a major peak having a density in sucrose of 1.09 and coincident with peak activities of 5'-nucleotidase and Mg2r-ATPase. In the electron microscope this fraction contained irregular, small vesicles free of other particles or organelles. Higher specific binding was found after labeling lysates rather than whole islets, perhaps due to slower penetration of WGA into the extracellular space of islets. Bound 125I-WGA could be displaced from the membrane vesicles by treatment with N-acetylD-glucosamine but not with D-glucose. The membrane fraction contained no significant amounts of DNA and RNA, less than 2% of the acid phosphatase and carboxypeptidase B-like activities of the whole lysate and roughly 5 % of the totalinsulin. It is concluded that 125I-WGA is useful as a specific marker for islet plasma membranes. Possible applications of this finding to the isolation, purification and further characterization of islet membranes are now under study. 165. Cation Dependent ATPases in Rat Islet Cell Membranes. G. L6ffler, W. Kemmler. Diabetes Research Unit and 3rd Med. Department, City Hospital, 8 Munich 40, K61ner Plata 1, FRG. The dependence of insulin secretion upon different cations has been demonstrated repeatedly. It was assumed, that the trigger for the secretory process is the transmembraneous transport of cations, especially Na +, K + and Ca + § which is catalyzed by cation dependent ATPases. We therefore tried to identify these enzymes in subcellular fractions of islets. Besides the high activity of a Mg § § ATPase, which works equally well with Ca -~ -Iinstead + §of Mg ~-/-, we were now able to characterize § a Na -K -ATPase, which is active only in the presence of Mg . Maximal activities are achieved if the ratio of Na + :K § is kept at 10:1. This activity is inhibited by ouabain at ~_d~se of 0.1 m M - 1 mM. Na-azide, wl~ich s~rongly inhibits the big -ATPase, h~sgo effect on the Na-r-Kn--ATPase. A Mgn- 7- dependent Ca n- n-ATPase similar to the calcium transport enzyme described in erythrocyte membranes or sarcoplasmic reticulu~, 9ould not be detected. However, it was possible to obtaina Can- -t-_stimulated ATPase activity in the presence of 6 mM Mg q- § 50 mM Na q- and 5 mM K +, when the N a + - K § was blocked by prior addition of I mM ouabain. We conclude from these results, that cation transporting enzymes exist in islet tissue and may be important for the secretory process. 166. Reversibility of Chemical Diabetes. A.W. Logie, J.M. Stowers and I. Dingwall-Fordyce. Diabetic Clinic, Aberdeen Royal Infirmary and University of Aberdeen, U. K. The sulphonylureas can reverse mild diabetes in experimental animals by inducing pancreatic beta cell hyperplasia. A trial was started in 1960 to see if such a result could be produced in patients with chemical (asymptomatic) diabetes. - Potential diabetic patients of near-normal weight have been tested under basal conditions with intravenous glucose (IVGTT) and included in the trial if chemical diabetes was found. Treatment was with Chlorproparnide in, a variable dose and assessment by sequential IVGTTs, at approximately annual intervals, having discontinued for three weeks. - 42 of the 446 patients developed overt diabetes and were

360

Abstracts

withdrawn from the trial. The IVGTT of all 446 patients improved to a mean normal figure, the better results occurring in those under 35 years at the start of treatment. After good improvement Chlorpropamide was discontinued in 82 patients for 1 - 2 years. If the IVGTT reverted to abnormal a further course of Chlorpropamide was given in 23 with subsequent return to normal glucose tolerance. A few subjects remain in remission for more than 8 years. The 49 deaths have been analysed in view of the UGDP experience. - The improvement in glucose tolerance noted in this study will be compared with the results of the control group treated by placebo. 167. Interaction between Glucose and Adrenergic and Cholinergic Drugs on Glucagon Secretion. Studies on the Isolated Perfused Rat Pancreas. A.-L. Loubati~res, M.-M. Loubati~res-Mariani, J. Chapal. Laboratoire de Pharmacologie et Pharmacodynamie, Facult6 de M6decine 34060 Montpellier Cedex, France. Glucagon secretion which was maximal at glucose concentrations up to 0.5 g/1, rapidly decreased at glucose concentrations ranging from 0.5 to 1.5 g/l. - At glucose 0.5 g/1 isoprenaline (0.05 ~tM) provoked a transient stimulation of glucagon secretion; this effect was suppressed by propranolol. A stimulation of the same type was observed with salbutamol, a more specific stimulator of the receptors. The isoprenaline induced stimulation did not occur at glucose 1.5 g/1. Epinephrine and norepinephrine (0.011 ~tM) did not modify glucagon secretion. Acetylcholine (0.5 9M) provoked a strong and lasting stimulation of glucagon secretion in the presence of the two glucose concentrations used (0.5 and 1.5 g/l). This effect was suppressed by atropine. - Conclusion: The acell of the islets of Langerhans of the rat appears to be endowed with fl-adrenergic receptors (/~ type receptors), the activation of which stimulates glucagon secretion only at low glucose concentration. In our experimental conditions we could not demonstrate the presence of a adrenergic receptors. The a cell is endowed with cholinergic (muscarinic) receptors which seem to have a more important function than the/3 adrenergic receptors. Their action does not depend upon the glucose concentration in the two concentrations studied (0.5 g/1 and 1.5 g/l). 168. A Possible Mechanism for Raised Insulin Production in Utero in Rhesus Affected Infants. C. Lowy, R.A. Basschus, A.P. Sadler, D. K. Yeoman. Department of Chemical Pathology and Metabolic Diseases, St. Thomas's Hospital, Lambeth Palace Road, London. S. E. 1, U. K. Hyperplasia of the pancreatic islets, hyperinsulinaemia and hyperinsulinuria are well recognised in Rhesus affected infants. Since the anaemic foetus may have an increased blood flow perfusing the placenta, we have investigated the uptake of foetal insulin by the placenta and transfer of glucose in relation to blood flow. Laparotomies were performed on pregnant guinea pigs and the umbilical vessels were cannulated. The foetus was discarded. The placenta was perfused with a solution containing 4 grams % albumin, 100 ~U of human or guinea pig insulin and glucose. Placental blood flow was varied from 0.8 to 3.0 mls per minute. - Foetal insulin uptake by the placenta varied from animal to animal ( 1 0 - 30%). The % uptake was relatively independant of the perfusion rate resulting in an absolute greater foetal insulin loss at the higher perfusion rate. Maternal foetal glucose transfer was dependent both on maternal plasma glucose concentration and foetal blood flow. Transfer of lmg/minute could be achieved either at maternal plasma glucose concentrations of 88 or 152 rag/100 ml and at corresponding foetal blood flow rates of 2.5 and 0.87 mls/minute. - In the anaemic foetus a higher clearance of insulin and an enhanced transfer of glucose may lead to compensatory increase in insulin production. 169. Action of Glucose and Insulinotropic Drugs on Glucose Induced Insulin Secretion. M. Lucas, J. Tamarit-Rodriguez, F. Sobrino, R. Goberna and L Tamarit. Departamento de Fisiologia y Bioquimica, Facultad de Medicina, Universidad Complutense, Madrid-3, Spain. The nutritional state is known to effect insulin secretory response to

glucose. We have studied the effect of preperfusion, in different conditions, upon insulin secretion in isolated and perfused pancreases of rats.The pancreases of rats (16 hours fasted) were isolated and perfused according to Sussman. The effluent was collected at two minutes interval. Pancreases were perfused for two different periods of 30 rain. The pancreatic insulin response provoked by 10 mM glucose was tested at the second period after 30 min of previous perfusion performed with: a) glucose free medium; b) glucose free medium plus i mM theophyline; c) glucose free medium plus 1 mM aminophyline; d) glucose free medium plus 0.15 mM sulphonylurea glipentide. - The same series of perfusions were performed adding 5 mM glucose during the first period. - The absence of glucose during the first 30 min diminished the insulin response to 10 mM glucose. The insulinotropic agents mentioned above do not compensate for the absence of glucose, moreover they inhibit significantly (p < 0.01) the pancreatic response to 10 mM glucose. - If 5 mM glucose is present during the first period these effects are reversed and insulinotropic agents potentiate the action of glucose. - Our data confirm the important relationship between initial glucose levels and pancreatic response to glucose. The insulinotropic effect of studied drugs may be conditioned for a normal glucose supply. The possible alteration of B-cell metabolism caused by glucose lack may interact with the normal mechanism of action of these drugs. 170. Influence of Somatostatin (SRIF) on Nicotinic Acid-Induced Changes in Blood Glucose (BG), Free Fatty Acid (FFA), Glucagon (PG), Growth Hormone (GH) and Cortisoi (Co) in Diabetic Subjects. A.S. Luyckx, P. J. Lefebvre. Diabetes Section, University of Li6ge, Liege, Belgium. It has been demonstrated that nicotinic acid (NA) infusion induces a decrease in plasma FFA and an increase in plasma PG, GH and Co in normal subjects. - The present study aimed at investigating the effect of NA in overnight fasted insulin-dependent diabetic subjects. - The metabolic and hormonal responses to NA were evaluated in the same subjects after and during an infusion of SRIF. For control tests, NA was given alone at a dose of 100 mg in 3 rain, followed by a infusion of 1 g NA/hr during 60 rain and then 0.5 g/hr during 30 min. When SRIF was used, 250 gg linear SRIF (Roussel Uclaf) were injected acutely followed by an infusion of 10 jxg/min for 75 rain, NA administration, was begun 30 rain after SRIF injection. - After 30 min, NA alone induced a significant decrease in plasma FFA ( - 272 ~tEq/I) and significant increments in PG (+ 29.4 pg/ml), GH (+ 5.6 ng/ml) and Co (+ 2.6 ~tg/100 ml). BG remained unchanged. , After 30 rain, SRIF alone induced a significant decrease in BG ( - 1 6 . 3 rag/100 ml) and significant decrements in PG ( - 34.4 pg/ml), GH ( - 1.6 ng/ml) and Co ( - 2.8 9g/100 ml). Plasma FFA remained unchanged. - SRIF did not modify the NA-induced fall in plasma FFA, but completely blocked the corresponding increments in PG and GH. On the contrary, the NA-induced ris6 in plasma Co persisted during SRIF infusion. 171. Intravenous Glucose Tolerance after Prolonged Exercise in Normal and Diabetic Men. The Role of the Liver. S. Ma~hlum, J. Jervell and E. D. R. Pruett. Institute of Work Physiology, Oslo, Norway, Med. Dept. B, Rikshospitalet, Oslo, Norway. The role of the liver in the elimination of glucose during an intravenous glucose tolerance test (IVGTT) after exercise was studied in 5 nondiabetic and 4 insulin dependent diabetic subjects. - They reported to the laboratory in the morning in the postabsorbtive state; the diabetic subjects also without their morning insulin dose. Catheters were introduced into a hepatic vein and the femoral artery. The subjects then worked continuously until exhaustion on the bicycle ergometer at a work load representing 70% of their maximal oxygen uptake. 15 min after the cessation of exercise 0.5 g glucose • kg- 1body weight was infused intravenously over a period of 8 minutes. Glucose dissappearance rate (GDR) was determined from blood sampled 34, 49, 64 and 79 rain after the start of the infusion. - Mean GDR was 0.38 and 1.65 % • min-1 for the diabetic and nondiabetic subjects respectively. Mean arterio-

Abstracts hepatic-venous glucose differences were small but negative throughout the whole postexercise period, indicating a release of glucose into the blood from the splanchnic region. - It is concluded that the liver is of no significance in eliminating glucose during an IVGTF after prolonged exercise in diabetic and non-diabetic man. 172. Rate of Glycogen Synthesis in Skeletal Muscle after Exercise in Normal and Diabetic Man. S. M~ehlum, O. Vaage, L. B. Enges~eter and L. Hermansen. Institute of Work Physiology, Oslo, Norway. The rate of glycogen synthesis in skeletal muscle after prolonged exercise was studied in 6 nondiabetic (ND) and 6 diabetic (D) subjects, the latter taking their regular insulin. Both groups exercised to exhaustion on the bicycle ergometer, the work load representing 70% of maximal oxygen uptake. During the recovery period (12 hrs) the subjects rested, ingesting a carbohydraterich diet. - Muscle glycogen content (MGC) was determined in biopsy samples from the lateral part of quadriceps muscle before exercise, immediately after and 2, 4, 6, 9 and 12 hours after cessation of exercise. - MGC decreased from 75.6 (ND) and 62.9 (D) mmoles glucosyl units • kg -1 w.w. at rest to 15.0 mmoles glucosyl units • kg -~ w.w. in both groups after exercise. After 4 hours of recovery MGC had increased to 43.6 (ND) and 40.6 (D) and after 12 hours to 62.4 (ND) and 55.4 (D) mmoles glucosyl units x kg -~ w.w. Glycogen synthesis rate during the initial 4 hours of recovery was rapid (i. e. 7.2 (ND) and 6.4 (D) mmoles glucosyl units x kg-! w.w. x hr -1) as compared with the last 8 hours (i. e. 2.4 (ND) and 1.9 (D) mmoles glucosyl units x kg -~ w.w. x hr-1). - There was no statistically significant difference in glycogen synthesis rate between the two groups. 173. Secretion of Insulin by Glycogen and Collagen in Vitro. V. Maier, J.J. Gagliardino, A. Goldmann and E.F. Pfeiffer. Dept. of Internal Medicine, Endocrinology and Metabolism, University of Ulm, FRG. It has not yet been elucidated whether the glucose molecule as such or some of its metabolites trigger insulin secretion. Hence, we started from the consideration that non-phagocytosable substances with carbohydrate structures might be helpful in solving this problem. - Pancreatic islets of rats were isolated according to Lacy and incubated in Krebs-Ringer albumin bicarbonate buffer in presence of tritiated leucine. - The secretion of insulin was stimulated by 3 mg/ml glycogen (60%) and compared to glucose. 14C-Glycogen was not metabolized to radioactive CO 2. - Normal collagen containing 0,5% carbohydrates did not stimulate insulin release. However, collagen from Actinia with a carbohydrate content of 22% ( . . . lys-OH-gal-glc) was active to the same extent as glycogen. - Proinsulin and insulin were not labelled by tritiated leucine in the presence of glycogen or collagen indicating that these substances were not degraded to glucose, which stimulates the incorporation of leucine to insulin. - Conclusion: A specific conformation of hydroxyl-groups is capable of stimulating the release of insulin; this lends further support to a receptor hypothesis. 174. Fasting-Induced Repression of Key Glyeolytic Enzymes in Pancreatic Islets. W.J. Malaisse*, A. Sener, J. Levy*. Laboratory of Experimental Medicine, Brussels University, Brussels, Belgium. The finding that both the metabolism and insulinotropic capacity of glucose are impaired in islets from fasted rats, whereas the response to glyceraldehyde is unaffected, led us to postulate that fasting inhibits the early steps of glycolysis in the B-cell (Diabetologia 10, 377, 1974). The present work aims at a further characterization of this metabolic abnormality. - At two ATP levels (0.1 and 5.0 raM) and over a range of glucose concentrations (0.1 to 100.0 raM), the velocity of glucose phosphorylation by islet homogenates from fed rats was compatible with the presence of high (glucokinase) and low (hexokinase) Km enzymes. The fractional contribution of hexokinase was reduced at the low ATP level. Mannoheptulose caused competitive-like inhibition of phosphorylation in islets from both fed and fasted rats. Fasting reduced the rate of phosphorylation by 18 to 33% when hexokinase was the prevailing active enzyme. Glucokinase activity (Vmax) was much more reduced (by 63%), but its Km was unaffected by fasting. The preferential re-

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pression of glucokinase could be statistically documented by comparing the velocity of phosphorylation at 0.1 and 100.0 mM glucose concentrations. - In islets from fed rats, phosphofructokinase activity could be characterized by dependency on ATP, inhibition by citrate, and relief from ATP inhibition by AMP. Under these various experimental conditions, the activity of phosphofructokinase was invariably reduced by fasting. - Our findings suggest that key glycolytic enzymes in the B-cell are susceptible to induction-repression regulation by environmental factors. The influence of fasting upon such enzymes might account for altered glycolytic flux and, hence, decreased calcium net uptake and subsequently reduced insulin release in response to glucose. (* Recipients of 1974 and 1975 Pfizer-Europe Travel Fellowships of the E.A.S.D.). 175. Evidence for Topographic Partition of Secretory Function in the Exocrine Pancreas. F. Malaisse-Lagae, P. Robberecht, A. Vandermeers and M. Ravazzola. Inst. of Histology and Embryology, and Biochimie Clinique, Geneva, and Laboratory of Experimental Medicine and Dept. of Biochemistry, Brussels, Belgium. In the pancreas of many species, distinct aeini surround the islets of Langerhans. Such periinsular halos are composed of large ceils and can be distinguished from the rest or teleinsular part of the exocrine pancreas by their tinctorial properties. This observation stimulated us to investigate the possible functional hetereogeneity between periinsular and teleinsular exocrine pancreas. - Hydrolases concentrations were measured in small fragments of teleinsular exocrine tissue, and in islets with attached periinsular exocrine tissue, respectively. The pancreas were derived from spiny mice, normal or diabetic rats. In order to correct for the variable amount of acinar cells present in each homogenate, the concentrations of amylase, lipase and chymotrypsinogen were expressed relative to each other. In each animal, we have calculated the ratio of teleinsular to periinsular values for the three relative concentrations. In the three groups of animals, 2 or all 3 of the computed ratios significantly differed from unity. For instance, the concentration of amylase relative to that of lipase was invariably higher in the teleinsular than in the periinsular exocrine tissue in pancreas from spiny mice, normal or diabetic rats. - These findings raise the possibility of a functional compartmentation of the exocrine pancreas, e. g. in modulating the composition of the pancreatic juice. 176. The Role of Cyclic-AMP Levels in Glucose-Stimulated Proinsulin Synthesis in Rat Islets of Langerhans. A. Maldonato. Institut de Biochimie Clinique, Geneva, Switzerland. Cyclic-AMP is of importance in glucose-induced insulin release (IR), whereas its role in proinsulin synthesis (PI) remains poorly defined. Accordingly, PI, IR and cAMP production were measured simultaneously in paired batches of pooled islets. - Production of cAMP was estimated according to Grill and Cerasi (1974) using 3 H-adenine incorporation into tissue and medium 3H-cAMP after 60 min at different glucose concentrations and with 0, 0.1 or 1 mM IBMX. The other batch of each pair was treated identically, 3H-adenine being omitted, and 3H-leucine being added at 30 min. Proinsulin was isolated by affinity chromatography. - IR and PI relations to glucose concentrations differed markedly: stimulation threshold for IR was about 1.25 mg/ml glucose, for PI already 0.5 mg/ml. Medium accumulation of 3H-cAMP paralleled IR. IBMX augmented Vmax and decreased apparent "Kin" of IR and 3H-cAMP responses. The phosphodiesterase inhibitor also enhanced PI, mainly at lower glucose concentrations (0.25-1.0 mg/ml), the relative effect of IBMX decreasing with increasing glucose. For each glucose concentration, any cAMP increase was associated with increased PI. However, changes in glucose not associated with cAMP stimulation did enhance PI. - It is concluded that, although glucose may control PI without altering islet [cAMP], the nucleotide does influence the setting of glucose control of PI. 177. Gonadal Steroid Effects on Plasma Lipoproteins: Alteration in the Insulin to Glucagon Molar Ratio. T. M. Mandour, A. H. Kissebah, V. Wynn. The Alexander Simpson Laboratory for Metabolic Research, St. Mary's Hospital Medical School, London, W. 2, U. K.

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Abstracts

The administration of 17-B-oestradiol or 5a-dihydrotestosterone to female rats readily induced a rise a plasma triglyceride concentration. We have found that pharmacological doses of either 17-Boestradiol (5 ~tg/kg) or 5 a-dihydrostosterone (4 mg/kg) injected into gonadectomised female rats increases the turnover rate of apo-VLDL peptides, the main carrier of plasma endogenous triglycerides. Progesterone (500 gg/kg), on the other hand, had no significant effect. - Since insulin and glucagon seem to exert antagonistic effects on the hepatic synthesis of plasma VLDL apoproteins, studies were performed to assess alterations in these hormones as a possible mechanism for the steroid effects. Oestrogen therapy lowered the levels of both insulin and glucagon in samples of portal vein blood of overnight fasted rats. The insulin to glucagon molar ratio (I/G), however, was increased 3 - 4 fold. Similarly dihydrotestosterone produced an increase in I/G ratio in portal vein blood though the absolute concentrations of these hormones were markedly increased. Progesterone, on the other hand, did not alter the I/G ratio despite mild elevation in the fasting levels of both insulin and glucagon. - The relation between the elevation in I/G ratio on one hand and the increased plasma flux of VLDL apo-protein on the other suggest that the effects of these steroids on plasma triglyceride level might be mediated by alteration in the portal blood I/G ratio rather than the absolute levels of these hormones. 178. Effect of Somatostation on Serum Insulin and Glucose in Normal and Obese-Hyperglycaemic Mice. F. M~irki, J. Motz and M. Schindlery. Research Department, Pharmaceuticals Division, CIBA-GEIGY Limited, 4002 Basel, Switzerland. The effect of a single subcutaneous injection of synthetic cyclic somatostatin on serum immunoreactive insulin and serum glucose was studied in normal albino mice and Bar Harbor obesehyperglycaemic mice (C 57 BL/6j-ob/ob). - In normal mice, somatostatin (1 mg/kg) decreased serum insulin to less than 30% of control within 10 minutes, with a gradual return to control values within 1 hr. The minimum effective dose was 0.1 mg/kg; it was reduced to 0.03 mg/kg by administration of a glucose load of 1 g/kg s.c. simultaneously with somatostatin. Serum glucose was not influenced by somatostatin. - In obese-hyperglycaemic mice with serum insulin levels more than 10 times higher than those found in normal mice, somatostatin in doses between 0.03 and 1 mg/kg decreased insulin to a degree comparable to that seen in normal mice. However, in contrast to the response shown by normal mice, serum glucose was lowered by as much as 50%. - Somatostatin treatment thus unmasks a difference in glucose homoeostasis between normal and obese-hyperglycaemic mice. 179. Improved Metabolic Clearance of Glucose during Infusion of a Carbohydrate Mixture Containing Glucose, Xylitul and Fructose. F. Matzkies, G. Berg, H. Heid. Forschungsabteilung ftir Ernfihrung und Stoffwechselkrankheiten an der Medizinischen Klinik der Universit/it Erlangen-Ntirnberg, Institut f/ir experimentelle Ern~ihrung e.V., Erlangen, FRG. In 1951, Strauss and Hiller showed a decrease in serum glucose during rapid fructose injection. We measured glucose clearance during long term infusion of a carbohydrate mixture. - Eight healthy subjects received a carbohydrate mixture containing fructose, glucose, xylitol (2:1:1) at a rate of 0.5 g 9 kg-1 9 h -a, 4 subjects glucose (0.125 g 9 kg-a 9 h-l), 4 subjects xylitol (0.125 g ' kg-1 - h -1) and 4 individuals fructose (0.25 g 9 kg-1 9 h-a) over a period of 6 hours. - The total clearance, metabolic clearance and k2-value of each substrate were measured when the substrates were given alone and in combination, respectivelyl - The total clearance of glucose increased during the infusion of the carbohydrate mixture from 14.7 ml 9 kg-a 9 rain -a to 41.4 ml 9 kg-a 9 rain< while lactate production remained unchanged. Renal elimination of glucose, fructose and xylitol were not altered. - Therefore, the carbohydrate mixture is thought to improve glucose utilization. 180. The "Dynamics" of Electrical Activity of Beta-Cells in Response to Glucose and Glibendamide. H.P. Meissner, Blani Atwater. I. Physiological Institute, University of Saarland, 665 Homburg, FRG.

It is well documented that insulin secretion in response to a continuous constant stimulation is biphasic with glucose and monophasic with sulphonylureas. To analyse further this behaviour the membrane potential (MP) of r-cells (mouse pancreas) und these conditions was investigated with microelectrode techniques. In zero glucose the MP was high and there was no electrical activity. Within 60 sec after changing to 16.6 mM glucose the membrane depolarized to a potential level of about - 30 mV and developed spike activity. The spiking lasted for more than 1 min; then the activity changed to a burst pattern. At the beginning the bursts were short, while later on their duration increased. The relationship between relative duration of bursts and time of glucose stimulation was biphasic and similar to the biphasic insulin release. After stimulation with glibenclamide in zero glucose there was also a depolarization superimposed with spike activity. Initially the spikes were similar in shape and frequency to those induced by glycose. With time the frequency decreased and the shape of the spikes was markedly changed. A burst activity was not observed. The glibenclamide effect was irreversible. The data indicate that the electrical activity of r-cells is well correlated with insulin secretion; they suggest that the secretion pattern may be influenced by the MP. 181. The Effect of Sodium on the Glucose Induced Electrical Activity of Beta-Cells. H.P. Meissner, Illani Atwater. I. Physiological Institute, University of Saarland, 665 Homburg, FRG. The e ffect of Na + removal on insulin secre[ion is controversial. To elucidate further the participation of Na -r ions in the secretion process the influence of Na + on the glucose induced electrical activity of ~cells was investigated. - The experiments were performed with microelectrodes on isolated islets of the mouse pancreas. The glucose-induced electrical activity is.characterized by a regular burst pattern. After replacement of Na + by choline this pattern was markedly altered. In most experiments removal of Na 7was followed by a strong hyperpolarization without activity, lasting about 1 rain. Then the membrane depolarized again spontaneously, and continuous spike activity appeared with frequencies up to 8/sec. The spikes originated fron"t a potential level which was more negative than in normal [Na ~ ]. With time their frequency dropped, the spikes became irregular in shape and longer. After long exposure to zero Na -r (more than 8 min) the electrical a_~tivity disappeared almost completely. - Re-introduction of Na was first followed by a marked depolarization with increased activity; then, the membrane hyperpolarized and the activity disapppeared for up to 5 rain. Finally the normal burst activity pattern was restored. - The data show that there ~ a multiphasic response of membrane potential to changes of Na T , which may be caused by passive and active ion movements. Since there is a close relation between electrical activity an~d insulin secretion, it is conceivable that changes of external Na T are accompanied by a multiphasic insulin release. 182. Sensitivity to Insulin of Fetal and New-Born Rabbit Diaphragm. P. Metzger, E.A. Brachet. Laboratoire de Physiopathologie, Universit6 Libre de Bruxelles, Belgium. The aim of this work was to determine the gestational age at which insulin modifies glucose utilization by rabbit diaphragm in vitro. The tissues were taken from rabbit fetuses of known gestational age (from 2] to 31 days), one-day-old rabbits and one-day-old premature rabbits (obtained by caesarean section on the 30th day). The hemidiaphragms were incubated in 2.0 ml Krebs-Ringer bicarbonate buffer containing 1 mg glucose and 0.4 gCi U-14C-glucose, with or without insulin (2 mU). CO 2 and glycogen were assayed for radioactivity after a 120 min incubation at 37 ~ C. - With the fetal tissues, the production of 14CO2 was increased by insulin on the 31st day of gestation only; the incorporation of glucose into glycogen was not modified by the hormone throughout the gestation. However, after normal or premature birth, both parameters were increased by 25% in the presence of insulin. Radioactivity in glycogen was significantly higher with the hemidiaphragms of premature of new-born rabbits than with the fetal tissues. - It is concluded that insulin begins to exert its effect on

Abstracts glucose utilization by the rabbit diaphragm only late in fetal life or after birth, be it normal or premature. Moreover, after 24 hours of extrauterine life, glycogen synthesis by the diaphragm is notably higher than during gestation. 183. Effects of Insulin and NSILA-S on the Isolated Perinsed Rat Heart and Their Binding Characteristics. C. Meuli. Metabolic Unit, Department of Medicine, University of Zurich, 8006 Zurich, Switzerland. Some biological effects and the binding properties of insulin and NSILA-S were compared by means of the perfused rat heart. NSILA-S (nonsuppressible insulin-like activity) is a polypeptide extracted from human serum which shares many metabolic effects with insulin, but which has a different structure. The activity of NSILA-S is determined using insulin as a standard, (NSILA-S: MW 7500, purest preparation 375 mU/mg). - The dose-response curves of glucose uptake by the perfused rat heart are very similar for insulin and NSILA-S on a molar basis. Both substances accelerate the transport of 3-O-methyl glucose through the plasma membrane. - Specific binding of 125-I. insulin to heart muscle is defined as total binding minus nonspecific binding. Nonspecific binding is determined in the presence of a excess of unlabelled insulin (100 mU/ml, 2.2 10-7 M). - Saturation of specific binding sites occurred at insulin concentrations between 3 and 5 mU/ml (2.2 and 3.6 10-s M). It was not possible to displace 125-I insulin from its binding site with other peptide-hormones with the single exception of NSILA-S, which showed some affinity for the insulin binding site. On the contrary, 125-I NSILA-S was displaced only by unlabelled NSILA-S. - These findings suggest the presence of two separate binding sites for insulin and NSILA-S. The interaction of each hormone with its specific binding site produces the same biological effect. 184. Pituitary and Adrenal Control of Pancreatic Glucagon Secretion via Plasma Free Fatty Acids (FFA) and Aminoacids (AA). P. Mialhe, C. Foltzer and R. Gross. Laboratoire de Physiologie Grn6rale, Universit6 Louis Pasteur, E R A 188, CNRS and INSERM Strasbourg, France. Hypophysectomy induces an important increase in the level of plasma pancreatic glucagon which is only partly due to the hypoglycemia of the hypox animals. The problem was to know if plasma FFA and A A were modified by the operation and could also contribute to the increased secretion of glucagon through feed-back mechanisms. - Blood was taken from ducks (7 to 9 in each group) prior to, and 12 days after hypophysectomy; these birds were then treated for 4 days with bovine growth hormone (BGH), corticosterone (B) or both together; blood samples were drawn every morning in the fasting state. - In hypox ducks, plasma FFA and A A were reduced by 47 and 20% respectively. During the treatment with BGH, B or both hormones, plasma glucagon dropped below the normal level, while A A were restored to normal. B alone did not influence the FFA level, BGH restored it to normal and B + BGH increased it beyond the normal. - Plasma FFA variations, mainly influenced by the absence or presence of growth hormone, play an important rrle in the control by the pituitary gland of glucagon secretion. 185. The Influence of Thyroid Hormones upon the Dynamics of Insulin Release by the Perfnsed Rat Pancreas. S. Milcu, D. Ghiea, E. Costiner, L. Simionescu*, M. Oprescu*. The Institute of Endocrinology of the Academy of Medical Sciences, Bucharest, Rumania. The effect of thyroid hormones on the dynamics of insulin secretion was studied in order to find evidence of their direct action at the level of pancreatic r-cells. - Using the method of perfusion of the rat pancreas "in vitro", the authors measured the discharge of insulin under the influence of a constant glucose infusion (controls) and under different experimental conditions. - Constant glucose infusion (60/nin/16.7 mM) and triiodothyronine (150 y or 300 7/100 ml) significantly increased insulin secretion. - Interruption of the triiodothyronine infusion at 30 minutes after beginning the experiment brought about a return to control values. - A marked

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decrease of insulin secretion was observed in the pancreases from animals pretreated with triiodothyronine for 12 days. - Constant glucose (60 min/16.7 mM) and thyroxine (450 y/100 ml) did not significantly modify insulin secretion. - Thyroidectomy lowered insulin secretion. - The data obtained suggest: - a) the direct action of triiodothyronine on t-cells. - b) the influence of triiodothyronine on the mechanism of insulin secretion induced by glucose. - c) the permissive action of thyroid hormones on insulin secretion. * International Atomic Energy Agency, Research Contract 1215/R1/R B. 186. Study of Mortafity over a Period of Thirty Years among Diabetics Filed in Bucharest. I. Mincu, C. Dumitrescu. Clinic of Nutrition and Metabolic Diseases Hospital Dr. I. Cantacuzino Bucharest, Rumania. The authors have investigated mortality statistics in the period 1942 - 1972 of 7202 diabetics out of the 21,116 patients filed at the Centre of Nutrition and Metabolic Diseases in Bucharest. General mortality has decreased slowly and progressively from 86.62% in 1944 to 25.25% in 1972 accompanied by an important decrease of deaths of patients under 40 years old. - The whole repartition, according to the causes of death, places firstly the cardiovascular diseases (50%), followed in decreasing order by neoplasms with various localizations ( 12 %), tuberculosis and other pulmonary affections (6%), hepatic cirhosis (6%), kidney diseases (5.8%), diabetic coma (3%), other digestive diseases (1.9%), accidents and suicides (1.9%), infections (1.5%). - The average duration of the disease over 20 years increased among diabetics in whom the disease appeared before the age of 40, and the average duration of the disease ranging from 0 - 1 0 years increased in percentage for diabetics in whom the disease appeared after the age of 40. - The number of deaths among insulinized diabetics decreased progressively, whereas the deaths increased among those treated by simple diet or by oral drugs. - But these percentages are in accordance with the general percentages yielded by the treatment applied to diabetics filed at the Centre of Nutrition and Metabolic Diseases. It seems that a certain kind of antidiabetic medication has not notably influenced the mortality rates associated with diabetes. 187. Intestinal Absorption and Urinary Excretion Rates of Calcium before and after Initiation of Insulin Therapy in Diabetic Patients. J. Mirouze or L. Monnier, C. Sany and H. Ollet. Clinique des Maladies Mrtaboliques et Endocriniennes Hopital SaintEloi-34059-Montpellier Cedex, France. Five diabetic patients with severe deficiency in endogenous insulin secretion, i.e. fasting blood glucose higher than 300 mg/100 ml, glucosuria higher than 100 gm/24 h and high ketonuria, were investigated before and after a few days of insulin therapy. In each subject, the intestinal absorption rate of calcium (Va) was determined by a double radiotracer technique according to the method previously described in 1969 by Birge et al. The urine was assayed for calcium concentration by using the complexometric method. We found a significant drop in Va after insulin therapy, beyond the 2nd hour. The total intestinal fractional absorption rate (i. e. Va at 6 th hour) decreased from 75.8 % total dose absorbed + 6.4 (mean + SEM) before insulin therapy to 66.2% TD + 7.6 after (P < 0.025). The urinary excretion rate fell significantly from 455 + 62.8 to 309 mg/24 h + 13.3 (P < 0.025). In one of ourpatients, the diabetes mellitus was related to hemochromatosis and the time course of intestinal absorption was significantly lower (P < 0.001) than in the four other diabetic patients. During severe ketosis, it seems that calcium metabolism is considerably accelerated and that the elevation in intestinal calcium absorption rate may be interpreted as an efficient compensatory mechanism for the high urinary loss of calcium. 188. Comparative Study of Plasma Triglyceride Removal in Diabetic Patients Treated with Oral Antidiabetic Agents or Insulin. J. Mirouze or M. Piperno, A. Crastes de Paulet. Clinique des Maladies Mrtaboliques et Endocriniennes Hopital SaintEloi-34059-Montpellier Cedex, France.

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Abslracts

An intravenous fat tolerance test (IVFTT) as previously described by Boberg et al. was performed in two groups of diabetic patientsGroup I: diabetic patients under insulin therapy (30 cases), with good (Ia: 9 cases), medium (Ib: 12 cases), or poor (Ic: 9 cases) control. Group II: diabetic patients treated with oral antidiabetic agents, with good 24 hours control (II a: 12 cases), or poor control, i. e. insulin dependent (II b: 7 cases). Eleven subjects of group I a, b, c and/or IIb were also investigated after a good therapeutic control had been reached. - The fractional removal rate of triglyceride (KTG) was calculated from the plasma disappearance curve. - In group IIb, KTG was low (0.70 x 10-2 + 0.01) as compared to control subjects (1.50 x 10-~ + 0.16. KTG increased from 1.02 X 10-2 + 0.15 in group Ic, and from 1.32 x 10-2 + 0.10 in group lb to 1.50 • 10-2 _+ 0.16 after a good 24 hours regulation had been obtained with insulin therapy. In diabetics treated with oral agents, KTG remained low despite good control (1.12 x 10-2 _+ 0.12). The results were confirmed by the lipid electrophoresis data. Even when the oral treatment is efficient in terms of blood sugar control, the lipid metabolism remains poorly controlled in many patients. - In such cases the lipid abnormalities can be reversed by insulin therapy.

189. Progression of Nephropathy in Long-Term Diabetes and Effect of Initial Antihypertensive Treatment. C.E. Mogensen. Second University Clinic of internal Medicine, Kommunehospitalet, 8000 Aarhus C, Denmark. The rate of progression of diabetic nephropathy and possible effect of antihypertensive treatment was studied. - Glomerular filtration rate (GFR), and renal plasma flow (RPF) and albumin excretion was measured in 10 young male long-term diabetics, with constant proteinuria, at approximately yearly intervals for 2 0 - 6 0 months. In all patients there was a fall in both GFR and RPF and a gradual increase in albumin excretion. The rate of fall in GFR varied between 0.24 and 2.14 ml/min/month, and was positively correlated with diastolic blood pressure measured at the end of the control period (p < 0.01). - In 8 Patients antihypertensive treatment with propanolol alone or combinded with hydrallazine was started. After a mean period of treatment of 47 days blood pressure fell in all patients from a mean prevalue of 159/101 to 143/93 (p < 0.01). In the same period there was a marked fall in albumin excretion in all patients, from 3547 gg/min to 2414 ~tg/min (p < 0.01). There was a small fall in GFR from 93.5 ml/min _+ 35.2 (SD) to 86.5 ml/min _+ 30.6 (SD) (p < 0.05). Serum albumin rose from 31.9 to 34.9 g/1 (p < 0.02). RPF was unchanged. - These results suggest that antihypertensive treatment may be of value in long-term diabetics with proteinuria since albumin excretion decreases. Further control studies will clarify whether end-stage renal insufficiency can be postponed by such a treatment. 190. Microtubule Function and the Regulation of Insulin Secretion. W. Montague, S.L. Howell and I. C. Green. Kings College Hospital Medical School, London SE5 8RX and School of Biological Sciences, University of Sussex, Falmer, Brighton, Sussex, U. K. In a variety of tissues microtubules are formed by the polymerisation of protein (tubulin) subunits. We have investigated the possibility that changes in the equilibrium between subunits and polymerized microtubules might occur in the B cell during insulin secretion. An assay system, based on the observations that 3H-colchicine binds specifically and quantitatively to subunits but not to intact microtubules, was used to estimate the number of subunits in islets of Langerhans incubated under different secretory conditions. - Incubation of islets with vinblastine or at 4 ~ C, which on morphological evidence may disaggregate microtubules into subunits, increased the number of subunits in islet extracts. D20, which promotes the stability of microtubules and appears to increase their number, caused a decrease in subunit concentration. When insulin release was stimulated by glucose or by increasing intracellular cyclic AMP concentrations there was a rapid reduction in subunit concentration; conversely, when insulin release was inhibited by removal of calcium from the incubation medium there was an increase in the number of subunits present. These results

indicate that changes occur in the equilibrium between subunits and microtubules during the process of insulin secretion, and further implicate microtubules in the mechanism of secretion by exocytosis.

191. A Possible Relationship between Plasma Membrane Organization and Glucagon Unresponsiveness in Fetal Hepatocytes. R. Montesano, M. Amherdt, E. Blazquez, B. Rubalcava and R. Unger. Inst. of Histology and Embryology, University of Geneva Medical School, Geneva, Switzerland, and VA Hosp., University of Texas, Southwestern Medical School, Dallas, Texas, USA. It is known that the administration of glucagon or db-cAMP to 20-day rat fetuses induces a premature enzymic differentiation in hepatocytes, while in 18 day fetuses only db-cAMP is effective. In order to assess a possible role of the plasma membrane in determining the apparent unresponsiveness of the fetal hepatocytes to glucagon earlier than 20 days, plasma membranes of hepatocytes were studied by freeze-fracture throughout fetal and neonatal life. Freezefracture splits membranes along their inner matrix revealing protein-containing particles. While the number of particles per Ixmz of membrane face (A-face) varies slightly from the 14th day (789 _+ 59) to the 17th day (982 + 33), there is a significant increase from 982 + 33 to 1393 + 31, (p < 0.005) between the 17th and the 21st day, at which point they reached the adult level. Since the particles are thought to represent functionally important proteins in the plasma membrane (e.g. enzymes, receptors), their increase between the 17th and the 21 st day of the fetal life might be somehow associated to the acquisition by the cell of the capacity to respond to glucagon. 192. Alanine Turnover and Disposal in Healthy Subjects. J.A. Morrhouse, R.H. Chochinov, K. Perlman. Winnipeg, Manitoba, Canada. Peripheral and central AV differences have suggested a unique role for alanine in gluconeogenesis. Specific disposal data have been lacking. Eight healthy subjects were infused with U-C 14alanine and with U-C 14 glucose. The mean alanine turnover rate was 368 + 2S.D. 80, the mean glucose turnover rate 467 + S.D. 57 ~tM/min/1.8 m. Alanine promptly equilibrated with lactate and appeared in serum albumin. Early alanine disposal comprised 49% to lactate, 28% to whole-body protein synthesis, with most of the remainder to non-circulating intermediates. Alanine disposal at equilibrium comprised 29% to glucose, 28% to protein, and 44% to oxidation (expired CO~). 10% of circulating glucose was derived from alanine, 30% of circulating alanine was derived from glucose.~: - Conclusions: (1) The alanine and lactate cycles are inseparable and nearly identical in function. - (2) Glycogen-derived pyruvate, not alanine, is the focal intermediate, readily undergoing either reduction to lactate or amination to alanine for transport and recycling. - (3) Only a minority (30%) of circulating alanine represents a preformed stored amino acid; its role is quantitatively similar to that of several other glycogenic amino acids. The majority is not functionally an amino acid, but is a glucose-derived intermediate without a storage compartment; its role is about 50% of and similar to that of lactate. - (4) Alanine does not have a singular role in gluconeogenesis. 193. Impaired Control of HGH Secretion in Hepatic Cirrhosis (HC) and in Portocaval Anastomosis (PCA). Its Role in the Pathogenesis of Diabetes Observed in Cirrhotic Patients. M. Muggeo, D. Fedele, A. Baiocchi, M. Molinari, A. Tiengo, G. Crepaldi. Department of Internal Medicine, Division of Gerontology and Metabolic Diseases, University Hospital, Via Giustiniani 2, 35100 Padova, Italy. The aim of this paper was to investigate the role of hypersecretion of HGH in the pathogenesis of hyperinsulinism and diabetes observed in HC and after PCA. 12 males affected with HC, 4 with PCA, and a group of controls underwent oral glucose loading (OGTT), arginine test (AT), and i.v. glucose loading (IVGqT). AT and IVGTT were carded out in tandem as a combined test of stimulation-inhibition of HGH secretion. 6 HC patients showed overt diabetes, while 6 others only glucose intolerance after OGTT. Insulin response to OGTT was higher in uormoglycemic and hyperglycemic H C Pa-

Abstracts tients (peak = 180 + 5 and 106 + 19 IxU/ml respectively)than in controls (64 + 5 gU/ml). HC patients ;howed higher HGH levels than did controls both fasting (4.44 + 1.07 vs. 0.70 + 0.51 ng/ml; p < 0.001) and after arginine (28.00 + 6.04 ng/ml vs. 15.88 _+5.05 ng/ml). I. V. glucose (90 min after the beginning of arginine infusion) inhibited HGH in controls (1.62 +_ 0.43 ng/ml), while in HC patients a paradoxical response to glucose was observed (34 +_9.06 ng/ml). This increment in HGH production revealed an alteration in metabolic control and in the autoregulation of HGH secretion. PCA construction aggravates these endocrine-metabolie disorders. In fact, in PCA patients a striking increase in insulin response to glucose (278 + 110 ~tU/ml) and higher HGH levels fasting (6.48 +_ 1.03 ng/ml), after AT (33.20 +_ 7.54 ng/ml) and after 1VGTT (41.31 ___ 17.86 ng/ml) were observed. Elevated HGH levels and the paradoxical increase after glucose could explain the diabeticlike behaviour observed in cirrhosis and after PCA.

194. Metabolism and Aspects of Hormonal Regulation of Isolated Mouse Hepatocytes. P. Mfiller, C. Patzelt. Laboratoires de Recherches M6dicales, Avenue de la Roseraie 64, 1211 Geneva 4, Switzerland. We have studied the overall metabolism and aspects of hormonal regulation of isolated mouse hepatocytes. - Isolated hepatocytes were obtained by adapting the method of Berry and Friend to mouse livers. - Many metabolic parameters indicated the viability of these cells: ATP content was stable; lipogenesis was stimulated by the presence of glucose or fructose; gluconeogenesis was increased by lactate. Furthermore, isolated hepatocytes secreted proteins and triglyeerides, a secretion that required microtubules as it was inhibited by eolchieine (10-5M). Ultracentrifugation and eleetrophoretic analysis indicated that secreted triglycerides were mostly VLDL. Glueagon was very effective (10-1~ in stimulating glyeogenolysis and inhibiting lipogenesis. Inhibition of lipogenesis by glucagon was unlikely to be related to increased lipolysis (via increases in intraceIiular fatty acyl-CoA, inhibitory to lipogenesis and lipolysis appeared unrelated processes. Glucagon (from 10-8M onwards) stimulated ureogenesis and ketogenesis, but inhibited triglyceride secretion. Cyclic AMP mimicked all these metabolic changes. Addition of insulin failed to reverse these cyclic AMP-mediated effects. Evidence for possible "disappearance" of insulin when added to hepatocytes or possible alteration of binding sites during preparation of hepatocytes will be presented. - It is concluded that glueagon modulates the overall metabolism of isolated hepatocytes, while insulin so far fails to do so. 195. Subcellular Distribution of Protein Kinase and Phosphorprotein Phosphatase Activities in Rat Islets. W. A. Muller, D. B. Martin, G. W. G. Sharp. Institut de Biochimie Clinique, Gen6ve, Switzerland. This study represents a preliminary survey of the subeellular localization, and some of the characteristics, of protein kinase and phosphoprotein phosphatase reactions - enzyme systems intimately involved in the action of cAMP on intracellular regulatory processes. - Rat islets were isolated by collagenase treatment, homogenized, and separated by repeated sequential centrifugation into four fractions: a pellet after a centrifugation of 800 x G for 10 rain (P0, presumed to contain nuclei, membranes, and large fragments; a pellet after a centrifugation of 7000 x G for 10 min (P2), presumed to contain mitoehondria and granules; a pellet after a centrifugation of 200,000 x G for 60 min (P3), presumed to contain microsomes and large ribosomes; and the ultimate supernatant, presumed to be cytosol (Cyt). - Protein kinase activity without added substrate (3zp precipitated by TCA after incubation with y-labelled AT32p) was found predominantly in P1, with a small amount also in P3 and Cyt, and less in P2. Stimulation of protein kinase activity by cAMP was best in the presence of exogenous substrate. Thus, with histone present, cAMP stimulated protein kinase activity was found in P1, P2, and Cyt, with negligible activity in P3. - Phosphoprotein phosphatase activity (measured as a decrease in 32pin protein after addition of excess ATP) was found in all fractions, most impressively in Cyt.

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196. Adrenal Function in Obob-Mice. P. Naeser. Department of Histology, University of Uppsala, Sweden. Obese-hyperglycemic mice (obob) were used to further elucidate the relationship between adrenal cortical function and diabetes mellitus. These mice are known to display a marked hyperadrenoeortieism. The first morphological signs of an enhanced adrenal function were recorded in 5 month old obese animals, which correlated with a pronounced hyperglycemia and hyperinsulinemia at this age. Raised serum cortieosteroid levels were observed in the obob-mice already at 2 months of age. - In an attempt to study the possible etiological significance of the hyperadrenoeortieism adrenalectomy was performed in young obob-mice. This treatment led to a lasting decrease of both the hyperglycemia and the hyperinsulinemia, indicating that the hyperadrenocorticism aggravates the insulin resistance of the obob-mice. In addition, the content of eorticosteroids per unit adrenal weight was found to be significantly raised by 5 weeks of age. - Taken together the results suggest that the hyperadrenocorticism in the obob-mice reflects an increased ACTH production. 197. Comparative Effects of Phenformin, Mefformin and Glibenclamide on Metabolic Rhythms in Maturity-Onset Diabetes. M. Nattrass, P.G. Todd, B. Lloyd, P. T-Smythe and K. G.M.M. Alberti. Faculty of Medicine, Chemical Pathology and Human Metabolism, General Hospital, Southampton S09 4XY, U.K. The biguanides, phenformin and metformin are known to cause lactic acidosis in a small number of patients. Their effects on blood lactate and other intermediary metabolites in stable diabetics during normal life are less well known. - A group of patients have been studied while taking successively phenformin, metformin and glibenclamide with at least one month on each regimen before testing. Blood samples were taken half-hourly from an indwelling intravenous cannula for 12 hrs with patients taking their normal diabetic diet. - Mean 12 hr blood glucose concentrations ranged from 6.2 to 9.9 mM on phenformin, 6.8 to 8.7 on mefformin, and 4.6 to 5.1 on glibenclamide. Mean blood lactate was significantly raised on both phenformin (1.3 to 2.5 mM) and metformin (1.2 to 1.9 raM) compared with normal values (0.8 to 0.9) on glibenclamide. In addition, lactate/pyruvate ratios and blood alanine were grossly raised on biguanide therapy, with moderate rises in ketone body and glycerol concentrations. Most values returned to normal on glibenclamide. No difference was found between the two biguanides. - Thus although hyperglycaemia was moderately well controlled by the biguanides there were several abnormalities in intermediary metabolism not evident during glibenclamide treatment. 198. HL-A Factors, Autoimmunity, Virus and Juvenile Diabetes Memtus. J. Nerup, O. Ortved Andersen, M. Christy, P. Platz, M. Thomsen, A. Svejgaard, F. Botazzo, A. Pouplard and D. Doniach. Med. Dpt. F., Gentofte Hospital, Med. Dpt. E., Frederiksberg Hospital, Steno Memorial Hospital, Tissue Typing Lab., Rigshospitalet, Copenhagen & Dpt. Immunology, Middlesex. Hospital. Med. School, London, U. K.. Recently we proposed that the inherited susceptibility to develop juvenile diabetes mellitus (JDM) is conferred by HL-A 8 and W15 associated, immune response genes responsible for a defective T-lymphocyte response to certain viruses, leading to B-cell destruction directly or through the triggering of autoimmune reactions. (Lancet 2: 864, 1974). - Further investigations revealed: 1) The LD-types 8a and W15 a are more strongly associated with JDM than the SD-types 8 and W15. LD-8a and/or W15 occurred in 75 per cent of patients compared to 25 per cent in the controls. Relative risks for LD-8 a and W15 a: 4.5 and 3.7 respectively. - 2) Islet cell antibody (ICA) and antipancreatic, cell-mediated immunity (APCI) was demonstrated in 55 and 34 per cent of the patients with JDM of short duration. - 3) ICA and/or APCI was present in 66 per cent of the patients, showing that autoimmunity is a common feature of JDM. - 4) ICA was found in 75 per cent of the LD-8 a

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positive diabetes compared to 36 per cent of the LD-8 a negatives. - 5) Antibodies to Coxackie B 4 virus were predominantly found in ICA positive sera. - These findings link together the genetic markers of JDM with both autoimmunity and viral infections, thereby supporting the original hypothesis.

199. Noradrenaline, Adrenaline and Dopamine in the Cardiovascular System and in the Pancreas in Diabetics. 13. Neubauer, N. J. Christensen. 2nd Clinic of Internal Medicine, Kommunehospitalet, _~rhus, Denmark. Noradrenaline, adrenaline and dopamine were determined by double-isotope derivative techniques in tissue specimens obtained at postmortem examination in 8 longterm diabetics and in 10 controls. - The noradrenaline content in the cardiovascular system was considerably reduced in the diabetics. In the heart, the radial artery, the posterior tibial artery and the femoral artery, the noradrenaline content averaged 6, 9, 12 and 20%, resepctively, of the corresponding values in the controls. - Adrenaline was present in small amounts in the cardiovascular system but the concentration was not reduced in the diabetics. - The dopamine content in the cardiovascular system averaged 11% of the noradrenaline content in the controls. There was a strong correlation between the dopamine content and the noradrenaline content of the tissues. The dopamine content was reduced in the diabetics but relatively less than that of noradrenaline. - In contradistinction to the findings in the cardiovascular system, the noradrenaline content of the pancreas was not reduced in the diabetics. - The pancreas, especially the body of the pancreas contained considerable amounts of adrenaline both in the controls and in the diabetics. The average adrenaline content was twice as high in the diabetics as in the controls. - It is concluded that the depletion of the noradrenaline stores in the cardiovascular system in the long-term diabetics may in part be responsible for their reduced survival rate in myocardial infarction and in the arteries contribute to the development of diabetic macroangiopathy. - Adrenaline may be implied locally in the control of the secretion of pancreatic hormones in normals as well as in diabetics. 200. Plasma Citrate: IvGTT (K-Values), Plasma Insulin and Free Fatty Acids (FFA) in Obese and Normal Persons. T. T. Nielsen. Medical Department III, Aarhus Amtssygehus, 8000 DK Aarhus C, Denmark. Citrate accumulates in t-cells of hyperglycemic mice and probably activates processes involved in insulin secretion. - 23 obese patients (> 150% of ideal weight, age: 18-38 years) and 19 persons of normal weight (age: 22-33 years) were studied. All persons examined had plasma glucose < 6,2 mmol/l. In the group of normals only persons with K-values > 1,10 were included. Plasma citrate and plasma insulin were followed before and during the IvGTT. FFA were determined in the fasting state. - Mean fasting plasma citrate in the obese group was 133 + 17 ~tmol/versus 122 _+ 17 ~mol/1 in the normals (p < 0,02). A negative correlation between fasting plasma citrate and K-values was demonstrated in both normals (R: - 0,74; p < 0.001) and obese (R: - 0,48; p < 0,05) as well as in the total group of normals and obese (R: - 0,73; p < 0.001). Plasma citrate was negatively correlated with the 5 min. response of plasma insulin during IvGTT in the obese (R: - 0.49; p < 0.05). Furthermore a positive correlation between fasting plasma citrate and plasma FFA was demonstrated for all persons (R: 0.39; p < 0,02). The concentration of plasma citrate did not change during IvGTI" in any of the groups. - The results suggest citrate to be associated with intravenous glucose tolerance and insulin release and sensitivity. 201. Selective Assay of Postheparin Plasma Lipoprotein Lipase and Hepatic Lipase in Diabetes: Relationship to Plasma Triglyceride Concentration and Transport. E. A. Nikkil~i, J. K. Huttunen, C. Ehnholm and M. Kekki. Third Department of Medicine, University of Helsinki, Helsinki, Finland. A new immunochemical method for selective measurement of postheparin plasma lipoprotein lipase (LPL) and hepatic lipase (HL) activities was applied to the study of 25 patients with untreated diabetes. The control material consisted of 25 age-matched healthy

volunteers. The production and removal of VLDL-triglycerides was studied by an endogenous glycerol labelling method and the elimination of exogenous lipids with the Intralipid test. In patients with severe insulin-deficient, ketotic diabetes the LPL activity was markedly decreased (13.6 _+ 1.8 vs. 21.3 + 1.1 in controls, p < 0.001) whereas the HL activity was normal (22.9 + 6.9 vs. 26.6 _+ 2.2 in controls). The fasting plasma triglyceride (TG) level was elevated in five of seven patients, but there was no correlation between the TG level and LPL activity. Upon insulin treatment the LPL activity rose to normal and plasma TG concentration decreased. In insulin-deficient but nonketotic juvenile diabetics the LPL was slightly reduced (17.8 _+ 1.0, p < 0.05) but the plasma triglyceride concentration was normal. The plasma VLDL-TG turnover rate and the Intralipid removal rate were also within the normal range. Of eight patients with adult-onset type diabetes and obesity five had moderate to severe hypertriglyceridemia and an increased production of VLDL-TG. The LPL activity was normal in all but one patient, who had gross hyperlipemia. On the other hand, the HL activity was higher than in controls (34.2 + 3.6.) - It is concluded that the lipoprotein lipase activity of postheparin plasma decreases in insulin deficiency and this may contribute to development of hypertriglyceridemia in insulinopenic diabetics. In diabetics with subtotal insulin deficiency the plasma triglyceride concentration and transport remain normal in spite of a slight decrease of lipoprotein lipase activity. In adult-onset diabetes the LPL activity is not reduced and the hypertriglyceridemia if present is mainly accounted for by an increased production of VLDL.

202. Inhibitory Effect of Somatostatln on Plasma Insulin Response to beta-cytotropic Stimulation. C. Noacco, S. Macor, F. Bertolissi, and A. Benedetti. Diabetes Unit, Udine General Hospital, Udine, Italy. To elucidate further the extent and the mechanism of the inhibition of insulin release by somatostatin (SRIF), glucagon (1 rag), tolbutamide (1 g) or glucose (1 g/rain for 50 min) plus glucagon (1 mg) were infused i.v. in 12 subjects. - The tests were repeated during a 75 min infusion of cyclic SRIF (6.6 ~xg/min). Blood glucose and IRI were measured in venous blood at 15 min intervals, from the beginning to 1 hour after the end of the infusion. - Beta cells stimulation was followed by a prompt rise of IRI with the expected BG changes. - During SRIF, glucagon induced a more pronounced (+ 100% vs. + 52%) and sustained BG rise; tolbutamide did not affect BG significantly. IRI response to both stimuli were suppressed until the end of SRIF infusion, IRI rose up to 18-66 ~tU/ml 15-30 min after. A more potent stimulation (glucose + glucagon) failed to evoke any appreciable IRI rise in spite of BG levels up to 460 mg/dl. A conspicuous IRI rise (up to 290 ltU/ml) occurred only 15 to 30 min after the end of the SRIF infusion. - In conclusion, SRIF inhibited completely glucagon, tolbutamide and glucose + glucagon-induced IRI release without affecting the glycogenolytic activity of exogenous glucagon. This transient blockade is effective also in presence of high glucose and glucagon levels in blood. 203. Diurnal Glucose and Free Fatty Acid (FFA) Profiles in Normal and Diabetic Women in Late Pregnancy. N. W. Oakley, M. D. G. Gillmer, M. Elphick, F. M. Brooke, D. Hull, R.W. Beard. St. Mary's Hospital Medical School, London, W. 2., England. Dept. of Child Health, Nottingham University Medical School, U. K. The macrosomia of infants of diabetic women is usually ascribed to fetal hyperglycaemia and hyperinsulinism but elevated maternal FFA levels have also been implicated. We examined diurnal changes in maternal glucose and FFA in late pregnancy and related them to findings in the neonate. - 24 normal women, 13 chemical diabetics (CD) and 13 insulin dependent (ID) were studied during the last trimester of pregnancy. Venous plasma samples for glucose and FFA were taken at hourly intervals during 24 hours on standard diet. Cord and 2 hour post delivery neonatal blood was also collected. - Diurnal plasma glucose was very constant (84.6 _+SD 6.7 mg%) in normal women and higher and more variable in CD (100.9 + 18.5 mg%). ID showed a similar mean value (106.0 + SD 21.6

Abstracts mg%) with diurnal hyperglycaemia and nocturnal hypoglycaemia. Mean diurnal FFA was higher in CD (0.77 +_ SD 0.34 mEq/l.) than normal (0.66 _+0.20 mEq/l.) but the difference was not significant. Mean diurnal FFA in ID (0.44 + 0.11 mEq/l.) was lower than for either normal or CD (p <. 005 and p <. 001 respectively). Mean FFA was not correlated with any birthweight function or neonatal glucose assimilation, but was inversely correlated with cord growth hormone (p <. 01). Mean diurnal glucose correlated positively with neonatal glucose assimilation (p <. 005) and insulin levels but just failed to correlate with any birthweight function. - Results suggest that, even close to the normal blood glucose range, maternal glycaemia directly affects neonatal carbohydrate metabolism, but provide no support for the theory that high maternal FFA levels are responsible for fetal macrosomia. 204. The Influence of Dimethylbiguanide (DMB) on Liver Blood Flow in Dogs. E.E. Ohnhaus, W. Berger. Department of Medicine, University of Basel, Switzerland. In previous experiments an increase in liver blood flow was found in rats using high pharmacological doses of DMB. In the present study the effect of therapeutic doses of DMB was investigated. Dogs were anesthetized with phenobarbitone, and the following vessels cannulated: carotid artery, jugular vein, hepatic vein, femoral vein, femoral artery and portal vein (for taking blood samples at different time intervals). A catheter was inserted in the lower part of the duodenum and a single dose of sodium chloride, 15 mg/kg DMB or 50 mg/kg DMB was given. Liver blood flow was measured utilizing the indocyanine green injection method before and 60 and 120 min after drug administration. Blood glucose, insulin, lactate and pyruvate were estimated before and 30, 60, 90, 120 and 150 rain after administration in samples from the different cannulated vessels. Following sodium chloride or 15 mg/kg DMB no change occurred in any of the parameters measured. Following 50 mg/kg DMB liver blood flow decreased significantly(51 ml/min/kg to 4 I. 3 ml/min/kg) and this decrease occurred simultaneously with a fall in blood glucose and an increase in lactate. In additional experiments sodium chloride or 15 mg/kg DMB were given daily for 8 days. After this pretreatment liver blood flow was significantly higher in the DMB treated animals (81 ml/min/kg) in comparison to the controls (60 ml/min/kg). After administration of 2 gm/kg glucose no further increase in liver blood flow was seen in the DMB treated animals, while an increase up to 81.3 ml/min/kg occurred in the controls. Based on these experiments after chronic administration of a therapeutic comparable dose of DMB an increased liver blood flow was found, and this might beof clinical importance in influencing drug elimination or prohibiting lactic acidosis. 205. Relations between the Abnormal Parenchymatous Basal Laminae and the Follicular Cells in the Pituitary of Diabetic Patients with Retinopathy. L. Olivier, R. Vila-Porcile, O. Racadot, B. Lesobre and J. Canivet. Department of Histology Facult6 de M6decine Pitir-Salprtrirre F 75634 PARIS CEDEX 13 and Department of NutritionEndocrinology Hopital St Louis F 75475 PARIS CEDEX 10, France. The pituitary of diabetics with retinopathy shows modifications of the capillary basal laminae and more marked alterations of the pericordonal parenchymatous basal laminae. - The present study points out the relations between the abnormal parenchymatous basal laminae and the follicular cells of the epithelial cords. - The pituitary glands from 11 diabetics with severe retinopathy, having undergone hypophysectomy, were studied by electron microscopy with glutaraldehyde-OSO4 fixation, Epon embedding, and compared with 6 non diabetic patients. - In the normal pituitary, glandular cells and "end-feet" from follicular cells rest on a single parenchymatous basal lamina. These "end-feet" occupy a small surface. Rarely, some digitations protrude from them, originating folds of the basal lamina and thus the anlage of its duplication. - In diabetic patients, follicular "end-feet" spread and exhibit numerous digitations, making up a network throughout the perivascular and intercordonal spaces, thus originating multilamellar branched basal laminae. The most external layers of these laminae have lost contact

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with follicular elements. - The most striking disturbance of the diabetic pituitary involves the follicular cells, responsible for an exaggerated production of basal lamina material. The mechanisms involved could be that suggested by VRACKO for the peripheral capillaries. These data establish the first observation of a non tumoral pathology of the follicular cells. 206. Abnormal Platelet Function and Diabetic Peripheral Nenropathy. B.C. O'Malley, F.E. Preston, W.R. Timperley, J.D. Ward. Royal Infirmary, Sheffield $6 3DA, U. K. Peripheral neuropathy is a serious complication of diabetes. The precise pathogenesis is unknown. Recent histopathological studies in our department have demonstrated fibrin in the vessels supplying the sural nerve in patients with diabetic neuropathy. This finding prompted us to study platelet function in the same group of patients. - Blood samples were obtained from 20 patients. Platelet aggregation was measured following the addition of ADP, collagen and adrenaline. The responses were assessed photoelectrically in a temperature controlled, constantly stirred non-glass system. The samples were also tested for spontaneous platelet aggregation. Spontaneous platelet aggregation was detected in one third of the samples studied, but in none of the normal controls. Enhanced ADP and adrenaline induced aggregation was observed in approximately 50%. - Enhanced platelet aggregation has been demonstrated in diabetic patients with peripheral neuropathy. The authors believe that this abnormality of platelet function may be of considerable importance in the pathogenesis of diabetic peripheral neuropathy. 207. The Production of Insulin by Isolated Fragments of Pancreas Directly Stimulated by Way of Their Vagal Nerve Fibre. Im. portance of the Glucose Concentration of the Medium. A. Orsetti, F. Passebois. Service d'Exploration Physiologique des Hormones Hopital Saint-Eloi - 34059 MontpeUier Cedex, France. The original preparation of pieces of rabbit pancreas isolated with their vagal fibres permits the study of the direct influence of the pneumogastric nerve on insulin secretion in vitro. - Method: a) Separation of the pancreas adherent to the posterior face of the stomach; b) Dissection of the vagus a long the left lateral border of the oesophagus; c) section of the tail of the pancreas with the vagus; d) Incubation of the fragment in oxygenated Krebs solution at 37 ~C with varying concentrations of glucose: 1.0.1.5 and 3.0 mg/ml); e) Stimulation by rectangular signals: 5 volts, 20 cycles per second, 1 millisecond duration for a period of 20 minutes. - At a concentration of 1 mg/ml insulin production with stimulation is negligable; there being no significantdifference between the control groups and those receiving stimulation. On the other hand, at 1.5 mg/ml insulin secretion with stimulation is notably augmented: + 47% after twenty minutes (p < 0.001). At 3 mg/ml vagal stimulation does not reinforce the production of insulin which is already high in the controls (no significant difference between these two groups). All elevations of insulin production under vagal stimulation are blocked by atropine. The addition of propanolol and phentolamine protect the preparation from adrenergic effect during the stimulation of the vagus. - C o n c l u s i o n s : Direct cholinergic influences are optimal at a glucose concentration of 1.5 mg/ml. 208. Diabetic Ketoacidosis and Somatostatin. H. Orskov, K. G. M. M. Alberti, S. E. Christensen, Aa. P. Hansen, J. Iversen, K. Lundb~ek and K. Seyer-Hansen. Second University Clinic of Internal Medicine, Kommunehospitalet, 8000 Aarhus C, Denmark and Faculty of Medicine, Chemical Pathology, The General Hospital, Southampton S09 4XY, U. K. The effect of somatostatin was studied in 9 juvenile non-obese diabetics, after withdrawal of insulin, in two experimental situations when their total plasma CO 2 was between 12 and 20 meq/l. In six patients a somatostatin infusion was started 36-48 hours after the latest insulin administration. 250 gg somatostatin was given as a bolus injection followed by an infusion of 250 gg/h for 4 hrs. Blood glucose fell in all patients during infusion, in three cases to nearly hypoglycaemic levels. After infusion blood glucose rose again. This findings was in accordance with the suppression of the elevated

368

Abstracts

plasma pancreatic glucagon to nearly zero levels, with a high rise after ceasing infusion. The plasma growth hormone behaved as plasma glucagon. Acetoacetate and hydroxybutyrate, however, did not change appreciably during or after discontinuation of somatostatin. Nor did FFA and glycerol show any significant changes. Three patients were studied in the same state of metabolic disorder. In these patients soluble insulin was infused (1 U/h) for 9 hrs. During two 2-hr intervals somatostatin infusion was added after a bolus as before. The action of insulin began within half an hour and induced a fall in all parameters except in plasma growth hormone. There was no change in the patterns of betahydroxybutyrate, acetoacetate, FFA or glycerol when somatostatin was added, or after withdrawal of somatostatin. - In conclusion: Earlier experiments have shown that plasma glucagon rises early in diabetic ketoacidosis, long before other insulin antagonists. It has been suggested that the elevated plasma glucagon found in diabetics may be harmful and of importance in the genesis and development of ketoacidosis. - In the present experiments, carried out in patients with significant ketoacidosis, somatostatin induced the expected fall in plasma glucagon and blood glucose. However, the fact that there were no significant changes in blood ketones, FFA and glycerol indicated that the short-acting somatostatin available at present has no obvious place in the future treatment of overt ketoacidosis.

209. The Mechanism o[ the Glomerular Enlargement in Long-Term Diabetes: Compensatory Hypertrophy versus Excessive Deposition of Basement Membrane Material. R. Osterby, H.J. Gundersen. University Institute of Pathology and the Second University~ Clinic of Internal Medicine, Kommunehospitalet, 8000 Arhus Denmark. Although thickening of the glomerular capillary wall sets in at the time of onset of diabetes and progresses steadily over the years, kidney function-i, e. glomerular filtration rate- is elevated for many years in most diabetics. This paradox was the object of the present study. - Autopsy kidney specimens from 5 long-term diabetics and 3 controls were cut into serial sections. Applying stereological methods, estimates of various morphological parameters were obtained in 25-50 individual glomeruliin each subject. - In each of the diabetics a negative correlation was obtained within individual glomeruli between the degree of diabetic glomerulopathy (volume of PAS-positive substance per unit volume of the tuft) and the size of the tuft (ruft (~ = -0.29, 2p < 0.001). The mean glomerular volume was increased in the diabetics (2.17 Mill ~t3 against 1.19 Mill ~t3, 2p < 0.05), while the estimated total volume of glomerular capillaries was the same in the 2 groups. The enlargement of the tuft was closely correlated with cellular hyperplasia. - The finding.of the lowest degree of diabetic glomerulopathy and an excesswe number of ceils in the largest glomeruli indicated that the enlargement, at least partly, was compensatory rather than due to deposition of basement membrane material. This enlargement was most likely important for the maintenance of kidney function in diabetics. 210. Studies on intracellalar Cyclic AMP and Lipolysis in Human Adipose Tissue. J. Ostman, P. Arner, Department of Internal Medicine, Huddinge University Hospital, S-141 86 Huddinge, Sweden. To determine the relationship between the level of cyclic AMP and the rate of lipolysis in human adipose tissue. - Subcutaneous fat biopsies were incubated under various experimental conditions. Glycerol release per fat cell was taken as index of lipolysis. Cyclic AMP was determined by a protein binding assay with theophylline present as phosphodiesterase inhibitor. - The maximal rise (at 10 rain) in tissue cyclic AMP level and the glycerol release were interrelated both 1) in experiments where increasing concentrations of isopropyl-noradrenaline were used and 2) when data from several experiments were taken together. The beta-adrenergic blocking agent propranolol reduced tissue cyclic AMP when added simultaneously with or after isopropyl-noradrenaline, but lipolysis was diminished only on the former occasion. In adipose tissue obtained from obese subjects submitted to total starvation for one week noradrenaline diminished lipolysis significantly, apparently due to decreased production of cyclic AMP. These noradrenaline effects were reversed by re-feeding for one day. - It is concluded

that the adenylate cyclase system does not play the key role for catecholamine induced lipolysis in human adipose tissue and also that the maximal intracellular level of cyclic AMP (at 10 min) determines the rate of triglyceride breakdown.

211. Serial Blood Glucose/Plasma Insulin Estimations in Normal and Diabetic Patients Following Chlorpropamide and Glibenclamide. D. R. Owens, J. Seldrup, K. Wragg, M. Addison, K.J. Shetty, K. Davies. Medical Unit, University Hospital of Wales, Heath Park, Cardiff, U. K. Studies were undertaken to define the blood glucose/plasma insulin profile in normal and non-obese maturity onset diabetic subjects and observe the changes following two sulphonylurea compounds. - Five days hospitilization was involved with each subject receiving four isoealoric meals per day. During the last two days serial estimations (half hourly for 12 hours and at 24 hours) were performed following placebo and the test preparation. Each normal subject (n = 5) underwent the procedure twice, as opposed to diabetic subjects who reveived placebo and glibenclamide only. In normals glibenclamide (5 rag) chlorpropamide (250 rag) produced early hypoglycaemic responses between 1-2 hours, with maximum plasma insulin increases at this time. The hypoglycaemic response to glibencclamide lasted approximately 7 hours, whereas following chlorpropamide a more consistent lowering was observed for 12 hours. The insulin response was normalized by 1 and 9 hours following chlorpropamide and glibenclamide respectively. - Contrary to the findings in normal subjects, glibenclamide (5 and 10 mg p.o.) in all diabetic subjects (n = 5) produced a consistent dose related hypoglycaemic effect for ut to 24 hours. This response could not be simply ascribed to observed changes in plasma insulin values. - This method allows detailed characterisation of anti-diabetic agents, but illustrates limitations in extrapolation from normals to diabetics. 212. Examination of Renal Split Function in Nephropathies of Diabetics Using Nuclear Medicine Clearance Procedure and Serial Gamma Camera Scintigraphy. H.W. Pabst, P. Heidenreich, P. Bottermann, K. Kempken, G. H/Sr. II. Med. Klinik, 8 Miinchen 80, FRG. Quantitative determinations of split renal function by clearance procedures and gamma camera serial scintigraphy using radiopharmaceuticals were performed in order to get detailed information concerning loss of glomerular and tubular function in diabetics. - Renal clearance determinations in more than 100 patients after simultaneous administration of 131I-hippuran (ERPF) and 99mTc-DTPA (GFR) using whole body clearance principle. Calcula9tion of filtration fraction (FF). - Serial gamma-camera scintigraphy using 131I-hippuran. Parameters were compared with clinical and laboratory data of diabetics. - Long-term diabetics showed a simultaneous decrease of ERPF and GFR as well as a delayed intrarenal hippuran transport. Diabetics with inflammatory lesions of the kidney presented only a reduction of ERPF. FF was increased (0,25). In diabetics with hypertension and retinopathy we found the most severe reduction of ERPF and GFR. - The findings are discussed concerning severity, duration, complications and therapy of diabetes mellitus. Our methods are useful to determine impairment of renal function in short-term and long-term diabetics and are capable of supporting diagnosis and therapy. 213. The Effect of Metabolic Regulation on Microvascular Albumin Permeability in Short-Term Diabetics. H.-H. Parving, J. Noer, T. Deckert, P.-E. Evrin, S. L. Nielsen, J. Lyngsoe, M. Rorth, P. Aa. Svendsen, J. Trap-Jensen and N.A. Lassen. Department of clinical Physiology, Bispebjerg Hospital DK-2400 Copenhagen N.V., Denmark. The purpose of the present study was to determine the microvascular albumin permeability in short-term juvenile diabetics in good and poor metabolic regulation. - The daily urinary albumin and flz-microglobulin excretion rates were measured by sensitive radioimmunoassays in nine insulin dependent juvenile diabetics (duration of diabetes less than 1.5 year). Transmicrovascular escape rate of albumin, TER, was determined from tlae initial disappearance of intravenously injected 125I-labelled human serum albumin.

Abstracts The measurements werecarried out in the same patient during good (average plasma glucose = 6.3 mmol/1) and poor metabolic regulation (average plasma glucose = 14.4 mmol/l). - Urinary albumin excretion was significantly increased (+ 125 %) in patients during poor metabolic control; average 16.0 mg per 24 hours, compared with a mean of 7.1 mg per 24 hours during good metabolic control. Urinary fl2-microglobulin excretion rate increased in all but one patient during poor metabolic regulation (+ 64%). TER increased significantly from an average of 5.7 to 6.9% per hour during poor metabolic control (+ 21%). - These findings of increased microvascular albumin passage are compatible with the hypothesis that diabetic microangiopathy is caused by an increased extravasation of plasma proteins, with subsequent deposition in the vascular wall - i.e. the concept of plasmatic vasculosis. 214. Diabetic Retinopathy in Idiopathic Haemochromatosis. Ph. Passa, F. Rousselie, J. Canivet. Department of NutritionEndocrinology Hopital Saint Louis, F-75475 Paris Cedex 10, France. It is generally stated that patients with diabetes due to haemochromatosis are not prone to angiopathy. The aim of this study was to investigate in such patients the incidence and severity of retinopathy and the plasma growth hormone response to various stimuli. - This was carried out in 22 .patients with diabetes due to idiopathic haemochromatosis, usxng fundus examination, retinography and fluorescein angiography. The plasma growth hormone response to insulin hypoglycemia and arginine infusion was determined. - In these patients retinopathy was present in 9 of 22 cases and thus appeared of similar prevalence as in genetic diabetes according to patient's age and duration of diabetes. However retinopathy remained of mild degree. Plasma growth hormone response was significantly lowered, as compared with normal subjects matched for sex and age and with diabetic subjects matched for age and duration of diabetes. - It may be that the mild degree of the retinopathy is related to the diminished plasma GH levels. 215. Insulin Receptors in Lymphocytes from Normal and Obese Persons Correlated with IVGTT (K-values), Serum Insulin and Fat Cell Size. O. Pedersen, H. Beck-Nielsen, J.P. Bagger and N. Schwartz Sorensen. Medical Department III, County Hospital of Aarhus, DK 8000 Arhus C, Denmark. The specificity, sensitivity and kinetics of insulin binding to lymphocytes are similar to those of the major metabolic sites of insulin action - hepatocytes and adipocytes. - 125I-insulin binding to lymphocytes isolated from peripheral blood was studied using an improved technique, which includes separation of lymphocytes from suspensions by centrifugation through silicone oil. - Our present study includes 21 healthy persons (20-40 years old, 90-110% of ideal weight) and a comparable group of 23 obese subjects (minimum 150% of ideal weight). In the obese fewer receptor sites per lymphocyte were found (p < 0,01) than in the normals. - No correlation was found between bodyweight and insulin receptors. However, insulin binding was negatively correlated with fat cell size. An inverse relationship was demonstrated between fasting serum insulin and the number of receptors. (p < 0,001). K-values (IVGTT) were positively correlated with insulin binding (p < 0,01). - Insulin receptors were also studied in 9 obese hyperinsulinemic persons before and after 10 days of fasting. An average increase in insulin binding of 50% was observed with a simultaneous decrease in serum insulin to normal values. - The results indicate that obesity complicated by hyperinsulinism is associated with a decrease in the numbers of insulin receptors compared to the state of normal bodyweight. This finding may in part explain the decreased insulin sensitivity of the hyperinsulinemic obes% 216. Effect of Diet, Sulphonylurea and Placebo Therapies on Glucose Tolerance, Blood Metabolites and Insulin Secretion in Diabetics. J.R. Perkins, B.J. Hay, Susan L. Judd, K. Hilary Quine, T. E. T. West and P.H. S6nksen. St. Thomas' Hospital Medical School, London SE1 7EH, U.K.

369

Although sulphonylurea drugs are widely used in maturity-onset diabetic patients, who do not respond to diet alone, the mechanism of the hypoglycaemic action is disputed. We have studied the effect of sulphonylurea treatment on glucose tolerance, glucosestimulated insulin release and blood metabolites in 19 non-obese, diabetic patients. - Each patient underwent a 100 gm., 5 hr. oral glucose tolerance test, 1. On diagnosis. 2. After diet therapy alone (mean duration 2 months). 3. After 4 months diet + sulphonylurea treatment. 4. After up to 4 months diet + placebo treatment. Fasting blood glucose and glucose tolerance, which had improved marginally on diet therapy alone, improved further on sulphonylurea treatment, but both relapsed to pre-sulphonylurea levels on placebo. Insulin concentrations were similar on diagnosis and after diet alone or diet + placebo, but were higher after diet + sulphonylurea. Ketone body and glycerol concentrations were lowest after sulphonylurea treatment. Lactate, pyruvate, cholesterol, triglycerides and growth hormone concentrations and lipoprotein patterns showed no substantial changes. - These results suggest that the improvement in fasting blood glucose, glucose tolerance and blood metabolite concentrations in this group of patients were the result of increased insulin secretion, secondary to treatment with sulphonylureas. 217. The Natural History of Diabetes Mellitus: Results of a Retrospective Study. R. Petzoldt, H. Frerichs, H.K. Selbmann, K. ~berla, W. Creutzfeldt, K. Sch6ffling. Zentrum der Inneren Medizin, Abteilung fiir Endokrinologie Universit~it Frankfurt/Main; Medizinische Klinik, Universitiit G6ttingen; Institut fiir Informationsverarbeitung, Statistik und Biomathematik, Universit~it M/inchen, FRG. A controlled study has been carried out on 1302 diabetics, based on retrospectively collected data of the outpatient clinics of Frankfurt and Goettingen. The study analyzes some 11000 directly controlled years of 773 female and 529 male diabetics. - In 37.6% of the patients with available data the birth weight is higher than 4.5 kg. The symptoms at manifestation of diabetes were: thirst (70.3%), pol)uria (56.5%), weakness (49.2%), no symptoms, i.e. diagnosis by chance (33.1%), loss of weight (26.1%). At manifestation the mean overweight was about 15-20 kg (or about 25% in male and about 45% in female diabetics). - Of the numerous data at the end of the study the following results seem to be of special interest: overweight 84.6% (more than 20% overweight 44.1%), hypertension 46.7%, macroangiopathy* 91.8%, diabetic neuropathy* 81.0 %, diabetic nephropathy* 41.8 %, diabetic retinopathy 29.1%. The diabetics were subdivided into four treatment groups (diet 73, insulin 134, sulfonylureas 346, sulfonylureas and biguanides 286). The insulin treated patients had significantly more frequent retinopathy (58.2%). - In the course of the study 158 patients (18.8%) of the four treatment groups mentioned died. The insulin treated patients had the longest duration of the disease and showed the highest death rate. - Further data of the study will be presented. * (9 1 symptom) 218. The Artificial Pancreas: Application to Experimental and Clinical Research. E.F. Pfeiffer, Ch. Thum, W. Beischer, C. Meissner, Gy. Tam/ts Jr. and A.H. Clemens**. Department of Internal Medicine, Endocrinology and Metabolism, Center of Internal Medicine and Pediatrics, University of Ulm, FRG. An artificial endocrine pancreas has been developed, comprising an on-line glucose analyzer, a special microcomputer/controller, preprogrammed for glucose controlled insulin infusion, a novel insulin and glucose delivery system, and a printer/plotter for documenting the control parameters selected and the control results achieved. Insulin deficient diabetics, including juvenile patients, were controlled up to 3 days and maintained within the normal glucose range without any food restrictions. The rate dependent infusion of insulin during the rise of blood glucose was found to be of particular importance. In automated therapy of coma and precoma (14) computer-controlled insulin infusion (600 to 1000 mU/min) revealed a significantly more rapid normalization of metabolism

370

Abstracts

than could be achieved by conventional methods. Further experience was gained during complicated diabetes related surgery (12), including Caesarean section, and in pancreatectomized as well as hypophysectomized subjects. The apparatus was also used to profile the actual insulin requirements in response to various types of oral antidiabetic agents and after administration of somatostatin, as well as for evaluation and treatment of hypoglycemic attacks. The metered amounts of exogenous insulin were compared to a) the immunomeasurable insulin (IMI) of the peripheral vascular system, b) the proinsulin and c) to the C-peptide. Some insight could be obtained into the behaviour of endogenous and exogenous insulin even when antibodies were present. ** Life Science Instruments, Miles Laboratories, Elkhart, Ind., USA

219. Effect of Phenformin on the Level of Free Amino Acids in Plasma of Healthy Subjects. D.M. Piniewska, J. Jarz~bifiski and A. Czy~yk. Department III of Internal Diseases and Laboratory of Drug Analysis, Medical Academy, Warsaw, Poland. In two groups of diabetics with maturity-onset diabetes a significant rise in plasma alpha-amino nitrogen level was observed following phenformin administration. In the present study this phenomenon was investigated in more detail, determining the effect of phenformin on plasma level of free amino acids. - In 10 healthy subjects 15 free amino acids were determined in plasma by the method of the ion-exchange chromatography after an overnight fast, before and after 3 days' administration of phenformin in a daily dose of 150 rag. - A statistically significant rise was found in plasma levels of alanine (0.395 vs 0.306), histidine (0.116 vs 0.105), isoleucine (0.071 vs 0.061), leucine (0.128 vs 0.117) and lysine (0.302 vs 0.275) while the level of arginine fell (0.077 vs 0.095) all values calculated in micromoles per ml. - It is to be stressed that the highest rise after phenformin was in alanine level. The results will be discussed taking into account the influence of phenformin on both the glucose-alanine cycle and gluconeogenesis. 220. Assembly and Disassembly of Microtubules in Islets. D.G. Pipeleers, M.A. Pipeleers-Marichal, D.M. Kipnis. Metabolism Division, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA. Tubulin, the subunit of microtubules, was demonstrated in pancreatic islets by immunofiuorescence, gel electrophoresis and colchicine binding activity. The amounts of polymerized (microtubules) and depolymerized tubulin were measured by centrifuging islet homogenates in microtubule stabilizing medium and measuring colchicine binding in supernatant and pelleted fractions. Electron microscopy of the pelleted fraction demonstrated numerous microtubules, many of which were in close contact with granules. Islets, obtained from fed rats, contained 0.22 _+ 0.02 ~tg tubulin/~tg DNA, 34.9 -+ 2.1% of which was polyrnerized. The degree of tubulin polymerization decreased 20% in islets from 72 hr. fasted rats and increased 34% after incubation with 300 rag% glucose; a further increase of 8% was noted in the presence of 5 mM theophyline. Total tubulin remained unchanged under these conditions. These alterations in the degree of tubulin polymerization were paralleled by corresponding changes in insulin release, in particular the secretion of newly synthesized hormone (3H-leucine labelled). Colchicine also inhibited the conversion of proinsulin to insulin. It is concluded that microtubule assembly is modulated by physiologic factors affecting insulin release and may be involved in the intracellular transport and conversion of newly synthesized hormone. 221. Diabetic Retinopathy, Nephropathy, Neuropathy. Relation to Duration and Control. A statistical study in 4.400 diabetics. J. Pirart and J. P. Lauvaux, C. Eisendrath. H6pital Saint-Pierre and Clinique Cesar de Paepe. Brussels, Belgium. In a prospective study conducted from 1947 to 1973 the natural history of diabetes and its complications and the possible role of giycemic control on their development and evolution has been

assessed. Examination for complications was performed at least once a year. Methods and criteria were standardised and coded data submitted to statistical analysis (C. D. C. Computer). Among 4.400 diabetics, 2.795 cases were seen from onset (initial cohort); of these 547 were followed for 5 years, 219 up to 10 years and 29 up to 20 years. - The incidence and prevalence of the three complications run a parellel course. They are closely related to known duration and to glycemic control, assessed each year and expressed as a cumulative value for the whole duration. These relations were found in each sub group (initial cohort, age, sex, severity of diabetes). There was no correlation with family history and a poor correlation, if any, with either sex or severity of diabetes. - It is concluded that retinopathy, nephropathy and neuropathy are true complications of diabetes, with chronic hyperglycemia as a common actiological factor.

222. Growth Hormone Secretion in Chemical Diabetes; Differences Related to Sex, Body Weight and Serum Lipids; Effect of Clofibrate; Effect of Metergoline. A. E. Pontiroli, G. Viberti, A. Tognetti and G. Pozza. Cattedra di Patologia Medica dell' Universit~t degli Studi di Milano, Ospedale San Raffaele, 20090 Milano-Segrate, Italy. Growth hormone (GH) has been suggested to be involved in the pathogenesis of diabetes mellitus and in the development of diabetic microangiopathy. However, GH secretion has been found to vary according to the stage of diabetes. In chemical diabetes (C. D.), GH release has been evaluated, until now, only in response to oral glucose. In the present study GH response to arginine infusion was evaluated in C.D. subjects of normal body weight and obese. - In C.D. subjects of normal body weight GH release was slightly but not significantly reduced in comparison to normal subjects: in obese subjects, either C.D. or with a normal glucose tolerance, GH release was blunted. The well-known sex-related differences in GH secretion were observed in C.D. subjects. - In normal subjects, serum triglycerides levels higher than 172 mg% were associated with a blunted GH release. No such relationship was observed in C.D. Both clofibrate, an hypolipemic agent, (3 g/day for 10 days) and metergoline, an anti-serotoninergic agent (12 rag/day for 3 days) significantly increased arginine-induced GH release in normal subjects; in C.D. a slight but not significant decrease in arginine-induced GH release was observed following both treatments. - These results suggest that GH secretion is impaired in chemical diabetes. 223. Impairment of Insulinogenesis and Liver Glycogen Depletion in a Model of Prolonged Gestation. B. Portha, G. Rosselin, L. Picon. Laboratoire de Physiologie Animale, Universit6 de Paris VII and Unit6 INSERM U. 55, H6pital Saint-Antoine, Paris, France. Prolonged pregnancy was obtained in the rat by subcutaneous injections of Progesterone (Pg) 2.5 mg each day, on day 20-21-22 post coitum (d.p.c.). Comparison was made between the fetuses obtained after two extra days of gestation and those taken on day 21.5 p.c. just before normal delivery, both obtained by caesarean section. The 23.5 fetuses exhibit a 60% decrease in liver glycogen, a 50% fall in plasma immunoreactive insulin (IRI) while pancreatic IRI content is increased by 60%, as compared to the 21.5 fetuses. Blood glucose (BG) level is the same in both groups. These modifications are more likely related to the prolonged gestation than to Pg per se: a similar pattern of Pg-three days-injections followed by caesarean delivery on day 21.5 p.o. does not affect plasma and pancreatic IRI or liver glycogen as compared to the untreated 21.5 fetuses. Delayed delivery determines a high mortality rate (50%) during the first 24 h. of extrauterine life. The insulinogenesis which takes place in the two days following normal birth is not obtained by two extra days in utero: IRI content in the 23.5 fetuses is 103 mU _+ 11 per pancreas (7.8 mU/mg) against 229 mU + 17 per pancreas (15.3 mU/mg) in the 2 day old neonates (23.5 d. p. c.). This defect in insulin content persists after birth: IRI content on day 5 after birth (28.5 d.p.c.) is 65 % of the values observed in the 7 day old normal neonates (28.5 d.p.c.) although both groups have the same body weight and similar plasma IRI and BG values. These data suggest

Abstracts that prolonged gestation induces sustained alterations in the endocrine pancreas and an acute depletion of the fetal liver glycogen. These observations are of peculiar importance in view of the metabolic adaptation to extrauterine life.

224. The Relationship of Hyperglycaemia to the Risk of Cardiovascular Death. K. Py6r~l~i, A. Reunanen and A. Aromaa. Division of Epidemiology and Preventive Medicine and Research Institute for Social Security, Social Insurance Institution, Helsinki, Finland. Information concerning the relationship of decreased glucose tolerance or hyperglycaemia to the risk of cardiovascular death is still controversial. We have studied this relationship in a population comprising 17,169 men and 15,615 women aged 30 or over who participated in a multiphasic screening examination between February 1967 and May 1972. As a part of this examination an oral glucose tolerance test was carried out. The glucose dose given was 60, 75 or 90 g according to body surface area. Plasma glucose was determined from a venous blood sample taken 1 hour after glucose load. - Until the end of 1972 (median follow-up time 3 years) 644 men and 329 women died; the cause of death was cardiovascular disease in 363 men and in 201 women. Analysis of the causes of death by quintiles of 1-hour plasma glucose showed that in the age group 50-69 of both sexes high plasma glucose levels at the initial examination were associated with a statistically significant increase in cardiovascular mortality; coronary mortality and cerebrovascular mortality showed similar, though not separately significant trends. In persons aged 70 or over cardiovascular mortality failed to show any consistent trends with plasma glucose level. 225. Adenosine 3', 5'-Monophosphate (cAMP) and Insulin Responses to Glucose in Isolated Pancreatic Islets from Fed and Fasted Rats. A. Rabinovitch. Institut de Biochimie Clinique, Geneva, Switzer/and. A possible involvement of the adenylate cyclase-cAMP system in the impairment of insulin secretion associated with fasting was assessed by comparison of glucose-induced insulin release and [3H] cAMP accumulation in islets of fed and 48 hr fasted rats, after 1 hr preincubation of islets with [3H]-2-adenine. 30 sec. after incubation with glucose 26.7 raM, plus 3-isobutyl-l-methylxanthine (IBMX) 0.1 mM, there was a significant increase in [3H] cAMP accumulation islets from fed rats (p < 0.05), reaching a maximum i(+n 300%) between 2 and 10 rain (p < 0.001). By contrast [3H] cAMP in islets from fasted rats was not significantly increased until 1 rain (p < 0.01) and the increases were significantly less than in islets from fed rats at 1 min (p < 0.02) and at 2 rain (p < 0.005). Similarly, insulin release from islets of fasted rats was significantly less than from islets of fed rats between 2 rain (p < 0.05) and 30 rain (p < 0.001). Glucose dose-response studies in 10 rain incubations indicated a decreased sensitivity of glucose-induced insulin release as well as [3H] cAMP accumulation, or release, in islets of fasted rats. However in the presence of a high concentration of IBMX (1.0 mM), glucose-induced insulin and [3HI cAMP responses in islets of fed and fasted rats were similar. These studies demonstrate that decreased cAMP responses to glucose may account for the lesser sensitivity to glucose observed in the pancreatic beta cell during fasting. 226. Decreased Adenosine 3', 5'-Monophosphate (cAMP) and Insulin Responses to Glucose in Isolated Pancreatic Islets of Diabetic Chinese Hamsters. A. Rabinovitch. Institut de Biochimie Clinique, Geneva, Switzerland. A possible involvement of the adenylate cyclase-cAMP system in the impaired secretion of insulin associated with the diabetic state was assessed by comparing glucose-induced insulin release and [3H] cAMP accumulation and release in islets of normal, and glucosuric non-ketotic Chinese hamsters after i hr preincubation of islets with [3H]-2-adenine. Glucose dose-response studies, in 10 min incubations with and without 3-isobutyl-l-methylxanthine (IBMX) 1.0 mM, indicated a decreased sensitivity of insulin release for islets

371

of diabetic hamsters, with a glucose k m~ 25 mM compared to - 15 mM for normal islets. Similarly, in the presence of IBMX 1.0 mM, [3H] cAMP in normal islets increased from 409 + 50 (dpm/25 islets/10 min, means + SEM) in the presence of glucose 3.3 raM, to 590 + 47 (p < 0.05) with glucose 8.3 mM, 661/+ 72 (p < 0.02) with glucose 27.8 raM, and 811 + 95 (p < 0.005) with glucose 55.4 mM. There were no significant increases in diabetic islets. Furthermore, insulin release from perifused hamster islets stimulated with glucose 27.8 mM was about three fold greater (both phases) in normal than in diabetic islets, and [3H] cAMP efflux was increased about 50% (p < 0.01) in normal but not significantly in diabetic islets. There results suggest that defective glucose-induced cAMP formation in the beta cell may play a role in the impairment of insulin release associated with diabetes in the Chinese hamster.

227. Portal and Peripheral Blood Concentrations of Insulin and Exocrine Pancreatic Secretion in Response to Pure (99%) Cholecystokinin-Pancreozymin (CCK) in Man.* S. Raptis, H.C. Dollinger, W. Schlegel, A.S. Nadjafi. Department Internal Medicine Endocrinology and Metabolism, Section Gastroenterology, Department of Surgery II, University of Ulm, 79 Ulm/D, Steinh6velstrat3e 9, FRG. Impure CCK is known to stimulate insulin as well as exocrine pancreatic secretion in man. By contrast, pure CCK has been reported to have no influence on insulin release. The present study describes the effect of pure (99%) CCK on insulin release and exocrine pancreatic function compared with impure (10 %) CCK in normal subjects. - In 5 subjects, the effect of 2 Ivy-dog units/kg of impure and pure CCK on insulin release and exocrine pancreatic secretion were studied in randomized order. In 5 additional subjects, the influence of pure CCK on glucose assimilation was investigated. In 7 subjects receiving pure CCK the insulin concentration in the peripheral and portal blood (by umbilical catheterisation) was determined. - Compared with the impure preparation, pure CCK caused similar effects on exocrine pancreatic function, whereas the peripheral blood concentration of insulin was not significantly altered. Following pure CCK a significant improvement of glucose assimilation (2,17 _+ 0,6 to 3,4 + 0,2) and an increase in glucose-induced insulin secretion was observed (77 _+ 9 gU/ml and 112 + 11 ~tU/ml, respectively). Although the peripheral blood concentration of insulin was not affected, pure CCK led to a significant elevation (50%) of insulin concentration in the portal blood. - In conclusion, pure CCK stimulates insulin release and causes improvement of glucose assimilation. Since pure CCK induces a smaller increase in insulin secretion which is eliminated by the liver, no significant changes in peripheral blood concentration of insulin can be found. The higher elevation of insulin concentration in the portal as well as peripheral blood after i.v. administration of impure CCK seems, therefore, to be due to contaminating substances, such as gastric-inhibitory polypeptide (GIP). * Supported by Deutsche Forschungsgemeinschaft (SFB 87 Endokrinologie) Bonn - Bad Godesberg. 228. Mapping of Concanavalin-A Binding Sites at the Surface of Endocrine Pancreatic Cells in Monolayer Culture. M. Ravazzola, B. Blondel, A. Perrelet, and L. Orci. Institute ot Histology and Embryology, University of Geneva Medical School, Geneva, Switzerland. Carbohydrate residues are essential constituents of the cell surface antigens, enzymes or receptors. They have a strong affinity for plant lectins, such as concanavalin-A (ConA), and the mapping of lectin binding sites by electron microscopy has been an important tool in the study of surface topography in several cell types under various experimental conditions. After tagging ConA with hemocyanin for shadow casting and scanning electron microscopy, the topography and the mobility of ConA-binding sites were studied in pancreatic islet cells in monolayer culture. At 37 ~ C, most cells showed ConAbinding sites grouped in patches of variable size. Morever, binding sites at 37 ~ C tended to occupy the central part of the cell surface. At 4 ~ ConA-binding sites occurred as tiny clusters, randomly distibuted on the cell surface. After fixation, binding sites were random and dispersed. - It is concluded: a) that the distribution of ConA-binding sites on islet cell membrane is temperature

372

Abstracts

dependent; b) that clustering of the binding sites probably results from the cross-linking properties of ConA on mobile receptors; c) that the mobility of binding sites is restricted at low temperature and abolished by fixation. 229. Mechanisms of Insulin Resistance in Diabetes. G. M. Reaven, H. Ginsberg, C. Javorski, G. Kimmerling, and J. Olefsky, Department of Medicine, Stanford University School of Medicine and Veterans Administration Hospital, Palo Alto, California, USA. We have used a continuous infusion of glucose, insulin, epinephrine and propranolol to assess insulin resistance in adult diabetics. With this technique, endogenous secretion is inhibited, similar levels of exogenous insulin are achieved in all subjects, and the steady state plasma glucose level during the infusion measures insulin resistance. Mean (+ SE) glucose levels were 106 + 6 mg% in 63 normal subjects, 234 + 7 rag% in 28 chemical diabetics, and 344 + 15 rag% in 20 patients with fasting hyperglycemia. To further define this insulin resistance we used a constant infusion of 2-3H glucose to measure: 1) hepatic glucose production; and 2) efficiency of glucose uptake; fasting and during the infusion, in a subset of patients. Basal hepatic glucose production, which fell approximately 75% during insulin infusion in normais, and was comparably inhibited in chemical diabetics, was not suppressed by insulin in patients with fasting hyperglycemia. Efficiency of glucose uptake, which rose fourfold from basal levels in normals during the infusion, increased significantly less in patients with chemical diabetes or fasting hyperglycemia. Thus, insulin resistance in chemical diabetes is characterized by a defect in insulin-mediated glucose uptake, while insulin suppression of hepatic glucose production remains relatively intact. In patients with fasting hyperglycemia insulin does not stimulate glucose uptake or inhibit glucose production norm-

ally.

230. Effect of Insulin in Vivo on Cyclic AMP Levels in Adipose Tissue from Diabetic and Non-Diabetic Subjects. J. P. D. Reckless, D.J. Galton. Diabetes Research Laboratory, St. Bartholomew's Hospital, 51-53 Bartholomew Close, London, E.C.I, U. K. Impaired inhibition of lipolysis by insulin may lead to increased plasma fatty acid flux in adult diabetes. Insulin has been shown to inhibit lipolysis in adipose tissue in vitro. While the intracellular second messenger mediating the action of insulin on lipolysis is not known, changes in cyclic AMP levels due to insulin are observed. Altered cyclic AMP responses to insulin in diabetes may contribute to increased fatty acid flux. - Levels of cyclic AMP were therefore studied in adipose tissue from 22 obese subjects before and during an insulin tolerance test (0.3 units/kg). Needle biopsy tissue samples were immediately frozen in liquid nitrogen before extraction and assayed for cyclic AMP. - Eleven non-diabetics showed a fall in cyclic AMP levels from 0.063 _+ 0.012 pmol/mg (before insulin) to 0.039 + 0.005 at 15 rnin (after insulin) (p < 0.05), and to 0.036 + 0.005 at 30 rain (t7 < 0.02). Cyclic AMP levels in eleven diabetic subjects were 0.057 _+0.009 pmol/mg before insulin, 0.048 + 0.007 at 15 min, and 0.049 -4--0.007 at 30 min, and fails were not significant. - I n vivo administration of insulin lowers cyclic AMP levels in adipose tissue from non-diabetics and may be one mechanism of the antilipolytic action of insulin. The fall in cyclic AMP levels in tissue from diabetics is small, and not significant, suggesting resistance in the diabetic to this effect of insulin. 231. Opthalmologic Study of the Effect of a New Antidiabetic Treatment on the Course of Diabetic Retinopathy. F. Regnault, Hopital Cochin Paris, France. The demonstration of platelet and endothelial changes in diabetic retinopathy has led to the development of a new antidiabetic agent, Gliclazide, having hemobiologieal properties. The effect of this drug on the course of non proliferative diabetic retinopathy is at present being studied by the mean of a controlled longitudinal multicentric trial. - Every six months, clinical and opthalmological (retino- and angiography) measurements are carried out. The classification of retinal vascular abnormalities detected on an-

giography enables a statistical evaluation of the changes observed in treated and control patients. - After more than 18 months of treatment (2 to 3 years in a third of the cases), preliminary results concerning 258 opthalrnological cases show a signicantly greater percentage of improvement as well as a significantly lower percentage with deterioration in the gliclazide-treated group as compared with the control group. - At this stage of the trial, early stages of the diabetic retinopathy were favourabley influenced by the tested treatment. 232. Comparison of Insulin Binding to Liver Membranes in Three Species of Genetic Diabetic Rodents. R. Renner, W. Kemmler and K.D. Hepp. Diabetes Research Unit and 3rd Med. Dept. Schwabing City Hospital, 8 Munich 40, K61ner Platz 1, FRG. Several groups have shown a considerable decrease in total insulin binding to liver membranes from obese hyperglycemic mice which suggested a decrease in insulin receptors and a correlation with hyperinsulinism and insulin resistance. It seemed of interest to extend binding studies to other strains with different types of genetic hyperglycemic syndroms such as the Chinese hamster, and the KK-mouse. - Binding of lasI-insulin was studied at equilibrium in presence and absence of unlabelled insulin (104 - 1 Ix/ml). The membrane bound hormone was separated by centrifugation through silicon oil or by the conventional filter technique. - In confirmation of the work of Kahn et al., a marked decrease in total binding capacity was observed in crude membranes from ob/ob mice, as compared to the non-diabetic controls. In contrast, no significant difference was found in severely diabetic Chinese hamsters and obese, hyperinsulinemic and hyperglycemic KK mice, in comparison to their normal littermates. However, when membranes were further purified according to Neville, total binding capacity was increased by ca 100% in the diabetic hamsters as compared to their normal controls. The results suggest that in purified plasma membranes but not in crude preparations, total insulin binding reflects the state of insulin in blood. 233. Regulation of Cyclic AMP Dependent Protein Kinase in Human Skeletal Muscle in Vivo by Epinephrine and Insulin. Aa. Roch-Norland, R. Horn, E. Walaas, O. Walaas. St. Eriks Hospital, Stockholm, Sweden, and Institute of Medical Biochemistry, University of Oslo, Norway. Previous studies from our laboratory have demonstrated that regulation of glycogen metabolism in muscle in v i t r o b y epinephrine and insulin is mediated by control of cyclic AMP dependent protein kinase. The aim of the present work has been to investigate whether the same control mechanism is operating in intact human muscle in vivo. - Cyclic AMP and cyclic GMP were determined in muscle biopsies after administration of epinephrine or insulin. Protein kinase was assayed by incorporation of (3~p) from (32P)-7-ATP into h i s t o n e . - Administration of epinephrine increased,cyclic AMP 2-3 fold but had no effect on the level of cyclic GMP. Simultaneously the cyclic AMP dependent form of protein kinase was decreased. Insulin did not influence the level of cyclic nucleotides. Nevertheless, the cyclic AMP dependent form of protein kinase was increased. - It is concluded that epinephrine activates protein kinase due to an increase of the level of cyclic AMP. Insulin convert this enzyme to an inactive form by a mechanism which is still unknown. 234. Platelet Aggregation in Diabetic Patients Treated with Oral Hypoglycemic Agents. J. Zurro, H. Romero, A. Marafion, S. Duran, E. Romero B., Facultao De Medicina, Valladolid, Spain. The effect of several oral hypoglycemic drugs on platelet aggregation has been tested in a longitudinal study carried out on in 120 adult diabetic patients. - Groups of 20 patients were treated with glibenclamide, glycacyde, biguanide, gliboruuride, glypicide, and glypentide. Per cent of maximal aggregation, rate of aggregation, index of aggregation and minimal dose capable of doubling the ADP curve (Born's method) were determined before and 15,30 and 45 days after treatment. Total lipids, cholesterol and triglyceride blood levels were also estimated. In all cases the vascular damage

Abstracts was evaluated. Correlation studies between all parameters measured show that diabetic hyperaggregation was significantly decreased by all agents tested. In the group of patients treated with glycacyde platelet aggregation was significantly decreased (p < 0.01). We compare these results with the other hypoglycemic agents. 235. Proteolytic Activities in Skeletal Muscle of Diabetic Rats. H.-J. R6thig, N. Stiller, H. Reinauer. Biochemical department of the Diabetes-Forschungsinstitut D 4 Diisseldorf I, Auf'm Hennekamp 65, FRG. In insulin deficiency skeletal muscle mass decreases. Therefore, we investigated the insulin dependent proteolysis in skeletal muscle. The proteolytic activities in the acid (pH 3.0-4.0) and in the alkaline (pH 9.0-10.0) range were measured by splitting of exogenous substrates (C14-1abelled hemoglobin) and endogenous substrates. - The alkaline proteinases are associated with the myofibrillar fraction. In streptozotocin diabetic rats we found increased proteolytic activities in the alkaline range, whereas the activity of acid proteinases was unchanged. Four days after the injection of streptozotocin the alkaline proteolytic activity was 50% higher than in the controls, after 20 days of diabetes the activity was 300% higher. The proteinase activity could be reversed to the normal range by insulin. Adaptive behaviour of the myofibrillar alkaline proteolytic activity was also seen in fasted and cortisone treated animals. - In cases of diabetes the myofibrillar proteins undergo proteolysis by defined proteinases thus delivering amino acids for gluconeogenesis in the liver. 236. Cells lmmnnofluorescent to Anti-Growth Hormone Release Inhibiting Factor (Somatostatin) in Endocrine Pancreas and Digestive Tract. C. Rufener, M.P. Dubois. Inst. of Histology and Embryology, University of Geneva Medical School, Geneva, Switzerland, and I.N.R.A., Station de Physiologie de la Reproduction, Nouzilly, France. Using a rabbit antiserum to synthetic growth hormone releaseinhibiting factor (GIF), and sheep anti-rabbit immunoglobulin conjugated to fiuorescein isothiocyanate, immunofluorescent cells were detected in rat and dog islets of Langerhans, normal and diabetic (juvenile) human islets, rat endocrine pancreatic monolayer cultures (ML) and dog digestive tract. In normal islets, the anti-GIF fluorescent cells were less numerous than anti-insulin or anti-glucagon fluorescent cells. In islets of juvenile diabetic patients, only positive cells for anti-GIF and anti-glucagon were detected. In ML, a variable number of anti-GIF fluorescent cells was found in the endocrine clusters. Anti-GIF fluorescent cells were sparse in the intestine and in the gastric fundus of the dog, more numerous in the antrum, where they could be distinguished from anti-gastrin fluorescent cells by serial sectioning. Anti-GIF fluorescence was not affected by adsorbing the anti-serum with an excess of glucagon or gastrin, but was abolished by an excess of GIF. Since GIF appears to inhibit the release of various polypeptide hormones, it is concluded that GIF-producing cells could be local modulators of secretion in a number of organs. 237. Effects of Insulin and Non-Suppressible Insulin Like Activity (NSILA) Upon Adenylate Cyclase Activity of Rat Liver Plasma Membranes. A. Rutschmann, A. Jakob. Metabolic Unit, Department of Medicine, Kantonsspital Zurich, Switzerland. NSILA mimics the metabolic response of several tissues to insulin. In rat epididymal fat pads and perfused rat livers insulin inhibited hormonally stimulated cyclic AMP release. NSILA had a comparable effect on adipose tissue. Rat liver plasma membranes were prepared by a two phase system, containing dextran and polyethylene glycol. Adenylate cyclase activity was measured by the rate of conversion of 32p-ATP to 32P-cyclic AMP. Mouse livers were perfused with Krebs bicarbonate buffer in a non-recirculating system. Three preparations of NSILA with different biological activities per mg protein (estimated by adipose tissue assay) were compared: A 2.2 mU/mg, B 30 mU/mg, C 70 mU/mg. Prep A

373

inhibited basal glucagon (10-7M) and NaF (10-3M) stimulated adenylate cyclase by 70%, prep B by 40%. Prep C was not inhibitory. The concentration of NSILA was 1 mU/ml in all the assays. Insulin (1 and 0.1 mU/ml) did not influence basal and glucagon stimulated adenylate cyclase. In peffused livers prep B decreased glucagon stimulated cyclic AMP release into the perfusate by 50%. Oxidized glutathione (10-3M) inhibited glucagon stimulated membrane adenylate cyclase by 58%. The three NSILA preparations equally displaced 125I-labelled NSILA from liver membranes. An interaction of prep C with the membranes occurred despite the lack of an inhibitory effect on adenylate cyclase. From our experiments we conclude that NSILA and insulin do not inhibit adenylate cyclase. In impure preparations oxidizing contaminants may be responsible for the inhibitory effects that were obtained. 238. Ultrastructural Changes and Histo-Cytochemical Cation Patterns of the B-Cells Associated with the Action of Diazoxide and Diphenylhydantoine. H. Schiller, G. K16ppel and G. Bommer. University of Hamburg, Institute of Pathology 2000 Hamburg 20, MartinistraBe 52, FRG. Diazoxide and diphenylhydantoine (DPH) produce a temporary hyperglycemic syndrome in man and animals. The effects of diazoxide and DPH on the secreting and synthefizing apparatus and on cation distribution patterns of B cells in mice were examined by histochemistry, electronmicroscopy and electron cytochemistry. - Histochemically the calcium content of islet cells was determined using the glyoxal-bis-(2-hydroxyanil) method. For ultrastructural localization of cations the pyroantimonate method was performed. Elemental composition of precipitates at some typical cell sites was determined by energydispersive X-ray analysis; they were found to be rich in calcium. - DPH and, particularly, diazoxide cause a hypergranulation, intracellular granulolysis and a cation pattern of the "inactive type" Despite of presence of a hyperglycemic stimulus the light and electronmicroscopic calcium content of B cells was low compared with the ion overloaded state after treatment with sulfonylureas. Diazoxide and DPH do not suppress resynthesis of secretory granules and pre-degranulated B cells suggesting that mainly the granule release is impaired. - On the basis of these findings the model is discussed that the inhibitory effect of diazoxide and DPH on insulin secretion may be induced by a blockade of transmembraneous cation transport, which seems to be essential for insulin release. 239. Long-Term Effects of Sulfonyinreas (SU) on the Function of Pancreatic Islets in Rats. H. Schatz, J. Sieradzki, D. Steinle, S. Raptis, C. Nierle and E.F. Pfeiffer. Dept. of Internal Medicine, Endocrinology and Metabolism, University of Ulm, FRG. SU, especially tolbutamide (T) inhibits insulin biosynthesis in vitro. It was the aim of this study 1.) to evaluate the significance of this effect in vivo and 2.) to study late effects of SU in vitro. Methods: 1.) T or glibenclamide (G) were administered to rats for 10 months. Estimations: Insulin, protein and DNA content of their islets (collagenase). Release and synthesis of proinsulin and insulin after incubation of the islets with 3H leucine for 3 h. 2.) Islets of untreated rats were incubated for 2 h with T or G, followed by incubation for 2 h with 3H leucine and glucose alone. Proinsulin and insulin were separated on Sephadex G 50. Results." 1.) Weight, blood glucose and serum insulin were significantly higher in rats treated with T and G than in controls. Insulin content was significantly lowered after T not after G. No significant differences existed in insulin release from the islets. 3H leucine incorporation into proinsulin and insulin was significantly reduced after G, not after T. When incorporated cpm were expressed per unit of insulin content however, a relative increase in biosynthesis during these 3 h became apparent after T in vivo, in contrast to G. 2.) Following islet exposure to SU in vitro, proinsulin and insulin synthesis remained reduced during the subsequent 2 h. Conclusions: SU increase serum insulin levels after long-term administration which might explain the weight gain. T inhibits insulin synthesis in vivo, as judged by the reduced insulin content, in contrast to G. However, this effect appeared to be quickly reversible (judged from the relative increase of insulin

374

Abstracts

synthesis in vitro after T in vivo). The secretory capacity of the islets was also not disturbed. After exposure to SU in vitro, no rebound was demonstrable during the subsequent 2 h, which might partly compensate for both reduced synthesis and increased release. Long-term administration of SU increases insulin levels without serious alteration of the beta-cells. T and G rather appear to modify the function of the beta-cells in some different way in order to provide - possibly by an indirect mechanism - sufficient amounts of hormone for increased secretion. 240. 5'-Guanylylimiflodiphosphate: A Modulator of GlucagonInduced IRI Release from Rat Pancreatic Islets. P. Schauder, R. Ebert. Division of Gastroenterology and Metabolism, Department of Medicine, University of G6ttingen, FRG. Glucagon-stimulated insulin release is initiated by binding of the hormone to a specific receptor in the B-cells, followed by activation of adenylate cyclase and elevation of cyclic adenosine 3', 5'-monophosphate. The signal transfer from the hormone receptor to the catalytic unit of the enzyme is poorly understood but seems to involve purine nucleotides. Thus, GTP is known to amplify the glucagon-induced activation of adenylate cyclase from plasma membranes in rat liver. We have investigated whether glucagonstimulated IRI release from isolated rat pancreatic islets is amplified by a GTP-analogue. - Islets, isolated from pancreas of fed male Wistar rats (200-300 g) by collagenase digestion, were incubated for 45 min in 500 ~tlKRB-buffer (BSA 1 mg/ml) with the following additions. A: Glucose (8.12 or 16.6 mM). B: Glucose (8 or 16.6 mM) plus 5'-Guanylylimidodiphosphate (0.1 or 0.01 mM). GMP-PNP is a synthetic analogue of GTP resistent to cleavage by hydrolases. C: Glucose (8 or 16.6 mM) and glucagon (5 ~tg/ml). D: Glucose (8 or 16.6 raM) and glucagon (5 ~tg/ml) plus GMP-PNP (0.1 or 0.01 raM). E: Glucose (12 raM) and theophylline (0.5 mM) with or without GMP-PNP (0.1 raM). - GMP-PNP (0.1 or 0.01 mM) markedly enhanced the glucagon-inducedbut not the glucoseor theophylline-induced IRI release, suggesting that GTP might be a modulator of glucagon-induced IRI release. 241. Gastric Inhibitory Polypeptide (GIP): Stimulation of Insulin Release in vitro. P. Schauder, H. Frerichs, J. C. Brown. Division of Gastroenterology and Metabolism, Department of Medicine, University of G6ttingen, FRG. Gastric inhibitory polypeptide (GIP) has been suggested a likely candidate for the factor which via stimulation of insulin (IRI) r e lease enhances assimilation of orally administered glucose. A f t e r i.v. injection, GIP has been shown to increase IRI release, t h e plasma levels of GIP being in a range similar to that reached after oral administration of glucose. We have investigated whether GIP stimulates IRI release also after administration in vitro. Islets, isolated from pancreas of fed male Wistar rats ( 2 0 0 - 3 0 0 g) by collagenase digestion after i.p. injection of 0.6 ml pilocarpine (0.2% w/v) to decrease activity of pancreatic proteases, were incubated for 45 min in 500 ~1 KRB-buffer (BSA 1 mg/ml) with the following additions. A: Glucose (8 or 16.6 mM). B: Glucose (8 raM) plus GIP (10 ~g/ml). C: Glucose (16.6 raM) plus GIP (10 or 1 ~tg/ml). D: Glucose (16.6 raM) plus 5'-Guanylylimidodiphosphate (0.1 mM). GMP-PNP is a synthetic GTP-analogue resistent to cleavage by hydrolases. GIP (10 or 1 ~tg/ml) markedly enhanced glucose-induced IRI release. GMP-PNP had no additional effect on IRI release stimulated by glucose (16.6 raM) plus GIP (10 ~tg/ml). - The concept is supported that GIP might be part of a enteroinsular axis system. Inability of GMP-PNP to enhance the effect of GIP (10 ~tg/ml) on glucose-induced (16.6 mM) IRI release suggests that the secretory mechanism is already maximally stimulated or that GIP-, unlike glucagon-induced IRI release, is not modulated by GTP. 242. The Role of Intramuscular Glyceride Concentration in Control of Glucose Oxidation by Rat Diaphragm "in Vitro". Ch. Schindler, J.-P. Felber. Division de Biochimie Clinique, D6partement de M6decine, H6pital Cantonal Universitaire, 1011 Lausanne, Switzerland.

We attempted to demonstrate that a high intramuscular glyceride concentration was the most important factor in the control of glucose utilization by muscle in vitro. - For this purpose we proceded to a change of diet. After weaning, two groups of rats were fed for 4 weeks, a high-fat carbohydrate-poor diet or a carbohydrate-rich diet. A third and a fourth group received the high carbohydrate diet for respectively 26 and 22 days and then the high fat diet for respectively 2 and 6 days. - After 2 days of high-fat diet, plasma FFA level and muscular glyceride concentration were already similar to those found in control fat-fed rats. 2 days on high-fat diet led up to an impairment of in vitro glucose oxidation similar to that found in fat-fed controls. - This study suggests that the increase of muscular glyceride concentration (following the elevation of plasma FFA) and a greater availability for oxidation of fatty acid proceeding from stored glyceride, are the most important factors in the control of glucose utilization by muscle in vitro and perhaps in vivo in certain cases of obesity. 243. Immunogenicity and Crossreactivity of Pancreatic Insulin Contaminants. J. Schlichtkrull, J. Brange, Lise G. Heding, K. J~rgensen, An. Volund. Novo Research Institute, Novo All6, DK-2880 Copenhagen, Denmark. Conventional crystalline insulin was fractionated according to molecular size into the protein components a, b, and c. The a-component, which has the highest molecular weight, is chemically unrelated to insulin but contains a small fraction of proinsulin and insulin-like antigenic sites, which may explain the high immunogenicity of a-component with regard to the formation of antibodies against insulin. Patients treated with conventional insulin developed IgG against insulin, proinsulin-specific antigenic sites and a-component antigenic sites not cross-reacting with insulin or proinsulin. Thus, the immunogenicity of insulin preparations cannot be assessed only by insulin antibody estimates. - Rabbit and human anti-(beef/pork)-a-component IgG showed species crossreactivity, e.g., reactivity with (I-125)-a-component of human . pancreatic origin. The formation of antibodies against a- and b-components in rabbits was not eliminated by a single gel chromatography of crystalline insulin, its elimination required a radical removal of these contaminants by controlled chromatography. In patients, no antibodies against a-component pancreatic protein or proinsulin specific sites could be detected after treatment with insulin subjected to controlled chromatography. 244. Intrarenal Localization of Glycolysis. Schmidt, U. and W.G. Guder. Enzyme Lab., Medical Policlinic, Basel, Switzerland and Institute of Pathology, Tiibingen, FRG. To get more insight into the antomical sites of glucose metabolism along the nephron, we determined the four controlling reactions of the glycolytic sequence in single tubular portions from rat renal tissue. These include hexokinase (HK), phosphofructokinase (PFK), glyceraldehyde-3-P dehydrogenase (GAPDH) and pyruvate kinase (PK). Male rats which had free access to food and water were killed, the kidney removed and prepared for microdissection (N. Y. Acad. Sci. 2 4 2 , 489 (1974)). The following structures were isolated: glomerulus (G), proximal convoluted tubule (PC) and straight (PS) portion, thick ascending limb (AL), distal convolution (DC) and collecting duct (CD). Activities were expressed in moles reduced or oxidized pyridine nucleotide/kg dry wt/hour at 37 ~ C. G HK 0.7 PFK 0.3 GAPDH 3.4 PK 26.2

PC

PS

0.5 0.6 43.2 4.3

1.0 0.9 20.3 18.8

AL 7.8 18.0 77.6

DC

CD

6.2 3.0 12.0 138.3

58.7 231.2

The strong GAPDH in the PC points out the bidirectional function, e.g. gluconeogenesis is the main function in the PC. The high HK, PFK and PK in the portions of the distal tubule support the assump-

Abstracts tion that exogenous glucose is the main energy source for transport processes. Nevertheless, the constancy of glycolyfic capacity in PC suggests that a significant amount of glucose utilization is present in this segment.

245. Binding of 1251-Labelled Nonsuppressible Insulin-Like Activity to Isolated Fat Cells; Evidence for a Specific Acceptor Site Different From the Insulin Receptor. E. Schoenle, E.R. Froesch. Metabolic Unit, Department of Medicine, Kantonsspital, Zurich, Switzerland. Does 125I-labelled nonsuppressible insulin-like acitivity bind to isolated fat cells? If yes, are there two binding sites, one for NSILA-S and one for insulin or just one single site through which both hormones exert their identical metabolic effects? - Isolated fat cells from epididymal fat pads of normal fed rats were incubated for 60 min at 24 ~ C together with 125INSILA-S or with 125IInsulin and with different amounts of cold NSILA-S or cold insulin. Bound and free label were separated by filtration of the cells on Millipore filters. Binding was compared to the stimulatory effect of both hormones on glucose-1-1 C-oxidation in fat cells. - Between 0.5 and 1% of labelled NSILA-S (0.5 - 1 ~tU/ml) was bound to isolated fat cells. Binding was saturable and very sensitive to small concentrations of cold NSILA-S (KD - 2 - 3 ~tU/ml. Insulin up to 200 mU/ml did not displace labelled NSILA-S. In contrast, labelled insulin was displaced by both cold insulin (K D ~ 320 ~tU/ml) and cold NSILA-S (KD - 160 ~tU/ml). - Our findings suggest that adipose tissue, like heart muscle contain specific acceptor sites for NSILA-S different from those of insulin and not accessible to it, whereas the insulin acceptor displays affinity for both hormones. This raises the question whether NSILA-S exerts its insulin-like effects on adipose tissue by interaction with its own or with the insulin acceptor or with both. 246. Oxidation Rate of Plasma Free Fatty Acids in Maturity Onset Diabetics. Effect of Metformin. J. Sch6nborn, K. Heim, U. Rabast and H. Kasper. Medizinische Klinik der Universit~it, 87 Wiirzburg, FRG. Oxidation rate of plasma free fatty acids (FFA) and oxidation of carbohydrates was estimated in 15 maturity onset diabetics before and after 7 - day treatment with metformin, to relate quantitatively, metformin induced reduction of FFA - oxidation to carbohydrate oxidation and to the hypoglycemic action of the d~ug. - The estimation of the FFA-oxidation rate was based off the asymptotic (steady state) value of CO 2 specific activity, the FFA specific activity and the CO 2 output according to Borth et al.: J. Clin. Invest. 51, 1537 (1972). Calculation of carbohydrate-oxidation was based on indirect calorimetry. - Metformin reduced significantly the concentration, turnover and oxidation of FFA, the mean decrease being 18.37 and 34% respectively. The mean decrease of the FFA - oxidation rate was 93/~Eq/min being equivalent to 0.234 Cal/min on the basis of a FFA - MW of 270.4. Simultaneously there was a mean increase of carbohydrate oxidation (0,171 Cal/min) being equivalent to an additional oxidation of 60 g/day of carbohydrate. Thus the bloodsugar lowering action of biguanides is presumed to depend mainly on the decreased FFAoxidation and the increased carbohydrate-oxidation. 247. Relationship between Carbohydrate Tolerance (CHT), Secretion of Insulin, Proinsulin-like Material (PLM) and Insulin Sensitivity of isolated Fat Cells in Man. B. Schulz, D. Michaelis, S. Knospe, I. Neumann, H.-D. Gottschling, H. Bibergeil, Central Institut for Diabetes, 2201 Karlsburg/ Greifswald, GDR. The relationship between the biphasic insulin secretion pattern and the glucose metabolism in the substrate utilizing tissues are still obscure. - Therefore a 2-hour glucose infusion test was performed in 115 healthy subjects, 289 normal weight and 242 obese persons. In 28 subjects the 1-C 14-glucose oxidation to 14CO 2 stimulated by insulin was investigated using collagenase isolated fat cells. Insulin and PLM (enzymic method) were measured radioimmunologically. - The normal weight and obese persons showed a decrease of the early insulin response with increasing hyperglycemia. The augment-

375

ation of body weight was associated with elevated insulinemia in normal, borderline and pathological carbohydrate tolerances. The hyperinsulinemia of obesity was not due to increased PLM concentrations. There was no correlation between behaviour of PLM and CHT. Insulin stimulated glucose metabolism in isolated adipocytes increased to 192 + 21% in subjects with normal CHT. The insulin sensitivity was reduced in asymptomatic (127 + 5.8%) and overt diabetics (114 + 4.6%) despite no significant diminution of insulin secretion in the asymptomatic state. There was a significant negative correlation (r = -0.579) between CHT and insulin sensitivity in vitro. - The results suggest that biphasic insulin secretion pattern as well as peripheral insulin sensitivity are important factors in maintaining normal CHT.

248. Quantitatinn of Apolipoprotein B in Man. P. Segal, P. S. Roheim, O. Pinto, E. Gilon, The Metabolic Research Unit, The Chaim Sheba Medical Center, Tel-Hashomer, Israel. Hyperlipoproteinemia (HLP) is a major risk factor in Atherosclerosis. It is aslo highly prevalent in diabetes, where increased levels of very low and low density lipoproteins (VLDL and LDL) are often found. Apolipoprotein B (ApoB) is the major constitutent of LDL and VLDL protein, and has an important role in the regulation of tissue lipid metabolism. We studied the effect of metabolic and pharmacologic perturbations on ApoB levels in normal subjects and patients with abnormal lipid metabolism. ApoB was measured by quantitative immunoelectrophoresis (QIME). Whole serum and 1.006 infranate samples were electrophoresed on agarose containing goat anti-human ApoB. Rocket shaped precipitin lines were formed, the height of which was proportional to the amount of antigen. Nonobese normolipemic subjects served as controls, and the results are expressed as percent of control values. LDL contains 93 +4% of plasma ApoB in normal subjects, and VLDL only 7+4%. Patients with type IIa HLP had a 65% increase of plasma ApoB, which was normally distributed between LDL and VLDL. Plasma ApoB in patients with type IIb HLP was increased by 27%, but the distribution between LDL and VLDL was normal inspire of their hypertriglyceridemia. Patients with type IV HLP had a slight increase of plasma ApoB, but only 75% of it was found in LDL and 25% in VLDL. More than half of ApoB in a type III HLP patient was found in VLDL. Induction of lipemia, and low CHO diet, in type IV patients do not change significantly the total amount of ApoB, but rather it's distribution between LDL and VLDL. LDL ApoB levels are inversely related to the degree of lipaemia. Clofibrate acts in a similar manner. Conclusions: 1) QIME is a simple and reliable tool in the quantitation of ApoB. 2) Type IIa patients have increased amounts of normal LDL. 3) Type IIb patients have an abnormal composition of VLDL. 4) Induction of lipaemia in type IV patients probably inhibits the conversion of VLDL into LDL. 5) Diabetes per se does not alter lipoprotein compsotion. 249. Regulation of the Glucagon Release by Mouse Pancreatic Islets Maintained in Tissue Culture: Long-Term Effects of the Glucose Concentration of the Culture Medium. K. Segerstr6m, G. Lundquist, and B. Petersson. Department of Histology, University of Uppsala, Uppsala, Sweden. A deficient regulation of the glucagon secretion from the A2-cell has been documented in human diabetes mellitus. It remains unclear whether this defect represents a primary genetic disorder at the cellular level or is secondary to the diabetic state. It would, therefore be of interest to investigate whether prolonged alteration of the extracellular glucose concentration might influence the sensitivity of the A2-cell to the regulatory action of glucose on glucagon release.. - For this purpose isolated mouse pancreatic islets were mailltained in tissue culture for one week at glucose concentrations of 3.3, 6.1 or 16.7 mM. At the end of the culture period the glucagon secretion was measured in experiments lasting for 60 rain. - When the acute incubations were performed at 5.6 mM glucose, islets cultured at 16.7 mM glucose had the highest rate of glucagon release. Furthermore the glucagon secretion of such islets could be markedly suppressed by a high glucose concentration, which was at variance to the other two groups of islets. Arginine strongly stimulated glucagon release from all groups of

376

Abstracts

cultured islets and the stimulatory action seemed to be enhanced as the glucose concentration of the culture medium increased. - It is concluded that exposure of isolated pancratic islets for one week to a high glucose concentration causes an exaggerated glucagon response to both arginine and glucose. It remains unclear to what extent this is due to the high insulin concentration present in the culture system. 250. Glucose Effects on Calcium Fluxes in Pancreatic Islets, Using Lanthanum to Distinguish between Superficial and lntracellular Pools. J. Sehlin. The University of Ume~, Department of Histology, S-901 87 Ume~i, Sweden. Fluexes of 45Ca2q- were studied in islets from ob/ob-mice. La 3+ blocked both influx and efflux of 45Ca2+; in studies of intracellular 45Ca2+ the islets were washed with 2 mM La 3+ for 1 hour after incubation. The uptake of 45Ca2+ in islets ixposed to 3 mM D-glucose reached an apparent equilibrium after 2 hours at which time the intracellular concentration of exchangable Ca 2+ was about 7 mmol/kg dry weight (with 2.6 mM Ca 2+ in medium). Raising the D-glucose concentration to 20 mM enhanced the 45Ca2+ uptake whether or not the islets had first been equilibrated with the isotope. The effect could not be reproduced with equimolar L-glucose. The rate of release of intracellular 45Ca2+ was the same whether the islets had been preloaded with 3 or 20 mM D-glucose; halftime for release was 30 min. Thus, 45Ca2+ that had been taken up in response to 20 mM D-glucose appeared to be released much more slowly than the bulk of intracellular 45CaZ+. - It is concluded that 20 mM D-glucose caused a net uptake of Ca 2+ into the fl-cells. This uptake was probably not regulated at the level of the plasma membrane but more likely reflected an increased affinity of some intracellular compartment for the ion. 251. A Computer Evaluation System for the Study of the Natural History of Diabetes. H.K. Selbmann, W. Kreutzfeld, K. Sch6ffiing, K. Oberla. Univ. Mfinchen, Inst. f. reed. !nformationsverarb., Statistik, Univ. G6ttingen, Med. Klinik und Poliklinik, Univ. Frankfurt, Zentrum der Inneren Medizin, Abt. Endokrinologie, FRG. A retrospective study of 1302 diabetics was conducted in the outpatient departments of the Universities of Frankfurt and G6ttingen. The patients were observed every year, over a period starting usually with the year of diabetes recognition, and ending with the closing date of the studyin 1971. So approximately 11,000 years of diabetic life directly observed are available for statistical analyses. An efficient evaluation of such large volumes of data can only be done by computer information systems. The presented system SAVOD-L allows the statistical evaluation of the data in a dialogue with the computer. The immediate answering of an open list of questions in form of frequency information, histogrammes, crossclassification tables or survival curves enables an intensive exploitation of the data. - The features of the information system are demonstrated on a display terminal located in the congress area. This terminal is connected with the computer of the medical faculty of the University of Munich. So the participants of the congress have the opportunity to ask questions directly of the information system. 252. Elevations of Sorbitol and Myoinositol in Diabetic and Nondiabetic Patients Who Have Impaired Renal Function. C. Servo, E. Pitk~inen. IV Department of Internal Medicine, University of Helsinki, SF-00170 Helsinki, 17, Finland. We studied the mechanisms responsible for elevations in concentrations of sorbitol in diabetes and myoinositol in uremia. Concentrations of polyols were determined by gas-liquid chromatography in plasma, red cells or cerebrospinal fluid (CSF), or in all three; 10 diabetic patients with nephropathy, 10 non-diabetic uremic patients and 14 non-diabetic patients on dialysis were studied. Levels of sorbitol were significantly elevated (p 0.01) in CSF of diabetic patients and in red cells of both diabetic and dialyzed patients, an observation not previously reported. During dialysis red cell sorbitol levels rose by 20%. - Concentrations of myoinositol, normally elevated in CSF, were highest in plasma of

almost all patients, a finding which in uremic patients correlated negatively with creatinine clearance. During dialysis, plasma levels fell by 45 - 80% and red cell levels by 0 - 15 %. - Our results show that difference in osmolar gradients between red cell and plasma polyols were too small to cause intracellular damage and that elevations in levels of CSF-sorbitol were caused by synthesis in situ and not by the uremic state. Elevations of myoinositol in uremia were likely to result from tissue retention and impaired degradation in the kidneys and seem therefore to be an index of kidney damage. 253. The Role of Pancreas in HyperHpaemic Rats. A. A. Shabaan, V. Marks. Department of Biochemistry, University of Surrey, Guildord, Surrey. GU2 5XH., U. K. Experimental hyperlipaemia produced in the rat by cobalt chloride (COC12) injection can serve as a model for studying the mechanism and the factors involved in human hyperlipaemia. - Rats were made hyerlipaemic by subcutaneous injection of CoC12. Tissue distribution of cobalt was studied by injection of radioactive cobalt (57Co). Hepatocytes from cobalt chloride treated and control rats were studied and compared in monolayer tissue cultures. - The pancreas contained more cobalt than any other tissue apart from the liver and kidneys and the islets showed evidence of histological damage. Glucose uptake by hepatocytes isolated from the CoCl2 treated rats was unaffected by addition of either insulin or glucagon in contrast to hepatocytes isolated from untreated controls. CoCI2 induced hyperlipaemia is associated with abnormal control of carbohydrate metabolism in the liver, possibly secondary to islet cell damage. 254. Effect of Insulin on Activity of Lipogenesis Enzymes and Growth of Cultured Human Flbroblasts. E. Shafrir, E. L. Bierman. Department of Medicine, University of Washington School of Medicine, Seattle, Washington, USA. Since little is known about the regulation of lipogenesis in human tissues, the capactiy of fatty acid synthesis in human skin fibroblasts has been assessed. The fibroblasts, obtained from normal adult donors, were grown to confluency in a modified Dulbecco's medium. The medium was then replaced with a fresh one containing 10 % fetal calf serum made lipoprotein free by ultracentrifugation at d = 1.25 and free fatty acid free by charcoal absorption. From 20 9U/ml to 20,000 ~tU/ml of purified single component insulin (Lilly) were added and the cells incubated for 3 days. Proliferation of cells was discernible even at the lowest insulin concentration and a linear relationship between log of insulin concentration and cell growth was apparent. In the presence of 2,000 ~U/ml growth was increased by 28 to 45% with various cell strains. - The activity of the rate-limiting enzyme of fatty acid synthesis, acetyl-CoA carboxylase was determined in 100,000 g supernatant fraction, by fixation of 14CO2 after maximal activation with citrate. The activity ranged from 0.15 to 0.80 nmol 9 rain -1 9 -a of soluble protein in various cell strains compared to 1 and 24 nmol 9 min-1 9 mg-~ in human subcutaneous and rat epididymal adipose tissues, respectively. Increasing amounts of insulin induced a gradual rise in enzyme activity independent of the increase in cell number. At 2,000 vU/ml for 3 days, the activity rose 1.5 to 4-fold when expressed per cell, or per protein or DNA content. Likewise, insulin induced increases in the activity of other regulatory enzymes of lipogenesis, ATP-citrate lyase, NADP-malate dehydrogenase and fatty acid synthetase. The activity of the enzymes and the extent of induction tended to decrease after multiple passages of cells in culture. The presence of fatty acid synthetic enzymes and their amenability to insulin induction, in a fashion similar to that in liver and adipose tissue of experimental animals, render the cultured fibroblasts a convenient model living system for the study of regulation of lipogenesis in man. 255. A Possible Role of Glucagon in the Mechanism of Somatostatin - Induced Inhibition of Insulin Release. J. Sieradzki, H. Schatz, C. Nierle and E. F. Pfeiffer. Dept. of Internal Medicine, Endocrinology and Metabolism, University of Ulm, FRG. Alexander von Humboldt-Fellow 1974/75. Supported by Deutsche Forschungsgemeinschaft, SFB 87 Endokrinologie.

Ab~ra~s Somatostation inhibits insulin release in vivo and by the isolated perfused pancreas, however it is without effect on isolated islets. Studies to find an explanation for this discrepancy should also bring insight into the mechanism involved in the action of somatostatin on the pancreatic beta cell. Methods: Islets were isolated from rat pancreas by collagenase. They were incubated at 200 mg/100 ml glucose for two hours, in the absence or presence of somatostatin (100 ng/ml) and/or glucagon (5 ~tg/ml). Immunologically measurable insulin (IMI) was determined in the medium before and after 1 and 2 h of incubation: Insulin biosynthesis was estimated from incorporation of 3H-leucine into the proinsulin and insulin fractions following incubation of islets with 3H-leucine. - Results: Glucoseinduced release and biosynthesis of insulin were not affected by somatostatin. On the other hand, somatostatin exerted a significant inhibitory action (p < 0.001) on glucagon-potentiated insulin secretion (mean _+ SEM, ~tU/2h/10 islets: glucose and glucagon 1253 _+ 92; +somatostatin: 786 _+ 76). Insulin output in the presence of glucose, glucagon and somatostatin was significantly smaller (p < 0.05) than in the presence of glucose alone (1104 + t26) or glucose + somatostatin (1061 _+ 122). Conclusions: The failure of somatostatin to inhibit glucose effects on the beta cell in isolated islets might be due to the isolation procedure of the islets. Collagenase could have damaged mainly the alpha cells which are known to be located more at the periphery of the islets. After addition of glucagon, the inhibitory action of somatostatin was partly restored. Thus a possible role might be apparent for the hormone of the alpha cells, or the cyclic-AMP system, in the mechanism of somatostatin induced inhibition of insulin release. 256. Inhibitory Effect of Insulin on Glucagon Secretion in the Goose. G. Sitbon, P. Mialhe. Laboratoire de Physiologic Grnrrale - Universit6 Louis Pasteur - Strasbourg - ERA 188, CNRS and INSERM, France. Previous studies had pointed to 1) a glucose-glucagon and 2) a glucose-insulin feedback, operating under physiological variations of glucose concentration: the present series was designed to see if insulin exerted a direct effect on glucagon secretion. - Small doses of insulin, at 4 concentrations were infused or injected IV into 50 fasted geese and peripheral blood samples taken shortly afterwards. Insulin and "GLI" were measured in radioimmunological techniques. The non-specific glucagon antibody used, measured mainly pancreatic glucagon in the goose. - While 12.5 mU insulin/kg infused over 10 rain was ineffective, 25 mU/kg provoked a significant drop in "GLI" at the 6th minute. A drop in glycaemia was only observed later and explained the return of the glncagonaemia to normal. - With 50 mU insulin/kg over 10 min, the same phenomenon occurred, but in a more pronounced form. The inhibitory effect was still evident at 10 min, despite the glycaemia being appreciably lower than at the time zero. - A rapid injection of 200 mU insulin gave similar results, but after 10 minutes the resulting hypoglycaemia restored the "GLI" to above basal levels and masked the inhibiting effect of insulin. - l n s u l i n inibits glucagon secretion in the goose: this effect is secondarily masked by the increased glucagon a~ociated with the hypoglycaemla. 257. Citrate in Plasma and Urine during Starvation and Diabetic Coma. N. Schwartz Sorensen, T. Toftegaard Nielsen. Medical Department III, Aarhus, Amtssygehus, 8000 DK-Aarhus C, Denmark. Citrate is a regulator of key-enzymes of glycolysis, gluconeogenesis and de novo synthesis of fatty acids. - 1) Citrate levels in plasma and urine were determined daily in 10 obese persons during 10 days of total fasting. In all patients a continous rise was found in plasma citrate from 138 + 12 ~tmol/1to 227 + 18 ~tmol/1 (p < 0.01) on the 10 th day of fasting. During the same period the excretion of citrate in urine decreased considerably from 3.34 + 0.20 mmol/day to 0.20 + 0.07 mmol/day (p < 0.01). These changes were reversed on refeeding. - 2) Citrate levels in plasma and urine were studied in 8 persons of normal weight during short term fasting (8. a.m. to 9. p.m.). Again an increase in plasma citrate (109 + 8 to 142 + 9 ixmol/1, p < 0.01) and a simultaneous decrease in the excretion of citrate in urine (0.12 + 0.02 to 0.06 + 0.01 mmol/hr, p < 0.01)

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were found. - 3) Two patients with ketotic diabetic coma showed very high plasma citrate levels and extremely low urinary citrate excretion. Citrate values became normal during insulin treatment. - The results support the supposition that citrate is a key-substance in the control of glycolysis and gluconeogenesis. 258. Metabolism of Skeletal Muscle during Rest and Exercise: Studies on the Lower Limb in Normal and Diabetic Subjects. E. Standl, Th. Dexel, H.-U. Janka, H.J. Kolb, H. Czempiel, H.-G. Henftling. Diabetes Research Unit and 3rd Medical Department, City Hospital Schwabing, 8 Munich 40, K61ner Platz 1, FRG. Conflicting data exist on the metabolic behaviour of skeletal muscle in diabetics, especially during exercise. A careful reexamination was made taking advantage of the large muscle mass of the human lower limb. 7 normal and 8 juvenile diabetic subjects were studied under resting conditions, during a submaximal work load and a post exercise period. In order to minimize interference with adipose tissue metabolism, venous blood was collected from a catheter inserted into the popliteal vein under x-ray control. Muscle blood flow was estimated by recording the local clearance of 133Xe. In contrast to the normals where glucose was consistently taken up by muscle, particularly during exercise, glucose utilization was not uniform in the diabetics. 24 hrs after insulin withdrawal there was a net muscle efflux of glucose, even during exercise, in most of the diabetics; only 2 showed an uptake of glucose. In addition, lactate production during exercise was considerably less in diabetics, reflecting decreased glycolysis. Measurements of FFA, glycerol, fl-hydroxybutyrate, acetoacetate and O2-consumption indicated that insulin-deficient muscle mainly utilizes fatty-acids and ketonebodies as an energy source. The observed changes were partially restored towards normal by acute insulin administration (75 l~U/kg administered over 15 minutes) into the femoral artery. 259. Regulation of Adipose Tissue Pyruvate Dehydrogenase Activity in Diabetes, Starvation and Fat Feeding. D. Stansbie, R . M. Denton and P. J. Randle. Department of Chemical Pathology, Bristol Royal Infirmary, Bristol and the Department of Biochemistry, Medical School, University of Bristol, U.K. Mammalian pyruvate dehydrogenase exists in phosphorylated (in-. active) and non-phosphorylated (active) forms interconverted by an ATP linked kinase (pyruvate, TPP and ADP inhibited) and a phosphatase (Mg 2 + and Ca 2 + activated). Insulin acutely increases the proportion of enzyme in the active form in rat adipose tissue. In this study, the extent to which longer term changes in adipose tissue pyruvate dehydrogenase activity in vivo were the result of alterations in the ratio of active to inactive forms of the enzyme, or to variations in total enzyme activity, was investigated. - Alloxan diabetes and starvation (48 hr) markedly reduced the proportion in the active form without greatly decreasing the total activity. This reduction is still evident after subsequent incubation with insulin in vitro suggesting that there is some persistent alteration in adipose tissue metabolism in these conditions which affects pyruvate dehydrogenase interconversion; but no decrease in pyruvate dehydrogenase phosphate phosphatase activity was found. - Feeding rats a balanced diet with 40% fat for 21 days, results in a marked reduction in total pyruvate dehydrogenase activity in adipose tissue but not in heart, skeletal muscle, liver or kidney. A parallel decrease, but less marked, was also observed in adipose tissue pyruvate dehydrogenase phosphate phosphatase activity. 260. Metabolic Effects of Light and Antidiabetic Drugs on the Stimulated Diaphragm of Rats. J. Sterne, F. Pecard. Aron, 116, Rue Carnot, 92151, Suresnes, France. A model of electrically stimulated diaphragm, pulsating in a fluid containing the same concentration of glucose as the blood has been developed. - It is more sensitive and its respiration is better than that of the unstimulated diaphragm in the Warburg apparatus and the ratio between input of glucose and output of lactic acid is higher than in unmoving diaphragms.- Differences in the input of glucose according to the exposure to light were observed: the diaphragm

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metabolizes twice as much glucose during the summer than during the winter. This difference disappears, wheneven during the summer - the animals are kept in darkness. Insulin and biguanides are less active in diaphragms of animals exposed to light than in those kept in the dark. - With insulin, the release of lactic acid increases proportionally to the dose, but the lactic/pyruvic acid ratio does not change. - With biguanides, the increase of lactic acid is greater than that of pyruvic acid. The results are different with different biguanides in a way which parallels the clinical findings. 261; The Effect of Arginine Infusion upon Insulin Secretion during lntra-Abdominal Operation. W. Stremmel, H. Kiimmerle, J.D. Kruse-Jarres. Department of Surgery University of Freiburg, 78 Freiburg i. Br., FRG. Hyperglycemia and glucosuria are known metabolic disorders after trauma. One essential pathogenetic factor of the impaired peripheral glucose ntilisation is inhibited insulin secretion under stress situations. - It was the purpose of our study to examine the effect of arginine upon the inhibited insulin secretion during intra-abdominal operation in man. - The basal and glucose stimulated insulin secretion was studied in 30 patients. Arginine was infused (0.5 g/kg bw.) for 30 minutes. Insulin was determined radioimmunologically. 0.33 g/kg glucose i.v. was given during the operation. - During a 30 minute intraoperative observation period the blood glucose increases from 104 to 147 mg%. The insulin levels increase from 8 to 14 ~tU/ml. During arginine infusion glucose increased to 172 mg% and the insulin concentrations to 34 ~tU/ml. After the intraoperative intravenous glucose load blood glucose increased correspondingly but the insulin levels remain low. The simultaneous infusion of arginine produced high insulin levels about 100 ~tU/ml from 4 to 30 min after the glucose load. - The results show that the stress induced inhibition of insulin secretion can be abolished by arginine. It could be considered that arginine has a more useful role in postoperative or posttraumatic infusion therapy. 262. Alterations in Retinal Capillaries of Chronically (12 months) Diabetic Rats. P.-P. Studer, V. Trapp, E. Siegel, F. H. Schmidt, W. A. Muller. Institut de Biochimie Clinique, Institut d'Histologie, University of Geneva, Genbve (Switzerland) and Medical Research Laboratories of Boehringer GmbH, Mannheim, FRG. This study was undertaken to analyse quantitatively changes in the retinae of chronically (streptozotocin) diabetic rats maintained on low and high carbohydrate diets and to compare these retinae to those of control animals fed a high carbohydrate diet. - Retinae were prepared by trypsin digestion and mounted on slides. Nuclei of retinal capillary cells were counted per 2.5 cm of capillary. Periodic determinations of plasma glucose and insulin, as well as pancreatic insulin content at the time of sacrifice, confirmed the diabetic state. - A significant (p < 0.001) reduction in the number of perimural cell nuclei was found (47%, high carbohydrate diet; 60%, low carbohydrate diet) when compared to controls. Furthermore, the number of endothelial cell nuclei was also diminished to 61% (p < 0.001) and 89% (p < 0.05) respectively. - The number of perimural cell nuclei was significantly correlated (r = 0.40; p < 0.02) with the concentrations of plasma insulin measured at the time of sacrifice. - These data demonstrate that 1) chronic streptozotocin diabetes may bring about significant alterations of retinal vessels in this laboratory rodent, 2) it is possible to quantify with precision these retinal changes, which allows for rigorous statistical evaluation, and 3) the composition of the diet may influence this process. 263. The Relation of Maternal Fasting Blood Glucose Levels to Chemical Diabetes and Infant Birthweight. H. W. Sutherland, J.M. Stowers. Department of Obstetrics and Gynaecology, University of Aberdeen and Diabetic Clinic, Aberdeen Royal Infirmary, Scotland. To examine the hypothesis that maternal fasting blood sugar levels play a role in the determination of fetal birthweight in those with and without chemical diabetes in pregnancy. Can the estimation of

fasting blood sugar (F.B.S.) levels be used to screen for chemical diabetes in pregnancy? - 2002 antenatal and 500 6-8 week post partum intravenous glucose tolerance tests (IVGTTs), expressed as incremental indices, performed in potential diabetic women were analysed by computer in relation to maternal F.B.S. levels (mean of 3), standardised centile birthweights and obstetric data. - In women with chemical diabetes in pregnancy, birthweights expressed as centile birthweight were not increased. However, previous delivery of a high centile birthweight (> 95 ile) baby had a high predictive value for chemical diabetes. The F.B.S. levels correlated with the centile birthweight particularly in the potential diabetics (with normal IVGTTs), increasing levels being associated with increasing birthweights. There was a high proportion of chemical diabetes (50%) in the 44 women with fasting blood sugar levels below 50 mg/100 ml (2.8 mMol/1). - High infant birthweight is not commonly associated with abnormal IVGTTs during pregnancy. Infant birthweight correlates with maternal F.B.S. levels. F.B.S. value is an unreliable screening procedure for chemical diabetes in pregnancy. 264. Electromyographic Investigations in Subclinical Neuropathy in Diabetes Mellitus. W. Tackmann, H. J. Lehmann, W. Rabe. Neurological Clinic of the University of Essen and Universitfitsnervenklinik Marburg, FRG. A group of 33 diabetic patients was investigated. Physiological examination revealed clinically moderate signs of neuropathy in four patients; eleven had only mild symptoms. Eighteen patients had no impairment of the peripheral nervous system. More detailed information about nervous function in clinically apparent healthy subjects could be abtained by electrophysiological examinations of peripheral motor- and, especially, sensory nerves. - Motor nerve conduction velocity was slowed in median and peroneal nerves of 21 patietlts (64%). In sensory median and in sural nerves conduction velocity was diminished in 15 subjects (45%). - A more sensitive method seems to be the stimulation of sensory nerves with frequent electrical stimuli. Using 10 stimuli trains of increasing frequency (100-500 cps) early changes in the mode of nerve conduction could be demonstrated in 73% of median sensory nerves and in 84% of sural nerves of the patients with a suspected diabetic neuropathy. So the application of frequent stimuli might be a useful tool for the detection of clinically inapparent nerve involvement. 265. Effects of D-Glucose Anomers on Insulin Release and Glucose Metabolism in Pancreatic Islets. I.-B. Tfiljedal. Department of Histology, University of Ume~, Umeh, Sweden. To test the substrate-site hypothesis of glucose recognition in pancreatic/3-cells, the effects of a-D-glucose and/3-D-glucose on insulin release were compared with those on 3-O-methylglucose transport, 3H20 production from D-(5-3H) glucose, and islet glucose-6-phosphate. Islets microdissected from non-inbred ob/ob-mice were used. Perifusion of islets showed that 9 mM a-D-glucose was a more effective stimulus to insulin release than equimolar t-D-glucose; the difference was observed whether or not secretion was potentiated with 2 mM theophylline. In contrast, a-D-glucose was no better than ,6-D-glucose in inducing countertransport of methylated glucose, raising the glucose-6-phosphate or diluting 3H20 arising from D-(5JH)-glucose. Instead, 3 rain after exposure to the glucose anomers, the glucose-6-phosphate concentration was higher in islets incubated with the flanomer than in those incubated with the a anomer. Initial studies also suggested that production of 3H20from a-D-(5-3H)glucose was not faster than that from equilibrated, labelled glucose or a partially purified preparation of t-D-(5- 3H)glucose. - The results speak against the hypothesis that the recognition of glucose as insulin secretagogue only depends on the sugar being a substrate for the glycolytic pathway in the/3-cells. 266. Inhibitory Effect of Somatostatin on the Release of Insulin in Isolated Rat Pancreas. J. Tamarit, M. Lucas, J. Tamarit-Rodriguez and R. Gobema. Departamento de Fisiologia y Bioquimica, Facultad de Medicina, Universidad Complutense. Madrid-3, Spain.

Abstracts Somatostatin (Somatotropin release inhibiting factor, SRIF) is a potent inhibitor of the secretion of various hormones, specially GH, Insulin (I) and Glucagon. In order to characterize the physiological significance of its direct action on the liberation of I, we have studied its effect on the isolated and perfused rat pancreas, with SRIF in various concentrations. We used the pancreatic preparation of Sussman et al (1966) with a medium of perfusion (Krebs buffer pH = 7.35) supplemented with 0.5 % of Albumin and equilibrated with 95 % of O 2 and 5 % of CO:. The rate of perfusion was 2.5 ml/min, at constant pressure and at 38 ~ C. Insulin was measured by radioimmunoassay according to Melani et al (1965). The peffusion was divided into two phases: in the first, we utilized buffer with glucose 2.67 mM (G), for 15-30 minutes, and in the second phase we utilized buffer with glucose 16.7 mM and various concentrations of SRIF. The results (ng/ml/hour), with the number of perfusions in brackets, were: SRIF = 0; IRI = 926.84 _+ 90.73 (9). SRIF = 2 ~tg/ml; IRI = 223 + 20 (5). SRIF = 50 ng/ml; IRI = 64.86 + 3.59 (5) SRIF = 5 ng/ml; IRI = 192.58 + 47.62 (5). - The SRIF showed a significant inhibitory action on the secretion of Insulin at any concentration; the inhibition depends on the interaction between the concentrations of SRIF and the first phase of pancreatic response. Previous perfusions with theophylline 1 mM and Ca + + 8 mM, does not block the inhibitory action of 50 ng/ml of SRIF: IRI = 107.34 + 19.46 (6). If the rat pancreas is perfused in the first phase with SRIF (50 ng/ml) the effect of glucose 16.7 mM in the second phase is inhibited (IRI = 117.22 + 13.33). - In summary, SRIF has an inhibitory action on the secretion of I, at levels of some possible physiological significance, and this inhibitory action is neither blocked by Ca + + nor by Theophylline. 267. Insulin and Glucagon Secretion after Somatostatin in lnsulinoma. G. Tamburrano, F. Fallucca, C. Mirabella, D. Andreani. Cattedra di Medicina Costituzionale ed Endocrinologia Universitfi di Roma, Italy. Somatostatin (GIF) has been shown to reduce insulin as well as glucagon secretion elicited by different stimuli in normals. - In this research the effect of GIF on blood glucose (BG) plasma insulin (IRI) and glucagon (IRG) levels has been studied in 2 patients with proven insulinoma. - An arginine test (ATI') was performed a few days before and soon after rapid administration of GIF (cyclic form, 250 micrograms I.V. over a 30 seconds period). - In one of these patients the effect of a more prolonged infusion of GIF (250 micrograms I.V. in 30 seconds followed by an infusion of 500 micrograms in 60 min) on BG, IRI, IRG levels was also investigated. - A reduction in plasma IRI and IRG levels was observed in both patients during the GIF + ATT, when compared to the ATT alone. - GIF infusion for 60 min. produced a marked decrease in plasma IRI values (to about 50 % of fasting levels). IRG changes were less clear. - These data seem to indicate that GIF may inhibit the secretion of pancreatic hormones in islet cells adenomas. 268. Exercise Tolerance in Insulin Treated Juvenile Diabetes Meilitus. J. Tatofi, K. Nazar, A. Wigniewska, E. Kowalik. 3rd Department of Internal Diseases, Warsaw Medical School and Laboratory of Applied Physiology, Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland. For determining the safe and therapeutically effective amount and type of physical exercise, the individual criteria of exercise tolerance and simple methods for its practical estimation are needed. - In the male insulin treated juvenile diabetics, maximal oxygen uptake [V0 max] was estimated and metabolic responses to exercise, bloo~ glucose [King-Garner], lactate [Barker Summerson rood. by Strom], pyruvate [Friedman rood. Rindi, Ferrari], plasma FFA [Dole, Meinertz], plasma ketones [Engfeldt], at the work load of 30 [low load], 50 [medium load] and 75 [high load] per cent of V02max were studied. - Low values of respiratory exchange ratio at all the work loads were observed. Blood glucose concentration decreased during exercise at low and medium work loads whereas at the high load there was an increase in glucose level. Plasma FFA decreased during exercise at the low load, remained unchanged in the medium

379

load, and rose at the high work load. Plasma ketones did not change significantly at any level of the load applied. Blood lactate and pyruvate increased significantly during exercise at all the loads studied. - The exercise utilized for therapeutic training purposes should not exceed 50 per cent of V 0. max whereas the work loads permissible for juvenile type diabetics m vocational work should be smaller then 30 per cent of V02 max. .

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269. lmmunospecificity Studies with Chemically-Modified Insulin. J.H. Thomas, D.I. Dron, R.H. Jones. Departments of Biochemistry and Medicine, St. Thomas's Hospital Medical School, London SE1 7EH, U.K. Eighteen insulin analogues made available by D. Brandenburg (Aachen) and D.G. Lindsay (Sussex) were used to study the immunospecificity of 4 selected antisera raised against porcine insulin or pro-insulin in guinea-pigs. - The analogues were specifically substituted at the A s, B 1, or B29 positions, or included a synthetic At-B 1 or A1-B29 crosslink. Using ~esI porcine insulin, they were investigated by a standard double-antibody radioimmunoassay technique to permit calculation of binding affinity and capacity. The three antisera raised against insulin showed different binding characteristics with respect to the analogues. One distinguished between insulin and analogues with B1 modifications, a second between insulin and materials modified at A t and B29, and the third, in common with the antiserum raised against pro-insulin showed no discernible pattern of specificity. - Antisera prepared against insulin differ in their specificity for sites on the surface of the molecule. Hence the apparent immuno-reactivity of a chemically modified insulin will be critically dependent on the antiserum used. Some insulin antisera are suitable for the estimation of analogue concentrations in biological fluids, whereas others are sufficiently discriminating to allow assay of insulin in the presence of the analogue. 270. Control of Glucagon Secretion in a Case of Islet Cell Tumor Which Produced Glucagon, Insulin, and Gastrin. A. Tiengo, E. Marchiori, R. Nosadini, D. Fedele, C. Garotti, M. Muggeo, G. Crepaldi. Department of Internal Medicine, Division of Gerontology and Metabolic Diseases, Via Giustiniani, Padova, Italy. The existence of a stimulation or suppression mechanism of glucagon secretion is still debatable in glucagonomas and in multiple hormone producing pancreatic adenomatosis. - A patient who had undergone gastrectomy for Zollinger syndrome and who suffered from insular adenocarcinoma, accompanied by hepatic metastases, presented high fasting levels of pancreatoglucagon (IRG) (2500-3200 pg/ml which had been measured with the specific antiserum K30), insulin (IRI) (160-240 ~U/ml and gastrin (HSG) (150-180 pg/ml). High levels of glucagon were found in the extract of hepatic metastases. Glucagon stimulation and suppression tests were carried out. After arginine, IRG increased greatly reaching a peak of 10 ng/ml, while IRI rose to 330 ~tU/ml. Following oral glucose loading (OGTT of a diabetic type) and i.v. glucose, a paradoxical increase in IRG was observed. Insulin that was administered alone (4 U) or during glucose infusion induced a significant decrease in IRG (from 3.3 to 1.2 ng/ml and from 3.9 to 1.9 ng/ml respectively). Infusion of calcium (4 mg/kg/h) produced an increase in IRG, IRI and HSG. The insulinopoietic hyperglycemic effects of exogenous glucagon were normal. Hyperglucagonemia induced by islet cell tumor did not seem to be suppressed by glucose administered either per os or i.v., while it is suppressed, to some extent, by high levels of exogenous insulin. Despite pre-existing hyperglucagonemia, a-cells seem to be hyperresponsive to arginine. The metabolic effects of exogenous glucagon seem to be normal. 271. A Re-Assessment of Small Vessel Disease in Diabetic Neuropathy. W.R. Timperley, J.D. Ward, F.E. Preston, T. Duckworth, B. O'Malley. Department of Neuropathology, The Royal Infirmary, Sheffield, U. K. Controversy continues as to the exact role of vascular disease in the causation of diabetic neuropathy. With increased understanding of

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Abstracts

the balance between the blood coagulation systems and fibrinolysis and the demonstration of fibrin in small vessels in the brain and other organs in patients dying in diabetic coma, it seemed that a re-assessment of small vessel disease in diabetic nerve was indicated. - Twenty-five patients with diabetic neuropathy were studied and sural nerve biopsies performed. Sections were stained with haematoxylin and eosin, Martius-Scarlet-Blue, Solochrome cyanine and osmium tetroxide. Two patients were also studied at post-mortem. - Vessels plugged with fibrin were seen in eight cases. In two cases fibrin was tracking into the vessel wall. Four cases showed older thrombus in vessels and in two of these areas of necrosis were seen. Two had a preceding episode of disseminated intravascular coagulation. One case studied at post-mortem showed fibrin, red-cells and plasma infiltrating the vessel wall. Another showed focal haemosiderin deposits and demyelination. - Vascular mechanisms clearly play some part in diabetic neuropathy. The phasic nature of the clinical symptoms may be a manifestation of variations in thrombus in small vessels.

hypoglycaemia normal and obese subjects have marked suppression of plasma insulin to less than 1.5 gU/ml if the plasma glucose reaches 35 mgs. %. Twelve insulinomas had distinctly impaired suppression of insulin secretion in response to hypoglycaemia, with plasma insulin remaining above 6 ~tU/ml (normal subjects mean plasma insulin 8 to 1 ~tU/ml, insulinomas 22 to 21 ~tU/ml). Suppression of human C-peptide levels during insulin induced hypoglycaemia gives less distinction between normals and insulinomas than does plasma human insulin assay (normal subjects C-peptide 0.27 _+0.5 (+ 1 S. D.) to 0.11 _+0.03 pM/ml, insulinornas 0.30 _+ 0.07 to 0.20 _+ 0.08 pM/ml). This is because of decreased metabolic clearance rate of C-peptide, and because of insulinomas secreting proinsulin rather than insulin and C-peptide. One insulinoma in response to a glucose fall from 48 to 25 rag. % had normal suppression of C-peptide (0.22 to 0.10 pM/ml) with little suppression of plasma "insulin" (32 to 29 ~U/ml) because of cross reacting proinsulin. Thus C-peptide response to induced hypoglycaemia is not a reliable test unless accompanied by a proinsulin assay.

272. Hypersecretinaemia in Maturity Onset Diabetes Correctable by Dietary Measures. E.R. Trimble, K.D. Buchanan, D.R. Hadden, D.A.D. Montgomery and J.A. Weaver. Department of Medicine, The Queen's University of Belfast and The Metabolic Unit, Royal Victoria Hospital, Belfast, U. K. Secretin has structural and biological similarity to glucagon in that it stimulates insulin secretion, is suppressed by oral glucose, is elevated during starvation and is lipolytic. It would appear relevant to study this hormone in diabetes mellitus. - Immunoreactive secretin (IRS) levels were measured by a sensitive radioimmunoassay during standard 50 g oral glucose tolerance tests (GTT) in 22 newly diagnosed maturity onset diabetics. The studies were repeated after six months' treatment with a diet of restricted carbohydrate. Healthy subjects also had an oral GTT performed and acted as controls. - The diabetics had significantly elevated fasting IRS levels (M +_ SEM pg/ml) at diagnosis over controls (diabetics 131 + 22, controls 48 + 16 p < .0125), but these levels returned to normal following the dietary treatment (50 + 16 which was significantly less than the pre-treatment values - p < .005 but not different from the control values). In the diabetics there was a positive correlation between the fasting glucose at diagnosis and the IRS levels (p < .05). Suppression of plasma IRS during the GTT was noted in diabetics both before and after treatment and in the controls. - It is concluded that high circulating levels of IRS are found in untreated diabetics and return to normal following dietary measures. It is possible that this hormone by virtue of some of its metabolic actions, may play a role in the diabetic syndrome.

275. Two Disorders of Deficient Glucose-Induced Insulin Secretion - a "Quantitative" Defect (Low Vmax) in Latent Diabetes, and Decreased Sensitivity of B-Cells (High Kin) in Women who Produced LFD Babies. R.C. Turner, E. Harris, S. Bloom. Nuffield Department of Medicine, Radcliffe Infirmary, Oxford, U. K. The first phase insulin response to four i. v. glucose loads (0.05, 0.1, 0.2 and 0.5 g/kg), and glucose response to i.v. 0.025 u/kg insulin has been measured in normal (N) and obese (O) women, latent diabetics (LD - who had gestational diabetes) and in women who have had a LFD. Seven out of the 13 LD had an overall, decreased insulin secretion. Ten out of 18 LFD had a decreased insulin response to a small glucose load, but normal response to a high load. They had raised basal glucose (BG) levels (p < 0.005) which may be the cause of fetal over-nutrition. O tend to have larger babies than N, and this may also be due to their raised BG, The BG (p < 0.01) and Kg (p < 0.001) correlate with the insulin response relative to the basal insulin (B) better than with absolute insulin levels. All but 2 LD have decreased insulin response/BI, and this is a better diagnostic discriminant than Kg. All but 2 LFD had a reduced insulin response/BI to a small rise in plasma glucose. LFD had increased sensitivity to i. v. insulin (p < 0.05), O decreased sensitivity, and LD normal or increased sensitivity compared with similar weight controls. There is no difference between the groups in the suppression of plasma pancreatic glucagon following i.v. glucose.

273. Oestrogen Induced Hypertrigiyceridaemia: Induction of Hepatic Lipogenic Enzymes. B.R. Tulloch, S.K. Afolabi, A.H. Kissebah, C. White. The Royal Infirmary, Manchester, M13 9PL. Post-Graduate Medical School, London, U.K. It has been suggested that the increased insulin levels found in pregnancy and oestrogen therapy contribute to the accompanying hypertriglyceridaemia. - Ovariectomised rats were studied following oestrogen therapy with assessment of hepatic lipoprotein synthesis undertaken in vivo and in vitro. - Oestrogen treated animals exhibited increased secretion of triglyceride into plasma, while, in the presence of acetate, liver slices prepared from these animals showed increased incorporation of 14Clabelled amino acids into lipoproteins. - Stepwise examination of the pathways of hepatic lipoprotein synthesis has demonstrated a consistent increase in the enzymes acetyl Co A carboxylase and fatty acid synthetase. Since Km values are unaltered, it is suggested that oestradiol causes an induction of these hepatic enzymes in vivo. 274. C-Peptide, Proinsulin and Insulin Responses to Fish-Insulin Induced Hypogiycaemia in the Diagnosis of Insulinomas. R.C. Turner, E. Harris, L. Heding. Radcliffe Infirmary, Oxford, Novo Research Institute, Copenhagen, Denmark. Insulinomas, like other autonomous endocrine diseases, can be diagnosed by suppression tests. In response to fish-insulin induced

276. Kinetics of the Early Insulin Response of the Isolated and Perfused Rat Pancreas to Pharmacological and Metabolic Stimulus. P. Vague, G. Rahmahandridona, C. Dicampo-Rougerie and F. Mahmoud. Faculte de Medecine de Marseille, France. The collection of the venous effluent every 15" allowed us to observe that the Insulin response to tolbutamide (250 Ixg/ml) occurred immediately while that to d-glucose-(3 mg/ml) or 1-1eucine (2 mg/ml) was delayed by 90". The response to glyceraldelhyde (3 mg/ml) or to alpha ketoisocaproate (2 mg/ml), occurred earlier than that to glucose or 1-1eucine. This suggests that these two stimuli have to be metabolized to initiate insulin release. - The response to glucagon (2 ~g/ml), if the medium contained low concentration (0,7 mg/ml) of glucose (or glyceraldelhyde) was almost immediate. However glucagon alone elicited no response. Addition of low concentration of glucose (or glyceraldelhyde) allowed a delayed response. The immediate effect of glucagon on insulin release appeared to be dependent on the metabolism of glucose. When the medium contained a low concentration of glucose (or glyceraldelhyde), raising the glucose concentration promoted an immediate insulin discharge. This suggests the possibility that it may be possible to demonstrate kinetically two actions of g]ucose on the B-cell. The one, apparent after some delay, mimicked by glyceraldelhyde, necessary for glucagon induced insulin release, is probably mediated by the metabolism of the sugar. The second initiates rapidly the insulin discharge but appears dependent on the first action. The combination of the "substrate site" and "regulatory site" hypothesis in the mechanism of glucose induced insulin release appears probable.

Abstracts 277. K Value and Insulin Secretion during i.v.g.t.t, in Children with and without Family History of Diabetes. P. Vague, G. Ramahandridona, J. Vague. Faculte de Medecine de Marseille, France. We have previously observed than in a given individual the relative sensitivity of the pancreas to glucose may be appreciated by comparing the insulin response to intravenous glucose (iG) to that to tolbutamide (iT). In adults the iG/iT ratio was independant of age and relative body weight (RBW). It was frequently low in the offspring of diabetics and might be considered as a constitutional marker of the predisposition to diabetes. - In 35 children (age 5 to 17, RBW 89 to 196%) without family history of diabetes, the K value (during an I.V.G.T.T.) was negatively correlated with age and R.B.W. but not to the parameters of insulin secretion, iG/iT was independant of age and R.B.W. - In 38 offspring (age 7 to 17, R.B.W. 81 to 189%) of maturity onset diabetics the K value was correlated with iG among the normal weight children, with iG/iT in the whole group, iG/iT was not correlated with age and weight and was frequently in the low range. - In 30 offspring (age 4 to 17, R.B.W. 83 to 130%) of juvenile diabetics, the K value was not correlated with iG or iG/iT. - It is suggested than in young subjects the variation in assimilation of I.V. glucose is dependant on many factors, the insulin secretion being a determinant only in patients with impaired B-cell function. These subjects are more often encountered among offsprings of maturity onset diabetics. 278. Chromatographic Pattern of Plasma Glucagon lmmunoreactivity from a Patient with a Glncagonoma Syndrome. I. Valverde, H. Lemon, R. H. Unger. Madrid, Spain and Omaha, Nebraska and Dallas, Texas, USA. To characterize the nature of the extremely high circulating levels of glucagon immunoreactivity (GIR) present in a patient with the clinical picture of the glucagonoma syndrome, whole plasma was filtered on Bio Gel P-30 and eluates assayed with 30 K antiserum. - Fasting plasma GIR levels oscillated between 2400 and 2700 pg/ml. On gel filtration this GIR was separated into four fractions as those previously described in plasma from normal subjects. The fraction eluting with the plasma proteins ("big plasma glucagon") amounted from 160 to 260 pg (corresponding to one ml of plasma). The 9000 MW fraction ("proglucagon") ranged from 455 to 965 pg. About 750-1320 pg eluted in the "true glucagon" area (3500 MW). A minor part of the GIR appeared in the < 2000 MW zone (25-120 pg). - During an OGTT, total plasma GIR changed from 2400 to 2080 pg/ml. This slight decline was observed to be accounted for a parallel reduction of the 3500 MW fraction (from 1320 to 995 pg). The other three fractions did not seem to be affected by the oral glucose load. - It is concluded that: (1) plasma from a patient suspected of glucagonoma contains the same four GIR fractions as plasma from normal subjects; (2) an increase in both "true glucagon" (3500 MW) and "proglucagon" (9000 MW) fractions accounts for the hyperglucagonemia of this patient; (3) the small suppression of plasma GIR during an OGTT affects only the "true glucagon" fraction. 279. Light and Electron Microscopic Study of the Endocrine Pancreas of the Rat during Normal Pregnancy and during Diabetic Pregnancy. F.A. Van Assche, L. Aerts. Laboratory of Gynecological Physiopathology, University Hospital (K.U.L.) - Leuven, Belgium. What are the morphological features in the endocrine pancreas and in the B-cell during normal pregnancy? In experimental diabetic pregnancy is the endocrine pancreas and specifically the B cell, able to adapt to the metabolic derrangement? - Morphometric study (light and electron microscopic (T.E.M.) of the endocrine pancreas at the end of normal pregnancy and at the end of experimental diabetic pregnancy (30, 40, 50 mg/kg streptozotocin). - During normal pregnancy an increased percentage of endocrine tissue and a pronounced B-cell hyperplasia was present. In the B-cell increased granulation was observed. - These adaptative features were less pronounced in experimental diabetic pregnancies. - The morphologic changes in the endocrine pancreas during normal pregnancy indicate a marked hypertrophy of the total B-cell mass.

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In the B-cell itself increased granulation is present (due to the increase of the light (pale) granules), indicating hyperinsulinism of the individual B-cells. - The endocrine pancreas in experimental diabetes is unable to adapt during pregnancy. 280. Evaluation of the Antidiabetic Sulfonylurea Compound Glibornuride. R.G.A. Van Wayjen, A. Van Den Ende. Metabolic unit of the Department of Internal Medicine, Hofpoortziekenhuis, Woerden, The Netherlands. We have compared the antidiabetic activitiy, side effects and toxicity of glibornuride, compared with tolbutamide and glibenclamide, in a double-blind cross-over study of 24 weeks in 43 patients, divided at random into 2 groups, in which gliboruuride was compared respectively with tolbutamide (A-B) and glibenclamide (C-D), each drug given during 4 times 3 weeks. Clinical data, body weight, complaints and side-effects were noted every 3 weeks during each visit to our out-patient department. Simultaneously, extensive laboratory determinations were done e. g. blood glucose, cholesterol, triglycerides, liver- and kidney function tests and urinary excretion of glucose and ketone bodies. - The conclusions of this study were: 1. Glibornuride failed to show any side-effects. 2. Glibornuride did not possess any organ-toxicity. 3. Treatment with glibornuride resulted in a satisfactory regulation of all 43 diabetic patients without signs of hypoglycaemia. 4. The antidiabetic activity of glibornuride in comparison to that of tolbutamide and glibenclamide has been evaluated statistically. The results of this statistical evaluation will be communicated. 281. Plasma Insulin Responses to Glucose Infusion in Obese NonDiabetic and Diabetic Patients. J. Veleminsk~. Institute of Endocrinology, Lubochfia, Czechoslovakia. The glucose infusion test was performed in 10 control persons, 31 obese non-diabetic subjects and in 10 obese insulin-nondependent diabetics. After a priming injection of glucose of 500 mg/kg, glucose was infused during 60 min at a rate of 20 mg/kg/min. Blood samples were drawn at 0, 3, 5, 10, 20, 30, 40, 50, 60, 80, 100 and 120 rain. In healthy persons there was a biphasic plasma insulin curve with the first peak 3 min. after the start of glucose infusion and a second one after 60 min. In 20 obese subjects with a normal OGTT, both the initial and the late insulin responses to glucose infusion were higher than in control persons. In 11 obese subjects with mild glucose intolerance the initial phase of insulin response was subnormal while the later phase was similar to that of obese subject with a normal OGTT. In overt mild diabetes the initial phase of insulin levels was markedly diminished but a higher, though delayed, rise of insulin levels could be shown during the second hour. - Obesity, accompanied by a higher insulin requirement and hyperinsulinemia due to insulin resistance, may induce a prediabetic state. The first sign of this period is a defective release of insulin during the initial phase of glucose infusion corresponding to a border-line O GqT. 282. Equilibrium Exchange of 3-0-Methylgincose in Isolated Fat Cells. Concentration Dependence and Effect of Insulin. J. Vinten, J. Gliemann. The Institute of Medical Physiology C, University of Copenhagen, Denmark. The aim was to characterize the sugar transport system of isolated rat epididymal fat cells in the absence and the presence of insulin. This was done by measuring the intracellular distribution space for the non-metabolizable sugar analog 3-0-[14C]methyl-D-glucose at stationary distribution and by measurement of the efflux of this tracer under conditions of equilibrium exchange. For the rapid recovery of samples of fat cells during tracer efflux a modification of the previously described oil flotation technique (J. Gliemann, K. Osterlind, J. Vinten and S. Gammeltoft. Biochim. Biophys. Acta (1972)) was developed. It was found that the sugar equilibrates with the intracellular 3H20 space and that insulin is without influence on the size of the distribution space for the sugar. The half-time for 1 mM 3-0-methylglucose self exchange at 37 ~ C was about 50 sec whereas the half-time for L-glucose (1 raM) self exchange was more than 50 min. Insulin (0.7 ~M) increased the tracer efflux rate 10-25

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fold. At 25 mM 3-0-methylglucose, phloridzin (5 mM) inhibited the exchange process about 20 fold both in the absence and in the presence of insulin. Analysis of the concentration dependence of the exchange rate showed that the half-saturation constant for 3-0-methylglucose equilibrium exchange was about 5 mM and that insulin had no effect on this parameter, whereas it increased the maximal transport capacity. 283. Urinary Albumin Excretion during Exercise in Juvenile Diabetes. A Provocation Test for Early Abnormalities. E. Vittinghus, C. E. Mogensen. Second University Clinic of Internal Medicine Kommunehospitalet, 8000 Aarhus C, Denmark. The aim of the study was to develop a provocation test for early glomerular abnormalities. - Urinary albumin excretion during exercise was measured with a radioimmunological method in a group of 13 young male diabetic patients and in a comparable control group. The duration of diabetes was 2-18 years. They had no proteinuria (Albustix (R)) and no other signs of renal disease. There was no difference in the basal albumin excretion. In the diabetics the average albumin excretion was doubled during exercise with a work-load of 600 kpm/min for 20 min, namely from 9.1 ~tg/min to 18.7 ~tg/min (p < 0.005). No significant change was seen in the controls. In some experiments RPF and GFR were also measured. There was no difference in the renal hemodynamic response to exercise between normal and diabetic. - These results strongly suggest that abnormal glomerular filter properties are present in patients with relatively short duration of diabetes, that is in patients who are known to have a thickened glomerular basement membrane. 284. Angiographicai Findings in Peripheral Artery Disease (PVD): a Controlled Study on the Degree and Localisation of Sclerotic Lesions from Diabetes and Hyperlipoproteinemias (HLP) in Relation to Non-Metabolic Risk Factors. K.H. Vogelberg, P. Berchtold, H. Berger, F.A. Gries, H. Klinger. Diabetes Research Institute, Second Medical Department and Department of Statistics, University of Diisseldorf, FRG. Specific aspects of diabetes and HLP as risk factors in PVD are little known. The degree and localisation of PVD in these diseases were investigated in relation to obesity, hypertension and smoking, by angiography in 121 inpatients with clinical signs of PVD (15 diabetics, 75 with HLP, 31 without metabolic diseases). - PVD in diabetics and type IIb was twice and in type IV five times as frequent as in controls. Type IV patients revealed more than 2 additional risk factors and were 5-10 years older than type IIa patients. The degree of sclerotic lesions was directly related in all patients with a number ' of risk factors (p < 0,05), in type II with hypertension (p < 0,01). In addition: type IIa with cholesterol and type IIb with smoking (p < 0,05); In diabetics and type IV plus abnormal glucose tolerance with age (p < 0,05). - The sclerosis in type II (IIa IIb) affected predominantly the pelvic vessels, in diabetics and type IV the distal arteries (X 2 = 60, 15, p < 0,001). The degree of sclerosis in pelvic and distal arteries was correlated with the cholesterol/triglyceride ratio (t7 < 0,05). Smoking aggravated pelvic lesions in type IV. Hypertension augmented the lesions in distal vessels of type II patients. - The results emphasize a higher risk for PVD in diabetes with type II than with type IV. Type IV HLP favours PVD predominantly by favouring the effect of other risk factors. 285. Rate of Insulin Degradation in Different Organs of Diabetic Rodents. H.J.v. Funcke, H.J. Kolb and E. Standl. Diabetes Research Unit and 3 rd Medical Department, City Hospital Schwabing, 8 Munich 40, K61ner Platz 1, FRG. The influence of relative and absolute insulin deficiency on enzymatic activity responsible for the degradation of insulin was studied in liver, kidney and heart muscle. Several rodents, e. g. streptozotozin diabetic rats, two species of spontaneously diabetic mice (KK, C 57 BL) and Chinese hamsters were chosen and compared to nondiabetic controls. Insulin degradation was followed by a TCA-precipitation technique with 125I-insulin as marker. The total insulin concentration in the test was 2 mU/ml. - Streptozotozin-diabetes in

the rat caused a significant decrease of insulin degrading activity (p < 0.005) in liver and heart muscle but not in kidney. KK mice showed about a 3-fold decrease in liver activity (p < 0.001) but not in the other organs. In contrast, C 57 BL mice exhibited a significant decrease (p < 0.005-0.001) in all three organs tested. Severely diabetic Chinese hamsters failed to show a significant difference in therate of insulin degradation in liver, kidney and heart. - In view of different results among hyperinsulinemic as well as insulin deficient groups the hypothesis that serum insulin regulates insulin degrading activity cannot be confirmed. 286. The Role of Calcium in Insulin Action. V. Insulin, Calcium, c-GMP and the Regulation of Protein Synthesis in Adipose Tissue. N. Vydelingum, A.H. Kissebah, V. Wynn. Alexander Simpson Laboratory for Metabolic Research, St. Mary's Hospital Medical School, London, W2 INJ, U. K. We have previously proposed a hypothesis to explain the mode of insulin effects on lipolysis, lipogenesis and glycogen deposition in adipocytes based on alteration in intracellular calcium distribution. In this study we provide evidence that calcium ions might also mediate the effects of insulin on adipose tissue c-GMP levels and protein synthesis. - The incorporation of 3H-leucine into total proteins was measured in isolated fat cells incubated with and without insulin, c-GMP concentration was determined on aliquots of these incubates by a radioimmunoassay technique. - When calcium ions (1.25 mM) were present in the medium, insulin at concentrations between 10-100 IxU/ml produced an immediate rise in fat cell c-GMP levels associated with a progressive enhancement of protein synthesis. Omission of exogenous calcium from the medium partially reduced the basal rate of protein synthesis while depletion of endogenous calcium by EGTA markedly inhibited this effect despite elevation of c-GMP levels. Under these latter experimental conditions insulin neither induced a further rise in tissue c-GMP nor stimulated protein synthesis. - In conclusion we suggest that c-GMP might mediate the insulin effects on protein synthesis though calcium ions are necessary for the nucleotide to induce this effect. Furthermore calcium ions seem to play a role in the transmission of signals from the insulin receptor to the enzymes regulating c-GMP concentration and hence stimulation of protein synthesis. 287. Chemical Composition of Human Glomerular Basement Membrane in Diabetes Mellitus. P. Wahl, D. Deppermann, Ch. Hasslacher. Medical University Clinic, Heidelberg, FRG. In order to characterize the glomerular basement membrane in diabetes, basement membranes have been isolated from kidneys of normal and diabetic subjects according to a method described previouly. Kidneys from 11 diabetics and 15 nondiabetics were individually analyzed for amino acids and carbohydrates. There were no statistically significant differences in basic amino acid composition and carbohydrate content between nondiabetics and diabetics. These results did not confirm the observations of Spiro, who found a higher content of hydroxylysine and a higher glucose concentration in diabetic basement membranes. They are however in accordance to the findings of Westberg and Kefalides. These different results may be due to the technique of isolation, which can influence the chemical composition of basement membrane preparation. 288. Muscle Nitrogen Repletion after Protein Ingestion: Key Role of Branched Chain Amino Acids and Evidence of a New Defect in Diabetes. J. Wahren, P. Felig. Serafimer Hospital and the Karolinska Institute, Stockholm, Sweden and Yale University School of Medicine, New Haven, Conn., USA. In fasting man, muscle continuously releases a-amino nitrogen, primarily as alanine. To determine the source and mechanism of nitrogen repletion in muscle, leg and splanchnic amino acid exchange were determined in 7 normal subjects and in 6 insulin dependent diabetics before and for 180 min after a protein meal (lean beef, 3 gm/kg). In normals, protein intake resulted in splanchnic amino acid release (168 Ixmol/min), 93% of which was

Abstracts branched chain amino acids. This persisted for 180 min and was accompanied by hyperaminoacidemia, most marked for valine (90% rise), leucine (150% rise) and isoleucine (124% rise). Leg exchange of these amino acids reverted from a basal net output to a net uptake (valine 28 + 8 9mol/min, leucine 26 + 6, isoleucine 14 + 4) which persisted for 180 rain, accounting for 65-100% of total leg amino acid uptake. Alanine release from the leg (basal 34 + 7 ~tmol/min) and splanchnic uptake persisted after protein feeding. In diabetics, arterial branched chain amino acids were 45% higher than controls in the basal state (p < 0.02) and rose 50% more than controls after protein intake (p < 0.025). Splanchnic output of branched chain amino acids after protein was equal to that of controls. In contrast, leg uptake was reduced by 65% (p < 0.02) with no uptake occuring beyond 60 rain. As in controls, leg output and splanchnic uptake of alanine persisted throughout. - It is concluded that repletion of muscle nitrogen occurs after protein intake despite on-going alanine release, by selective splanchnic escape and muscle uptake of branched chain amino acids. In diabetes muscle uptake of branched chain amino acids is reduced, leading to hyperaminoacidemia and indicating defective protein as well as glucose tolerance in this disorder.

289. Intestinal Disorders Disguised by Treatment with Biguanides. W. Waldhfiusl, R. Dudezak and F. Pesendorfer. I. Med. Univ. Klinik, Spitalgasse 23, A-1090 Wien, Austria. Biguanides (B) are widely used as oral antidiabetic agents, and are known to cause gastrointestinal symptoms if administered in relatively large doses. This study reports 4 patients who were seen within a period of 12 months, who complained of gastrointestinal discomfort coincident with B treatment. Buformin, metformin and phenformin had been administered for 7 months to 10 years. In all patients the clinical complaints of slight abdominal discomfort and/or nausea were regarded initially as a consequence of B administration. After a delay of 3-6 months a thorough clinical check-up provided roentgenologic evidence for the presence of carcinoma of the stomach. This diagnosis was supported by gastroscopy in three of the 4 patients. Surgical exploration and post mortem examination proved the presence of gastric cancer in two individuals. One patient surgically explored presented with chronic adhesive cholecytstitis, and with intestinal metaplasia of the gastric mucosa, but without neoplasia of the stomach. - In conclusion, it appears that gastrointestinal symptoms associated with chronic administration of B might disguise the existence of both benign and malign gastrointestinal disease. Proper and early clinical exploration has to be performed in all individuals treated with biguanides and complaining of abdominal discomfort.

290. Provocation of Postural Hypotension by Insulin. B. M. W. Smith, P. J. Watkins and B. Page. King's College Hospital, Diabetic Dept., Denmark Hill, London, SE5 9RS, U. K. This study aimed to investigate the effect of insulin on blood pressure in diabetics with autonomic neuropathy and, in particular, to discover whether it aggravates postural hypotension, thus causing confusion between hypotensive and hypoglycaemic symptoms. Eight patients with advanced autonomic neuropathy were studied by measuring blood pressure changes in supine and erect postures before and after insulin administration. - In all, systolic and diastolic pressures fell considerably after insulin, especially after tilting to the vertical position. Five of the patients fainted when upright with systolic blood pressures less than 50 mm. HG. This hypotensive effect of insulin occurs whether it is administered intravenously, intramuscularly or subcutaneously. The effect is almost immediate after intravenous insulin and may last for several hours. It coincides with a falling blood glucose level, but occurs before hypoglycaemic levels are reached and may even be present when the blood glucose level is high. Diurnal variations of postural hypotension have been recorded in some patients on their normal insulin regime when the standing blood pressure decreases with the onset of insulin action and increases again as the latter declines. The mechanism for this hypotensive effect of insulin has not yet been elucidated. - Some of our patients were unable to differentiate between symptoms of hypoglycaemia and hypotension. This may account for some episodes of sudden loss of consciousness without warning usually attributed to hypoglycaemia.

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291. Activity in Adipose Tissue and Liver of Pyruvate Dehydrogenase (PDH) and ATP : Citrate Lyase (ATP : CL). L. Weiss, K. Kreisel, M. Haslbeck and O.H. Wieland. Inst. Clin, Chemistry, 3 rd Med. Dept. Schwabing City Hospital and Diabetes Research Unit, 8 Mfinchen 40, K61ner PI. 1, FRG. The activity of both enzymes in rat and human was studied to get insight in the capacity of lipogenesis from carbohydrates. In fed rats (< 220 gm) PDHa (the active portion of PDH) in adipose tissue amounted to 7.52 mU/mg prot. (80% of total PDH), ATP : CL to 15.74 mU/mg prot. The activity in liver was 0.97 mU/mg prot. for PDHa (20% of total PDH) and 0.74 for ATP : citrate lyase respectively. In starvation only 10% was found as PDHa in adipose tissue and liver, while ATP : CL was slightly lowered only in adipose tissue. In adult animals (500 gm) PDHa in adipose tissue was decreased to 0.57 mU/mg prot. (30% of total PDH) and ATP : CL to 0.4 mU/mg protein. In livers of adult rats the activities remained at the same level. In human adipose tissue, independently from age, total PDH was 2.02 mU/mg prot. In the livers of adult humans, during adsorption, P D H a was 1.09 mU/mg prot. (40% of total PDH). The.activity of ATP : CL was 0.4 mU/mg prot. in adipose tissue and 0.71 mU/mg prot. in liver. No differences were seen between children and adults. - These results indicate that in the adult rat, only the liver has a substantial capacity for the conversion of glucose to fatty acids. In addition the data clearly demonstrate that a young rat is a poor model for human metabolism.

292. The Role of Insulin in Oestrogen Induced Hypertriglyceridaemia. C. White, B.R. Tulloch, D. Wain. The Royal Infirmary, Manchester, M13 9PL, U.K. Apparent insulin resistance accompanies oestrogen administration, and may result in increased hepatic very low density lipoprotein synthesis. In oestrogen treated rats we have observed concurrent increase in the liver lipogenic enzymes acetyl Co A carboxylase and fatty acid synthetase. - In these experiments hepatic lipoprotein synthesis was studied in vivo and in vitro in oestrogen treated, ovarieetomised rats following saline or streptozotocin, injection. Streptozotocin therapy led to biphasic changes in plasma triglyeerides with maximal increase in the partially diabetic animals. Homogenised liver preparations possessed lower levels of fatty acid synthetase, whereas acetyl Co A carboxylase was little effected by streptozotocin injection. - The studies suggest that the increased insulin levels resulting from fl-oestradiol therapy may predominantly affect the fatty acid synthetase step.

293. On the Regulation of Pyruvate Oxidation in the Human Central Nervous System (CNS) during Fasting. M. Wicklmayr, G. Dietze. 3 rd Medical Department, Schwabing City Hospital, Munich, FRG. Metabolic balances of human CNS measured during various periods of fasting (15- (n = 9), 60- (n = 5) and 120- (n = 7) hours) showed increasing rates of lactate production (- 5.91 + 0.63, - 9.17 + 2.0, 14.23 + 1.47 ~tmoles/100 g • min.). Since cerebral glucose utilization declined simultaneously (19.61 + 1.23, 12.24 + 2.91, 11.31 + 1.42 ~tmoles/100 g • rain.) there was a greater proportionate change of glucose metabolism to lactate production (15%, 38%, 63%). This could be due to a reduction of PDH-activity which is probably caused by a 10- and 20-fold augmentation of cerebral ketone body utilization (2.22 + 0.62, 27.6 + 3.16; 44.23 _+ 4.38 ~moles/100 g X min.). This was underlined by a significant correlation between cerebral ketone body extraction and the estimated rate of anaerobic glycolysis (y = 12.32 _+ 0.39 y; r = 0.93, p < 0.05).

294. Changes of Individual Serum Free Fatty acids (F.F.A.) and Fatty Acid Synthesis of Adipose Tissue during Experimental Hyperglycemia and Hyperinsulinemia. A. Wirth, C.C. Heuck. Medizinische Poliklinik der Universit~it Heidelberg, Klinisches Institut fiir Herzinfarktforschung, H e i delberg, FRG. Previous experiments have revealed that chronic hyperinsulinism and hyperglycemia in the rat had marked effects on fatty acid

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Abstracts

synthesis in the liver. This investigation should elucidate the action insulin and glucose in vivo on the synthesis of fatty acids in adipocytes and their individual changes in the serum. Rats (300 g to 400 g) received high doses of glucose by intravenous infusion for several days. Blood was collected in intervals, F.F.A. were quantitatively and qualitatively determined by GLC. F.A. from adipocytes were measured. Adipocytes were investigated by electronmicroscopy. Intense accumulation of glycogen was observed. Serum F.F.A. concentration decreased. The pattern of F.A. composition changed towards shorter chain length and monodesaturated F.A. both in serum and adipocytes. The observations may be important in respect to F.A. mobilisation, as the removal of short chain F.A. is facilitated, in respect to intracellular triglyceride formation due to intracellular supply of glycerophosphate through glycogenolysis and glycolysis, in respect to atherosclerosis, as a high percentage of lipids in fatty streaks are esters of monodesaturated F.A. 295. Time-Related Changes in Insulin Release, Cyclic AMP and Glucose Oxidation in Rat Pancreatic Islets during Glucose Stimulation. G.H.3. Wolters, W. Konijnendijk and P.R. Bouman. Endocrine Research Unit, Department of Pharmacology, University of Groningen, The Netherlands. The aim of this study was to investigate whether the rate of glucoseinduced insulin secretion might be related to time-dependent changes of glucose oxidation and cAMP levels in the islet tissue. Pancreatic islets of fed rats were incubated for 5, 15, 30 and 60 rain in glucose, 2.5, 7.5 and 15 mM. Islet cAMP and insulin release were measured by radioimmunoassay. In separate experiments the rate of glucose oxidation was measured continuously by fractional collection of 14-CO z at 5 rain intervals over 120 min of incubation. Glucose 15 mM caused a more than 2-fold rise of cAMP over the initial 15 min, which preceded the major increase of insulin secretion. Glucose 7.5 mM caused moderate secretion from 30 min onward, preceded by a minor but persistent elevation of islet cAMP. Glucose 2.5 mM had no such effects. At all glucose concentrations tested, half of the final rate of glucose oxidation was reached within 5 min of the start of incubation. This was followed by a less rapid increase, the steady state being reached after 20 min at glucose 2.5 mM, after 40-50 min at glucose 7.5 mM and after 60-75 min at glucose 15 raM. - These observations suggest that time-related changes in the cAMP level and the rate of glucose oxidation may play a key role in shaping the time-related pattern of insulin secretion during glucose stimulation. 296. On the Mechanism of the Stimnlatory Effect of Na-Lactate on Insulin Secretion in Dogs. M. Zaccaria, S. Pedrazzoli, E. Zago, N. Sicolo, G. Federspil, C. Scandellari. Istituto di Semeiotica Medica, Universith di Padova, Istituto di Clinica Chirurgica, Universith di Padova, Italy. In a previous report we found that in dogs Na-Lactate administered in peripheral veins at physiological concentrations, stimulated insulin secretion and slightly raised blood glucose levels. The present research has been undertaken to ascertain the mechanism by which Na-Lactate stimulates insulin secretion. - Experiments were carried out on anesthetized dogs fasted overnight. Lactate was perfused into a peripheral vein over 20 min at dose of 20 mg/Kg/min. Plasma insulin was measured in the pancreatico-duodenal vein. - The results obtained show that: 1) Diazoxide completely blocks Na-Lactate stimulatory effect on insulin release. 2) Epinephrine infusion diminishes basal insulin values and slightly inhibits Lactate-induced increase of insulin secretion, in spite of the clearcut blood glucose increase. 3) During Propranolol infusion, at a dose not reducing basal insulin levels, no significant increase of insulin release was observed. 4) Ten dogs were injected via the pancreatic artery with small and increasing amounts of Na-Lactate. This administration failed to modify glucose concentrations in the pancreatico-duodenal vein; only the highest doses capable of increasing blood Lactate levels, also induced a significant increase of insulin release from the pancreas. - Present results suggest that the Na-Laetate effect can probably be explained by stimulation of adrenergic receptors of the beta-ceils; other mechanisms cannot, however, be excluded.

297. The Role of Circulating Insulin Levels in the Lipolytic Response of Rat Adipose Tissue (AT) to Catecholamines in Vitro. J. Zapf, M. Waldvogel. Metabolic Unit, Department of Medicine, Kantonsspital Zurich, Switzerland. AT from diabetic rats is between 5 to 10 times more sensitive to catecholamines than normal AT. The effects of insulin treatment (diabetic rats) and of fasting and refeeding (normal rats) on epinephrine sensitivity were investigated. - Glycerol- and FFA release and tissue FFA were measured 30 rain after addition of different concentrations of epinephrine or other lipolytic hormones to pooled AT preincubated in KRB-albumin buffer containing 2 mg/ml of glucose and anti-insulin serum. - 1. AT from fasted rats exhibits increased sensitivity to epinephrine, which is reduced by refeeding. 2. Insulin treatment for 24 h of diabetic rats restores the lipolytic response of AT to near "normal". 3. Hypersensitivity of diabetic AT is also observed with isoproterenol, which is not taken up into adrenergic nerve endings, but not with ACTH, glucagon or dibutyryl cyclic AMP. - 1. Insulin-deficiency causes hypersensitivity of AT to catecholamines, but not to other lipolytic hormones. 2. Insulin "normalizes" the increased lipolytic response. 3. Circulating insulin levels seem to modulate the responsiveness of AT to catecholamines. 4. Our findings suggest that lipolytic hypersensitivity is caused by a postsynaptic mechanism involving a change in the affinity of the catecholamine receptor. 298. Receptor Binding at Nonsuppressible Insulin-Like Activity (NSILA-S) to Chicken Embryo Fibroblasts (CEF) and Its Effect on DNA-, RNA- and Protein Synthesis. J. Zapf, M. Waldvogel, M. M~ider and B. Morell. Metabolic Unit, Department of Medicine, Kantonsspital Zurich, Switzerland. NSILA-S purified from human serum stimulates DNA-synthesis and growth of chicken embryo fibroblasts at molar concentrations one hundred times below those of insulin. This finding suggested the presence of a sensitive and specific membrane acceptor for NSILA-S in these ceils and led us to look for other metabolic effects possibly related to hormone-acceptor interaction. - Specific binding of 125INSILA-S to CEF was determined by incubation of cells in the presence and absence of an excess of cold NSILA-S. DNA-, RNA- and protein synthesis in the presence of fetal calf serum, NSILA-S and insulin were measured by the incorporation of 3H-thymidine into DNA, of 3H-uridine into RNA and of 14Cleucine into protein and compared to stationary controls. - 1251 NSILA-S binds specifically to CEF. Binding is highly sensitive to small concentrations of cold NSILA-S (KD 2 - 4 ~tU/ml) and shows saturation kinetics. Insulin is between 50 and 100 times less potent in competing for labelled NSILA-S. NSILA-S stimulates DNA-, RNA- and protein synthesis in CEF over the same concentrations range at which specific binding and displacement are observed. Our findings suggest that the effects of NSILA-S on DNA-, RNAand protein synthesis in CEF are mediated by interaction with a specific high affinity, low capacity membrane aeceptor site. 299. Increase o| Insulin Releasing Activity from Duodenal Mucosa after Administration of Giibenclamide to Rat and Man. A. Zermatten, W. Heptner, B. Delaloye, C. Geser, E. Figueroa, R. Srchaud and J.-P. Felber. Division de Biochimie Clinique, Drpartement de M6decine, Hrpital Cantonal Universitalre, 1011 Lausanne, Switzerland, Hoechst AG, Medical Department, Frankfurt, FRG. The purpose of this work was to study the possible effect of glibenclamide on the release of insulinogenic activity from duodenal mucosa in rat and man. - Duodenal mucosa was removed (in man through a multiple blind biopsy canula; in rat after decapitation) after oral administration of a glibenclamide solution. The duodenal extracts were tested for their action on promoting insulin release in an in situ rat pancreas preparation as described previously (Horm. Met. Res. 6, 272, 1974; Lancet 2, 187, 1974). - Results showed a significant increase of Duodenal Insulin Releasing Activity (DIRA) after oral administration of glibenclamide, both in rat an in man. A control injection of a glibenclamide solution in amount exceeding five-fold the maximum level detected in duodenal mucosa extract showed no effect on insulin release. - In man,

Abstracts glibenclamide given per os enhanced DIRA before any peripheral insulin increase or blood lowering effect. - These experiments suggest that glibenclamide may exert its insulinogeniceffect at least in part through gastrointestinal mediators. (W. Heptner et al., Acta diab. lat. 6 suppl. 1, 105, 1969). 300. Carboxypeptidase B- and Trypsin-Like Activities in Isolated Rat Pancreatic Islets. H. Ziihlke (Karlsburg, GDR), A. Lernmark (Ume~t, Sweden), D. F. Steiner (Chicago, Illinois, USA). Department of Biochemistry, University of Chicago, Chicago, Illinois, USA. Carboxypeptidase B (CPB)-like and trypsin-like enzymes are believed to play an important role in the conversion of proinsulin to insulin in the islets of Langerhans. The measurement and characterization of these activities require highly sensitive and specific assay procedures. Therefore, ultra-micro methods using hippuryl-L-arginine, Cbz-Gly-Gly-Arg-4-methyl-fl-naphthylami-

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de, 3H-bradykinin and a heptapeptide with the partial sequence of porcine proinsulin Pro-Lys-Ala-[14C]-Arg-Arg-G1u-Ala were developed. Measurements were carried out with homogenates of islets isolated from pancreas by digestion with collagenase and centrifugation on Ficoll gradients. By means of different stimulators and inhibitors (EDTA; 2-Mercaptoethanol, ME; soybean trypsin inhibitor, STI; N-ethyl maleimide, NEM; diisopropyl-fiuorophosphate, DFP), the following results were obtained: islets of Langerhans contain carboxypeptidase B-lille and trypsin-like activities. The CPB-like activity possesses a pH-optimum of 5.5 - 6.0; it is inhibited by EDTA; ME and DFP have no effect. The trypsin-like activity has a pH-optimum of 6.0; STI and DFP have no effect; ME stimulates, whereas NEM inhibits. It is concluded that the activities measured have properties suggestive of catheptic proteinases and therefore may be of lysosomal origin. Whether these or similar enzymes are involved in the conversion of proinsulin to insulin will require additional study.