Organization Section �9 Abstracts 57

Eighth Annual Meeting of the European Association for the Study of Diabetes

Madrid, Spain, September, 6 - -8 , 1972

Abstracts

Tissue Oxygenation in Severe Diabetic Ketoacidosis. K.G.M.M. Alberti, J . Darley, Pauline M. Emerson and T. D.R. Hockaday. Radcliffe Infirmary, Oxford. Tissue oxygenation has not been studied in diabetic coma, yet may be an impor tant factor in recovery. In 51 subjects in diabetic ketotic coma we have determined [lactate]/ [pyruvate] (L/P) and [3-hydroxybutyrate]/[acetoacetate] (3 HB/ACAC) ratios (which under normal conditions reflect tissue cyCosol and hepatic mitochondrial redox state); arterial pH and pO2; and red-cell 2,3-diphospho- glycerate (2,3-DPG) (an important determinant of haemoglobin affinity for oxygen). -- Before t reatment L/P and 3 HB/ACAC ratios were elevated at 19.0~:0.9 and 3.7q-0.3 (normal, 10.6 and 1.8); p i t was 7.14q-0.03, and 2,3-DPG greatly decreased at 2.18q-0.36 (normal, 4.5). Acidaemia causes a fall in 2,3-DPG, pH correlating with 2,3-DPG (r, 0.88; p < 0.001). L /P ratios correlated negatively with 2,3-DPG ( p < 0.01) and pH (p < 0.05). However, there was no correlation between 3 HB/ACAC and 2,3-DPG or pH. pO 2 did not correlate with any other parameter. On t rea tment L/P ratios fell (15.1 ~0 .7 at 5 h, 13..5 -V 0.6 at 24 h) while 2,3-DPG rose more gradually and was still significantly decreased at 24 h. Increased in t ra - cellular [H +] raises L/P and 3HB/ACAC. I t is suggested that the raised L/P ratios are due to both increased [H+] and decreased oxygen release at peripheral tissues result- ing from decreased 2,3-DPG and diminished peripheral perfusion.

Monolayer Cell Culture of Neonatal Rat Pancreas: An Ultrastructura| and Autoradiographic Study. M. Amherdt, A.A. Like, B. Blondel, E. Marliss, C: Well- helm, L. Orci. Inst i tutes of Histology and Biochimie Clinique, Geneva. Pr imary monolayer cultures of enzymatically dissociated neonatal rat pancreatic ceils yield an endocrine cell population which maintains hormone biosynthesis and shows insulin and glucagon secretory responses to classical modulators. Since these cultures are ex~ensively utilized in physiological studies, the fate of the cells introduced into culture was studied.:Suspensions of pancreatic ceils dissociated by t rypsin and collagenase and monolayer cultures aged 14 h to 19 days were studied by light and electron microscopy (EM). Small clusters of epithelioid cells first recognized a t 14 h increased in size with time, frequently merging to form large sheets of cells surrounded by fibroblastoid cells. Endocrine B (insulin producing) and A (glucagon synthesizing) cells maintained their differentiated ultrastructure, vir tual ly indistinguishable from ceils fixed in situ. The quant i ty of rough endoplasmic reticulum (RER), size of Golgi and number of secretory storage granules of both endocrine cell types correlated with hormonal synthesis and release data. Autoradi0- graphic studies using aH-leucine emphasized the sequen- t ial involvement of the RER, Golgi and secretory granules in insulin production. EM evidence of B and A:cell replication was documented by ceils undergoing mitotic division, autoradiography and cytochalasin-B induced binucleate cells. Presumptive serotonin, gastrin and D- cells were also identified. In contrast, exocrine cells under-

went progressive dedifferentiation giving rise to a popula- t ion of degramllated cells rich in free ribosomes, poor in R E R and containing numerous microfilaments. Th~ monolayer chlture is a valuable model for studying hormone secretion and mechanisms controlling pancreatic

�9 endocrine cell replication. I t is also of value for studying pancreatic exocrine cell differentiation and possible nee- formation of endocrine cells.

Antibodies to Proinsulin in Diabetic Patients Treated with Porcine Insulin Preparations. O. Ortved Andersen, Steno Memorial Hospital, Copen- hagen. I n most diabetic patients t reatment with commercial insul in preparations containing 1 to 2 per cent proinsul in primarily results in formation of antibodies reacting with both insulin and proinsulin. Later on, formation of anti- bodies reacting specifically with proinsulin also seems to occur. In some plasmas from patients treated with porcine insu l in it has been possible to demonstrate formation of antibodies reacting with proinsulin and intermediate forms, bu t not with monoeomponent insulin, dimer forms or C-peptide from porcine insulin. No reaction between bovine proinsulin and antibodies to porcine proinsulin could be demonstrated. The observations indicate pro- insulin having considerable significance for the ant ibody formation in diabetic patients treated with usual available insulin preparations.

Structure and Metabolism of Isolated Pancreatic Islets in Tissue Culture. Arne Andersson, Department of Histology, The Univer- sity of Uppsala, Uppsala, Sweden. Isolated pancreatic islets from adult mice were cultured for up to 12 days in a simple in vitro system, which allow- ed detailed comparisons between the metabolism of non~ cultured and cultured isolated islets in subsequent short term experiments. As evidenced by both light and electron microscopy there was a very good morphologic preserva- t inn of the cultured islets and no overgrowth of fibro- blastoid cells. The cultured B-cells, however, became degranulated, which was in contrast to the well-granu- lated As-cells. Mitoses were observed as well as incorpo- ration of aH-thymidine into nuclear DI~A of the islet cells. -- After culture for 6 or 12 days in a medium con- raining 1.2 mg/ml glucose both the insulin release and the respiratory rate responded to glucose in a manner very similar to tha t of non-cultured islets. The rate of glucose oxidation was related to the glucose concentration of the culture medium in tha t the KM-value decreased with an increasing glucose level. The insulin content of the cul- tured islets decreased within one week after explantation. This was not due to an impaired abil i ty for insulin bio- synthesis in the cultured islets as evidenced by the obser- vat ion that these islets incorporated aH-leueine into both proinsulin and insulin to the same extent as non-cultured mouse islets. -- I t is suggested that this cultt/re method should be useful for providing new information on the long-term effects in vitro of various factors on the struc- ture and metabolism of the pancreatic B-cell.

58 Organization Section �9 Abstracts DiabetoIogia

Prognostic Significance of Hyperglycaemia in Acute Myo- cardial Infarction. F.M. Antonini, C. Fumagalli , E. Pctruzzi, G. Bertini, S. 5iori, P. Tinti. Inst i tute of Gerontology, University of Florence, Italy.

In 319 patients with acute myocardial infarction, a clinical diabetic condition has been confirmed in 27.1 ~ of the women and 11.5~ of the men. Clinically it has be~n possible to detect more frequent complications of a haemodynamic nature, and a higher death rate in a group of patients comparable to the diabetic group. -- In order to evaluate the significance of the altered glucose metabo- lism during the acute phase of myocardial infarction, the fatty acid composition of subcutaneous tissues, plasma phospholipids and cholesterol esters was asse~sed. In 8 subjects admitted during the first hour of the illness, the following parameters were examined every 3 h for a period of 48 h: blood glucose, NEFA, glucagon, eortisol, and urine eatecholamine levels. -- Our results with these cases indicate a more prominent relationship between hyperglycaemia and haemodynamic complications than between a latent diabetic condition and such compli- cations.

Islet Metabolism, ATP Concentration and Insulin Release. S.J .H. Ashcroft, L.C.C. Weerasinghe and P.J . Randle. Dept. of Biochemistry, University of Bristol, U.K.

The ATP concentration of mouse islets incubated without exogenous substrate shows a gradual fall which can be prevented by glucose or mannose (20 raM) or leucine (2.5 mM) ; glyceraldehyde (5 raM) is as effective a~ glucose (5 raM); fructose or N-acetylglucosamine (20 raM), pyru- vate (10 raM), hydroxybutyrate (2 raM) are less effective; galactose (20 raM), acetate (10 raM), octanoate (2 raM) have no ATP-maintaining ability. Islets oxidise glucose, manuose, glyceraldehyde, leucine, and, less readily, N-acetylglucosamine and glucosamine; galactose, how- ever, is poorly metabolised. Mannoheptulose inh ib i t s oxidation of glucose but not of glyceraldehyde. Insul in release is stimulated by glucose, mannose, leucine, gly- ceraldehyde or glucosamine but not by fructose or N-acetylglueosamine. The latter, however, potentiated effects of glucose or glyceraldehyde (5 raM) or leueine (2.5 raM) on release; the potentiatory effects are inhibited by mannoheptulose which also blocks glucose- bu t not leueine- or glyeeraldehyde-stimulated release. Glucose- stimulated insulin release is depressed in parallel with ATP concentration by metabolic inhibitors. However there is no correlation between islet ATP concentrations and insulin release rates with various combinations of initiators and potentiators of secretion and sulfonylureas stimulated insulin release bu t decreased islet ATP concen- tration. -- These data suggest that the association be- tween metaboli te and seeretagogue activities of sugars may not depend solely on their use as a fuel. The specifi- city of the secretory responses to sugars may be inter- preted on the basis of a two-site glueorecept~r model.

Structure/Function and Structure/Immunoreactivity Rela- tionships of the Glucagon Molecule and Related Synthetic Peptides. R. Assan*, 1~. Slusher***, B. Guy-Grand*, F. Girard**. H6tel-Dieu*, and Facult4 des Sciences,** Paris-France, Universi ty of Columbia***, Missouri, U. S. A.

Several synthetic glucagon-related peptides have been studied with respect to biological activities in vivo and in vitro and immunoreactivity. Glucagon [1 -- 29] (natural or synthetic) injected in man induces glycogcnolysis,

* H6tel-Dictl and ** Fac. Sci. Paris-France *** Univ. of Parma, Italy.

lipolysis, and insulin secretion; on the other hand the [1--23] synthetic peptide induces a rise of free glycerol, but no hypcrglycaemia or insulin secretion; similarly [I--23] is lipolytic in vitro, on rat and human adipose tissues: The lipolytic potency of [1--23] on rat adipose tissue is enhanced by addition of theophylline. As pep- tides shorter than [1--19] have no activity at, all, the structure responsible for lipolysis seems contained inside the [19--23] amino-acid sequence while the structure necessary for glycogenolytic and insulinogenic actions must be located inside the [24--29] amino acid sequence. -- The radioimmunological behaviour of these peptides towards several distinct anti-glueagon immunesera makes it possible to determine at least two chemically-defined antigenic sites: one (C-terminal portion of the molecule) is bound by a specific anti-glucagon [pancreatic] serum. The second site (N-terminal + central portion) is bound by a less specific ant iserum; digestive extracts and purified enteroglucagon preparations competitively in- hibit the binding of labelled [1--23] with this cross- reacting antiserum.

Glucagon Release Induced Respectively by Arginine and by Na Lactate in the Periused Rat Pancreas. R. Assan*, E. Soufflet**, J.l~. Attali*, G. Ballerio***, J. Boillot*.

Rat pancreas -}- duodenum blocks were dissected and per- fused with Krebs-Ringer-Biearbonate -}- Albumin 0.5% A- s constant glueos~ concentration (80 mg~ Flowrate (2 to 3.5 ml/mn) temperature (-{-39~ and perfusion pressure ( + 2 to + 5 emHg) were carefully mor~itored throughout each perfusion (96 mn). -- Arginine infusion, at constant flow rate (24 mg/mn), induced a strong diphasic glueagon secretion: an early short and sharp peak reached 1500 pg/mn in the very first few seconds of st imulation; it was followed, despite continuation of arginine infusion, by a subsequent decline ( ~ 700 pg/mn) ; then by a secondary sustained release beginning at 10th rain, reaching rapidly a high output (1500 pg/mn) ; it was not exhausted at the end of a 44 min-long stimulation. Glucap;on output went down to zero as soon as arginine st imulation ceased. Subsequently arginine pulses (31 rag) were able to elicit acute peaks of secretion (1500 or 2000 pg/mn). These data support the concept of a bicompart- mental storage system for glucagon. -- In other identical preparations, infusion of Na d. 1. (q-) lactate (0.37 raM/ ran) induced a glucagon release, significant, bu t not so dPowerful (1000 pg/mn) as that induced by Ar~inine, and

isappearing at the end of lactate infusion. Subsequent pulses of Na lactate induced peaks of glucagon secretion (maximum 500 pg/mn). -- Thus, a direct effect of lactate on alpha-2 cells probably contributes to the glucagon release measured in Ra t (in r ive) after Na lactate or lactic acid infusions.

* H6tel-Dieu and ** Fac. Set. Paris-France *** Univ. of Parma, Italy.

Further Studies on the Role of the Adenyl Cyclase-eAMP - Phosphodiesterase System in the Mechanism of Insulin Secretion. T. Atkins, A.J. Marry. Universi ty of Aston in Birming- ham, Gosta Green, Birmingham, England.

The presence of an adenyl cyclase system has been established in normal and obese islet tissue, sensitive to adrenaline (B-receptor activation) and HB 419 but not to glucose 1. The reduction in islet adenyl cyelase activity induced by membrs ne active agents Ca 2+, alloxan, phlorid- zin and ouabain reinforced cytoehemical evidence for a

1 Atkins, T., Matty, A.J . : J. Endocr. 51, 67--78 (1971).

Vol. 9, No. 1, 1973 Organization Section �9 Abstracts 59

localisation of cyclase activity at the B-cell plasma membrane and the B-cell/capillary junction. Insulin- ogenic aminoacids lcucine and arginine and a crude secretln preparation were without effect, while glucagon stimulated the cyclase act ivi ty of both normal and obese islets. -- Islet cAMP-phosphodiesterase activity was insensitive to glucose, adrenaline and Ca 2+ but stimulated in obese by imidazole. ~ A meehanis~n for the secretion of insulin via the glucagon st imulat ion of B-cell adenyl cyclase will be discussed.

The Effect of Age on Aspects of Normal and Obese Hyper- glycaemic Mouse Islet Morphology and Metabolism. T. Atkins, A.J . Marry, C.J. Bailey. University of Aston in Birmingham, Gosta Green, Birmingham, England.

At the climax of the Birmingham obese mouse hyper- glycacmic syndrome (20--25 weeks), the prevail ing hyperglycaemia and hyperinsulinacmia was accompanied by B-cell hyperplasia and the appearance of hyaline/ fibrinoid filled lacunae. In addition, adenyl cyclase and eAMP-phosphodiesteraee activities, glycogen, ATP, Ca 2+, Mg 2+ and total RNA contents of obese mouse islets were significantly greater than values recorded for the islets of normal lean l i t termate controls. Islets from 40--45 week old obese mice showed a reduced B-cell volume. At this age islet size was maintained by an increased contr ibution of the lacunae to the total islet volume. There was evidence of endocrine to exocrine cell transformation and a reduction in the severity of hyperinsulinaemia in the presence of a maintained hyperglycaemia. The reduced obese islet B-cell volume (at 4 0 - 45 weeks) was consonant with significant reductions in obese islet adenyl eyclase and cAMP-phosphodiesterase activities, ATP, Ca 2+ and total RNA (Mg 2+ unchanged), bu t an increased glycogen content. The relevance of these observations to the hyper- secretorY behaviour of obese islets will be discussed.

The Effect of Amiloride on Insulin and Glucagon Secretion from the Isolat.ed Perfused Rat Pancreas. A. Aynsley-Green, K.G.M.M. Alberti and S.R. Bloom. Radcliffe Infirmary, Oxford and Middlesex Hospital, London.

We have previously reported the insulin st imulatory effect in the whole rat of the diuretic amiloride hydro- chloride. Like arginine, it contains a guanidine group. This communication reports its effect on the isolated perfusod rat pancreas. -- An amiloride pulse (1.3 Iz-M) caused an immediate increase in insulin secretion (peak increment of 34 [~U]min) in the absence of glucose, while decreasing glucagon output by 30%. In the presence of 5 mM glucose (itself without effect} an identical pulse resulted in a peak insulin increment of 213 (zU/min, whilst decreasing glucagon by 50~ . Insul in secretion following a glucose pulse was pot gntfated 100 fold by a simultaneous amiloride pulse. In the absence of glucose, amiloride infusion (2 /zM/min) caused only a 2 fold rise in insulin, but 60% fall in glucagon. Adding glucose (5 mM) resulted in a 32 fold increase in insulin, affeeting both first and second peaks. Mannoheptuloso (14 raM) or removal of calcium or potassium from the medium inhibited the amiloride effect while ouabain (1 rrLM) inhibited both phases of amiloride induced insulin secretion. Amiloride like axginine, requires glucose for a maximum effect on insulin secretion, and may operate through an effect on sodium or potassium exchange, bu t in contrast to arginino it inhibits glucagon release.

Adrenergic Receptor Sites Modulating the Insulin Response to Arginine in the Rat. R.A. Bacchus, L.G. Meade and D.R. London. St. Tho- mas' Hospital, London and Queen Elizabeth Hospital, Birmingham.

Adremdine, and to a lesser extent noradrenaline, inhibited the insulin response to an intravenous infusion of arginine. Alpha-adrencrgic blockade with phentolaminc or thymox- amine enhanced the insulin response. When isoprenaline was infused with arginine there was a greater rise in insulin than could be accounted fo~ by adding together the changes found when either substance was infused separately. When the bcta-adrenergic blocker propranolol was given there was a diminution in the changes in hormone level following injections of the drug/in doses of between 0.5 mg/kg and I mg/kg; however, at a dose of 10 mg/kg there was an increase in insulin levc!s that was assumed to be due to the quinidine-like action of the drug. When prostaglandin E 1 was given there was an increase in the effect of arginine on insulin levels. From the result of these experiments it was concluded that, in a manner analogous to that demonstrated by others for glucose, the effect of natural ly occurring catecholamines was to reduce the insulin response to arginine and that this effect was produced by st imulation of alpha-adrencrgic receptor sites.

Influence of Renal Failure on the Pharmacokinetics and Hypoglycemic Effect of 3 Sulfonylureas. L. Balant, G. Zahnd, B. Pctitpierre, J. Fabre. Policlinique de m~decine, Department of Medicine, University of Geneva.

Patients with renal failure (RF) may exhibit severe hypo- glycemia after sulfonylureas (SU). Therefore the pharma- cokinetics and hypoglycemic effect of chlorpropamide, glipizide and glibenclamide have been studied in patients with various kinds of nephropathy. A single dose of chlor- prooamide was given to 14 controls and 2 0 patients (GFR 1--130 ml/min). Chlorpropamide was determined according to the method of Toolan and Wagner. Serum T/2 was 36.6 4-11.8 h in controls, but prolonged up to 200 h in RF; This increa~e was related to the decreased ur inary clearance of chlorpropamide, which correlated more with impairement of tubular function than with reduction in GFR. The delay in excretion was associated with higher serum concentration of metabolites. In 2 cases of RF, symptomatic hypoglycemia appeared although the serum concentrations of chlorpropamide were lower than therapeutic levels. Similar results were obtained with glipizide and glibenclamide studied by administration of the 14C-labelled compounds. -- The accumulation of SU in the body increases the risks of severe hypoglycemia in RF, bu t I~F enhances the hypoglycemic response to SU even if the blood concentration of the drug is relatively lOW.

Influence of Nicotinic Acid on the Rates of Transport and Oxidation of Plasma Glucose in Man. E.O. Balasse and M.A. Neef. Metabolic Unit, Department of Medicine, St Peter's Hospital and Laboratory of Experimental Medicine, University of Brussels.

The aim of the present work was to study the role of plasma FFA in the control of glucose metabolism in mail under basal conditions. -- A technique of priming injee- t ion-contlnuous infusion of 1-14C-glucose (with serial sampling of blood and expired air) was used to measure the rates of t ransport and oxidation of plasma glucose in 3 normal postabsorptive, 3 obese postabsorptive and 2 obese starved females. Each subject was studied for 210 rain under 2 experimental conditions (i) in the basal state (ii) after acute lowering of plasma FFA concentra- tion obtained by i.v. administrat ion of nicotinic acid. -- The normal postabsorptive subjects had the highest control values for fractional removal rate of glucose (0.91% per min), for glucose transport (1.92 mg/Kg min) and for the fraction of total CO~ production which derived from glucose (19.8~ wherea~ the lowest control figures

60 Organization Section , Abstracts Diabetologia

(respectively 0.53% per rain, 0.89 mg/Kg rain and 11.0%) were observed in the starved group. -- Relative changes induced by nicotinic acid were similar in the 3 groups : on an average, plasma concentrations of FFA, glycerol and ketches decreased by about 60% whereas the fractional removal rate of glucose and the rate of glucose transport increased by 24 • 4 (S.E.M.) ~o and 27 ~ 5% respectively without si~ozfificant changes in glycemia nor in I R I con: centration. A 30 =t= 7% increase in the contribution of plasma glucose to total C02 production was also observed. -- These data indicate that a decreased availabil i ty of FFA to tissues enhances plasma glucose uptake and oxidation; hypoglycemia is prevented through a stimula- t ion of hepatic glucose output.

Evaluation of Fluorescein Angiographic Changes in Juve- nile Diabetes. L. Barta, G. Brooser. Maria Molnar. First Dept. of Pediatrics, "Semmelweis" University Medical School, and Dept. of Ophthalmology, Post-Graduate Medical School, Budapes t , Hungary.

I n 111 diabetics whose illness had manifested itself before the age of 14, microaneurysms were seen in nearly half of the cases. The moment of appearance of the microaneu- rysms was not in close connection with the durat ion of diabetes in the patients whose ret inopathy had been established by fluorescein angiography, in contrast to the cases in which the ret inopathy had been detected by ophthalmoscopy. In 70 patients, fluorescein angiography was repeated after one year. In 17 patients, with negative ophthahnoscopic findings, the progression of ret inopathy was established. Negative retinal findings turned to positive mainly in the patients with diabetes lasting be- tween 0 to 5 years, i n 10 cases we could not detect the previously established microaneurysms, bu t there were mostly microaneurysms in other parts of the fundus. Such disappearance of microaneurysms was noted in 7 of 10 patients with diabetes of less than 5 years duration, and it never was observed after 10 years. In some of the cases, no microaneurysms were found even after 15 years of diabetes. Differences in genetic factors seem to play a decisive role in juvenile diabetes. The non-diabetic parents of 25 diabetic children were also examined by fluorescein angiography and multiple microaneurysms were detected m some of them.

"Enteroglucagon": A Common Receptor Site with Pan- creatic Glucagon in Rat Liver Plasma Membranes. D.P. Bataille, P. Freychet, P. Ki tabgi and G.E. Rosselin Inserm U. 55 Centre de Recherches Inserm, I-ISpital St Antoine, Paris -- France.

Extracts of porcine jejuno:ileum that contained glucagon- like immunoreactivi ty (GLI) were studied with respect to both st imulation of adenylate cyclase (AC) and binding to receptor sites of pancreatic glucagon (PG) in plasma membranes of rat liver .When these extracts were filtered on Bio Gel P 10, the GLI eluted as a single peak of which about 65% was recovered between the insulin and the PG markers ("A" fraction) and 35% overlapped the PG marker ("B" fraction). Both the "A" and "B" fractions st imulated AC and inhibited the specific binding of mono- I~sI-PG in rat liver plasma membranes. On the basis of their GLI, relatively small amounts of the "B" fraction were about as effective as PG in st imulating AC and in inhibit ing the binding of ~25I-PG: 2.6 ng GLI of the "B" fraction produced the same effect as 2.0 ng of PG. Larger amounts of both "B" and "A" fractions failed to stimu- late AC a n d to inhibit the binding of 125I-PG in direct proportion to their respective GLI: half maximal inhibi- t ion of ~25I-PG binding required about twice as much "B" fraction (12 vs 5 ng/ml) and 120 times as much "A" fraction (600 vs 5 ng/ml) as PG. -- These findings indicate

tha t porcine "enteroglucagon" is a heterogeneous mixture of different molecules tha t shares a common receptor site with PG in the liver.

Oxidation of Fatty Acids and Ketone Bodies in Isolated Pancreat ic Islets~ Christian Berne, Department of Histology, University of Uppsala, Uppsala, Sweden. Both fa t ty acids and ketone bodies have been shown to cause insulin release, bu t their mechanism of action as insulin secretagogues is unknown. I t is not even known to what extent they are metabolized by the B-cells. The aim of the present s tudy was to examine the oxidative metabolism of fa t ty acids and ketone bodies in pancreatic islets from obese hyperglycemic mice by measurements of the 14CO2 production from labelled substrates. I t was demonstrated that both medium-chain and long-chain fa t ty acids were oxidized by the B-cells. At a concentra- t ion of 0.6 mM both palmitate and oleate were oxidized at rates corresponding to the total degradation of 3.1 and 2.1 mmoles per kg dry weight and hour, respectively. Octanoate was metabolized about 15 times faster. Aceto- acetate and D,L-beta-hydroxybutyrate were als0 oxidized by the B-cells and th e enzyme responsible for their inter- conversion, beta-hydroxybutyra~e dehydrogenase (EC 1.1.1.30), displayed a considerable activity. -- The results indicate tha t both fat ty acids and ketone bodies can serve as metabolic fuels for the B-cells. The possibility tha t the metabolic utilization of fat ty acids and ketches may be of importance for their action as insulin releasing agents is now being investigated.

Pancreatic Actions and Extrapancreatic Effects of Sulpho- nylureas. J. Beyer, U. Cordes, G. Sell, C. Rosak, K. SchSffling. Dept. of Endocrinology, Center of In ternal Medicine, Johann- Wolfgang Goethe University, F rankfur t /M, W-Germany.

Various investigations have shown that besides their established insulin releasing action, sulphonylureas possess peripheral, extrapancreatic effects when administered in therapeutic doses. I t is not clear, however, whether these effects, are beneficial as regards diabetes. Studies on the mechanism of action of pancreatic and extrapancreatic effects have revealed parallels between the mechanism of action upon the islet cell and the peripheral cell, respec- tively. In earlier investigations, an influence of alpha- and beta-receptor blocking agents on sulphonylurea-induced insulin secretion had been shown. Preinhibit i0n of the beta-receptors by a propranolol-like beta-receptor block- ing agent (KS 592) decreases the sulphonylurea-induced insulin secretion. Preinhibit ion with an alpha-receptor blocking agent (phentolamine) leads to a potentiat ion of the sulphonylurea effect. -- Our further studies indicate tha t the effect of sulphonylureas on the islet cell can be best explained by a simultaneous st imulat ion of alpha- and beta-receptors. These results have been confirmed by studies on the isolated adipose tissue Of the rat. Similar effects may be assumed to influence hepatic glucose output.

Peripheral Release of Insulin during Acute Muscular Exercise in Normal Man. * B. Birkner, J. Henner, P. Wagner, F. Erhardt , P. Dieterle. Second Medical Clinic, Universi ty of Munich, Germany.

The forearm preparation was used in all studies. Seven healthy male subjects were submitted to a standardized exercise for 5 rain after a 14 h fast. The exercise took place on an adjustable hand ergometer between 30 and 35 min after the test had started. Blood flow was measured by intramuscular injection of 13~Xenon. Simultaneous with-,

�9 Supported by 'Deutsche Forschungsgemeinschaft'.

Vol. 9, No. 1, 1973 Organization Section �9 Abstracts 61

drawal of arterial and venous blood samples at regular intervals was done for the assessment of g'~ucose, lactate, free fat ty acids (FFA), glycerol, and irmnunorcactive insulin (IRI). The uptake of glucose and FFA showed a significant rise during muscular work. Concurrently, the production of lactate and glycerol was significantly enhanced. During exercisd, the mean arterial IR I concen- t rat ion showed a slight decrease whereas the average venous concentration rose. The mean arterio-vcnous (a--v) insulin differences were slightly positive at rest and in the recovery period. The a - - v differences became negative during muscular work and at the sixth minute after stopping exercise. Statistically significant changes in the a - - v insulin differences were observed. As the a - - v insulin gradients became negative during exercise, some insulin was displaced from peripheral receptor sites into the venous ~blood. I t is concluded tha t the enhanced uptake of substrates is at least partially accounted for by this shifted insulin.

Pancreatic Glueagon Levels in the Calf. S.R. Bloom, N.J .A. Vaughan and A.V. Edwards. Mid- lesex Hospital, London, and Dept. Physiology, Cambridge, England.

Pancreatic glucagon (P.G.) activates glycogenolysis and activates and induces enzymes necessary for gluconco- genesis and may therefore have an important ~ole in the neonate. I t has recently been reported that the rise of P.G. after bir th in infants of human diabetic mothers is impaired. I t seems important , therefore, to establish the normal neonatal P. G. physiology and investigations have been undertaken in newborn calves. -- P.G. was measured by radioimmunoassay. An antiserum cross reacting little with enteroglucagon was used and detected changes of 25 pg/ml plasma with 95% confidence. -- Calves under 24 h old had a mean fasting P.G. level of 125 pg/ml (SE 25, N 4) and a plasma glucose of 84 mg/100 ml. Calves botwecu 2 and 18 days had a mean fasting P.G. ot 1.124 pg/ml (SE 241, N 11) and glucose of 83 rag/100 ml. Calves between 19 and 35 days had a mean fasting P.G. of 126 pg/ml (SE 31, N 17) and glucose of 86 mg/100 ml. Following intravenous Insulin, 0.1 U/kg, P.G. rose to a peak of 280 pg/ml (SE 45, N 4) at 120 rain in calves under 24 h, to a peak of 2100 pg/ml (N 3) at 90 min in 5 day calves and to a peak of 316 pg/ml (SE 52, N 4) at 45 rain -- 30 day calves. The poor P.G. response in the first 24 h may be similar to the impaired early insulin response to hyperglycaemia. -- Splanchnie nerve st imulat ion in 30 day calves produced a P.G. peak of 3450 pg/ml (N 3) at 10 rain but insulin hypoglycaemia with splanchnic nerves cut produced a P.G. peak of 499 pg/ml (SE 69, N 4) which did not differ significantly from intact ani- Inals.

Hypertrophy, Hyperpl~sia and Neoplasia of the Endocrine Pancreas. L. Boquist. Ins t i tu te of Pathology, University of Umeh, S-901 87 Ume~, Sweden.

In light and electron microscopic studies, hypertrophy of fl-eells was found in Chinese hamsters and duct-ligated rats treated with alloxan, and in obese Mongolian gerbils with or without associated hyperglycemia. The hyper- trophic cells were large and had one, or seldom two, often peripheral nuclei with a coarse chromatin pat tern and prominent nucleoli. The cytoplasm exhibited sparse granulation, well developed Golgi complex and endo- plasmie reticulum, and a moderate number of mito- chondria. -- Hyperplasia of endocrine cells with the appearance of large islets, was found in obese Mongolian gerbils and some alloxan-treated Chinese hamsters, but not in harnsters with spontaneous diabetes. These islets were mainly, or exclusively, composed of fl-cells with

signs of high activity. Occasional adenomatous, giant islets were seen in some alloxan treated Chinese hamsters and obese Mongolian gerbils. They were composed of cords of sparsely granulated fl-cclls separated by sinus- oidal blood vessels. In human insulomas fl-, %-, ~2-, and agranular cells were identified. -- Increased functional demand on tile endocrine pancreas seems to play a major role in the devclopment of hypertrophy, hyperplasia and adenomatous transformation of the pancreatic islets.

Kinetic Properties of Rat Liver Phosphofructokinase and its Function in the Hepatic Glycolysis and Gluconeogenesis Regulation. I. Brand, H.D. S61ing. Department of Clinical Bio- chemistry, Clinic of Internal Medicine, University of G6ttingcn, G6ttingen, W-Germany. One of the regulatory steps in hepatic glucose metabolism is catalysed by phosphofructokinase (PFK) and fructose- 1,6-diphosphataso (FDPase). In t h e aim to study the kinetic characteristics of liver PFK, the enzyme was purified to homogeneity (about 5000-fold) from rat liver. P F K was inhibited by various metabolites already known from studies with muscle PFK. In addition, ADP exerted a very strong inhibitory effect ( K i = 2 • 10-4M), which could not be overcome by other adenine nucleotides. The only mctabolite capable of relieving the ADP inhibit ion was NH4+ at physiologic concentrations. -- The inhibi- t ion by citrate with the liver enzyme was 10 times lower in comparison to the muscle enzyme. Citrate does not seem to play an important role in the regulation of liver PFK, especially since the inhibitory effect is abolished by very low concentrations of 5'-AMP (0.025 raM). Studies on the effect of the "energy charge" of the adenine nucleotides on the purified enzyme are in progress.

Structure-Function Relationships of Chemically Cross- linked, Homogenous Insulin Derivatives. D. Brandenburg, J. Gliemann, H.A. Ooms, W. Puls, A. Wollmcr. 'l)cutsehes Wollforschmagsinstitut', Aachen; Universi ty of Copenhagen; Free University of Brussels; Bayer Farbenfabriken, Elberfeld, and Teehnische Hoch- schule, Aachen. Beef insulin was reacted with activated esters of aliphatic dicarboxylic acids in organic solvent under dilution conditions. Subsequent fractionation of the reaction pro- ducts by gel filtration and ion exchange chromatography gave 8 homogeneous insulin derivatives. These contained an intramolecular crosslink of 2-- 10 carbon atoms length between the amino groups of glycine-A1 and lysine-B29. The CD-spectra at pH 8 in the far UV show that the three-dimensional structure of the derivatives is only partially altered in comparison to insulin. V~rhile the hypoglycemic activity of the crosslinked derivatives in the rat shows to be between 32% (glutaryl insulin) and 100~) (azelaoyl insulin), the in vitro act ivi ty (fat cell assay) is reduced to maximally 12~ in all instances. The radioimmunoassay shows the reactivity of all the deriva: t i res to be considerably decreased. The results will be discussed with regard to the three-dimensional insulin model.

Deterioration of Tolerance to Glucose and Progression of the Microangiopathy. R.A. Camerini-Davalos, J .M.B. Bloodworth, Jr.*, B. Limburg, and W. Oppermann. Diabetes Center, Dept. of Medicine, New York Medical College, New York, N.Y. 10029. Basement membrane width (B.M.W.)was determined in 25 control subjects, in 32 genetic prodiabetics, in 19

* Dept. of Pathology, University of Wisconsin, Madison, Wisconsin.

62 Organization Section �9 Abstracts Diabetologia

chemical diabetics, and in 4 nontrcatcd overt diabetics. Mean B.M.W. for controls was 1250 A-4-60 (S.E.M.), 1292 A • for genetic prediabeties, 1491 A~-83 for chemical diabetics, and 2530 A for overt diabetics (p < .05 between control-chemical diabetics, control-overt dia- betics, genetic prcdiabetics-chemical diabetics and be- tween genetic prcdiabetics-overt diabetics). Six (19~ genetic prediabctics showed B.M.W. above the mean plus 2 S.D. of cent.tel subjects of same age. A significant difference (p < .02) was found when subjects with normal tolerance to glucose -- normals and prediabetics -- were compared with chemical diabetics. When the mean B.M.W. of 20 chemical diabetics treated with oral compounds was compared with the mean B.M.W. of controls, no difference was found, (previously described difference abolished by treatment). When the mean B.M.W. of 8 chemical diabetics, with both parents diabetic, treated with oral compounds, was compared with 10 non-treated chemical diabetics with both parents diabetic a significant differ- ence was found (p < 0.05).

The Establishment of a Hormonally Sensitive Organ Culture for Adult Rat Liver. A.K. Campbell & K. Siddle. Department of Chemical Pathology, The Welsh National School of Mc~iicine, Heath Park, Cardiff.

Adult rat liver has been cultured in basal Eagle's medium, supplemented with 2 mg/ml glucose, in 95~o O2/5~ CO~ at 37~ Light and electron microscopy, together with measurement of ATP and ion content have shown that a zone of cells at the gas-tissue interface appears to remain viable for at least 6 days. The effects of hormones on three parameters have been investigated (i) tissue and medium cyclic AMP levels, (ii) changes in the glucose-ATP phos- photransferase isocnzyme pattern, (iii) tyrosine trans- aminase. The effect of glucagon and insulin on cyclic AMP levels in fresh cultures has been compared with effects on liver cultured for 2 and 6 days. After 15 min incubation with 1.2 • 10-SM glucagon tissue cyclic AMP levels reached a maximum and then decrease<l. This maximal st imulation decreased over the 6 days in culture. 3.6 • 10-~M insulin appeared to inhibit the effect of 2.9 • 10aM glucagon on 2 day cultures, but had no significant effect on fresh cultures. In the absence of insulin a large increase in type I I and a decrease in type IV (glucokinasc) glucose-ATP phosphotransferasc was detectable. 7.2 • 10-~M insulin was unable to prevent these changes. 20 [~m dexamethasone phosphate caused a 3--5 fold increase in the act ivi ty of tyrosine trans- aminaso within 6 h when added to 2 and 6 day ciHturcs.

A New Approach to the Treatment of Diabetic Retino- pathy. J.M. Cafmdell, J. Barraquer, A. MuiSos, C.D. i-Icrcdia. Clinica Barraquer. Barcelona, Spain.

The destruction of the pi tui tary gland, in spite of its severity, is the best t reatment of progressive diabetic retinopathy in young patients, with no other complica- tions and with sufficient intelligence and education to follow a life-long treatment regime for hypopituitarism. Continuous administration of medroxyprogesterone ace- tate produces an inhibitory effect upon pitui tary activity, which affects the secretion of STH, ACTH, and gonado- trophins. On the basis of these premises, 64 diabetic patients with ret inopathy of degree I I to IV were given this gestagen. A dose of 15--25 mg/day was administered and the duration of t reatment varied between 6 and 20 months. A f a r three months of treatment, hemorrhagic activity was diminished, the progressive course of the retinopathy was confined and the ophthalmoscopic images were improved. Favourable effects were folmd in 53 patients and confirmed within the subsequent months.

The 11 failures noted belonged to the group with final forms of retinopathy. The treatment must ' be followed without interruption. A sudden deterioration of the ret inopathy was soon in two patients who stopped t reatment after 5 and 15 months, respectively. -- De- crease of ACTH secretion in five patients was accompanied b~ symptoms of adrenal hypofunction. The suppression of gonadotrophic activity frequently causes impotefice in male, and amenorrhea (or continuous spotting) in female patients. In seven patients with insulin-dependent dia- betes, the dose of insulin had to be reduced.

Intra-Hematic Glycolysis in vitro: on the Path to a New, Earlier Method in Diabetes Detection ? J. Castillo-Olivares, J. Guijo, L.F. Pallardo. Ins t i tu te of Diabetology, Madrid, Spain.

Under appropriate conditions, glucose uptake by human blood cells was measured in samples obtained during a

s t anda rd oral glucose tolerance test from three groups, namely: control subjects, prediabetics and subjects with chemical diabetes. Glucose uptake was expressed as micro- reel. of glucose disappearedper gr. of haemoglobin and hour of incubation. -- The analysis of the data are pre- sented. -- The significance of the data is studied in respect to their corresponding fasting values *in each group. At every point, the values in the control group were lower, and only at the 120th min was there a signifi- cant difference (p < 0.05). By contrast, in both predia- betics and subjects with chemical diabetes the levels after challenge were higher than the corresponding fasting levels. In th~ prediabetics, these differences were signifi- cant at 60, 120 (p < 0.025) and at 180 rain (p < 0.01). Significant differences in the group with chemical diabetes appeared at 30 (p < 0.05), and at 60, 120 and 180 rain (p < 0.005). The increments occurring in prediabetes and chemical diabetes were correlated with the decreases in controls at corresponding points. Highly significant differ- ences wore found: for prodiabeties at 60, 120 (p < 0.01) and at 180 rain (p < 0.025), for chemical diabetes at 30 (p < 0.05) and at 60, 120, and 180 rain (p < 0.005). Suggestions for a test for earlier diabetes detection are presented.

Insulin Response to Oral Glucose in Normal, Diabetic and Prediabetic Subjects -- A Dose-Response Study. E. Cerasi, S. Efendi6, R. Luft. Department of Endocrino- logy, Karolinska Hospital, Stockholm.

We have recently demonstrated that the diminished insulin response to glucose in prediabetic and mildly diabetic subjects is due to decreased sensitivity of the fl-cells to glucose, very high i.v. glucose doses overcoming this insensitivity and releasing adequately insulin (Dia- betes 21 : 224, 1972}. Since blood glucose had to be elevated to above 500 rag/100 ml, the physiologic implication of this finding was not clear. In the present experiments, blood glucose was raised moderately by using various doses of oral glucose. Each subject served as his own control. At all dose levels the prediabetic and diabetic subjects had lower insulin responses. The glucose-insulin dose response curves after oral glucose show the same pat tern as after i.v. glucose, i.e. a shift to the right of the curves in the diabetic and prediabetic groups. In these experiments, however, only subtle changes in blood glucose concentration were required to produce dramatic changes in plasma insulin. Our results show that the decreased sensitivity of the prediabetie -- diabetic E-cell can be demonstrated also with oral glucose administra- t ion. I t is emphasized that insulin response to oral glucose cannot be compared between groups unless blood glucose is identical (statistical significance is of no value) or complete dose-response studies are performed.

Vol. 9, No. 1, 1973 Organization Section. Abstracts 63

Hepatic Sensitivity to Endogenous Insulin in Prediabetes. E. Cerasi, J. Wahren, R. Luft, P. Felig. Depar tment of Endocrinology, Karolinska Hospital, Depar tment bf Clinical Physiology, Serafimer Hospital, Stockholm; De-' partmen~ of In ternal Medicine, Yale University, New Haven.

The mechanism whereby normal glucose tolerance is mainta ined in spite of diminished insulin secretory capacity in prediabetes is not clear. To examine hepatic sensitivity to endogenous insulin in prediabetes, arterial and hepatic venous blood samples were obtained from prediabeties (unaffected monozygotic twins of known diabetics and subjects with a subnormal insulin response to glucose infusion but normal glucose tolerance) a n d healthy controls. Basal splanchnic glucose output was 30% lower iIx the prediabetics. During a 45 rain glucose infusion (2 mg/kg/min) splanchnic glucose production fell to a significantly greater extent in the prediabetics, de- clining to less than 5% of basal output , as compared to 30% of basal output in the controls. -- Conclusions : Basal splanchnic glucose production is reduced in prediabetes, and in association with small increments in endogenous insulin falls to a greater extent than in controls. The data suggest an increase in hepatic sensitivity to insulin in prediabetes which may account for normal glucose tolerance despite decreased insulin response.

Increased Levels of Plasma Noradrenaline and Vasoconstric- tion in the Hypometabolic State. The Effect on Capillary Fragility. Niels Juel Christensen. The 2nd Clinic of In terna l Medicine, Kommunehospitalet , Aarhus.

Plasma noradrenaline is considerably elevated in diabetics hypophysectomized during the t rea tment of ret inopathy (J Clin Invest 51:779, 1972). Vasoconstriction secondary to hypometabolism has previously been suggested to be at least partially responsible for the normalization of capillary fragility and the decrease in capillary perme- abil i ty occurring after the operation. The following ob- servations support this hypothesis: (1) Prel iminary ex- periments have shown that cutaneous blood flow is diminished in hypophysectomized diabetics (skin temper- ature 31.5~ against 33~ in controls). When the same degree of vasoconstriction was induced in non-operated diabetics with retinopathy, the number of petechiae induced by venous stasis decreased from 60/3 cm ~ to less than 10. (2) In 6 pat ients with hypometabolism due to lack of thyroid hormone, plasma noradrenaline was increased approximately threefold from 0.21 ng/ml to 0.59 ng/ml. (3) I t is demonstrated by the l i terature tha t the hemodynamic changes are very similar in myxedema and after hypophysectomy (q- cortisone and thyroid hormone). The question, is raised as t o whether hypo- thyroidism may also be beneficial to diabetic retinopathy.

Plasma Noradrenaline and Plasma Adrenaline in untreated Diabetics, during Fasting and after Insulin Administra- tion. Nieis Juel Christensen. The 2nd Clinic of In ternal Medi- cine, Kommunehospitalet , Arhus.

Employing a precise and sensitive double-isotope tech- nique plasma noradrenaline and plasma adrenaline were measured in 23 normal subjects and in 14 diabetics during various metabolic conditions, t) Poorly controlled dia- betics showed a rise in plasma noradrenaline during resting conditions which was correlated to the degree of metabolic derangement. High adrenaline levels in plasma were seen only in pat ients with moderate to severe keto- acidosis. -- 2) During exercise ketotie diabetics demon- strated very largo increments in plasma catecholamines compared to normals. -- 3) During fasting plasma

noradrenaline rose from 0.18 ng/ml to 0.40 ng/ml in 4 normal non-obese subjects. No change was observed in plasma adrenaline. -- 4 ) D u r i n g insulin hypoglycemia high plasma adrenaline levels were only seen in subjects in whom the blood glucose concentration declined to values below 20 rag%. plasma noradrenaline rose with decreasing blood glucose concentrations even in diabetics in Whom hypoglycemie levels were not reached. 1~o correlation was observed between plasma adrenaline and increase in pulse rate during insulin hypoglycemia.

Immunoelectrophoresls Applied to the Study of Serum Proteins from Diabetics. A.H. Christiansen, A. Volund. Novo Research Inst i tute, Copenhagen, Denmark.

Further methodological investigations have indicated that the IgG is the essential insulin-binding protein in sere of diabetics treated with insulin. Hence, we have revised our earlier s tatement tha t other serum proteins such as albumin, fl-lipoprotein, ~2-maeroglobulin and IgM are capable of binding significant amounts of added insulin. -- I t will be demonstrated that IgG is heterogeneous in regard to eleetrophoretic migration and that both the anodic a n d the cathodic par t bind insulin to a varying extent. -- A comparison of insulin binding in sera from diabetics can be obtained by addit ion of a fixed amount of t2aI-insulin to the sera and estimating the percentage of added insulin bound to the IgG. Although this method makes it possible to rank the sera for routine purposes, it does not determine the maximum insulin binding capa- city. -- A method for the estimation of the maximum insulin binding capacity of IgG will be described and the results compared with the results obtained by the routine method.

Inhibition of Phenformin-Induced Ketogenesis by Dinitro- phenol. J . J . Connon, E. Trimble. Department of Medicine, The Queen's Universi ty of Belfast.

Phenformin causes a moderate increase in ketone body production in normal subjects. We have previously shown a marked increase in the rate of ketogenesis by the pe r- fused rat liver when phenformin is added to the perfusate. Fur ther studies have been carried out to establish the mechanism of phenformin-induced ketogenesis. -- Ketone body production by the isolated rat liver was increased forty-fold by the 20 mgm% phenformin. Ninety-five per cent of this increase was due to 3-hydroxybutyrate. Various substances were added to the perfusing medium in order to reverse the increased ketogenesis. The addit ion of 200 mgm% glucose had no significant effect (47 • 4.0 ~moles/G/60 mins control v. 44 ~moles/G/60 mins). One thousand ~ units/ml of insulin was added without effect on ketogenesis (40~:2.7 ~moles/G/60 rains control v. 35 ~ 1.9 ~moles/G/60 rains). However, 1 mM dinitro- phenol, an uncoupler of oxidative phosphorylation pro- duced a marked decrease in 3-hydroxybutyrate produc- t ion (41~: 2.0 ~moles/G/60 rains control v. 15 ~: 2.6 tzmoles/G/60 mins). -- We have previously suggested that phenformin produces inhibit ion of ghmoneogenesis in the perfused liver by affecting mitochondrial permeability and hence flux of NADH across the mitochondria. I t is reasonable to suggest that the increase in ketone-body formation due to phenformin is also due to an effect on mitochondria.

Adrenocortical Secretion in Obesity and Diabetes. G. Copinsehi, R. Leelercq, O.D. Bruno. Laboratories of Experimental Medicine and Clinical ChemistrjG and Department of Medicine, University of Brussels, Brussels, Belgium.

64 Organization Section �9 Abstracts Diabetologia

The possible relationship between adrenocortical function on the one hand, and food intake or carbohydrate metabo- lism on the other hand, was studied in 165 obese subjects (30 men and 135 women) who were submitted by groups to one or several investigations. The cortisol secretion rate (CSR) was similarly increased in diabetic and non-diabetic obese subjects, and there was no relation between CSR and oral glucose tolerance. However, a significant nega- tive relationship was evidenced between CSR and K values found in the I.V. glucose tolerance test. Significant positive correlations were found between CSR on the one hand, and the usual dietary intake of proteins, lipids or carbohydrates On the other hand. Those correlations were independent of the body mass. CSR decreased significantly following an l l - day total starvation, bu t not after a diet containing relatively normal a m o u n t s of proteins and only negligible amounts of lipids, carbohydrates and cMories. I t is concluded that the increased cortisol secre- t ion frequently encountered in obese subjects is very likely to be, at least partly, related to a chronic protein over- feeding. This hypercorticiSm in turn might be one factor responsible for carbohydrate intolerance in obesity.

Various Effects of Glibenclamide, Tolbutamide, and Insulin on Epinephrine Secretion Following Adrencrgic Blockade. U. Cordes, G. Sell, J. Beyer, E. Haupt, K. Sch6ffling. Dept. of Endocrinology, Clinic of Internal Medicine, Johann-Wolfgang-Goetho University, Frankfurt /Main, W-Germany.

The authors demonstrated in recent investigations that glibenclamido and tolbutamide-induced insulin secretion is potentiated by the blockade of adrenergic alpha- receptors, and inhibited by an a~Irenergie beta-receptor blockade. An a t tempt was made to elucidate the question whether the modified insulin secretion following adren- ergic blockade may be due to simultaneous release of epinephrine and a new sensitive and specific spoctrofluoro- metric method was applied. Thus the influence of intra- venously administered glibenclamide (50 t~g/kg) and tolbutamido (15 mg/kg) on blood glucose and epinephrine levels was studied in dogs following adrenergie receptor blockade. Additional experiments were performed with insulin (0.25 iu/kg) in order to exclude an influence of the receptor-blocking substances on epinephrine levels. -- After sulphonylurea administration, an increase in epin- ephrine levels was observed within 30--60 rain. When insulin was given, the epinephrine levels increased earlier. The extent of epinephrfile secretion was not influenced by alpha- nor by beta-blockade. In contrast, epinephrine secretion following sulphonylurea administra- t ion was excessively enhanced by alpha-receptor blockade, while no effect on epinephrine levels could be observed during beta-receptor blockade. -- The direct influence sulphonylureas may exert on the adrenergic alpha- and beta-receptors of the pancreatic B-cell will be discussed in the light of ~heso experiments.

In vitro Studies of the Rate of Proinsulin/Insulin Turnover in Human Insulinomas. C. Creutzfeldt, N.S. Track. Division of Gastroenterology and Metabolism Depaxtment of Medicine, University of Goottingen, GFR

5 " 10 mg pieces of human insulinoma tissue (total weight 150--200 rag) excised from 5 patients were incubated in Krebs-Ringer bicarbonate buffer supplemented with glucose (3.0 mg/ml), tritiated-loucine (100 t~C/ml) and an amino acid mixture (20 lzg/ml) for a 15 min pulse period. One third of the tissue was immediately homogenised in 0.3 M sucrose (pH 6.0), one third after 20 min chase (with L-lcucine (375 ptg/ml)) and the remaining third after 80 rain chase: Subeelhilar fractions (cell debris, mitochond-

ria, secretory granules, mierosomes and S-100) were prepared by centrifugation. The S-100 fractions contained ro~ughly 50% of the I R I ; the remaining 50% was distri- buted between the secretory granule and mitochondrial fractions. The secretory granule fractions were fraetionat-

e d by sucrose gradient u!tracentrifugation. At the three time intervals examined, a peak of radioactivity and I R I was located at 1.60 M sucrose. ]Endogenous proinsulin and insulin was released throughout the incubation. Newly synthesised proinsulin was detected in the 15 rain pulse medium- in 3 of the tumours; radioactive insulin was present in the 20 and 80 rain chase media. -- The ident i ty of the subeellular fractions was confirmed by the ultra- structural analysis. I n tumours with typical fl-granules, both turnout cells and the secretory granule 'fractions contained three types of granules identical to those found in the normal human B-celL The presence of Some secretorygranules in the mitoehondrial fractions suggests tha t these granules may be heavier than those in the sub- sequent fractions. Autoradiographicalty, few grains were found overlying granules in the 15 min pulse secretory granule fraction. After 20 min chase, there was an even distr ibution of grains with most granules labeled. -- These data support the conjecture that human insulinomas have the capacity for a more rapid turnover of proinsulin and insulin than normal pancreatic islet tissue, in particular, pertaining to release.

The Fasted Obob Mouse: Model for the Metabolic Response t o Fasting in Man. G.S. Cuendet, C.B. Wollheim, D.P. Cameron, L. Balant,

W . Stauffacher, E.B. Marliss. Ins t i tu t de Biochimie Clinique, University of Geneva (Switzerland).

Man, lean or obese, adapts to prolonged fasting by con- serving vital protein and mobilizing expendable fat for his energy requirements. An animal model with these responses would permit more detailed metabolic studies, in largo numbers of genetically homogeneous individuals. : - Lean mice (C57-B1/6--t-/~), 22 g (10~o fat), survived only 4 days of fasting. Though fat was mobilized (blood glycerol 460, BOHB 1362 AcAc 499 ~zM), protein mobiliza- t ion (urea N excretion) increased progressively. At 3 days, no visible fa t remained, and a striking fall in ketones and glycerol, and rise in plasma alanine and glutamine occurr- ed. By 1 day, plasma glucose and I R I had fallen to a plateau (70.5 mg~o, 0.24 ng/ml). Survival beyond 4 days was achieved by fat refeeding. -- Obese mice (C57-B1/6-

o ob/ob), 35 g (30 ~o fat), survived fasting more than 28 days. Marked ketonomia developed and persisted (BOHB 3331, Ac,kc 589 iLM). By 5 days, protein mobilization decreased 2/3. Plasma alanine fell transiently, glutamine rose 64%; both were stable after 8 days. Plasma glucose and I R I fell progressively from 270 and 2.1 to 48 mg% and 0.26 ng/ml by 16 days. Abundant fat stores persisted through- out. Similar responses occurred in dbdb, goldthioglucose- treated and spiny mice. -- Conclusion: (1) Lean mice have insufficient fat to survive prolonged fasting. (2) Obese mice convert to fat-derived energy sources and conserve protei n . The adaptive mechanism relates part ly to falling I R I and changing blood substrate levels. (3) An absolute defect in fat mobilization in obob is excluded. (4~ Similar- ities between obob and human responses warrant further s tudies of mice as a fasting model.

Comparative Evaluation of the Effect of Antidiabetic Bigu- anide Dervativcs on the Blood Lactate Level. A. Czyzyk, B. Lao, W. Bartosiowiez and Z. Szczepanik. Department I I I of In ternal Diseases, Medical Academy, Warsaw, Poland.

I t has been suggested that out~ of different antidiabetie bi- guanide derivatives phenformin induces particularly mark-

Vol. 9, No. 1, 1973 Organization Section �9 Abstracts 65

ed increase in blood lactate level. Observations were made on diabetics chronically treated with various b i~mnide derivatives and in short time tests in healthy subjects, in whom an increase in blood lactate was induced by oral ethanol administrat ion or intravenous fructose infusion. -- In diabetics treated with biguanide derivatives the highest blood lactate level was obtained upon the use of phen- forrain/raean value ~ 1.53 • 0.13 raM/l / ; the respective values for buformin and metformin were 1.22 • 0.12 mM/1 and 1.29 ~ 0.13 raM/1. - - I n healthy subjects the degree of potentiat ion of the increase in alcohol or fructose induced hyperlactaeidaemia w a s similar for all the biguanide derivatives studied. -- I t is concluded that there are no dissimilarities in the mechanism underlying the rise of blood lactate level, as induced by various biguanide derivatives. However, upon therapeutic appli- cation of these drugs slight differences in the intensity of the above phenomenon are observed.

Insulin Response to Intravenous Glucose Administration in Normal Subjects, Mild Maturity-Onset Diabetics and Subjects with a "Lag-Storage" Curve. E.De Nobel, A. Van ' t Laar, R .A.P . Koene, Th.J . Ben- raad. Department of Medicine, Universi ty of Nijraeg.en.

A large dose of glucose (50 g per 1.73 m ~ body surface) was injected intravenously in 3 min. Plasma insulin and glucose values were measured every minute after the start of the injection. In normal subjects the initial insulin peak was reached after 3 or 4 rain. This peak was repro- ducible in the sarae subject as to t ime and height. In mild raaturity-onset diabetics the initial insulin peak was significantly lower than in the normal subjects. There was vir tual ly no overlap between normal and diabetic sub- jects, when the initial insulin response was expressed as "insulinogenic index" [A insulin ) . x ~ . -- A group of subjects

who had repeatedly shown lag-storage curves in oral glucose tolerance tests, was examined with the same technique. Their insulin response to intravenous glucose was not different from that in the normal subjects. These results suggest that the lag-storage curve should not be considered an early sign of diabetes.

Application of a New Method Measuring Hepatic Blood Flow to Metabolic Balance S~udies in Human Liver. H. Re- gulation of Glucose Output by Lactate and Insulin. G. Dietze, K.D. Hepp, M. Wiekmayr , H. Mehnert. Dia- betes Research Uni t and 3rd Medical Clinic, Schwabing City Hospital, Munich, Germany.

The arterial and hepatic venous concentrations of insulin, glucose, lactate, pyruvate, alanine, urea, and glycerol were measured in 8 healthy resting human volunteers in the post.absorptive state. ~Substrate determination and simultaneous measuremen~ of total liver blood flow with the new ~aaXenon inhalation technique were performed during the following periods: control (0--30 rain), lactate infusion (30--120 rain, 0.03 mMoles/kg rain), and after insulin injection (0.05 U/kg at 60 rain). Glucose produc- t ion was measured at 8.43 i 1.04 rag/100 g/rain, which, calculated per 1500 g total liver weight, is equivalent to a basal production of 180 g/day. On the basis of stoiehio- metric calculations including the substrates alanine, lac- tate, pyruvate, and glycerol, lactate represented the major gluconeogenic precursor in the post-absorptive state. During the lactate infusion period, hepatic glucose production was increased about two-fold (16.33 ~: 2.42 rag/ 100 g/rain), while lactate uptake rose to about eight- fold. Under the influence of insulin, the utilisation of gluconeogenic precursors decreased only slightly in com- parison to the remarkable decrease of the glucose output (9.52 4- 2,1 rag/100 g/rain).

Analysis of Insulin Binding Antibodies in Serum. K. Dixon, P.D. Exon, H.R. Hughes, D.W. Jones. De- par tment of Clinical Chemistry, General Hospital, Bir- mingham, England.

Diabetic patients treated with insulin develop antibodies to insulin. New methods of studying these antibodies in serum will be described. Serum is treated with charcoal at pH 3.5 to remove endogenous and therapeutic insulin which would otherwise interfere with the measurement of the binding of ~2~I-insulin to the insulin antibody. A series of dilutions Of the insulin free serum is incubated with physiological levels of ~2~I-insulin for 18 h at pH 7.40. Charcoal is added to remove free 12~I-insulin and 1251- insulin bound ~o the insulin ant ibody is measured in the supernatant. The amount of free 125I-insulin is the differ- once between the bound l~I-insulin and the total amount added to the serum dilutions. The values for free and bound insulin in each diJution of serum are used to calculate the maximum binding capacity (S) and the affinity constant (K) of the antibody. The relationship between free insulin and tota l insulin in the serum in vivo can be simulated mathematically using the individual values of K and S for each patient. The influence of the properties of insulin binding antibodies and their abili ty to buffer fluxes of insulin into the vascular space will be discussed.

Modulation of Glucose-Induced Insulin Response by Arginine in Normal, Diabetic and Prediabetic Subjects. S. Efendid, E. Cerasi, 1%. Luft. Department of Endocrin- ology, Karolinska Hospital, Stockholm.

We have previously suggested that arginine induces insulin release by raodulating the insulinogenic signal of glucose in the/?-cell. In order to further characterize this modulation, arginine infusion (150 mg/kg priming ~-10 rag/kg/min for 30 rain) was followed by a glucose infusion test with varying doses of glucose (100, 250, 500 and 1000 mg/kg as priming, followed by 5, 10, 20 and 40 rag/ kg/min for 60 rain, respectively). Each subject served as his own control. In normal subjects, insulin response to glucose was enhanced by arginine to the same degree in all hyperglycemic levels. -- Arginine thus seems to be a multiplicative potentiator of the glucose effect on the t-cell. I n prediabetie and diabetic subjects, arginine alone elicited a lower insulin response. The potentiat ing effect of arginine on glucose induced insulin release (in per cent) was similar to that found in the controls. However, since glucose itself is less insulinogenic in these subjects, the plasma insulin response to arginine ~ glucose was also less than in the normal group. Our findings indicate that the mechanisms responsible for the potentiat ing effect of alginine are not affected in prediabetes -- diabetes.

Lipoprotein Lipase in Human Adipose Tissue. R.S. Elkeles, Department of Medicine, Welsh National School of Medicine, Heath, Cardiff,

Lipoprotein lipase (LLA) (Clearing Factor Lipase) has been studied in human adipose tissue obtained from routine surgery. In contrast to previous work, LLA was found both in acetone other extracts and by elution with heparin. Activity was found in subcutaneous, omental and breast tissue. Like the enzyme i n rat adipose tissue, it was inhibited by Protaraine Sulphate and 0.5 MNaCL However, there was some 30 times less activity in human than in rat adipose tissue on a weight basis. A mono- glyeeridase was also demonstrated in acetone ether ex- tracts and found to be present in elution medium in- dependently of the presence of heparin. Further, this enzyme, like that found in rat adipose tissue, was not inhibited by Protamine Sulphate or 0.5 MNaC1. When LLA was measured using two different triglyeeride

Diabe~ologia, Vol. 9 5

6~) Organization Section �9 Abstracts Diabetoloyia

emulsions as substrate, Intralipid and Ediol (containing 1.5 ~ monoglyceride), fa t ty acid release and glycerol fa t ty acid ratio were higher with Ediol. I t was concluded tha t the different results obtained with the two substrates could be explained by the act ivi ty of the monoglyceridase and that Ediol, which has been used in previous work on this subject is an unsuitable substrate for the assay of LLA in human adipose tissue.

Insulin Antibodies and the Control of Diabetes. P.D. Exon, K. Dixon, M.G. FitzGerald, J .M. Malins. Diabetic Clinic, General Hospital, Birmingham, England.

Insulin antibodies modify the effect of injected insulin and we suggest that they buffer insulin in serum (see Dixon et al, Abstract No. 69). The control of the patients ' diabetes was assessed from clinical features. The patients were classified according to the degree of control. -- The relationship between the ability of the insulin antibodies to buffer insulin and the diabetic control in the various groups will be described. -- Patients whose insulin anti- body is theoretically an adequate insulin buffer have stable diabetes. Patients with unstable diabetes have antibodies that are poor insulin buffers or they have very small amounts of antibody.

Failure of Calcium to Increase Insulin Secretion during Intravenous Glucose Tolerance Tests in Premature Infants. A. Falorni, F. Massi-Benedotti, G. Gallo, S. Maffei. Inst i tute of Clirdcal Pediatrics, Universi ty of Perugia, Perugia, I t a ly .

The presence of calcium is reported to be necessary for insulin release from beta cells and is said to increase the insulin response to glucose. -- Intravenous glucose tolerance tests (IVGTT) were performed in three groups of premal~ure infants of different age: infants aged 1 day, 7 days, and 30 days. After 24 h, the same tests were per- formed at the end of a 30 rain intravenous infusion of calcitun (20 mg/kg body weight) given in the form of CaClz. After the infusion, the levels of serum calcium ++ increased to the normal values seen in adults. -- The comparison between the two tests did not show any difference in each group as to blood glucose and plasma insulin levels at any t ime during the tests and this applies also to the index of peripheral glucose assimilation. -- These findings suggest that the low sensitivity of neonatal pancreas to glucose may not be corrected by calcium and does not seem to be a coru~equenco of the newborn's physiologic hypocaleaemia.

Influence of Triglycerides on the Insulin Response to Arginine. D. Fedele, A. Tiengo, M. Muggoo, P. Fabris, G. Crepaldi. Clinic of Internal Medicine, of the University, Policlinico, Padova, Italy.

The hyperinsulinism of patients with endogenous hyper- triglyceridemia is still under discussion as to whether it is related to hypertriglyceridemia itself, as a factor inducing peripheral insulin resistance, or to overweight, often present in these patients. -- The influence of plasma triglyceride (TG) concentrations on the insulin (IRI) response to arginine infusion (25 g in 30 rain) was studied in 10 lipemic patients (type IV and V) with very high triglyeeride levels (~ 1293 :t: 162 rag%) and after clofib- rate t reatment (r~ 314 :k 45 mg~/o). In both conditions, the I R I response to arginine was already present after 5 rain and reached its maximum at 30 min. -- As there are no differences between the basal plasma levels, the mean I R I response is significantly higher after 30 and 60 min when TG are more elevated. The max imum I R I response and the mean increase above b,, -~al values are significantly higher when hypertFiglyccridemia is more marked ( IRI

Mx. response 48 :L 5 vs. 34 =L 4 ; ~l I R I 32 • 4 vs. 20 =k 3). A positive correlation exists between basal TG levels and the maximum IIr response (p < 0.05). A preliminary study has been carried out using arginine infusion in 4 normolipemic subjects, in both basal conditions and hypertriglycerideraia induced by Intralipid infusion (0.33 g/min). The results are not uniform even though tlm I R I response to arginihe in 2 subjects is higher during ex- perimental hypertriglyceridemia. -- Our results are m accordance with the hypothesis that TG levels might affect the insulin response.

Transplantation of Isolated Islets of Langerhans into Pancreateetomized Rats. K. Federlin, K. Helmke, M. SlijepSevi~, E .F . Pfeiffer. Center of Internal Medicine, Universi ty of Ulm, Germany.

Rats weighing 120--150 g were pancreatectomized ac- cording to the method of Foglia with our own modifica- tions. Occurrence of diabetes was proven by daily control of glycosuria and repeated glucose tolerance tests. Only animals demonstrating marked glycosuria for 10 days or more were chosen for transplantation. Islets were isolated from pancreatic tissue of homologous rats using col- lagenase according to Lacy and Kostianovsky (1967). After each isolation, a sample of islets was tested by incubation with 300 m g % glucose for 60 min. Trans- plantat ion of 100--1000 islets was performed by either subcutaneous injection or by implantation of a millipore chamber into the peritoneal cavity. -- Nineteen out of 44 animals showed a transitory disappearance of glycos- uria one to five days after transplantation. Glucose toler- ance curves of transplanted diabetic animals showed an increase in the K-value to normal levels in 18 out of 50 animals. With these parameters, the islets show no apparent behavioral differences, whether transplanted subcutaneously or in a millipore chamber. I t is therefore suggested that immunological factors at this early stage do not influence the islet function; however the short duration of this function may be caused by insufficient blood supply. -- The results demonstrate that isolated islets after transplantation secrete insulin and are able to respond, at least temporarily, to glucose stimulation.

Evidence for a Direct Effect of Fat on the Function of Pancreatic ~-Cells. J .P . Felber, J . Oul~s, Ch. Schindler and V. Chabot. D~- partement do Biochimio Clinique, H6pital Cantonal Uni- versitaire, Lausanne, Switzerland.

A glucose-infusion test according to Cerasi and Luft (1967) was repeated in 7 normals and 7-1ow responders ("pre- diabetics") 2 h after the beginning of a lipid infusion (Lipofundine). While both glucose and insulin curves were higher during infusion in normals, there was a marked increase of insulinemia with no change in the glucose curve in low rcsponders, thus demonstrating a direct effect of fat on the pancreatic response to glucose. Both early and late insulin responses were higher, with disap- pearance of the early insulin deficiency of the low-re- spenders. -- In 5 other normal subjects OGTT was carried out 2 h after the beginning of a similar lipid infusion. Separations of the plasma of 60 and 120 min on a Sephadex column showed a marked decrease of the proinsulin (big insulin) peak in comparison with controls without fat infusion. The in vitro addition of fat did not modify the proportion of insulin. -- A preliminary study has shown a smaller proportion of proinsulin in pancreas from rats fed a high fat diet compared with control rats. -- Next to the known effect of fat on muscle, adipose tissue and liver, these three studies show that fat has a direct effect on pancreatic fl-cells by modifying the quality and the quant i ty of insulin secretion. This could have implications in the pathogenesis of diabetes.

Vol. 9, No. 1, 1973 k

Organization Section �9 Abstracts 67

Human Growth Hormone and Immunoreactive Insulin Levels in Human Bile. A. Fernandez-Cruz, Jr., E. Catal'~n, M. Luque Otero, A. Fernandez-Cruz. Catedra I Patologia General. Labo- ratorio Medicina Nuclear . JEN School of Medicine, Hospital Clinico San Carlos. Complutense Universi ty of Madrid, Spain.

Measurements of human growth hormone (HGH) by radioimmunoassay in biologic fluids have been confined mostly to assay of blood, except for an occasional report in urine and lymph. We have carried out experiments which have clearly demonstrated the excretion of insulin in bile. In order to find out whether human growth hor- mone is also present in bile, we selected two spontaneous bile fistulae after a cholecystectomy and gastrectomy, and determined HGH by the radioimmunoassay of Hales and Randlo using a double ant ibody technique. The identifi- cation of the hormones was clearly defined in the dilution curves ranging between 1 : 2 -- 1 : 128, using human growth hormone antibodies supplied by the Laboratory of Clinical Physiology of the Universi ty of Pisa (Sorin). -- The results showed extremely high levels of HGH with an average of 38 ng/ml. With the idea of determining the physiological significance of markedly elevated HGH, experiments were undertaken to s tudy the dynamics of the curves following both oral and intravenous glucose tolerance tests. The results will be discussed.

Human Bile Insulin Sephadex Fractions. A. Fernandez-Cruz, Jr., O. Garcia Hermida, M. Luque Otero, E. Catalan. 1. Chair of General Pathology, Hospital Cllnico de San Carlos. Facul ty of Medicine, Universi ty of Madrid, and Laboratory of Anatomy and Morphology, Ins t i tu te G. Marafon, C.S.I.C,, Madrid.

The significance of insulin sephadex fraetions in relation to insulin release was studied in two patients with spon- taneous bile fistulae by measuring the serum insulin and bile insulin following oral glucose, and intravenous glucose infused a t a constant rate. The results confirmed the presence of insulin in bile, and the changes during glucose tolerance tests do in fact indicate the different behaviour for oral glucose and glucose infusion. On the other hand, the findings clearly indicate the existence of two immunoreaetive components in human bile. The two correspond to what Roth (1968) described in human plasma as "big" and "l i t t le" insulin. The results provide no support for the concept tha t a large fraction of "big" insulin is related to the high levels of immunoreaetive bile insulin seen after oral and intravenous glucose admini- stration. -- The assessment of insulin in serum and bile was carried out according to the method C of Hales and Randle. Bile insulin was fractionated on sephadex G-50 columns. Eluates corresponding to porcine proinsulin ("big" insulin) and regular insulin were assayed for insulin by a modification of the radioimmunoassay of Yalow and Berson.

Hepatic Energy Metabolism: a Possible Link between Gluconeogenesis and Ketogenesis. J .P . Flat t . :Department of Nutr i t ion and Food Science, Massachusetts Ins t i tu te of Technology, Cambridge, Mas- sachusetts.

The conversion of fa t ty acids to ketone bodies is an ox- idative, and therefore energy-generating process. I n deeompensated diabetes up to 200 g of ketone bodies may be produced in one day. To form 200 g of ketone bodies 2.5 moles of oxygen are required which corresponds to the total daily oxygen .consumption of the human liver, i n studies ~ i t h perfused livers from fat-fed ketotic rats, 90 ~o of the oxygen consumption was accounted for by fa t ty acid oxidation to ketone bodies (H.A. Krebs et al.,

Biochem. J., 119, 525, 1970). Thus, under extreme con- ditions, ketogenesis appears to provide most of the energy used in the liver, suggesting that the rate of energy util isation could become rate-limiting f o r ketogenesis. Gluconeogenesis, by increasing the energy expenditure in the liver, can be seen to faci l i ta te ketogenesis. I t is proposed that this interaction, at the level of the energy metabolism in the liver, explains why pathological rates of ketogenesis are encountered in metabolic situations where gluconeogenesis is very intensive. Furthermore, the decline in ketogenesis during prolonged starvation, from 130 g/day after 5 weeks (O. E. Owen et al., J. Clin. Invest. , 48, 574, 1969) may be at t r ibuted to the progressive decline in gluconeogenesis as adaptat ion to starvation occurs and daily losses of 1orotein decrease. (Supported by U.S.P.H.S. Grant AM 14161).

Effects of Ketone Body Infusion on Hypoglycemic Reaction in Post-Absorptive Dogs. J, P. Flat t , G. Blackburn and G. Randers. Department of 3N'utri~ion and Food Science, Massachusetts Ins t i tu te of Technology, Cambridge, Massachusetts.

During prolonged s tarvat ion the brain derives the greater par t of its energy through oxidation of ketene bodies. I t is not clear whether the brain has the inherent abil i ty to use large amounts of ketone bodies instead of glucose, or whether it has to adapt to their use as they appear during starvation. We provoked severe hypoglycemia in post-absorptive dogs by injection of insulin (0.4--0.7 U/kg) while measuring the release of epinephrine occurred (3-- 5 x basal rate of 200 - 700 ng/min). This discharge was completely prevented by infusing DL-B-hydroxybutyrate or acetoacetate at rates sustaining circulating ketone body concentrations of 10--20 mg~/o . Arterio-venous differences of ketone body levels across the dog's head during hypoglycemia were quant i ta t ively comparable to arterio-venous differences in glucose levels before hypo- glycemia. Thus, the dog's brain is able to use appreciable amomlts of ketone bodies wi thou t prior adaptat ion to starvation. Insufficient energy production in the brain, rather than lack of glucose or of one its direct metabolic products, appears to be at the origin of the signals which cause epinephrine release during hypoglycemia. The presence of ketone bodies seems to protect the dog's brain against the ill effects of acute hypoglycemia. (Supported by U.S.P.H.S. Grant AM 14161).

Influence of Fructose, Sorbitol, and Xylitol on Gluconeo- genesis an.d on Glycogen Formation in the Liver. H. F6rster, I. Hoes, D. Lerche. Ins t i tu te for Vegetative Physiology, Universi ty of Frankfurt , Germany.

Glueoneogenesis from fructose, sorbitol and xylitol was investigated using the isolated perfused rat liver. 40-- 55 ~o of added fructose or sorbitol was transformed into glucose while 60--75% of the xylitol was transformed. The increase of hepatic lactate production was higher with fructose or sorbitol than with xylitol. The perfused rat liver was unab le to accumulate glycogen in normal amounts under these experimental conditions. The uptake of added substances was greatest with fructose and least with sorbitol. Continuous infusions of fructose, sorbitol or xylitol in narcotized and in non-narcotized rats effected an accumulation of glycogen in the liver while the concentration of glucose in the blood remained nearly constant. Up to 50% of fructose, sorbitol and xylitol were accumulated as glycogen in the liver after conversion to glucose. Glycogen was also formed in diabetic rats follow- ing the infusion of these substances. -- The results show that glueoneogenesis from fructose, sorbitol or xylitol results, in vivo, mainly in glycogen formation even when diabetic animals are used. I t is supposed that the appli- cation of these substances does not effect the change for

5*

68 Organization Section �9 Abstracts Diabetologia

the worse in carbohydrate metabol ism al though extensive conversion into glucose takes place in the liver.

Influence of Biguanldes on the Intestinal Absorption of Glucose. B. FSrster, I . Boos, 1~. Matth~us. Ins t i tu te of Vegetat ive Physiology, Univers i ty of Frankfur t , Germany. The inhibit ion of intest inal glucose absorpt ion is con- sidered to b e one of the main effects of biguanides. This hypothesis concerning the mode of action of buformin and phenfqrmin was verified by extensive investigations using different experimental models. I n r i v e experiments in rats showed tha t an inhibit ion of intest inal glucose ab- sorption did not occur unless sublethal concentrations of biguanides (10 mM) were administered. In contrast , how- ever, an increase of intest inal lacta te formation was observed when low biguanlde concentrations (1 raM) were used. The concentration of biguanldes was increased to 50 raM, using in vi tro preparat ions with sustained blood circulation. Especial ly phenformin provoked an increase of passive mucosal permeabi l i ty u n d e r these conditions. But even a t a biguanide concentrat ion as high as 50 raM, the active t ranspor t of glucose was not inhibited b y more than 50o//o. - - The glucose absorpt ion tested in an in vi t ro system without a blood Supply was not inhibited either, unless buformin a t high concentration was added to the mucosal solution. The best effects were observed when the buformin concentration was considerably higher t h a n the glucose concentration. - - I n there experiments, the effect of biguanides cannot be explained by an inh ib i t ion of intestinal glucose absorption. The inhibit ion of gastric emptying and the intest inal lacta te product ion are con- sidered to be of greater importance in the mode of action of these drugs.

The Glomerular Clearance of Exogenous Insulin. J .R .M. Franckson and B . A . Ooms. Laboratoi re de Chimie M&licale, Univers i ty of Brussels. The glomeruiar clearance of insulin was determined ac- cording to the method of Moneke and Yule. Anaesthet ized dogs mainta ined in normoglycaemia b y glucose compen- sation were infused with crystal l ine insulin and with l~aI-insulin (S. a. 1.5--3.0 mCi/mg, mean iodinat ion degree less than 0.2 I/Mol.) P lasma insulin levels ranging from 30 ~U/ml to 6 U/ml were tested. Creatinine and Jail- insulin were used to est imate the glomerular f i l trat ion rate. Analysis of the relationship between the ur inary outflow of insulin and its plasmatic concentration detected 2 par t s in the curve : an init ial splay extending to p lasma levels of 0.3 U/ml followed b y a s t ra ight line. - - The comparison between the glomernlar clearance ra te of insulin and the glomerular f i l t rat ion ra te demonstra ted the existence of an impor tan t sieving phenomenon, the value of the sieving coefficient averaging 0.58 fo r crystal- line insulin .and 0.40 for labelled insulin. - - These results do not support the concepts tha t exogenous insulin would circulate either as a free monomer (MW 6 00O) or as bound to p lasma proteins bu t under the form o.f a polymer, the sizes of the circulating part icles s tanding between tha t of

myog lob in (MW 17 000) a n d of ovalbumin (MW 42 000). The influence of iodinat ion is discussed.

A Novel Pancreatic Amylase InhibRor (Bay d 7791) . Experimental Studies on Rats and Clinical Observations in Normal and Obese Diabetic and Non Diabetic Subjects. H. Freriehs, H. Daweke, F. Gries, D. Griineklee, J . Hes- sing, K. Jahnke, U. Keup, H. Miss, B. Otto, W. Puls, D. Schraidt, C. Zumfelde. Medizinische Kliniken: Bremen- Nord, Diisseldorf, GSttingen, Wupper ta l . Pharmakologi- sches In s t i t u t : Bayer AG, Wupporta l .

A group of potent inhibi tors of pancreat ic a lpha-amylase was prepared from wheat b y aqueous alcoholic extrac-

t ions at .an acid pH. These inhibitors (BAY d 7791) are thermostable proteins (MW approx. 16000; 70~ they are specific for amylase; do not affect pretenses or l ipases; bu t are inact ivated by pepsin and trypsin. Their electro- phoretie pa t t e rn and excellent inhibi tory effect on alpha- amylase differs significantly from tha t of known wheat- derived amylase inhibitors. - - I n rats, 9 mg BAY d 7791 extended the t ime for complete intest inal hydrolysis of 300 mg of s tarch from 120 to 240 rain. Accordingly, the inhibitor markedly diminished and smoothed the hyper- glycemia following feeding of 2.5 g/kg s tarch; and reduced the concomitant fall of serum NEFA. This effect was dose-related. Absorpt ion of glucose, maltose and sucrose was not affected. -- The same dose-dependent inhibi tory effect of BAY d 7791 (1 to 4 g/subject), i .e. reduction and prolongation of hyperglycemia af te r ingestion of 100 g of raw starch, was seen in 79 volunteers (22 heal thy, 14 obese, 43 obese diabetic). BAY d 7791 also reduced the hydrolysis of s tarch adminis tered as a 50 g CB-equivalent in the form of bread, noodles, pota toes or rice in 71 diabetic or non diabetic obese patients . In each case, changes induced in the blood glucose response resulted in the ant ic ipated changes of serum I R I and N E F A . The five research groups engaged in this s tudy consider i t possible tha t amylase inhibit ion m a y represent a novel principle in the the rapy of obesi ty and diabetes mellitus.

Relation of Pyruvate Dehydregenase (PDH) Intercen- version to Pyruvate Decarboxylation in Isolated Perfnsed Rat Hearts. B. v. Funcke, G. LSffler, O. Wieland~ Diabetes Research Unit , City Hospi ta l , Miinchen-Schwabing.

Interconversion of P D H from the act ive (PDHa) to the inactive (PDHb) form and vice versa is subject to meta- bolic control. There is, however, l i t t le information regard- ing the correlat ion of P D B interconversion and the ra te of py ruva te oxidation. In the present s tudy, hear ts were perfused with 5 ~ glucose and then w i th one of the following substrates" 1 mM palmita te , 4 mM aceteaceta te (AcAc}, 10 mM D,L-f l -hydroxybutyra te (flHOB), 8 mM acetate (Ac) and 4 ml~ AcAc together wi th 8 mM Ac. P D B a accounted for 60~o of to ta l ac t iv i ty after 10--20 rain of glucose perfusion in fed ra ts and was reduced to near ly 30% (p < 0.60!) in the presence of the substrates mentioned. P D H a ir~activation b y pa lmi ta te was noted within 1 rain (24.99~-3.8~o, p < 0.02). Low levels of P D H a were observed in hear ts from s tarved (24.2 -~ 3.5~/o) and diabet ic (21.5• ra t s and these were not increased following perfusion with glucose. 20 rain of perfusion with insulin (3 mU/ml) exerted no effect on P D H a in diabetic hearts. Calculations showed tha t P D H a ac t iv i ty was near ly 4 t imes higher than formation of laCO~ from 1-1aC-pyruvate (1.96 ~: 0.2 to 0.58 ~Mol/g wet weight/ mln) in hear ts of fed ra ts perfused with glucose, bu t near ly 10 t imes higher in hear ts perfused with flHOB (1.22 Jr 0.4 to O. 13), or pa lmi ta te (1.02 :~ 0~4 to 0.1). I t was concluded tha t decarboxyla t ion of pyruva te in the beat ing ra t hear t m a y be almost completely inhibi ted under conditions of enhanced fa t ty ac.id oxidat ion, pa r t ly due to the inact iva . t ion of P D H a by interconversion and to the. feedback control by acetyl CoA or some other unknown effeetors.

T h e Effect of Adult Diabetes on the Levels of Glucese-6- Phosphate and L-Glycerol 3-Phosphate in Human Adipose Tissue During a Glucose Load. D.$. Galton, R. Gut tman. Depar tment of Medicine, Stl Bar tholomew's Hospi ta l , London .

Glycolysis m a y be reduced in adipose tissue of adul t diabetics and this may contr ibute to the hyperglycaemia. Two possible sites for this defect are a t the ent ry of glucose into the cell or a t react ion cata lysed b y phospho- fructokinase. I n au a t t empt t o dist inguish between them

Vol. 9, No. 1, 1973 Organization Section �9 Abstracts �9 69

we have measured the intracellular contents of glucose-6- phosphate (G6P) and glycerol-3-phosphate (GP) in adipose tissue during a glucose load. -- I n a group of 13 diabetic pat ients (mean fasting blood sugar 187 mgm~/o) the mean fasting levels of G6P and GP were 3.3 ~: 0.6 and 12.0 :]: 2.6 nmoles/g, wet wt. Corresponding values for a group of 9 obese non diabetic pat ients (mean blood sugar 97 mgm%) were: 3.64. 0.7 and 12.1 • 1.8 nmole/g. The levels of G6P and GP in adipose tissue were measured during an oral GTT (100 g). The tissue con ten t s of G6P were: 5.64-1.4, 6.64-1.6% 6.84-1.5 a and 6.94-2.5 (ap < 0.01), and 5.4 4- 1.2, 6.1 • 1.5, 4.6 4- 0.8 and 5.7 4- 1.9 n.moles/g for diabetics and non-diabetics respectively. The prolonged rise in t issue levels of G6P after an oral glucose load in diabetics does not therefore suppor t the h y p o - thesis t ha t glucose t ranspor t into the adipose cell is impaired, and is more in accord with a defect in glycolysis a t a s t e p dis ta l to hexokinase.

Insulin and Adaptive Regulation of Amino Acid Transport. G.C. Gazzola, R. Franchi , P. Ronehi, V. Saibene, G.G. Guidott i . Ins t i tu t i di Patologia Generale, Universi tk di Milano e di Cagliari, I t a ly . �9

In muscle tissues amino acids are transported: into the cells b y saturable processes of mediat ion. Although many of the kinetic aspects and propert ies of the t ranspor t systems involved have now been elucidated, the accom- panying regula tory aspects are stil l l i t t le known. The amino acids tha t respond to insulin belong essentially to the group t aken up b y the A system of mediat ion. A t ime- dependent adapt ive system for the t ranspor t of neutra l amino acids per ta ining to the A mediat ion is operat ive in chick embryo hear t cells : this system is subject to regula- t ion b y its subs t ra te molecules. Both hormonal and adapt ive regulations occur through a mechanism affecting the maximal veloci ty of t ranspor t . As a pre l iminary approach for deciding whether insulin and adapt ive regulations of amino acid t ranspor t are coupled processes, t ime- and amino acid-dependent changes of t r a n s p o r t ac t iv i ty were s tudied in a var ie ty of insulin-sensitive and -insensitive biological preparat ions. Muscle tissues includ- ing in tac t chick embryo hear t a n d isolated cardiac ceils, isolated ra t d iaphragm and mouse embryo hear t respond- ed to insulin and exhibi ted adapt ive regulat ion of amino acid t ransport . R a t l iver slices, Ehrl ieh ascites carcinoma (mouse), Yoshida ascites hepa toma (rat) cells and rabb i t reticnlocy~es were insulln-insensitive and failed to d isplay adapt ive regulat ion of amino acid t ranspor t . Exper iments designed to ascertain whether both control mechanisms act a t the same molecular site in muscle tissue are in progress (Aided b y U.S.N.I .H. Grant AM-05290).

The Significance of Hyperlipopr0teinaemia in Diabetes Mellitus. V. Gligore, N. Hincu, Rodica Tecuceanu. Second Medical Clinic, Univers i ty of Cluj, Rumania .

Our studies concerning l ipid metabol ism in diabetes mell i tus have been presented in previous papers. The aim of this work is to invest igate the significance of hyper- l ipoproteinaemia with special emphasis on prevent ion and t rea tment . The l ipoprotein types (Frederickson's criteria, i .e. the serum appearance, agar gel electrophoresis, t r i- glycerides, and to ta l serum cholesterol) were determined in 50 diabet ics and in a control group of 20 hea l thy individuals. I n addit ion, serum pseudoeholinesterase (SPC) ac t iv i ty , the incidence of angiopathy, and the body weight were es t imated in each pat ient . Types I I , H I , IV, V of hyper l ipoprote lnaemia (HLP) and the normal l ipoprotein pa t t e rn were present in diabetics. All eases wi th H L P also had angiopa thy and most of these pat ients were obese m a t u r i t y onset diabetics. The SPC ac t iv i ty was greatest in type IV of HLP . -- I n conclusion, H L P seems to be a

valuable criterion of the metabol ic disturbances and of the atherogenic humera l syndrome in diabetes mellitus. The evaluat ion of the H L P pa t t e rn in diabetics is an a id for the selection of therapeut ic and preventive measures in diabetes mell i tus especially with respect to vascular complications.

The Effects of Progesterone and Estrogen Treatment on Insulin Secretion after I .V. Glucose Tolerance Tests in Normal Subjects. R. Goberna, F. Garcia-Albertos, J. Tamari t -Rodriguez, E. dei Rio, R. Roea. Depar tments of Physiology and Biochemistry, Facu l ty of Medicine Complutense Uni- versi ty, Madrid, Spain.

The mechanism b y which inhibi tors of ovulat ion (estro- gens plus progesterone) impair glucose assimilat ion has n o t ye t been clarified. The purpose of the present analysis is to invest igate the actions of these hormones when adminis tered separate ly in normal subjects. A to ta l of 39 women was divided into three groups. The first one received estradiol (0.33 mg/kg i .m.), the second 17-OH- progesterone (5 mg/kg i.m.). The th i rd group served as control and was given olive oil. F ive days following t rea t - ment, I .V. glucose tolerance tests (IVGTT) were perform- ed and samples t aken a t 0,5, 15, 30 a n d 45 rain so as to assess glucose and insulin levels (by the glucose oxydase and/or Melani 's method, respectively). - - Progesterone had no effect on the assimilat ion of glucose nor on the secretion of insulin (IMI levels 5 rain following glucose adminis t ra t ion was 118 4. 12 in the progesterone t rea ted group and 106 -4- 10 in the control group). On the other hand, a significantly higher insulin secretion was noted in the group t rea ted with estrogens (202 4. 18 as compared to 106 -V 10 in the control group). Of the two hormones i t was found t ha t the estrogens were solely responsible for the hyperinsulinism.

Scanning Electron Microscopy of the Islets of Langerhans of Ducks and Chicken. Josd Gomez-Acebo. Ins t i t u t e Gregori0 Maranon. Velas- quez 144, Madrid, Spain.

Up to now, numerous studies on the s tructure and func- t ion of the islets of Langerhans and their different com- ponent cells h a v e appeared in the l i terature, while only scanty reports on the three-dimensional ana tomy of s~ngle cells, or ruptured cells, have been published thus far. The technique u s e d in visualising the islet cells in previous reports on scanning microscopy were not useful for the identification of the different types of cells and are not effective in correlat ing anatomical ' and functional studies. In th is report , an easy method is described for s tudying a l ternate pancreat ic sections wi th the l ight and scanning electron microscope, and making possible t h e easy identification of islet cells of chickens and ducks as well as the three.dimensional struc- t u r e of the interior of cells visualized. The use of the avian islets in these studies conferred the advantage of the special ar rangement of the islet cells in this species, thus making the identification of the proper cells easier. Our findings are presented and discussed and we believe tha t this way of visualizing the s t ructure of islet cells opens a new perspective for future anatomical studies in this field.

Effect of a Cholecystic Extract on Insulin Secretion in the Cat. A.V. Creeo, G. Fedeli , G. Ghirlanda, R. Feniei, M. Lucente. Ins t i tu te of Medical Pathology, Catholic Uni- vers i ty S. Cuore, Rome, I t a ly .

In tes t ina l hormones (gastrin, secretin, cholecystokinin, pancreozymin ) are known to be related to insulin secre-

70 Organization Section �9 Abstracts Diabegologia

rich. A study has been carried out in the cat to investigate if other substances, not yet tested, might influence insulin secretion. Insul in was measured in samples taken from the anterior mesenteric vein, foIlowing i.v. administrat ion of a purified concentrated extract of eholecystic mucosa (6.25 mg/kg and 12.50 mg/kg body weight). Insul in secretion was found to reach its peak about 90 or: 120 rain later and persisted above basal levels unt i l after 180 rain. Only an insignificant decrease in blood glucose was observed. The mechanism of action of the extract will be discussed.

Correlation between Glycogen Synthetase Activity and Blood Glucose Levels in Human Leukocytes. A. Gutman, G. Agam, N. Nahas. Dept. of Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

In contrast to earlier reports glucose-6-phosphate in- dependent (I) act ivi ty of glycogen synthetase was detect- able in granulocytes from both healthy controls and diabetic patients. When blood glucose levels of healthy controls were raised to values encountered in diabetics (glucose tolerance test) a severalfold increase in the activ- i ty of the I form of glycogen synthetase was found. This effect could not he ascribed to increased levels of circu- lating insulin, since administrat ion of insulin (0.1 U/kg i.v.) or tolbutamide was followed by a decrease in the act ivi ty of the I form. The low act ivi ty of the I form in diabetics is in line with the reduced incorporation of glucose into glycogen in whole leukocytes from these patients (4.3 vs. 6.7 vmoles/h • 101~ cells). Total glycogen synthetase act ivi ty in the patients was higher than in control subjects (2.9 and 1.9 ~moles/min • 10 l~ cells respectively). -- I t is suggested tha t the conversion of glycogen-synthetase D to I is stimulated by glucose. Insul in does not have a direct effect on this mechanism. However, the presence of insulin may be obligatory for s.ynthesis of the enzyme involved in the D to I echoer. a l e r t .

Lipolytic Responses of Human Adipose Tissue in Vitro to Natural [1--29] Gincagon and to Synthetic [1--23] Gin- cagon Peptide. B. Guy-Grand, R. Assan. Clinique Mddical and Service Diab6~ologie, HStel-Dieu, Paris, France.

A synthetic peptide with the [1--23] glucagon sequence has been recently demonstrated to be a potent lipolytic agent in man (in vivo) and on rat adipose tissue (in vitro), despite the loss of other biological potencies of the glu- cagon molecule. -- Regarding the absence of a clear-cut lipolyCic effect for [1--29] glueagon on human adipose tissue in vitro (as previously described by others), com- parative studies v~ere performed on human subcutaneous

tissue for these two peptides. -- Natural purified ~dipose glueagon --29] did no t significantly increase glycerol release at a concentration of 5.4 108 ~M/ml. In the same system, the synthetic [1--23] poptide induced a signifi- cant glycerol release: 2.08 ~ 0.3, 9.07 -4- 1.7, 12 -V 2, 19 ~ 3 J ~tM/g/2 h, respectively for control, and 1.38, 2.73, 5.5 tzM 10-6/ml concentrations. -- This lipolytic effect of [1--23] is potent iated by theophylline: 7.08:1:1.65 ttM/ g/2 h glycerol release for theophylline 10 -a M (alone) vs. 19 • 5.2"6 y2Cl/g/2 h for theophylline 10 -a M plus [1 --23] peptide 3.9 10 -6 ?M/ml. -- The in vitro data obtained are in good agreement with the lipolytie action in vitro of

�9 [1 --23] in man. Potentiat ion by theophylline suggests the action of c AMP. The possible significance of the dis- crepancy found between in vitro effects of [1--23] and [ 1 - 29] on human adipose tissue will be discussed.

Effect of Adipose Cell Size on Lipolysis of Human Adipose Tissue in Vitro. B. Guy-Grand, P. G azalis. Clinique M~dicale de l 'H6tel- Dieu, Paris, France. (Pr. H. Bour).

Basal and norepinephrine stimulated glycerol release was studied on omental and subcutaneous adipose tissue obtained from normal fasting females undergoing ab- dominal surgery before any glucose infusion. Incubations were carried out for one hour, at pH 7.4, in a medium containing 100 mg~ glucose and 2% albumine. -- The adipose cell size was measured microscopically on an aliquot. Omental cell size is smaller than and correlated with subcutaneous cell size (r = 0.87). -- Dose- action curves for norepinephrine show a lipolytic response for 0.1 ~g/ml and a maximal effect for 0.2 ~g/ml. The curves and absolute glycerol values for the two tissues are nearly identical if glycerol release is referred to lipid content of the sample: referred to 10 s cells, omental act ivi ty is much lower. Lipolysis/10 s cells is significantly correlated to cell size (lipid content/cell): r = 0.83, p < 0.001; r----+ 0.85, p < 0.001, respectively for control and maximal norepine- phrine st imulat ion; yet the slopes of the regression lines are different and there exists no correlation between basal vs. st imulated lipolytic activity. -- The physiological and pathological significance of these data will be discussed.

Free Amino Acids in Pancreatic Islets of 0bese-Hyper- glycemic Mice: Content and Formation from Glucose. E. Gylfe, B. Hellman. Department of Histology, Univer- s i ty of Umeh, Umch, Sweden.

Free amino acids were measured with a micro technique involving reaction with dansyl-chloride and separation with two-dimensional thin-layer chromatography. In- corporation studies were performed using 14C-labelled glucose and unlahelled dansyl-C1 whereas amino acid levels were determined with the aid of 14C-labelled dansyl- C1. Most amino acids occurred in greater amounts in the islets than in the exocrine pancreas. Leucine, for example, was present in tenfold amounts, suggesting that the fl-cells are equipped with binding sites for large amounts of this amino acid. Like nervous tissue, the pancreatic islets were characterized by the presence of 7-aminobutyric acid (GABA) and high levels of taurine. Incubat ion in the presence of glibenclamide did not affect the amounts of amino acids measured whereas high glucose reduced the levels of aspartic acid and GABA. Glucose carbon was incorporated into aspartie acid, g lu tamic acid, GABA, alanine and proline. Glibenclamide had no effect on this incorporation. With increasing glucose concentrations more glucose carbon was incorporated into alanine, glutamic acid and proline. The specific incorporation expressed as g-atoms glucose per mole amino acid was increased for all amino acids except proline.

Coxsackie B Virus Neutralization Tests in Newly Diagnosed Diabetic Patients: a Negative Report. D.R. Hadden, J .H. Conilolly, D .A.D. Montgomery, J .A. Weaver, Metabolic Uni t and Dept. of Microbiology, Royal Victoria Hospital, Belfast..

To prove an association between a Coxsackie Group B virus infection and the clinical onset of diabetes mellitus it would be necessary to (a) isolate the virus from the pat ient and/or (b) show a rising t i tre of hntibody to a particular Coxsackie B viruS. I n view of the variable period of t ime elapsing between postulated pancreatic damage by a virus and t he onset of symptoms of frank diabetes, one would not expect either of these criteria of association to be fulfilled. More indirect proof would depend on showing (a) an epidemiological association and/or (b) an excess of high antibody levels to a particular Coxsaekie B type above the "background" level observed

Vol. 9, No. 1, 1973 Organization Section �9 Abstracts 71

in the same population at the same time. -- In a pro- spective study of this po~ible association, among sixty patients whose newly diagnosed diabetes required ad- mission to hospital for stabilization, the profile of Cox- sackie B1--6 ant ibody titres did not differ significantly from that of a control group of non-diabetic hospital inpatients matched for age and sex. The "background" profile of Coxsackie B I - 6 ant ibody titres obtained from 100 hospital outpatients was similar. -- We have not found either an epidemiological association or an excess of high ant ibody titres to any Coxsackie B type in newly diagnosed diabetic patients.

Studies on the Biosynthesis of Glucagon in the Pancreatic Islets of Guinea-Pigs. Claes HellerstriSm, Simon Howell, Arne Andersson and John Edwards. School of Biological Sciences, The Univer- sity of Sussex, Brighton, Sussex, England and Depart- ment of Histology, The Universi ty of Uppsala, Uppsala, Sweden.

Isolated pancreatic islets from guinea-pigs were incu- bated in the presence of all-labelled amino acids, mostly tryptophan, which is present in the glucagon but not in the insulin molecule. Proteins in the islet homogenate were precipitated with TCA and the precipitate extracted with acid alcohol and alcohol-ether. Proteins were then fractionated with gel chromatography and with disc electrophoresis or immunoprecipitation with antiglucagon antibodies. Localization of t ryptophan containing cell components was performed with electron microscopic autoradiography. -- The results indicate incorporation of both tryptophan, leucine and isoloucine into proteins of molecular weight above 60000 as well as in a fraction of molecular weight about 18000. Little or no incorporation of labelled amino acids was found in the fractions con- taining bovine-porcine carrier glueagon during incuba- tions for up to 17 h. Incorporation into the glucagon region of the gel chromatograms appeared only when islets were maintained in tissue culture for 6 days in the presence of sH-t .ryptophan. Electron microscopy of the incubated islet cells demonstrated an excellent ultrastrue- rural preservation and the autoradiography showed a significant incorporation of SH-tryptophan in the secre- tion granules of the glucagon producing A-cells. (C. H. was in receipt of a Wellcome-Swedish Travelling Research Fellowship).

Do the Sulfonylureas Interact with the Plasma Membrane of the Pancreatic iff-Cells ? B. Hellman, J. Sehlin, I.-B. T/~ljedal. Department of Histology, Universi ty of Umeh, Umeh 6, Sweden.

Despite the extensive clinical use of the hypoglycemie sulfonylureas it still remains to be clarified by which mechanisms these compounds stimulate the pancreatic fl-cells. A fundamental question is whether the sulfonyl- urea effect is accounted for by drug interaction with the cell membrane or whether there is an intracellular receptor mechanism. The latter problem was approached by studying the uptake of radioactively labelled tolbutamide and glibenclamide by fl-cell-rich pancreatic islets micro- dissected from obese-hyperglycemic mice. Glibenelamide had a greater affinity to the islets than tolbutamide. In the presence of a lbumin tolbutamide equilibrated in a space approximately equal to that obtained with the extracellular markers mannitol and sucrose. Uptake of sulfonylureas was easily reversible. I t was depressed by serum albumin but not significantly affected by glucose, leucine or diazoxide. Sulfonylurea binding was increased after pretreatin~ the islets with compounds (p-chloro- mercuriphenylsulfonie acid, ant imycin A) which make the fl-cells permeable to sucrose. Neither of these compounds increased the binding of sulfonylureas to suhcellular

particles of homogenized islets. The restllts suggest tha t sulfonylureas are normally restricted to the outside of the fl-eclls and consequently stimulate insulin release by interacting with the plasma membrane.

The Influence of Phenformin on the Absorption of Various Food Components. W. Heptner, H.B. Neubauer. Pharmazeutisch-wissen- schaftliche Laboratorien, Farbwerke Hoechst AG, Frank- furt/M.-Hoechst, Germany.

The effect of phenformin on the absorption of various substrates has been studied in normal dogs and rabbits. After oral administrat ion of U-ltC-glucose, 13~I-trioleine or lalI-albumin phenformin causes delayed transfer of these substrates from the stomach into the duodenum which is evidenced by the fact that gastric content of the compounds two hours after application is 3--5 times higher. This effect was demonstrable after the admini- strat ion of 2 mg phenformin per kg body weight. The result could be confirmed when the transfer of ge~utro- graphin contrast medium was monitored by means of X-rays. Parallel to this, phenformin delays the accumula- t ion of radioactivity in the blood. Assessment of radio- act ivi ty excreted in feces revealed that the biguanide compound does not affect the amount of glucose absorp- tion but that it significantly diminishes fat uptake. -- I t is concluded that the main action of phenformin in dogs and rabbits consists in an inhibition of chyme transport into the small intestine (Creutzfeldt 1962, F6rster 1965) rather than in its effect upon the intestinal absorption of such different compounds.

Effect of Fasting on Insulin Biosynthesis and Release. A. Herehuelz, D.G. Pipeleers, W.J . Malaisse. Laboratory of Experimental Medicine, Brussels University, Brussels, Belgium.

The insulinotropie action of glucose is known to be reduced after fasting. The pancreatic tissue apparently keeps the "memory" of this nutr i t ional influence since the reduced secretory responsiveness to glucose can be demonstrated in vitro. In the present exiJeriments, pancreatic tissue or isolated islets removed from fasting rat, s ssmthc~uized much less (pro)-insulin and secreted much less in- sulin on exposure to glucose than tissue or islets removed from fed animals. The insulinotropic action of other sti- muli, such as fl-hydroxybutyrate or theophylline, was either unaffected or barely decreased after fasting. These findings are consistent with the idea that fasting reduces the rate of glucose phosphorylation and further metabo- lism in the beta cell, leading to a concomitant inhibition of both glucose-induced insulin synthesis and release. By contrast, fasting has little effect on the insulinotropic action of factors of which the preliminary site of act ion is thought to be independent of glucose metabolism. More- over, these data are compatible with the concept that keto acids play a significant role in the regulation of insulin release during starvation.

Effects of Streptozotocin on Liver Composition and Circu- lating Fuels in the Rat. E. Herrera, Elm:lio Montoya. Ins t i tu te G. Marafion. C.S.I.C., and Chair of Animal Physiology, Facul ty of Sciences, Universi ty of Madrid, Madrid, Spain.

To study the carbohydrate-lipid interrelationship in fed and fasted rats made diabetic by the administration of streptozotocin, male rats were sacrificed by decapitation three days after a single i.p. injection of 65 mg/kg of the drug (St), and compared with controls (C) injected with buffer. When fed, St animals show hyperglyeaemia, hy~orketonaemia and hypoinsulinacmia. Liver gl:veogen and citric acid concentrations are diminished, wl{ile the

72 Organization Section �9 Abstracts Diabetologia

concentration of DNA-P, phospholipids-P, proteins and acctyl-CoA in the liver are augmented in the rats receiv- ing the drugs vs. C. The response of C to 48 h of s tarvat ion was normal, while in St treated rats neither plasma insulin, liver phospholipid-P, proteins, glycogen, acetyl- CoA or citric acid change with food deprivation. Blood glucose is lower and blood ketches and liver DNA-P are higher in the fasted St rats than in the fed ones, but changes are smaller than in C, in such a way tha t the differences among the two groups are minimized in the fasted state. The turn-on failure of post-prandial insulin secretion may contribute to the metabolic alterations found in the fed St rats and such alterations are minimized when food is withheld and circulating insulin levels are similar to those in controls.

Dynamics of Pancreatic Blood Flow after Intravenous Glucose Administration as Determined by an Electro~ magnetic Flow Meter. It . Hommel, U. Fischer, E. Schmid, H. Fiedler, H. Bibcr- geil. Zcntralinsti tut for Diabetes "Gerhardt Katsch", Karlsburg, DDR.

I t is indispensable for the investigation of the dynamics- of endocrine pancreas function to calculate the insulin concentration to the corresponding blood flow. In Alsa- tian dogs, the portal vein or the splenic artery were eannulated to withdraw blood samples for assessment of I R I concentrations. The blood flow curves of the superior pimcreatico-duodenal artery and/or the pancreatico- duodenal vein were recorded by means of an electro- magnetic flow meter. -- Already with i.v. glucose application (IVGTT, 0.5 g,fkg) the blood flow in the superior pancreatico-duodenal artery increased by 15-- 35% extending for 20--25 min. In most experiments, up to 80 min, blood flow decreased to a trough below baseline. Simultaneously, a transient increase in blood flow is observed for 2 to 4 rain in the femoral artery. The corresponding control injections (1.0 ml NaC1/kg, 0.9%) did not show any changes in blood flow in both areas. Under these conditions, the dynamics of pancreatic blood flow consist both of the elevation of heart-minute-volume and of a pancreas specific elevation of the blood flow. Simultaneous recording of the insulin concentration per- mits us to quanti tat ively describe the dynamics of insulin secretion of the flow area studied.

Effect of Acetyl Choline on the Secretion of Glucagon and Insulin from the Isolated, Perfused Canine Pancreas. J. Iversen. Second Universi ty Clinic of Internal Medicine. Kommunehospitalet , 8000 .~rhus, Denmark.

During perfusion with a glucose concentration of 25 and 150 mg/100 ml the effect of infusions of acetyl choline on "g]ucagon and insulin release was investigated in 5 per- fusion experiments. -- Acetyl choline at a concentration of 10-- 100 izM stimulated release of glueagon both at low

. as well as at the high glucose concentration which in itself inhibits basal glueagon release. Glueagon was released in a monophasic pattern, resembling the release pattern found after catecholamines and in contrast to the biphasic pat tern obtained after gastrointestinal hormones and argininc, as previously reported from our labora- tory. -- Acetyl choline always stimulated release of insulin in a biphasic pattern at the high glucose concen- tration, while no consistent effect was obtained at the low concentration. Both at low and at high glucose concen- trations a pronounced rebound stimulatory effect was seen. -- Infusion of atropine at a concentration of 25 [zM completely abolished the st imulatory effect of acetyl choline on both glucagon and insulin release. -- The results reported suggest that the parasympathetic nervous system may play a direct role in the control mechanism

of the release of the pancreatic hormones during the process of food ingestion.

Effect of Different Non-Metabolizable and Metabolizable Hexoses on the Secretion of Insulin and Glueagon from the Isolated, Perfused Canine Pancreas. J. Iversen. Second Universi ty Clinic of Internal Medicine, Kommunehospitalct , Aarhus, Denmark. Release of insulin and glucagon was investigated following infusions of either 3-0-M-glucose, glucosaminc, galactose, mannose, fructose or glucose, with zero glucose in the perfusion medium. The hexoses were given at equimolar- concentrations of 8.3 m]~f for periods of 20 min. The combined effects of 3-0-M-glucose, glucosamine or glucose and of the adenyl-cyclase system were also investigated using glueagon, cyclic AMP (cAMP), or theophylline as a means of increasing intracellular cAMP. -- Release of insulin was stimulated by mannose and glucose, while no effect was seen after fructose, galactose, glucosamine or 3-0-M-glucose. Infusions of glueagon (2 ng/ml), cAMP (1 raM) or theophylline (1 raM) during perfusion with either 3-0-M-glucose or galactose also had no effect upon insulin release while an effect was always observed during perfusion with glucose. -- Release of glucagon appeared to be suppressed to some degree by all hexoses investigat- ed. The suppression exerted by glucose however, was faster and more pronounced than that of other hexoses. At the present t ime it cannot be excluded that the sup- pression of glueagon release by non-metabolized hexoses can be explained by traces of glucose (0.1 mM) present in commercial preparations, cAMP and theophylline in preliminary experiments released glueagon during per- fusion with galactose, 3-0-M-glucose and glucose. -- The results suggest that the insulin releasing effect of glucose is int imately associated with glucose metabolism. Further experiments are required to determine whether the glueagon-suppressing effectt of glucose is similarly depend- ent upon the glucose metabolism by the alpha cell.

Stimulation of Renal Gluconeogenesis in Methylacetamide Diabetes of the Rat. P.B. Iynedjian and G. Peters. Depar tment of Pharma- cology, Universi ty of Lausanne, Switzerland. The gluconeegenic capacity of renal cortical slices taken from rats made diabetic by oral administration of 8 ml per kg b.w. of methylacetamide (MA) was studied. In the absence of substrate in the incubation medium, kidney cortex of treated rats produced 32 4- 1.3 lzM glucose �9 g dry. weight -1. h-l, while that of controls released 21 4- 0.5 ~M. When the medium contained 10 rmM per L. pyruvate, u- ketoglutarate (a-KG) or fructose, glucose synthesis from these substrates amounted to 363 4- 11.6, 235 4- 7.7 and 6824- 13.4 ~M with slices from treated rats; the corresponding control values were 296 4- 9.4, 172 4- 5.0 and 599 4- 12.4 t~M (all differ~jlccs between MA treated animals and controls significant at p < 0.001). Gluconeo- genesis from 10 mM per L. glutamine in treated rats did not differ from that in controls (1384- 8.0 v.s. 1304- 3.7 ~M). Thus, in rats made diabetic by MA, in vitro renal gluconeegenesis from pyruvatc, ~-KG or fructose is stimulated by 22, 36 and 14~/o respectively, while gluco- neogenesis from glutamine is not accelerated. -- Support- ed by SNSF, Grant no 3.370.70.

About the Biological Activity or Inactivity of Monoiodo- insulin. C. Jaequemin, B. Lambert, B.Ch.J . Sutter. Laboratoires de Biochimie et de Physiologic animale et Centre de Biologic du Ddveloppement, Faeultd des Sciences, (F) 51-Reims.

lzaI-monoiodoinsulin has been prepared using an enzymat- ic procedure. The crude iodinated product, which contains

Vol. 9, No. 1, 1973 Organization Section �9 Abstracts 73

only 25% of monoiodinated molecules, exhibits a v e ~ poor biological ac t iv i ty (less than 30%). This discrepancy may be in line with the following facts: monoiodoinsulin binds to the hormonal receptors of the diaphragm and competes with nat ive insulin. Iodinated species have been separated from uniodinatcd ones by Ampholines-iso- electrofocusing. Complete analysis shows tha t an homo- gcneous product is obtained, in which the A19 tyrosyl residue is monoiodinatcd. Elimination of .Ampholinos from monoiodoinsuline has been performed by two ways : paper chromatography or ammonium sulfate precipita- tion. Both procedures do not give rise to a decrease of the biological ac t iv i ty of nat ive insulin. Nevertheless the first procedure yields a completely inactive product, whereas the second gives fully active insulin. -- The lack of act ivi ty, when observed, is not definitive and it is possible to reactivate the monoiodoinsulin. The mechan- ism of this reactivation has not been elucidated; however we speculate tha t the salting-out enables the molecule to recover the nat ive conformational state.

The Control of Glycogen Metabolism in Perfused Livers of Normal and Diabetic Rats. A. Jakob, J. Zapf, E .R . Froeseh. Metabolic Unit, Dept. of Medicine, Universi ty of Zurich, Switzerland.

The effect of constant concentrations of insulin, glucagon and glucose on hepatic glycogen metabolism was investi- gated by non-reeirculated pcrfusion of isolated rat livers. In the absence of substrates and hormones, oxygen con- sumption was constant over 90--120 rain of perfusion. ATP/ADP ratios were over 3. Addition of glucagon (1 • 10 -9 M) or cyclic AMP (2 • 10-' M) to livers of normal, fed rats resulted in a 4-fold st imulation of glucose pro- duction and in an increase of oxygen uptake by 15~/o. Phosphorylase ac t iv i ty also rose 4-fold after addition of glucagon. Insulin (2 • I0 -s M) part ial ly prevented the increase of glucose production and of phosphorylase activ- ity in response to glucagon. Tissue contents of UDPG fell by 30% and glucose 6-phosphate (G6P) rose 4-fold after glucagon. Addition of glucose (20 raM) resulted in a 50% decrease of phosphorylase and in a 9-fold increase of glycogen synthetase-a act ivi ty. Despite an increase of G6P, tissue UDPG fell by 50%. Glucagon completely suppressed the act ivation of synthetase by glucose, but the glucose effect on phosphorylase was essentially unimpair- ed. Alanine (I raM) was added as substrate in experiments with diabctic, fed rats to prevent an extreme decrease of G6P and to minimize fa t ty acid oxidation. Glycogen content was 1/6 of that in normal animals. Glucagon doubled phosphorylase act ivi ty, the glucose effect of phosphorylase was as in normal livers. In diabetic livers however, glucose completely failed to act ivate glycogen synthetase.

Experimental Suggestion for a Specific Insulitis Producing Antigen Different from Biologically Active Insulin. F.K. Jansen, G. Freytag, L. Herberg. Diabetes Research Insti tute, Dfisseldorf, and Pathological Insti tute, Univer- sity of Hamburg, Germany.

In our former experiments designed to produce an im- munological tolerance to insulin, the results of which have been reported last year, 6 different doses of once cristal- lized insulin and monocomponent (MC) insulin (NOVO, Copenhagen), were administered over 3 months 3 times a week to NMRI mice. We have now examined whether our immunizations produced immunological reactions in the islets of Langerhans, called insulitis, and whether the reaction of blood glucose to a glucose load had been affected. Both insulins, the less purified as well as the MC-insulin were able to elicit the insulitis but at different doses. 1 t ime crystallized insulin produced the insulitis at 1 and l0 ng, MC insulin at doses of 1, 10 and 100 ~g. There

is a distinct dissociation of the biological activity and the insulitis-producing act ivi ty of the insulin preparations, suggesting tha t the biologically active insulin molecule can not be the responsible antigen for insulitis. The insulitis-producing antigen may be a modified insulin or most probably an antigen of the islet unrelated to insulin. -- Alterations of the blood glucose response were seen but did not correlate well with the existence of an in- sulitis.

Blood Sugar and Plasma Insulin Response to Tolbutamide in the Morning and Afternoon. R . J . J a r re t t and I .A. Baker. Department of Medicine, Guy's Hospital, London SE1 9RT.

We have previously reported I tha t during oral glucose tolerance tests performed at three different times during the day the blood glucose levels were lowest and the plasma insulin levels highest in the morning. Possible explana- tions for this phenomenon are a) tha t there is a diurnal variat ion in the sensitivity of the pancreatic B cell to the glucose stimulus b) that insulin sensitivity varies diur- nally and c) tha t both a) and b) operate. -- In the present investigation, we have studied an al ternative B cell stimulant, namely, one gram of tolbutamide given by rapid intravenous injection. Two tests were performed upon healthy vohmteers on separate days at 09.00 and 15.00 h following a nine hour fast. -- The blood sugar fall after tolbutamide was consistently and significantly smaller in the afternoon test. Mean insulin levels following the tolbutamide stimulus were also lower in the afternoon, but the individual differences were less consistent than the differences in the blood sugar response. The results would support the hypothesis that the diurnal variat ion in glucose tolerance is due part ly to differential response of the B cell and part ly to variat ion in insulin sensitivity.

1 Jarre t t , R . J . , Balker, I .A., Keen, H. and Oaklcy, N.W. British Medical Journal (1972) 1, 199.

In Vitro Study of Ins-l in and Glucagon Release by the Foetal and Newborn Rat'Pancreas. C. Jarrousso, F. Rancon, G.E. Rosselin. I .N.S.E.R.M. U. 55, Centre de Recherche INSERM, H6pital Saint Antoine Paris.

Whole pancreas explants were removed from foetal rats on the 19th and 21st day after fecundation (19 and 21 DAF) and on the 3rd day after birth (3 DAB). Explants were diredtly incubated in Krebs-Ringer bicarbonate buf- fer for 90 rain at 37~ pH 7.5 with 0.5% bovine serum albumin. Immune-react ive insulin (IRI) and glucagon (IRG) were measured in incubation media and in ultra sonffied acid alcohol extracts of pancreas explants. In the presence of trasylol (1000 K.I.U./ml), both insulin and glucagon degradation during incubation were less than 20% at 19 DAF and 3 DAB. The mean I R I and IRG contents were respectively 1.5 :t: 0.3 mU/mg -- 1.6 :[: 0.5 ng]mg at 19 DAF for a 3.2 mg pancreatic weight (PW), 2.5 _ 0.6 mU/mg -- 8.3 :k 0.6 ng/mg at 21 DP~F for a 10.6 mg PW and 23 • 5 mU/mg -- 59 :[: 9 ng/mg at, 3 DAB, for 11.8 mg PW. Insulin and glucagon contents were cor- related to each other during the pancreas growth (r = 0.89 p < 0.01) and within the 3 DAB ( r = 0.73 p < 0.05). In the presence of glucose, there was a correlation be- tween insulin releases before and after incubation (r -- 0.61 p < 0.01) and between both these values and the pan- creatic I R I content ( r=0 .61 p < 0 . 0 1 and r - - 0 . 6 2 p < 0.01). Correlation between IRG release and IRG content was also observed (r = 0.54 p < 0.05). This system provides a useful tool to study directly the insulin and glucagon pancreatic cOntent and release at various s',ages of pancreas growth.

74 Organization Section �9 Abstracts Diabetologia

Relationships between Diabetic Retinopathy, Blood Pres- sure, Body Weight and Serum Lipids Levels. D. Job, G. Tchobroutsky, E. Eschwege, C. Guyot-Argen- ton, J .P . Aubry, M. Ddret. Unit~ de Recherches Statisti- que.~ de I 'INSERM, Villejuif, France. Chaire de Diab~to- logic et Clinique Ophtalmologique de l 'H6tel-Dieu de Paris, France. 53 and 92 insulin-dependent diabetics studied prospect- ix-ely and retrospectively demonstrate a direct relationship between the severity of retinal disease (assessed either quanti tat ively by microaneurysm counts or qualitatively} and three parameters: overweight, blood pressure and blood cholesterol level. These parameters are directly correlated with the duration of diabetes, but not with the age of the patient nor with the age of the o n ~ t of the disease. Weight, blood pressure, cholesterol level and insulin requirement all increase directly with the number of retinal microaneurysms. Almost all our patients were within normal limits for these parameters: only 2% were more than 20 per cent over their ideal weight and the diastolic blood pressure of all patients was under 105 mm Hg. I t suggests that microangiopathy and arteriosclerosis develop together early in the natural history of diabetes and it should be possible to find quant i ta t ive indices of the degenerative processes even if it is at an asympto- matic stage.

Glucose-Glucagon Feed-Back Mechanism in Normal and Diabetic Geese and Ducks. H. Karman and P. Mialhe.* Ins t i tu te of Physiology, University Louis Pasteur, Strasbourg, France. Geese rendered permanently diabetic or ducks rendered transiently diabetic by subtotal pancreatectomy, show a very low insulin plasma level bu t a subnormal, normal or high glucagon plasma level with a normal or high glyc- aemia. This observation leads to the hypothesis that the glucose-glucagon feed-back mechanism observed in nor- mal animals is no longer working. Glucose tolerance tests were then performed during or after diabetes, i.e. when plasma IRI was low or again normalised. While in normal apimals or in animals that had returned to a normal meta- bolic state after transient diabetes, plasma glucagon decreased after i.e. glucose; it did not change in diabetic geese or ducks. -- These results show that the glucose- glucagon feed-back mechanism does not work during diabetes and that it is probably insulin-dependent. * With the help of the C.N.R.S. and the I.N.S.E.R.M.

Factors Affecting Glucose and Ketone Body Utilisation by Human Adipose Tissue. A. Kissebah, B. TuUoch, Russell Fraser. Royal Post- graduate Medical School, London, W. 12, England. Util isation of C~ labelled glucose, aceto acetate or hydroxybutyrate by human adipose tissue fragments has been investigated. Most of the 14C from the ketone bodies was found in the esterified fat ty acids while that from glucose was found in the glycerol fraction. This incorpora- t ion of C H aceto acetate was increased 10 fold on adding 5 mM glucose. Furthermore, axlding insulin increased further this ketone body utilisation, provided glucose was present in the medium. Increased fat ty concentration in the medium raised the glucose utilisation bu t decreased fat ty acid synthasis from ketone bodies. -- Sulphonylurea did not affect either glucose utilisation or the incorpora- t ion of aceto acetate or B hydroxybutyrate into lipids. Inhibi t ion of lipolysis with nicotinic acid did not alter either glucose or ketone body utilieation. On the other hand, biguanides reduced the incorporation of these into tissue lipids. -- On adding VLDL and LDL from human serum, incorporation of glucose and of ketone bodies was reduced, but chylomicra from human serum or an artificial triglyceride emulsion had a stimulatory effect.

Purification and Properties of a c-AMP Protein Kinase from Human Adipose Tissue. A. Kissebah, B. Tulloch, N. Vydelingum, Russell Fraser. Royal Postgraduate Medical School, London, W. 12, England.

A protein has been isolated from the 50,000 G supernatant of human adipose tissue homogenate, and found to be similar to the protein kinases isolated from other tissues. The enzyme is capable of transferring in vitro P32 from labelled ATP to a histone. Data is available on the puri- fication steps, and the reaction kinetics. The enzyme activity was increased on adding 3'5' c-AMP, slightly on adding c-IMP, but not at all after c-GMP. -- Ca ++ and Mg ++ ions have been found to affect the kinase activity, and Ca ++ also to affecting the binding of c-AMP to the kinase. These ion effects may be involved in the cell's response to hormone stimulation, and the enzyme activ- ity in the diabetic state is under investigation.

The Effect of Oral and Intravenous Administration of Fat on the Function of the Endocrine and Exocrine Pancreas in Man. J. Kissing, S. Raptis, H. Dollinger, J. Faulhaber, G, Rothonbuchner, E.F. Pfeiffer. Department of Internal Medicine, Endocrinology and Metabolism, University of Ulm, W-Germany.

I t is well kno~n that orally administered glucose and amino acids via the st imulation of intestinal hormones effect higher insulin responses than nutr ients given intravenously. I t seems reasonable to assume that similar mechanisms are also involved in insulin secretory re- sponses to the administrat ion of fat. Hence, blood glucose (BG), immunologically measurable insulin (IMI), free fatty acids (FFA), free glycerol (FG), neutral fat (NF), and the exocrine pancreatic secretion (volume, bicar- bonate, lipase, amylase, trypsin) were assessed in 10 healthy volunteers following i.v. and oral administration of 40 g of soy bean oil (Intralipid/Vitrum). IMI increased to 32.2 =t= 4.1 [zU/ml after ol~l application while 35.0 -4- 3.8 ~U/ml were recorded upon i .v injection. The maximal insulin responses and the increase of the F F A (1284 =[= 101.5 mEq/1), FG (14.2 =t= 5.9 rage/o) and NF (374 -V 61.2 mg%,) following the i.v. fat administrat ion were found between 160--170 rain. BG decreased slightly after 200 min. In contrast, the maximal insulin responses after oral fat administrat ion were recorded after 75 min when F F A (578.8• 89.3 mEq/ l ) , FG (1.18-l- 0.11 mg% ) and NF (96.6 =j= 12.5 rag%) were not yet significantly increas- ed. The volume, bicarbonate, and enzyme contents follow- ing oral fat administrat ion were also elevated within 60 min. -- In summary, both i.e. and oral fat administra- t ion resulted in small bu t significant increases in IMI. The stimulation of insulin secretion by oral administration of fat, however, seems to be mediated by intestinal her- manes.

On the Importance of the Mitochondrlal Tricarboxylate Carrier for the Species Dependent Regulation of Hepatic Gluconeogenesls. J. Kleineke, H. Sauer, H.D. S61ing. Department o f " Clinical Biochemistry, Medical Clinic, Universi ty of G6t- tingen, Germany.

The capacity to synthesize phosphoenolpyruvate (PEP) iatramitochondrially is strongly species-dependent. Since the outward transport of PEP from the mitochondria into the cytosol involves a special carrie~ system, studies have been undertaken to determine the specificity of this system. These studies revealed that not only tricarboxyl- ates, PEP, and L-malate, bu t also D-2 and D-3-phospho- glyeerate, and synthetic P EP analogues including phenyl- P EP are transported by this carrier system. The non-

I'ol. 9, No. 1, 1973 Organization Section �9 Abstracts 75

physiological stereoisomer L-2-phosphoglyccrate likewise is transported. The KM values for the c i t ra te-PEP ex- change have been determined in various species. The results are compared with investigations on isolated per- fused rat and pigeon livers, in which several inhibitors of anion translocation and their effect on gluconeogenesis were studied. The role of anion translocation in the regulation of hepatic glueoneogenesis will be discu~ed.

Effects of Oral Antidiabetics on the Aggregation of Blood Platelets, in Vitro. J. Kloeze. Research Laboratories, Unilever, V]aardlngen, The Netherlands.

Clinical studies as well as a retrospective survey indicate that t reatment of maturi ty-onset diabetes with oral anti- diabetic agents increases the risk of cardiovascular com- plications. Generally, proce, qses such as adhesion and aggregation of blood platelets are involved in these complications. -- Moreover, the recent observation tha t tolbutamldc is able to increase the intracellular 3',5'- cyclic AMP concentration - - at least in the pancreatic fl cell -- may be relevant a.u the intracellular 3',5'-cyclic AMP concentration of blood platclets seems to determine their adhesion and aggregation tendency. -- Therefore, the effects of sulphonylurea and biguanide compounds on the aggregation of rat blood platelets were examined in vitro. -- I t appears that sulphonylureas inhibit the aggregation induced by adenosin diphosphate (ADP) as well as that induced by collagen in concentrations tha t occur in blood of patients treated with these drugs. -- Preincubation of these drugs with the platelet suspension increases the effect. The biguanides seem to have l i t t le or no effect. The relative potencies of vaxious commercial ly available oral antidiabetic agents will be shown. -- I t is concluded tha t the observed increase in cardiovascular incidents in diabetic patients treated with oral ant i : diabetics is probably not due to the effects of these drugs on platelet aggregation.

The Retinal Bloodflow in Diabetes. Eva M. Kohner, Barbara A. Sutcliffe, M. Tudball, C.T. Dollery. Royal Postgraduate Medical School, London W12, England.

Direct measurement of blood flow in retinal arteries is an experimental procedure. However an estimate of the retinal blood flow can be obtained by measuring the mean transit t ime between retinal arteries and their aecom: panying veins, and relating this to the cross-sectional are~ of the retinal vessels (volume flow = blood volume/mean transit time). The method developed by Hickam and Frayser (1965) has boon adopted to measure volume flow in the superior temporal segment of the retina. -- Using this method we have measured the volume flow in 7 normal vohmteers and in 30 diabetic subjects. The dia- betics were subdivided into those with no or background ret inopathy only and into those with proliferative lesions. -- The mean transit t ime was similar between normals and diabetics (3--4.5 see). The volume flow was significantly increased in those with no ret inopathy and background ret inopathy only, although there was a con- siderable overlap. Patients with proliferative lesio/~s had volume flows similar to that seen in normals (8.5 arbi t rary units). Successful p i tu i tary ablation and photocoagulation caused reduction of volume flow. -- The results indicate that contrary to previous belief blood flow in diabetic ret inopathy is increased, at least in the early stages. -- Ref. : Hickam, J .B . and Frayser, R. Invest igat ive Ophthalmology 4 : 876," ! 965.

The Influence of Pregnancy on the Course of Diabetic Retinopathy. A Prospective Study. "W. Korp, J . Neubert, t t . Br,meder, A. Lonhar(ti, R .E.

Levett . 3rd Medical Department for Metabolic Diseases, Krankenhaus dor Stadt Wien-Lainz, Vienna, Austria.

Fundus colour photographs (Zeiss fundus camera) were taken of all pregnant diabetic patients who had attended the clinic between October 1968 and April 1972. The fundus photographs were graded according to the method of Oakley eta l . (Diabetologia 1967, 3, 402). Ret inopathy was observed in 13 patients (mean age 26 =k_ 2.9 years, mean duration of diabetes 18.2 ~ 5.3 years) belonging to class C, D and F of P. White 's classification. The course of ret inopathy has been followed up to April 1972 and in selected eases fluorcscein angioretinography has been performed. Severe progression of diabetic ret inopathy was observed only-in eases exhibiting proliferative changes during pregnancy.

Relationships between Glycerolkinasc-Activity and Tri- glyceride-Synthesis in Rat Liver under Various Nutritional Conditions. T. Koschinsky, F .A. Gries. Second Medical Clinic, Uni- versi ty of Diisseldorf, Germany.

It, is well known from liver pcrfusion experiments by Heimberg et el. that triglyeeride (TG) and VLDL syn- thesis depend on substrate level of free fa t ty acids (FFA) and glucose. But it is unknown whether glycerol, and variations of glycerol kinase (GK) act ivi ty axe able to affect TG synthesis in liver. -- Experiments were per- formed on liver slices of male Wistar I I rats, which were divided into two groups: (A) six normal fed animals, (B) nine animals which were fasted for 48 h, and (C) 5 animals fasted for 48 h and refed for 16 h. Approximately 50 mg liver slices were pzeincubated for 30 rain and then in- cubated 60 min in 5 ml of buffer containing 1.3 rmM/1 FFA, 1 mg/ml glucose and 5 mM/l glycerol with 1 fzC glycerol per test system. The TG was separately ex- tracted from the incubation medium and liver slices, ac- cording to the method of Folch. GK act ivi ty was deter- mined by a modification of the me thod of Newsholme et el. As expected, the 3 groul~s of rats differed in relation to the total liver weight, N~-content/g liver and GK activi ty/rag N./min. The amount of newly synthesized TG correlated significantly (r ~ 0.8757; y = 0.5120 {- 0.3807 • with GK act ivi ty in the different metabolic situations. After 60 min incubation, 65O,/o of the newly synthesized TG was found in the incubation medium using rats of group A, 57~ with animals of group B, and 42o/0 with group C. The remaining TG was found inside the liver slices. -- In view of the previously de- scribed regulation of GK act ivi ty by insulin, the possible role of GK in the regulation of TG synthesis in rat, liver will be discussed.

The Influence of TheophylHne on L-Leucine and L- Phenylalanine Induced Insulin Release Studied with the Perfused Rat Pancreas. M.M.C. I,andgraf-Leurs, R. Landgraf, R. HSr]. II . Med. Clinic, Univ., Munich, Gorma.ny.

Recent data from our laboratory have demonstrated that leucine and phenylalanine provoke insulin release with opposite kinetic patterns. The leucine induced pattern was biphasie and comparable to that of glucose. When phenylalanine was porfused in combination with sub- s t imulatory levels of glucose the only consistent result was a dose dependent "off-effect". -- Theophylline (5 raM) by itself caused hormone release with a .~low onset reaching a very low plateau. When phenylalanine (5, J 0 or 20 raM) was perfused together with theophylline a rapid first phase of insulin release occurred. Theophylline potentiated the biphasie pat tern of insulin output induced by 10 mM leucine. In contrast, addition of theophylline during perfusion with 20 mM lcucine caused an immediate

76 Organization Scction �9 Abstracts Diabetologla

drop in insulin release, then a rebound effect, followed by a continuous decrease in insulin secretion. Subsequent removal of theophylline while continuing the leucine per- fusion resulted in a marked "off-effect". -- Theophylline, most likely through the adenylcyclase system seems to influence the transport into the fl-cell and/or the binding of amino acids to the cell membrane, depressing the secretory response to high concentrations of leucine and enhancing those of low concentrations of leucine and of phonylalanine.

Intestinal Hormones and Gut Glucagon-Like Immune- reactivity (Gut GLI) and Their Action on Lipolysis in Chicken Adipocytes. D.R. Langslow, Houghton Poultry Research Station, Houghton, Huntingdon, England.

Chicken adipocytes are very sensitive to the lipolytic action of pancreatic glucagon but insensitive to other hormones which are lipolytic in mammalian adipocytes. They therefore offer a useful system for investigating the possible lipol.~ic activity of gut GLI. Intest inal hormones have some effects on chicken adipocytcs. Both secretin and ch01ecystokinin-pancreozymin weakly stimulate lipo- lysis at high concentrations. Gastrin does not stimulate lipolysis and antagonises pancreatic glucagon-stimulated lipolysis at concentrations greater than 1 ~g gastrin/ml. Intest inal extracts from the pig, rat, duck and chicken, containing known amounts of gut GLI, have been assayed for possible lipolytic activity. Impure porcine gut GLI (LGH) was weakly lipolytic, but the lipolytie activity could be correlated with the known secretin content. This was confirmed by its action on rat adipocytes. Pure porcine gut GLI (Murphy R.F . et al., Biochem. J. 125, 61 P, 1971) possessed no lipolytie activity in concentrations up to 100 ng GLI/ml. Ra t gut extract (KDB) was weakly lipolytic "_but again the activity was probably due to seeretin. An extract of duck ileum (EK) was significantly active bu t the log-dose response of GLI could not be correlated with the effect of pancreatic glucagon. Crude extracts of chicken duodenum and je junum had little lipolytie activity and this did not correlate with their gut GLI content. The lipolytic activity and the GLI content of an extract of chicken pancreas corresponded closely. Hence it seems unlikely tha t gut GLI possesses any lipolytic activity in any of the four species investigated.

Depression of Glucagon Release by a Sulphonylurea in the Arginine-Stimulated Perfused Pancreas of the Rat. H. Laube, R. Fussg~nger, R. Mayer, E .F . Pfeiffer. De- partment of Endocrinology and Metabolism, Center of Internal Medicine, Universi ty of Ulm, "VV-Germany.

Radioimmunologically measurable glucagon (IMG) and insulin (IMI) were determined following infusion of arginine (5 mM) and a new hypoglycaemic sulphonylurea (Re-6) in the isolated pcrfused rat pancreas. After arginine administration, a marked rise in glucagon (1.8--3.1 ng/ml) and a biphasic pat tern of stimulated insulin release (180-- 285 izU/ml) were observed. Addition of Re -6 (10 Izg/ml) further accentuates the insulin secre- t ion (495 I~U/ml) bu t inhibits significantly glucagon release (2.9--1.4 ng/ml). The secretion rates of insulin and glu- cagon were thus inversely related during stimulation with Re-6. Sulphonylureas did not only depress glucagon release in the presence of low glucose concentrations, bu t also when the pancreas was actively stimulated by arginine.

The Effect of Starch and Sucrose Rich Diets on Serum Lipids and Insulin Release in Rats. H. Laube, R. Fussgs H. K16r, E .F. Pfeiffer. Depart- ment of Endocrinology, Metabolism and Nutrit ion, Center of Internal Medicine, University of Ulm, W-Germany.

The effect of carbohydrate-induced hyperlipemia and hyperinsulinemia has bc(,n shown by numerous workers. Few attempts, however, have been made to determine different effects of various types of carbohydrates. The purpose of this investigation thus was to measure and compare detailed effects of sucrose and starch as dietary lipemic and insulincmie agents. Both parameters seem to be high risk factors for the development of degenerative vascular diseases. After sucrose fecding, a marked hyper- insulinaomia has been established in rats. Glucose dis- appearance rate, however, was decreased in sucrose fed rats following OGTT. Within a few days, these animals exhibited a significant hyperlipemia with an increase in pre-fl-lipoprotcins (400 mg~/o). Serum lipoprotein electrophoresis demonstrated a broad and sharp band in the area of VLDL. F F A increased from 400--760 t~val/l, serum cholesterol, however, was not significantly different in sucrose or starch fed rats (75--80 rag%). -- The data seem to be of importance in regard of a possible association of sucrose consumption and degenerative vascular dis- eases. The postulated specific effect of sucrose might thus be caused by a st imulation of endogenous triglyceride production and an increase in basal and st imulated insulin release.

Presence of "Mini" Insulin in Peripheral Human Plasma E. Lhzaro. C~itedra de Fisiologia II , Facultad de Medicina, Universidad Complutense, Madrid.

"Mini" insulin (MI), an insulin immunoreactive compo- nent, has been fotmd present in the portal plasma of rabbits. When whole plasma is filtered through a G-50 Sephadex column the MI fraction appears in a MW region of about 3000. After i.v. seeretin injection, there is a clear-cut increase in the relative concentration of MI with regard to "big" and "li t t le" insulin. The present study was designed to detect the presence of l~fI in human blood after st imulation with seeretin. After an overnight fast, three normal volunteers were given 75 clinical units of secretin i.v. as a pulse. Blood samples were taken at 0, 2, 5, 10, 15, 30 and 45 rain after the injection. Insul in (IRI) was determined by the Yalow and Berson method. Total I R I increased as expected (from basal values of 5-- 12 [zU/ml to 33--64 IzU/ml at 2 rain) and returned to preinjection levels at 15 rain. Two ml aliquots of plasma, corresponding to the samples with maximal I R I concen- tration, were chromatographed on G-50 Sephadex columns and the eluates were analyzed radioimmunologically. Three immunoreactive insulin fractions were found, one appearing in the MW region of "big" insulin (percentages between 3 an 11%) and another in tha t of "li t t le" insulin (61--73%). A third component, analogous to the "mini" insulin found in rabbi t portal plasma, was obtained in a proportion of 23 -- 27 e/o. This finding indicates tha t "mini" insulin can be demonstrated in human peripheral plasma.

The Effect of Calcium and Magnesium on the in Vitro Release of Glucagon and Insulin from the Pancreas of Duct-Ligated Rats. V. Leclercq-Meyer, J. J. Marchand, W. Malaisse. Labora- tory of Experimental Medicine, Free Universi ty of Brussels, Brussels, Belgium.

The role of calcium and magnesium on glucagon (IRG) secretion was studied in vitro using the duct-ligation technique as previously reported from our laboratory (Nature, l~*ew Biology 231:248, 1971). The experimental design was improved in tha t the pieces of pancreas were incubated for as many as nine successive 30 min periods, thus allowing us to study several types of stimuli on the same piece of pancreas. Insulin was simultaneously deter- mined so as to assess the functional integrity of our pre- paration. Glucose (8.3 raM) waspresent in all media. -- Incubat ion in a calcium-depletedmedium enriched with

Vol, 9, No. 1, 1973 Organization Section �9 Abstracts �9

a calcium-chelating agent (EGTA, 1 raM) induced a marl~- ed increase in IRG secretion. This effect was seen both in the concomitant absence or presence of a potent stimulus of IRG secretion (arginino, 10 raM). When EGTA was omitted from the calcium-depleted medium, the stimula- t ion of IRG secretion persisted significantly, bu t was of much smaller amplitude, suggesting that either a suffi- cient amount of extracellular calcium was still present and able to prevent the full s t imulation of I IRG release, or that EGTA was also acting via another means, perhaps as a magnesium-chelating agent. The eff- ects of the absence of magnesium on I R G secretion has as yet not been documented in our experiments. However, increasing the magnesium concentration from 2 to 20 mEq/1 resulted in a clear inhibit ion of the arginine-sti- mulated IRG release, whereas decreasing i t from 20 to 2 mEq/1 restored IRG secretion to its control value. -- Our data therefore suggest that, in contrast to their; ef- fect on the beta cell, Ca ++ and Mg, might act as syner- gists upon the secretory process of the alpha cell, IRG release being enhanced at low levels of both cations.

Quantitative Histological Studies of the Fibrosis and the Number of Capillaries in Hearts from young Persons with Juvenile Diabetes Mellitus. Thomas Ledet. The Ins t i tu te of Pathology and the Second Universi ty Clinic of In ternal Medicine, Kommunehospi- taler, Arhus, Denmark.

I n a s tudy designed to evaluate the heart disease in diabetes, twelve hearts from young persons with juvenile diabetes mellitus and four heaxts from persons without diabetes have been used. -- Representative sections of the myocardium have been obtained by a systematic sampling technique. On every section a point grid has systematically been placed 10 times and the points on the connective-tissue and on the muscles have been counted.

T h e degree of myoeardio-fibrosis was expressed as the number of points over connective-tissue as per cent of the total number of points over connective-tissue plus muscle. -- The number of capillaries (PAS stain) per mm 2 myocardium have been counted on areas where the capillaries were cut at right angles. -- The degree of myocardio-fibrosis was positively correlated to the heart weight and to the durat ion of diabetes bu t not to age. The number of capillaries per mm 2 myocardium was negatively correlated to the heart weight, bu t not to age or durat ion of diabetes. -- The strongest correlation demonstrated so far in this preliminary study was the one between durat ion of diabetes and the spotted fibrosis of the myocardium.

Effect of Prostaglandin PGE 1 on Blood Flow and Insulin Output of Dog Pancreas in Situ. P.J , Lcf~bvre and A.S. Luyckx. Division of Diabetes, Ins t i tu t de M6deeine, Universi ty of Liege, Belgium.

Prostaglandins are now recognized as natural Compounds which may be of much greater physiological importance than hitherto accepted. Their action on insulin secretion has received little or no at tention, although several of their effects have been related to changes in intracellular cyclic-AMP, an event presumed to occur in the mechan- isms associated with insulin release. Overnight fasted anesthetized dogs were infused with prostaglandin PGE~ at a dose of 1 tzg/kg/min for 40 rain and compared with saline infused dogs. PGE1 induced a significant t ransient ,drop in arterial blood pressure (--31 mm Hg), and pan- creatico-duodenal vein (PDV) blood flow (40--50%). PDV plasma insulin rose significantly during PGE 1 infusion while total I R I output remained unchanged. The cessation of PGE 1 infusion was accompanied by a slight tendency of blood flow toward recovery and a highly

significant increase in insulin output ( + 141 4- 120 ~U/g pancreas/rain versus controls). No significant change in blood glucose was observed during PGE 1 infusion, moder- ate hypoglycemia being recorded at the t ime of maximal insulin output. These results (1) confirm the relative independence of insulin output from gross changes in pancreatico-duodenal blood flow, and (2) demonstrate a significant increase in insulin release as a consequence of PGE1 infusion.

Biochemical and Ultrastructural Evidence for a Role of the Microtubular System in the Release of Trigiyeerides by the Perfused Mouse Livgr. Y. Le Marchand,.F. Assimaeopoulos, A: Singh, M. Am- herdt, Ch. Rouill6r, and B. Jeanrenaud. Laboratoires de Recherches Mgtlicales and Ins t i tu t d'Histologie et d'Embryologie~ Geneva, Switzerland.

Previous studies from other laboratories have indicated that triglyeeride rich particles are included within vacuoles which arisd from the Golgi complex, to then be secreted outside the liver cells as triglyceride containing lipo- proteins. Little is known, however, about the mechanisms by which these particles move intraeellularly, and leave the hepatocytes. I n the present experiments, livers from normal fed Swiss mice have been perfused in situ with a Krebs-Ringer bicarbonate buffer containing bovine albu- min serum and red blood cells. I t was found that the output of triglycerides was linear over a 2 h perfusion time, and was markedly stimulated by the addition of 1.5 mM oleate. Of further interest was the observation that colchicine (10 -t M), a substance known to disperse microtubules, markedly decreased the output of triglyeer- ides at a t ime when neither the uptake nor the oxidation of oleate by the liver were modified. The addit ion of vinblastine, another anti-mitotic agent (2 x 10 -4 M), resulted in a complete and rapid (within 30 rain) cessation of the secretion of triglyeerides. Perfusion of livers in the absence of calcium also resulted in a marked decrease in the output of triglycerides. In the presence of mitotic- spindle inhibitors, "lipoprotein" particles could no longer be observed in the Disse spaces, and vacuole containing particles were then frequently seen within the hepato- cytes. These experiments strongly favour the concept tha t the triglyceride rich "lipoprotein" particles are transport- ed inside the hepatocytes and egress across the cell mem- brane via a process involving the mierotubular system. Supported by FN Switzerland, grant ~qo 3.552.71.

Growth Hormone Plasma Levels in Normal Subjects and Diabetics with and without Retinopathy, during Glucose and Insulin Administration. G. Lenti,* R. Frezzotti**, G. Angotzi*, A.M. Bardelli**, G. Pagano*. *Department of In terna l Medicine, **De- par tment of Ophthalmology, Universi ty of Siena, Italy.

An'oral glucose load (1 g/kg body weight), followed after 180 min by i.v. insulin injection (10 U) was performed in 8 non-diabetic subjects, 6 adult diabetics without re- t inopathy, and 6 adult diabetics with severe ret inopathy (with hemorrhages and proliferation). Plasma glucose, free fat ty acids (FFA), imm~moreactive insulin (IRI) and human growth hormone (HGH) levels were assessed over 4 h at 0, 30, 60, 90, 120, 150 and 180 min after glucose; + 5, + 10, + 20, + 30, + 45, + 60 min after insulin~ The results obtained were compared in the different graups. The differences between normals and the two diabetic groups were as expected as regards glucose, F F A and I R I levels. HGH plasma concentrations after glucose were about l - - 2 ng/ml both in normals and in diabetics without retinopathy. After insulin injection, plasma t t G H in- creased to 6.5 d=0.5 ng/ml i n normal subjects and to

78 Organization Section �9 Abstracts Diabetologia

5 .2+0 .8 ng/ml in diabetics without retinopathy. I n contrast, patients with ret inopathy showed an irregulax but higher increase in HGH levels, be Jr'during the whole test or in the post-insulin phase (up to 15--20 ng/ml, and up to 50 ng/ml in one patient). -- The possibility of an abnormal HGH release to different stimuli in diabetics with ret inopathy is suggested as well as the introduction of a positive hypothesis for the study and t reatment of diabetic vascular lesions.

Serum Insulin Response to Maximal Insulinogenic Stim- ulus in Normal and Diabetic Subjects. G. Lenti, G. Pagano, A. Basetti-Sani, M. Galli. Depart- ment of In ternal Medicine, University of Siena, Italy.

Intravenous injection of ~2 rag glucagon in normal subjects increases serum I R I levels to 50-- 60 [zU/ml within 5 min. After i .e. glucose injection (priming dose infusion t ech - nique), serum I R I levels show a double peak over a 120 rain period. Apparently, ghmose enhances insulin release as well as insulin synthesis and intracellular storage. If glucagen is given right after glucose admini- stration as above, I R I levels rise up to 400--600 tzU/ml. This high insulin response seems to indicate an increase in the so-called insulin reserve. -- On the other hand, I R I increase is poor with glucose in prediabetic and insulin- i~dopendent diabetics, while it is maintained with glu- cagon (administered immediately after glucose) although I R I levels a t ta in a peak of only 150--250 [zU/ml. The difference between normal and diabetic subjects is statisti - ta l ly significant, Finally, in severe diabetic patients (insulin-dependent), I R I levels do not change after the glucose-glucagon procedure. -- I t is suggested that the docre~sod---~.esponse to a glucose stimulus in diabetes may be duo to an impairment of the glucose receptor of the be~a cells, as well as to reduced synthesis and reserve of insulin.

Glucose-Stimulation of Phospholipid Metabolism in Pan- creatic ~-Cells. A. Lernmark, G. Fex. Departments of Histology and Physiological Chemistry, University of Ume~, Ume/~, Sweden. Biosynthesis, storage and release of insulin are processes which engage various types of membranes in the fl-cells. Cell membranes are rich in phospholipids, which presum- ably are important for membrane function. The metab- olism of phospholipids we~s therefore studied in resting and actively secreting pancreatic islets. Microdisseeted islets from obob-miee were used since they contain more than 90~o h-cells. Their content of phospholipid was 115 pmol/ ~ dry weight,. Islet phospholipids. . consisted of 56~o phos- phatidyleholine, t 3 ~o dlphosphatldylglycerol -k phosphat- idle acid, 11~o phosphatidylethanolamine, 12 % phosphat- idylinositol plus phosphatidylserine, and 8 % sphingomye- lin. Exposing the islets to 20 ml~ glucose for 5 rain stimulated insulin release but had no effect on the incor- poration of 3-"P-orthophosphate into the phospholipids. After 60 or 120 rain the st imulated insulin release was accompanied by an increased a~-P-labelling of the phospho- lipids. Most of the label occurred in the combined phos- phatidylinositol/phosphatidylscrine fraction (70~o) and in phosphatidyleholine (14~o). These resultJs suggest tha t glucose stimulates phospholipid synthesis in t.,he fl-cells and that t h i s effect is particularly striking m specific classes of phospholipid.

Monocomponent Insulin, A Clinical Trial after one Year~ R. E. Levett, W. Korp. 3rd Medical Department for Meta- bolic Diseas(~. Krankenhaus der St, adt Wien-Lainz, Vienna, Austria. The antigenicity of monoeomponent insulin was investi- gated in 15 patients not previously treated with insulin

(Group A) and in 12 patieuts treated With conventional insulin preparations for two to 15 years (Group B). Ig-G- antibodies were determined by the method of Christiansen (Hormone and Metabolic Res., 1970, 2, 187) and total and free insulin by the method of Heding (Hormone and Met.a-' belie Res., !969, 1, 145). 50% of the patients in group A exhibited a transitory increase of Ig-G-insulin antibodies two to seven months after the onset of insulin treatment. Ig-G-antibody levels, however, decreased below 0.05 mU/ ml in all patients by the end of the observation period. Levels of bound insulin rose from 0 to 48 4- 30 ~zU/ml in group A, but showed a decrease from 135 i 53 ~zU/ml to 80 q- 34 ,~U/ml in group B. Both groups exhibited minor or inconstant changes in daily insulin requirements. Re- sults of this study are compared with observations on a control group treated with monospecies pork insulin (n = 24) and -with conventional insulin preparations (n = 88).

The Biological Properties of an Intramolecular Cross- Linked Insulin Derivative. D.G. Lindsay and O. Logo. School of Biological Sciences, University of Sussex, Brighton, England and Forschungs- laboratorium der Schering AG, Berlin, G.F.R.

The brief half-life of parentally administered insulin re- quires the use of depot preparations of the hormone. The addition of ioreign substances such as protamine or Surfen or the administrat ion of Lento forms of insulin have the disadvantage of potentiat ing the immunogenicity of in- sulin. Preparation of a pure intramolecular cross-linked insulin in which the amino groups of glyeine A i and lysine B 29 are joined by a succinyl bridge and testing of this derivative in the rabbi t blood sugar assay has recently been achieved. This derivative shows 60 % of the biological activity of insulin as judged by the initial fall in blood glucose level but has a significantly prolonged duration of action when injected intravenously. The action could be further prolonged by intramuscular injection in a non- depot form. The prolongation of action of this derivative compared with other modified insulin preparations indi- cates that this is not a geuerM property of amino-modified insulin derivatives.

The Effect of Placental Lactogen on Glucose-Influced In- sulin Release. C. Lopez-Quijada, L. Chiva. Ins t i tu te "G. i~Iarafion", C.S.I.C., Velasquez 144, Madrid, Spain.

That placental lactogen induces insulin secretion in vivo has recently been reported (Life Sciences, 1972). The possible effect of placental lactogen (Lederle) in the in- sulin secretory process was investigated with the same experimental conditions in rabbits by examining its action upon gkmose-indueed insulin secretion. -- Glucose injec- t ion (0.5 mg]kg weight) resulted in a .prompt insulin re- sponse. When placental lactogen (5 mg/kg weight) and glucose were injected simultaneously, no significant changes in b!ood glucose levels were observed as compared to" those after glucose alone. -- Plasma insulin levels at five minutes after the combined injection were higher than after glucose alone and the total insulin response appeared to be an additive effect of the two stimulating agents. -- I t is suggested that the increase in the secretion by the beta cell during pregnancy could, at least in part, be due to the st imulat ion of the endogenous production of chorio- nic growth homnone.

"Antidiabetes-Like" Action of the Chlorella Pyrenoidosa on Experimental Diabetes in Rats. C. Lopez-Quijada, M. Rodriguez-Lopez. Ins t i tu te "G. Marafion ', C.S.t.C., Velasquez 144, Madrid, Spain.

White Wistar rats weighing approximately 200 gr. were made diabetic by means of intraperitoneal injection of

Vol. 9, No. 1, 1973 Organization Section �9 Abstracts 79

alloxan. Their diabetic state is confirmed by studying in- sulin in plasma and sugar in the urine: Twontyfour hours after intraperitoneal injection of the algae, glycosuria and polyuria of diabetic rats has disappeared. Some rats main- tained this normal condition indefinitely or unti l given a new injection of alloxan; other animals returned to nor- mal but reaequired diabetic symptoms within a few days and a new injection of chlorella is necessary to temporarily restore normality. Chlorotla injected into normal and alloxanized rats does not modify significantly the plasma insulin levels. If at the end of the experiments the ab- dominal cavity of the rats is examined, we find chlorella associated with connective and adipose tissue, forming green nodules of variable size. These nodules consist of giant cells of algae.

On the Antilipolytic Action of Insulin and Prostaglandin E 1 �9 E.G. Loten. Department of Clinical Biochemistry, Uni- versity of Otago Medical School, Dunedin, New Zealand.

Both insulin and prostaglandin E a are antilipolytic in rat adipose tissue by virtue of their abil i ty to decrease cyclic AMP levels. In these experiments, the effect of insulin on cyclic AMP breakdown was studied using homogonates prepared from isolated fat ceils which had been pretreated with the hormone. Such homogenates displayed kinetic evidence of two cyclic 3 ' 5' nueleotide phosphodiesterases with different K m values. Insul in was found to increase the Vmax of the low K m enzyme. This effect may explain the abil i ty of insulin to inhibit lipolysis stimulated by a variety of hormones, methyl-xanthines or exogenous cyclic AMP. -- Prostaglandin E 1 under conditions when it strongly inhibits lipolysis stimulated by theophylline, had no effect on phosphodiesterase activity either when added to intact fat cells or to fat cell homogenates. Insul in ex- erted its usual effect on phosphodiesterase of fat cells from the same pool. This suggests that insulin and pros- taglandin E 1 act by different mechanisms. Further sup- port for this hypothesis was obtained from experiments in which it was shown that the two agents could have addi- tive effects on the lipolytic process. -- This work was carried out during the tenure of a New Zealand Medical Research Council Training Fellowship.

Studies of Propranolol and Hypoglyeaemic Sulphonamides on Insulin Secretion and Glycaemia. A.L. Loubatibres, M.M. Loubati~ros-Mariani, G. Ribcs, J. Chapal. Laboratory of Pharmacology and Pharmaco- dynamics, Facul ty of Medicine, Montpellier, France.

From the oxperimente we have carried out i n vitro with tolbutamido on the isolated and perfused rat pancreas as well as i n r ive with glibenelamido on the normal, anesthet- ized dog, it can be seen that propranolol, by its beta adrenorgic blocking action, does not modify the insulin- secretory action of these hypoglycaemic sulphonamides. According to the pharmacological arguments provided by our experiments, the beta aclrenergic receptors do not seem to be directly involved in the st imulating action of these sulphonylureas on the beta cells of the islets o f Langorhans.

Analysis of the Stimulating Action of Tolbutamide on the Insulin Secretion using Mannoheptulose and Diazoxide. M.M. Loubati~res-Mariaaai, A.L. Loubati~res, J. Chapal. Laboratory of Pharmacology and Pharmacodynamics Facul ty of Medicine, Montpellier, France.

The experiments were carried out on the isolated and per- fused rat pancreas. Tolbutamide alone, in the absence of glucose in the porfusion medium, provokes a st imulation of insulin secretion which takes place in two phases: the first, rapid and important phase is expressed by a peak in

insulin secretion, the second phase is less intense but prolonged. In the presence of glucose (1.5 g/l) in the per- fusion medium, the level of the secretion during the second phase is considerably higher. -- In the presence of mannoheptulose, the rat pancreas perfused with a nmditun containing glucose, responds to the addition of tolbuta- mide as it would do m the absence of glucose. In the absence of glucose from the perfusion medium, manno- heptulose does not modify the insulin secretion provoked by tolbutamide. -- Diazoxide is refractory to the two phases of insulin secretion provoked by tolbutamide, whether it be in the presence or the absence of glucose. -- In conclusion, the st imulating action of tolbutamide has two components: the one independent of the presence of glucose in the' medium that bathes the beta "cells; the other, on the contrary, is dependent on glucose. Diazoxide can be considered a direct antagonist of tolbutamido, while mannoheptuloso is but an indirect antagonist, its action being mediated by its effect on the metabolism of glucose.

Influence of Mother's Age, Parity, Obesity, and Diabetes Upon Birth Weight. J. Lubetzki, J. Duprey, C1. Sarnbourg. Policlinique, 1-16- pital Ambroise Pard, Boulogne, France.

A retrospective statistical study was undertaken of the birth weight of 953 newborns of 667 women, who were divided into 4 groups : normal (206 women, 285 newborns), non diabetic obese (250 women, 380 newborns), non obese diabetic (45 women, 75 newborns), obese diabetic (166 women, 213 newborns). -- In each group, global and partial correlations were calculated between birth weight, pari ty and mother's age. The birth weight in the 4 groups were also compared. -- RemdXs: Advancing mother 's age. is not responsible for foetal macrosomia. On the contrary, in the diabetic groups (obese or non obese), a significantly negative partial correlation was found between mother's age and birth weight. -- A positive partial correlation was fotmd between parity and birth weight in the 4 groups. -- Foetal macrosomia is found in diabetic women, but also in obese women. There is no additive effect of the two dis- eases. There exists no statistically significant difference between the birth weight of newborns of diabctic and obese non diabetic women.

On the Mechanisms of Exercise-Induced Glucagon Secre- tion in Rats. A.S. Luyckx and P.J . Lefebvrc. Division of Diabetes, Ins t i tu t d~ Mddeeine, University of Liege, Belgium.

Overnight fasted male albino rats were subjected to forc~,d swim (FOSWI) for 60 rain in tepid water. Blood for gh~- eose, free fat ty acids, insulin, and glueagon measurements was obtained by cardiac puncture immediately after exo~'ciso or after a 60 rain rest period in control animals. The FOSWI-induced rise in plasma glucagon was of pan- creatic origin since it was significant with an antiserum highly specific for pancreatic glueagon (mean increase + 118 pg/ml, p < 0.01), very similar with an anti.,w.rum weakly crossreacting with gut GLI (q- 93 pg/m], p < 0.05) and absent with a non-specific antiserum. The glucagon rise was more important in hypophysectomized rats (+501 pg/ml, p < 0.001). Intraperi toncal glucose injec- t ion (0.2 g/kg) decreased plasma glueagon in resting ani- mals (--91 pg/ml, p < 0.01) but did not abolish t.be FOSWI-induced glueagon secretion (+ 133 pg/ml, p < 0.001 versus glucose-treated resting animals). Adrena l ectomy reduced the glucagon response to FOSWI ( d- 34 pg/ ml, p < 0.05), whereas propranolol protreatment (5 mg/kg intraporitonoally) completely blocked the glucagon rise. Phentolamine (25 mg/kg) increased blood glucose, plasma I R I and glucagon in resting animals, FOSWI did not further increase the already elevat~t glucagon levels ill

80 Organization Section �9 Abstracts Diabetologia

phentolamine treated rats. These results suggest tha t catecholamines and in particular the sympathetic inner- vation of the islets play a major role in exercise-induced glucagon secretion.

Evidence for a Specific Glucose Receptor by Removal of Sialie Acid from Isolated Pancreatic Islets of Mice. V. Maier, M. Hinz, H. Schatz, C. Nierle, E .F. Pfeiffer. Dept. of Endocrinology and Metabolism, Center of Inter- nal Medicine and Pediatrics, University of Ulm, Ulm, W- Germany.

The molecular basis of glucose as a signal for insulin re- lease is not yet elucidated. N-acetylneuraminic acid (sialic acid) is a functional consti tuent at the terminal non- reducing ends of gangliosides and glycoproteins which are located on the surface of cell membranes. ~-neuraminidasc (EC. 3.2.1.18) from Vibrio cholerae is able to remove sialic acid under mild conditions. -- Isolated pancreatic islets from mice were desialylated and incubated with 3H-leu- cine for 3 h at 5.5 mM and 16.6 mM glucose. Two effects were observed : (i) after disintegration of the islets, extrac- tion and gel filtration of proteins on Sephadex G-50, the radioactive incorporation rates into the proiusulin and insulin fractions were identical in normal and desialylated islets, i.e. no influence on biosynthesis was noted. (if) Secretion of insulin, however, from islets into the incuba- tion medium was inhibited to about 90% by removal of sialic acid. Mannose and galactose too were no longer able to stimulate desialylated islets, while the well known stimulatory effect due to pancreozymin, arginine, the- ophylline, and tolbutamide was not influenced in com- bination with 10 mM glucose. -- Conclu,~io~,: (1) Removal of sialic acid eliminates a specific receptor for glucose. (2) Glucose entry into the cell is not impeded. (3) The other stimuli presumably act via different pathways on insulin secretion.

Effects of Gliclazide and Diazoxide on 45Calcium Efflux from PeriIused Islets. W.J. Malaisse. Laboratory of Experimental Medicine, Brussels University, Brussels, Belgium. Various agents known to affect insulin release, such as glucose, thcophylline and epinephrine, cause dramatic and immediate changes in the radioactive efflux from perifuscd islets previously exposed to 46calcium. The aim of the present study was to extend these observations to gliclazide, a new hypoglyeaemic sulphonylurea, and dia- zoxide. At normal calcium concentration, gliclazide in- creased and diazoxide inhibited the release of calcium concomitant with insulin secretion. Under conditions known to abolish insulin release, i.e. in the absence of extracelhflar calcium or in the presence of D20, glicla- zide caused an immediate but t ransient decrease in 45Ca2+ efflux, this inhibition of the outward transport of calcium being observed both in the absence and the presence of glucose. In the presence of glucose, diazoxide provoked a rapid increase of ~6Ca 2+ efflux. -- These fin- dings are compatible with the concepts that (i) gliclazide and diazoxide modify the amotm~ of calcium accumu- lated in the beta cell by altering calcium transport ac- ross the coil membrane; and (if) the changes in calcium handling by the beta cell are sufficiently rapid to ac count for the subsequent changes in insulin release.

Effect of a VerapamU Derivative (D 600) on Calcium Uptake and Insulin Release by Isolated Islets. W.J. Malaisse, D.G. Pipeleers, F. Malaisse-Lagae, L. Orci: Laboratory of Experimental Medicine, Brussels Univer- sity, Brussels, and Inst i tutes of Histology, and Clinical Biochemistry, University of Geneva, Geneva, Switzerland. D 600, a methoxy-derivative of verapamil, is a highly

potent calcium-antagonistic inhibitor of excitation-con- traction coupling in both myocardium and myometrium. In view of the analogy between excitation-contraction coupling in muscle and stimulus-secretion coupling in the pancreatic beta cell, the effect of D 600 on glucose in- duced 4Scalcium net uptake and subsequent insulin re- lease by isolated islets was investigated. At concentrations ranging between 2 and 10 ~zM, D 600 provoked a dose- related inhibit ion of both glucose-induced calcium uptake and subsequent insulin release. The inhibitory effect of D 600 was more marked at subnormal (0.4 mEq/l) than normal (2.0 mEq/1) con. centrations of cxtracellular cal- cium. In preliminary observations, no obvious alteration of the ultrastructure of the beta cell was detected after exposure to D 600. These findings emphasize the key role of calcium in the stimulus-secretion coupling of glucose- induced insulin release.

A Peculiarity of the B-Cell Microtubules in Spiny Mice. F. Malaisse-Lagae, M. Amherdt, M. Ravazzola, W. Stauffacher, L. Orci, A.E. Renold. Inst i tutes of Histology, and Clinical Biochemistry, University of Geneva, Geneva, Switzerland.

A high incidence of carbohydrate intolerance is fmmd in the colony of spiny mice (acomys cahirinus) bred in Geneva. In spite of the large amotmts of insulin stored in their pancreas, both normoglycacmic and hyperglycaemic acomys are characterized by an abnormally low increase in circulating insulin in response to glucose, amino acids, or theophylline st imulation when compaxed to tha t seen in either Swiss, C57bl, ob/ob or DBM mice. A possible explanation for this anomaly was sought in the present stu~iy. -- Isolated islets of acomys, rats ; Swiss, C57bl, and DBM mice were incubated for 3 h in the presence of vincristine, and then examined by electron microscopy. While exposure to vincristinc caused crystalloid deposits of mlcrotubular protein in a high proportion of B ceUs in rats (74.3 • 2.8~/o), Swi~ mice (50.0 -i- 4.5~o), C57bl mice (46.2 • 5.0%) or DBM mice (27.4 • 9.6~/o), only a few crystal-containing B cells could be found in the acomys (4.9 + 2.1 ~/o)- The latter value was significantly lower than that found in normal rats and mice (p < 0.001), or even DBM mice (p < 0.05). Moreover, crystals found in acomys B cells were smaller than those in mice or rat B ceils.

A Study of the Relationships between OGTT and IVTr in 251 Patients. P. Manzano, E. Rojas-Hidalgo. Serivce of Diabetes and Nutrition. Clinica Puerta di Hicrro, Universidad Autono- ~aa de Madrid, Madrid, Spain.

Oral glucose tolerance tests (OGTT) and intravenous tol- butamide tests (IVTT) were performed in 251 subjects in order to correlate both tests. Diet, age, physical activity, site of sampling (capillary blood), and other factoFs were considered. -- The subjects were grouped as follows: Group A: Subjects with personal and/or family history of diabetes with normal fasting glycasmia levels; Group B: Subjects with changes suggesting diabetes, although showing normal fasting glycaemaa or" slight hypergly- caemia (not more than 20 mg~/o over normal range ac- cording to the Autoanalyzer method); Group C: Subjects with gastro-intestinal or liver disorders with normal fasting glycacmia or slightly hyperglycaemic. -- Both tests were evaluated on the basis of the following criteria: Studor, WHO, Fajans-Coan, and Danowski for OGTT evaluation, and Lange-Knick, Unger-Madison, and Da- nowski, for IVTT evaluation. -- The aim of the present work was to establish an accurate diagnostic correlation between OGTT and IVTT, and to discuss the criteria used for evaluating both tests.

Vol. 9, No. 1, 1973 Organization Section Abstracts 8t

Increased Alpha Cell Responsiveness to Arginine after Glucocorticoid Treatment in Man. J. Marco, D. Diaz-Fierros, C. Calle, D. Roman, M.L. Villanueva, I. Valverde. Clinics Puerto dc Hierro and Fundacion Jimenez Diaz, Universidad Autonoma de Madrid, Spain.

Since glueocortieoids (Get) and glucagon have common metabolic effects, they stimulate gluconeogenesis and may be regarded as diabetogenic, it seemed of interest to s tudy the activity of the alpha cell under the influence of Get t reatment . Young, healthy, non obese volunteers were given prednisolone (Prd) as follows: (1) in a single intra- venous dose of 100 mg over 5 min (n ---- 5), and (2) a 4-day treatment , with 40 mg/day orally (n----8). In order to st imulate glucagon secretion, arginine was employed at a dose of 468 mg/kg i.v. over a ten-minute period. Each individual served as his own control. Pancreatic ghmagon was measured by radioimmunoassay, using a highly specific antiserum (a gift from Dr. R.H. Unger). Insul in (IRI), blood sugar (BS) and amino-N were also determin- ed. The acute injection of Prd failed to modify the basal levels of plasma glucagon and insulin (range or the means: 105-- 135 pg/ml; 9-- 12 [zU/ml), BS rose by 8-- 13 ~/o, 135-- 150 rain after the injection. After oral Prd treatment, no change in the basal plasma glucagon concentration was seen (range of the means: 97--113 pg/ml) bu t a greater response to the arginine load was observed: + 117 pg/ml at 10 mill and + 97 pg/ml at 20 min (p < 0.01). This was accompanied by a statistically significant elevation of basal amino-N and overall BS and I R I levels. I t is con- eluded that : (a) a single large Prd dose i.e. is ineffective in altering basal plasma glucag0n values; (b) Gct pretrcat- ment markedly potentiates the iriitial glucagon response to arginine. A sensitisation of the alpha cell following t reatment might be a causal factor in this phenomen, either directly by Get or by a rise in aminoacidaemia, or

~ ossibly secondary to the diabetic state induced by these orlnones.

Streptozotocin Effects on B and A Cells in Monolayer Pan- creatic Cultures -- A Physiologic and Morphologic Study. E.B. Marliss, C.B. Wollheim, B. Blondel, L. Orci, A. Like, M. Amherdt, and ~W. Stauffacher. Ins t i tu ts de biochimie elinique et d'Histologie, Geneva.

Streptozotocin (SZ) has been employed in a system per- mitr ing s tudy of longterm effects in vitro. SZ was ad- ministered (1) directly to 2-day old rats (150 mg/kg i.p.) 24 h prior to enzymatic dissociation of pancreatic cells for culture, and (2) by addition to t homed ium of established cultures (0.1--5.0 mg/ml). -- I n rive injection produced hyperglycemia (180% of control) and reduction of pan- ereatic insulin (IRI) to lt)~o of control and of pancreatic glueagon (IRG) to 679/o of control. Cultures from treated animals showed low I R I content and release. Though IRG content was reduced by comparison with control cultures, this reduction was less than that of IRI , and IRG release was unaffected. -- SZ in established cultures caused a dose-related, permanent B-cytotoxic effect (with parallel I R I changes) between 0.1 and 1.0 mg/m], apparent a t 2. and maximal at 24 h. By contrast, A cells retained ap- parent normal ultrastructure. Again, reduction in IRG content with no change in release (except at highest doses) was observed. Nicotinamide and methyl-nicotinamide protected against all SZ effects on both I R I and IRG. -- Conclusions: ( 1 ) SZ exerts B-cytotoxic effects in vitro by a mechanism similar to tha t in rive. (2) Cultures poor in B cells may be produced by SZ either in rive or in vitro. (3) Though morphologic alterations occurred only in B cells, an effect on A cell IRG content was also observed. Wheth- er the latter is a direct effect or secondary to B-cytotoxi- city is not known.

Glucose Metabolism and Insulin-Glucagon Relationship in Newborn Infants of Normal and Diabetic Mothers. F: Massi-Benedetti *, A.L. Luycks* *, F. Fraeassini *, P. J. Lefebvre**, A. Falorni*. H uma n neonates were studied during the first hours of life. Blood glucose, portal plasma insulin and glucagon have been determined both at regular intervals up to 24 h after bir th and during an intraportal glucose load (IPGL) performed at the 24th h. A material presenting the im- munological chara~cteristic of pancreatic glucagon has been found in the portal plasma of both normal infants (NI) and infants 9 fd iabe t ic mothers (IDM). In NI, the glucose load was followed by an increase in portal gin- cagon in 5 out of 6 cases. On the contrary, a decrease in portal glucagon was observed in 4 out of 5 IDM. Signifi- cantly higher portal plasma glucagon was observed in the late phase of the IPGL in NI compared to IDM. Portal plasma insulin has been found higher in IDM both at the 24th h of life and during the early phase of the glucose tolerance test. In NI simultaneous injection of glucose plus insulin reproducing the hyperinsulinism of IDM, significantly reduced portal plasma glueagon. The be- haviour difference in ghmagon secretion between NI and IDM might be a consequence of the relative hyperinsulin- ism of IDM with insulin facilitating the entry of glucose into the cell thus presenting a more effective glucagon suppression.

* Is t i tuto di'Clinica Pediatrics dell 'Universitk di Perugia (It). ** Ins t i tu t de M6decine, Sccteur Diab6tologio Universit~ de Liege (B).

Insulin Secretion and Human Growth Hormone and Hy- drocortisone Behaviour in the Course of Combined Glu- cose-Arginine-Infusion in the Initial Phase after the Onset of Juvenile Diabetes. R. Menzel, D. Michaclis, I. Neumann, H. Bibergeil, B. Sehulz, W. Wilke, P. Wulfert, K. Kriimer. Central In- st i tute for Diabetes "Gerhardt Katseh", Karlsburg, DDR. A prolongation of the so-called remission phase may be of advantage for the prognosis of juvenile diabetes. These investigations were based on two groups of juvenile diabe- tics with onset at the age under 20, andbe tween 20 and 35, before treatment, and at short intervals during the first year of insulin medication. -- Control groups consisted of 20 healthy subjects and 11 long-term diabetics with onset under age 30 and more than 10 yrs' t rea tment with in- sulin. The levels of blood glucose, free fat ty acids, gly- cerol, hydrocortisone (serum) total immunoreactive in- sulin (IRI) and human growth hormone (plasma) were measured in the course of a combined glueoso-arginine infnsion. After the test, samples for determination of insulin antibodies were taken. In agreement with the improvement of glucose tolerance and fat metabolism during glueose-arginine infusion, restitution of insulin secretion was found. HGH and hydrocortieene seemed to have no influence on the remission phase. These results are discussed with regard to the characteristic behaviour of ant ibody ti tre and the management of diabetes.

Effects of Pancreozymin and Caerulein on Insulin and Glucagon Secretion in Normal Subjects and in Patients with Insuloma. G. Menzinger, F. Fallucca, F. Tamburrano, R. Carratu' , D. Andrcani. Cattedra di Endocrinologia, I I Clinics Me- dica, Universitk di Roma, I taly. Pancreozymin is known to stimulate insulin and glucagon secretion. Caerulein is an extractive deeapeptide with a spectrum of effects overlapping those of pancreozymin. Therefore changes in plasma insulin (IRI) and glucagon (IRG) were studied after pancreozymin and caerulein in normal subjects and in patients with insuloma. Rapid

Diabetologia, Vol. 9 6

82 Organization Section �9 Abstracts Diabetologia

!1 rain) intravenous injection of pancrcozymin (75 IDU) m 9 normal subjects was followed by a significant in- crease both of I R I and of IRG. After rapid intravenous administrat ion of cacrulein (10 ng/kg) in 6 normal subjects there was no increase of IRI , while there was a significant increase of IRG levels, which was even ~Tcater than after pancreozymin. I t appears tha t caerulein represents a more powerful stimulus for glucagon than for insulin secretion. -- In three out of 7 patients with insuloma there was a marked response of I R I levels to pancreozy- rain administrat ion: a similar behaviour of I R I values was observed in the same subjects, following caerulein. -- An increase of IRG levels was registered in 3 out of 4 patients after pancreozymin and in 1 out of 3 patients after caerulein. Patients with insuloma seem to differ from normal subjects in the response to these incretory

�9 stimuli.

Dynamics of Insulin Secretion During Infusion of Ketone Bodies in Dogs. P. Metzger, P. Franken, E.O. Balaese. Laboratories of Pathophysiology and of Experimental Medicine, Uni- versity of Brussels. Ketone bodies are known to stimulate insulin secretion and to induce hypoglycemia in vivo. The aim of the present work is to study the dynamics of insulin release during a steady hyperketonemia in dogs. -- Two groups of 5 normal dogs each were given a constant infusion of Na acetoacetate (55--128 izmoles/kg rain) for 60 rain. Levels of aceto- acetate, ~-hydroxybutyrate, glucose, IRI , FFA and gly- cerol were followed in arterial blood; IR I concentrations were also measured in the portal veonous blood. In one of the groups, glycemia was allowed to fall, whereas in the other one, hypoglycemia was prevented by a continuously adjusted glucose infusion. -- In the first group, steady hyperkstonemia (6--7 ixmoles/ml) was obtained from the 10th min onwards. In both arterial and portal venous blood, I R I levels rose sharply, a t ta ining a peak concen- t rat ion (about 300~ of baseline value) 5 min after t h e onset of the ketone infusion; thereafter, I R I levels rap- idly returned to control values. A prolonged fall in glu- cose (30%), FFA (40O//o) and glycerol concentrations (40%) was observed. -- I n the second group, glycemia was maintained at its normal level during the whole ex- periment and I R I concentrations remained high (about 300% of basal value) throughout the s tudy in both arterial and portal venous blood. The rise in ketone con- centration and the decrease in FFA and glycerol levels were similar to those observed in the first group. -- I t can be concluded that glucose exerts a permissive effect on the insulinotropic action of ketone bodies in r ive . This is in

eement with in vitro data showing that the sensitivity he ~-cells to ketones is markedly decreased a t low

glucose concentrations.

Patterns of Growth Hormone and Insulin Secretion in Protodiabetic Subjects. D. Michaelis, R. Michael, I. Neumann, B. Schulz, R. Menzel, W. Hildmann, E. Jutzi, H: Bibergeil. Central Ins t i tu te for Diabetes "Gerhardt Katseb", Karlsburg, DDR. I n order to assess the possible role of human growth hor- mone (HGH) in the pathogenesis of diabetes mellitus, the behaviour of the plasma levels of (i) HGH, (if) immunore- active insulin (IRI), and (iii) blood glucose were measured during glucose-induced st imulation of insulin secretion in 20 healthy and more than 50 protodiabetic subjects. A priming injection of glucose of 0.33 gm/kg body weight was followed by a glucose infusion of 12 mg/kg/min for 2 h. -- Healthy subjects without family history of diabetes demonstrated a mono- or biphasic reaction of HGH levels. More than 50 per cent of protodiabetic subjects

s h o ~ d either invariabili ty of HGH secretion or a para- doxical rise during hyperglycaemia. Tbose abnormal reactions of HGH secretion could not be related to the altered dynamics of insulin secretion but only to the degree of disturbed carbohydrate tolerance. 'I~e results support the hypothesis that tbe paradoxical increases in plasma levels of HGH are rather the sequel than the cause of carbohydrate intolerance.

Significance of the a and b-Components as to the Antigenic- ity of Different Porcine Insulin Preparations. J. Michl, S. Fankhauser, J. Sehlichtkrull. Medical Out- pat ient Department, University of Berne, Switzerland, and NOVO Research Inst i tute , Copenhagen, Denmark.

Three groups of non-diabetic psychiatric patients who were not previously insulintreated, underwent insulin shock therapy for 2 months. They were given the following in- sulin preparations: The patients of group I (n : 11) re- ceived monocomponent pork insulin; those of group I I (n = 10) were administered ten times recrystallized perk insulin containing 0.0% a-component and 12~/o b-com- ponent, mostly added. Group I I I (n = 11) had one-time crystallized pork insulin containing 9~ a-component plus 4O//o b-component. Two methods were used to quanti ta- t ively assess the antibodies against insulin. The patients of group I had no antibodies. An increase in the ant ibody titre was observed in 3 patients of group I I (30%), while the same was true in 8 patients (73O/o) of group I I I . The increase in antibodies against insulin is not significant when group I is compared to group I I (p = 0.10), while the difference between groups I and l I I and I and II , is statistically significant (p < 0.02 and p < 0.05, respectively). A good correlation exists between the two methods, except for 2 cases in group II . -- Conclusions: Monocomponent pork insulin does not show antigenicity under the conditions of these tests, while, on the other hand, the a-component and/or the combination of a- and b-components does not seem to have more impact as re- gards the antigenicity of the insulin preparations than the b-component by itself.

Analyse de la Lipacidemie et de L'Insulinemie lors des Surcharges en Glucose, Fructose ou Galactose .chez des Sujets Obeses, Maitres ou Norrnaux avee ou sans Diabete Sucre. J. Mirouze, A. Orsetti, Y. Vierne, N. Arnoux. Cliniquo des Maladies m4taboliques et endoeriniennes, Montpellier, France.

37 malades sent r@partis en 6 groupes: los uns ne sent pas diab6tiques (poids normal -- surpoids et sous-poids re- speetivement de 327 q- 45 et 281 • 35 g/kg de poids id6al). Los autres sent at teints de dial~te discret (poids normal -- surpoids et sous-poids respeetivement de 522q-81 et 281 • 35 g/kg de poids id~d). -- De tous los rdsultats pr~ent~s et diseut~s, il convient de souligner l ' identit~ de la r6ponse insulin@mique chez tousles sujets sans diab~te chimique quel que soil le poids, alors que chez los ob~sos diab6tiques, le fructose ot le galactose enclenchent une r6ponso franche et durable, significative, non ou peu objeetiv~e ehez los maigros : fructose maxinmm 60e minute 75.23-t-12.47 ~tu/ml -- galactose maximum 30e rain 87.14 q-31.64 tzu/ml. De m6mo, la lipacid6mie chute de facon comparable choz tous los individus sans diab~to chimique, alors que chez los ob~ses diab6tiques, la lipacid6- mie de je6ne est significativement plus 61cede et l'abaisse- ment constant mais plus intense sous charge on glucose, tandis que chez los diab6tiquos maigros, le galactosc abaisse peu la lipaciddmie comparativement au glucose et au fructose. Dans cos derniers groupes, le galactose en- gondre trAs r i te une ~l~vation durable hautement signifi- cative de la m~llit6mie.

Vol. 9, No. 1, 1973 Organization Section �9 Abstracts 83

Congenital Malformation in the Offspring of Diabetic Women. L. Molsted-Pederson, Inge Tygstrup, /~ge L. Villumsen and Jorgen Pedersen. Diabetes Center, Obstetrical I)e- par tment and Laboratory of Paediatric Pathology, Rigs- hospitalot, Copenhagen.

The frequency of congenital malformation (c.m.) in the newborn infants of diabetic mothers have beer, consecu- tively and prospectively studied. Of 1435 infants (birth weight 1000 g or more) 8.1~o had c.m. In a consecutive control series of 8789 newborn infants of non-diabetic mothers born in the same hospital e.m. were found in 2,8~). No difference was fotmd in the average and distribu- t ion of maternal ages in the diabetic and non-diabetic group. -- In the newborn infants of diabetic mothers the over-all frequency and the frequency of major congenital malformations seem to be three to four times as high as in the general population. -- As previously reported from this centre the frequency of c.m. was increased in infants of mothers with vascular complications (White's classes D and F). -- By comparison of the different types of malformations in the diabetic and non-diabetic materials we did not find any "specific diabetic" malformation apart from 3 cases with the combination of sacral agenesis and severe malformation of the lower limbs.

The Mechanism of Action of Cyclic AMP in Rat Islets of Langerhans. iV. Montague and S.L. Howell. School of Biology, Uni- versity of Sussex, Fahner, Brighton, BN 1 9QG, Sussex.

The secretion of insulin and glucagon appears to be con- trolled at least in par t by the intrac011ular concentration of cyclic AMP in islets of Langerhans. We have shown that the effects of cyclic AMP in islet cells may be related to its abil i ty to activate a cyclic AMP-dependent protein phos- phokinase, this act ivat ion occurring as a consequence of the binding of the nuclootide to a specific receptor subuni t of tim enzyme. -- Protein phosphokinaso act ivi ty was investigated in homogonates prepared from islets of Lan- gerhans which had previously been incubated in the pros- ence or absence of agents known to affect the intracellular concentration of cyclic AMP. Theophylline, which raised cyclic AMP concentrations in the intact islets, increased the protein phosphokinase activity in the homogenates whereas adrenaline, which lowered cyclic .4~P levels, decreased the enzyme activity. These results indicate tha t alterations in the intracellular concentration of cyclic AMP in islets of Langorhans may produce changes in the act ivi ty of protein phosphokinaso in intact islet cells. -- I t is suggested that cyclic AMP may exert its effects on hormone release from this tissue by increasing the act ivi ty of a protein phosphokinase which in turn promotes the phosphorylation and act ivi ty of a rate determining com- ponent of the secretory mechanism.

Characterisation of "Incretin". A.J. Moody, G.S. Agerbak, F. Sundby. Note Research Inst i tute, Copenhagen, Denmark.

A revised bioassay for insulin releasing act ivi ty in vitro will be prescnted. I t consists of measuring the insulin out- put of pieces of the neonatal rat pancreas in the presence of different concentrations of fractions prepared from crude extracts of pork intestine. This system has proven to be more reliable than a similar bioaesay using adult rat islets. -- The basic parameters of the bioaseay are: 1. In- sulin output is not increased by glucose, panereozymin, secretin, V.I.P., glucagon or highly purified gut GLI. 2. Insul in output is increased by a factor present in extracts of pork intestine and by theophylline. 3. Insulin output is decreased by anox ia ' and by the ammonium ion. -- A comparison of the effects of secretin, pancreozymin and an

incretin-containing fraction at the same concentrations ( 1 -- l 0 ~g/ral) shows conclusively tha t the insulin releasing factor present in the extract is neither secretin nor pan- creozymin.

A Study of Growth Hormone (HGH) S,~retion in Diabetic Retinopathy. M. Muggeo, G. Crepaldi, D. Fedole, A. Baritussio. Cliniea Medica-Policlinieo, UniversitY, Padova (Italy). The suppression of HGH secretion by hypophysectomy is often efficacious in preventing the evolution of diabetic ret inopathy (DR). HGH is considered an important causal factor in the development of DR. However, not all the Authors agree that there is an increase in HGH secre- tion in juvenile diabetes with or without retinopathy. In 12 non-obese insulin-dependent diabetic patients w i t h hemorrhagic-proliferative diabetic rotinopathy the twen- ty-four-hour plasma H G H pat tern (with determination of plasma HGH, NEFA and glucose every three hours) and the HGH response to insulin induced hypoglycemia (0.2--0.3 u/kg pbw) have been evaluated. A comparison, with 6 controls and 10 acromegalic patients was carried out. There was no significant difference between the dia- betic and normal subjects during twenty-four-hour HGH pattern. The response to insulin in patients with DR was delayed and lower than in controls. This behaviour must be related to the delayed blood sugar fall in patients with DR in comparison with normal subjects, although the degrec of hypoglycemia was 'the same. In acromegalic pa- tients the HGH levels during twenty-four-hour pat tern and after insulin were higher than in other groups. Our results demonstrated that in DR there is no significant increase of HGH secretion in basal conditions and after insulin induced hypoglycemia, even if the plasma HGH levels could be considered higher in relation to the elevat- ed blood sugar levels.

Adrenal Function in the Obese-Hyperglycemic Syndrome (obob) in Mice. Peter Naeser. Deparments of Pathology and Histology, Universi ty of Uppsala, Uppsala, Sweden. Previous studies have demonstrated a marked hyper- plasia of the adrenal cortex in mice with the obese-hyper- glycemic syndrome (obob). In order to evaluate whether increased secretion of cortieosteroids contribute to the manifestations of the syndrome the effects of adrenalee- tomy have now been studied in obese animals of various ages, In addition, plasma levels of eorticostercids have been measured according to Lever (197 I) before and after induction of stress. I t was found that wi thin a week after adrenalectomy both insulin and glucose levels wore significantly reduced in obese mice and remained so for up to 36 weeks. In the lean lit termates the corresponding t reatment caused a less marked and more gradual reduc- tion in blood glucose levels whereas the immunoreactive serum insldin level remained unchanged. In no case was there any significant change in weight gain in adrenaloc- tomized animals as compared to s h a m operated controls. Obese mice of various ages consistently exhibited signifi- cantly raised levels of serum corticosteroids as compared to their lean littermates. Both obese and lean animals re- sponded to stress with a significant increase of serum cortieosteroids. Similar observations were made after in- jection of adrenocorticotropic hormone. The results con- firm the previous suggestions of a h:!(perfunction of the adrenal cortex in obese-hyperglycemm mice. They also indicate that the adrenal glands are of etiologic significance for the insulin resistance of these animals.

Insulin Kinetics in Normal and Diabetic Subjects. R. Navalesi; A. Pile, S. Lenzi, P. Cecehetti, G, Corsini, L. Donate. CNR Laboratory of Clinical Physiology, Uni- versity of Piss, Pisa, I taly.

84 Organization Section �9 Abst rac ts Diabetologia

Insulin metaboL "~., in man has been extensively s tudied using radioiodine labelled hormone and the radioimmuno- logical technique. Yet the experimental procedure has often been unsatisfactory, mainly due to denatura t ion of the labelled hormone, inadequate separat ion of p lasma ac t iv i ty fractions, and incorrect elaborat ion of the ex- perimental data. -- In the present s tudy the insulin meta- bolic clearance rate (MCR) was measured using a single injection of insul!n 125I a t the iodinat ion degree of 0.2 iodine a tom per molecule. MCR was calculated as the ra t io between t h e injected dose and the integral of the p lasma concentration of immuno-precipi table ac t iv i ty ; ex- changeable insulin body pool (EIBP) has been evaluated on the basis of the t r ans i t t ime dis t r ibut ion function and of the p lasma insulin concentration determined by the method of Hales and Randle. Interference of antibodies in diabetics previously t rea ted with insulin has been avoided. -- In 20 normal subjects, MCR was 671 • 136ml/min, and 757 ~ 184 ml/min in 10 insulin~-independent diabetics (IID), while i t a t ta ined 210-~ 104 ml/min in 8 insulin- dependent diabetics (IDD). E I B P was increased in I ID , and t o a greater extent in IDD.

Anti-Pancreatic Cellular Hypersensitivity in Diabetes Melli- tus. J. Nerup, O. Ortved Andersen, G. Bendixen, J . Egeherg, J .E . Poulsen. Steno Memorial Hospital , Depa r tme n t of Medicine TA, Rigshospital , and Ana tomy Depar tment B, Universi ty .of Copenhage/l, Denmark.

Using fetal Qalf pancreas ext rac t as antigen, 74 diabetics (group A: 20 with juvenile, unt rea ted diabetes or insulin- t rea ted for less than one year, group B: 34 juvenile dia- betics t rea ted with insulin more than one year, and group C: 20 matur i ty-onse t cases) and 31 controls were examin- ed for the occurrence of ant i -pancreat ic cellular hyper- sensi t ivi ty (APCH) by means of a leukocyte migrati~)n test (LMT). Resul ts : APCH was demonstrable in .24~/o of the patients . The antigen was organ-specific, species-non- specific and independent of insulin. APCH was predomi- nan t ly found in group A. -- Ra t s immunized with a single intracutaneous dose of fetal calf pancreas extract 'present- ed the following findings: A s ta te of specifically al tered immunologcal reac t iv i ty (APCH) was induced. Morpho- logical abnormali t ies ( lymphocytic infil tration and de- generative islet changes) were demonstrable, bu t the carbohydra te tolerance was unaffected. - - Da ta on the capabi l i ty of some viruses (Coxsaekie B4 and EMC) to cross-react with antigenic components of the endocrine pancreas in the LMT system will bepresen ted .

The Influence of Rat Weight and Cell Size on Lipogenesis and Lipolysis in Isolated Fat Cells from Fed Rats. J. Hoiri is Nielsen, F. Molgaard Hanson, J . Gliemann. Sten0 Memorial Hospital , Research Laboratory , Gentofte, and Ins t i tu te of Medical Physiology, C, Univers i ty of Co- penhagen, Denmark.

The influence of cell size on basal and insulin s t imulated conversion of [U-l~CJglucose to tr iglycerides by isolated epididymal fat cells was assessed using ad lib fed rats weigh- ing 100--500 g showing increasing mean cell size. -- Aft- er the incubation, aliquots of ceils from each animal were fixed with osmium tetroxide and fract ionated according to size by passage through nylon filters of decreasing mesh width. I t was found tha t within the same animal both the basal and the insulin s t imulated (10 a ~tU/ml)l ipogenesis per cell increased with cell size. The per cent increase due to insulin was equal for all cell sizes. - - Comparing fat cells of the same size from rats of different w~ight the basal lipogenesis was the same, while the insulin effect increased sl ightly with r a t weight from 100 to 250 g and then decreased markedly with ra t weight from 250 to 500 g. - - In contrast , ACTH st imulated (0.1 lzg/ml) If-

polysis increased with the mean cell size on the whole range of ra t weights. -- I t is concluded tha t factors other than the cell size per se (e.g. age, degree of obesity) determine the responsiveness of fat cells to insulin.

Insulin Biosynthesis: Effects of Metabolic Inhibitors. A. Niki, H. Niki, T. Koide, B . J . Lin. Depar tment of In terna l Medicine, Aiehigakuin Universi ty, Nagoya, Ja- pan and Depar tment of Physiology, Univers i ty of Toron- to, Toronto, Canada.

The effeCtS of metabolic inhibitors on the biosynthesis of insulin were studied i n vitro using isolated islets of Langer- hans from rats. Incorporat ion of 3H-leucine into proinsulin and insulin was used to indicate prolnsulin synthesis and insulin formation. Mannoheptulose (3 mg/ml) markedly inhibited the biosynthesis of insulin, a t either the low (1 mg/ml) or high (3 mg/ml) concentrat ion of glucose. 2-Deoxyglucose (3 mg/ml) inhibited the insulin biosyn- thesis induced by the low concentration of glucose (1 rag/ ml), while i t was ineffective a t the high concentration of glucose (3 mg/ml). Iodoaeeta te (0.1 raM) did not inhibi t the insulin biosynthesis induced by either 1 mg/ml or 3 mg/ml of glucose. -- However, when pyruva te (10 raM) was added to 1 mg /ml of glucose in the presence of 2- deoxyglucose (3 mg/ml), i t restored the insulin biosynthe- sis. I t would suggest t ha t 2-deoxyglueose inhibited only the bioenergetic effec t of glucose on the insulin biosyn- thesis, and the signal(s) to ini t iate biosynthesis might be located not beyond the level of glueose-6-phosphate. Moreover, glueosamine (3 mg/ml) unexpectedly did not modify the glucose-induced insulin biosynthesis ut the concentrat ion tha t is enough to inhibi t the secretion of insulin. Since both mannoheptulose and glucosamine are known to inhibit glucose phosphorylat i0n , the possibi l i ty could not be neglected tha t glucose itself, ra ther than its mvtabolites, triggers insulin biosynthesis.

Insulin Biosynthesis: Effects of Oral Hypoglycemic Agents. A. Niki, H. Niki, T. Koide, B . J . Lin. Depar tment of In ternal Medicine, Aichigakuin Universi ty, Nagoya, Ja- pan and Depar tment of Physiology, Univers i ty of Toron- to, TorQnto, Canada.

The effects of sulfonylurea ( tolbutamide and glibenclam- ide) and biguanide (dimethylbiguanide) on the biosynthesis of insulin were s tudied using the islets of Langerhans of male Wistar rats. The islets were isolated by collagenase digestion and incubated for 3 h in a medium containing 1 mg]ml or 3 mg/ml of glucose, with or wi thout the agents tested. The concentrations of Na-~olbutamide, glibenc. lamide and dimethylbiguanide were 0.1 mg/ml, 0.5 lzg/ml and 0.1 mg/ml respectively. Incorporat ion of aH-leucine into proinsulin and insulin during incubation was used to indicate the biosynthesis of insulin. - - Sulfonylurea, both to lbutamide and glibenelamide, inhibited the bio- synthesis of insulin a t the low concentration (1 mg/ml) of glucose, while a t the high concent ra t ion (3 mg/ml) of glucose, nei ther modified the insulin biosynthesis. When the islets were preineubated with glibenclamide for 30 rain, insulin biosynthesis did not occur during 2 h of in- cubat ion a t 1 mg/ml g l u t e n , whereas the insulin bio- synthesis was not modified a t 3 mg/ml glucose. Biguanide had no effect on the biosynthesis of insulin a t ei ther the low and high concentration of glucose. None of the oral hypoglycemic agents tested had the direct s t imulat ing effect on the insulin biosynthesis of the islets, a t least, in our experimental conditions.

Capillary Basement Membrane Thickness in Early Stages of Diabetes Mellitus. R.E . Nikkels, J . Terpstra. Diabetes Uni t of the Dept. of Metabolism and Endocrinology, U n i v e r s i t y Hospi ta l , Leiden, the Netherlands.

Vol. 9, No. I, 1973 Organization Section �9 Abstracts 85

The basement membrane thickness of capillaries in the quadriceps fcmoris muscle was examined according to the method of Siperstein in the following groups of pa t ien ts : 12 pat ients under 21 years with juvenile diabetes; 16 young pat ients with newly-diagnosed diabetes; 18 women with la tent diabetes (having an abnormal glucose toler- ance during pregnancy only); 10 persons with potent ia l diabetes (normal glucose tolerance, bo th parents having diabetes) ; and a control group of 22 subjects younger than 26 years with a normal cortison GTT and no family his tory of diabetes. We found no significant difference between normals and subjects wi th potent ia l or la ten t diabetes. There was also no significant thickening in the group with newly-diagnosed diabetes. The mean values for the nor- mal group were lower than the values repor ted by Am- herd t et aL (Acta Diab. Lat . 8 suppl. 1, 88, 1971): Our findings suggest t ha t the thickening does not precede the clinical onset of the dis turbance of the carbohydra te metabol ism.

Glueagon Biosynthesis in the Isolated Perfused Rat Pan- creas. A. Gay, R .H . Oakman and Norman R. Lazarus. Diabetes Research Unit , The Wellcome Foundat ion, T e m p l e H i l l , Dartford, Kent , England.

The isola ted ra t pancreas was perfused b y recireulation with Gey and Gey buffer - - 4~/o dext ran p H 7.4 containing aH- t ryp tophan (25 Ci/mmole) and 5 mM glucose. The pancreat ic duct was t ied to prevent exocrine secretion. After perfusion varying from 4.5 to 12 h, the pancreas was ext rac ted by the procedure of Kenny (J. clin. Endocr., 15, 1089, 1955). The ex t rac t was filtered on Sephadex G.50 Superfine in 1M acet ic acid (60 • 1.6 cm). A peak m~grat- ing in the elut ion posi t ion of bovine/porcine glucagon could be detected. The following charac te r i s t i cs were observed. 1. Elut ion posi t ion was identical wi th glueagon. 2. The elution posit ion corresponded with the glucagon immunoreac t iv i ty ext rac ted from the pancreas. 3. The amount of mater ia l synthesised was reduced b y the pres- ence of 15 mM glucose. 4. Synthesis is to ta l ly abolished b y cycloheximide (185 ~g/ml). 5: Synthesis increases wi th t ime and is not detected under 4 h incubation. 6. Strepto- zotocin t r ea tment of the pancreas while stil l in vivo, did not inhibi t in vitro glucagon synthesis and in addi t ion the streptozotocin abolished the sensi t ivi ty to glucose con- centra t ion (Fussganger ctal., Diabett)logie, 7, 478, 1971). A t ryp tophan containing protein of molecular weight less than the exclusion l imi t of Sephadex G-50 and larger than insulin is ex t rac ted along with the glueagon. This prote in is also ~veakly immunoreact ive and its relat ionship to glucagon will be discussed.

Evidence for the Existence of two Catabolic Pathways of Insulin in Vivo. H.A. Ooms, and J .R' .M. Franckson.Laboratoi re de Chi- mie Mg~licale, Universi td de Bruxelles.

Anaesthet ized dogs were infused with graded amounts of crystal l ine and labelled insulins by the "pr iming dose- constant infusion" technique. Catheters were inserted into the aor ta , the renal, por ta l and hepat ic veins, the thoracic duct , the main bile duct and the bladder. Hepat ic , por ta l and renal blood flows were est imated. Parameters s tudied included total , hepat ic and renal metabolic clearances o f cold and labelled insulins, urine, bile and lymph excretions, regional balances of radioiodine. Measurements were carried out in s teady s ta te ; a range of insulinemia from 30 ~tU/ml to 6 U/ml was tested. Insul in catabol ism b y the kidn,ey was pract ica l ly not influenced by the increase in concentrat ion and b y the iodinat ion of the hormone; the degradat ion of the molecule was rap id and complete leading to a release of free iodide balancing the up take of labelled insulin. The hepatic ra te of catabol-

ism was rapid ly al tered by increasing the p lasma level and b y iodination. The labelled insulin taken up was only par t ia l ly degraded to free iodide and par t ia l ly remained t rapped into hepat ic tissues. -- The physiological role of both pa thways which normal ly account for more than 70% of to ta l catabolism is discussed with regard to their chemical characteristics.

Early Effects of Alloxan and Streptozotoein on B-Cells: A Freeze-Etching Electron Microscopic Study. L. Orci, M. Amherdt , W. Stauffacher, A.A. Like, C. Rouiller, A .E . Rcnold. Ins t i tu tes of Histology, and Clini- cal Biochemistry, Univers i ty of Geneya, Geneva, Switzer- land. The diabetogenie ~gents al loxan (A1) and streptozotocin (SZ) induce B-cell necrosis within 4-- 10 h subsequent to their adminis t ra t ion: Al act ion is almost immediate ly irreversible, bu t t ha t of S Z can be prevented during t h e init ial 2 h. Although the B-cell p lasma membrane is con- sidered the p r imary site of Al action, convincing altera- t ions are not detected b y conventional electron micro- scopy during the early phase when functional al terat ions arc demonstrable. An a t t e mp t was thus made to visualize al terat ions in B-cell p lasma membrane by means of the freeze etching technique. This method reveals two fracture faces of p lasma membrane : i.e. face A, directedr the exterior of the cell, and face B, directed toward the cyto- plasm. -- Diabetogenic quanti t ies of A1 (200 mg/kg) and S Z ( 150 mg/kg) were administered int rapcr i toneal ly (i.p.) to normal spiny mice (acomys cahirinus), and the pan- creatic B-cells were s tudied a t t imed intervals~ In control B-cells, face A of p lasma membrane appeared s tudded with numerous (2297• ~2) randomly d is t r ibuted particles, averaging 85 A in diameter and thought to represent enzyme complexes . An irregular d is t r ibut ion and numerical reduc t ion of these part icles was noted within 10 min of exposure to A1 (702 • 45 ~ ) and 90 min

2 of exposure to S Z (913 4- 90 ~t ) .-- These results suggest t ha t a l loxan and streptozotocin induce s t ructura l 'd isarray a n d possible impai rment of B-cell p lasma membrane early in the course of events which lead to B-cell necrosis.

Exocytosis-Endocytosls Coupling in the Pancreatic B- Cell. L. Orci, F. Ma!aisse-Lagas, M. Amherdt , M. Ravazzola, C. Rouiller, A .E . Renold. Ins t i tu tes of Histology, and Clinical Biochemistry, Geneva, Switzerland. Emiocytosis in the B-cell invoives the incorporation of the secretory granule membrane into the p lasma membrane, leading theoret ical ly to an augmentat ion of the cell sur- face. In s t imulated ceils, the formation of microvill i might represent one mechanism b y which approximate constancy of the cellular volume is mainta ined despite augmented surface. I n addit ion, a compensatory reloca- t ion of segments of membrane from the cell surface into the intracytoplasmic space might occur during prolonged st imulation. In order to assess this phenomenon, we have invest igated here whether secretory ac t iv i ty m i g h t b e coupled with endocytosis, thus allowing for such a re- cycling of membrane consti tuents within the B-cell. -- I sola ted islets of the ra t were incubgted a t two glucose levels and for various lengths of t ime in the presence of horseradish peroxidase (HRP), a marker of endocytotic act ivi ty . An increased up take of H R P was detected a t high glucose concentration, and persisted after cessation of glucose st imulation. HRP-conta in ing vesicles moved to the Golgi area, where they accumulated in great number. - - The present s tudy indicates tha t s t imulat ion of the B- cell by glucose results in enhanced endocytotic act ivi ty . This exoeytosis-endocytosis coupling might represent a mechanism by which membrane consti tuents are re- cycled within the B-cell, under conditions of increased secretory ac t iv i ty .

86 Organization Section �9 Abstracts

The Role of Cell Size on the Lipolysis and the Insulin Sensitivity of Human Subcutaneous Adipose Tissue. J. Ostman, L. Backman, E. Cerasi, R. Luft, and D. Hall- berg. Huddinge and Karolinska Hospital, Stockholm.

Specimens of human subcutaneous adipose tissue were removed from subjects with marked difference in body weight, patients with recent onset diabetes mellitus of juvenile and adult type, and from obese subjects before and after intestinal shunt operation. The production of glycerol by sections of adipose tissue of the non-diabetics was under basal (glucose cone. 5 mM) as well as stimulated conditions positively correlated to the mean cell volume estimated according to the histiometric procedure. The maximal lipolytic responses to theophylline and dibutyryl cAMP, expressed as the relative change in the glycerol production above the basal values, were negatively re- lated to the mean cell volume. The addit ion of alpha- adrenergie blocking agent increased more markedly the noradrenaline-induced glycerol production by small fat cells than that by enlarged cells. Fat cells of insulin-de- pendent diabetics exhibited accelerated lipolysis, which was not enhanced further by the addition ofnoradrenaline or isopropylnoradrenaline. The antilipolytic effect of in- sulin (100 Iz U/ml) (glucose cone. 0) was strongly related to the mean cell volume when calculated as the percentage change below the vasal lipolysis. -- The findings clearly show that the ~ize of the fat cells has substantial influence on the lipolysis and the insulin sensitivity of human fat cells. The results might be of relevance to the understand- ing of the lipid metabolic situations in r ive in obesity and in insulin-deficiency.

Postischaemlc Polarography in Calf Muscle for Evaluation of the Peripheral Vascular Changes in Diabetes. K. Ostrowski, I I I r d Department of Internal Diseases, Medical Academy, Warsaw, Poland.

The method of postischacmic polarography was used, and the term "oxygen tension recovery time" introduced and defined as the time interval between the release of oc- clusion and the reaching by the oxygen tension curve of the point corresponding to 75% recovery from the drop during occlusion. -- The material comprised 171 subjects divided into the following groups : I -- control : 42 healthy subjects, I I -- 64 juvenile-onset diabetics, I I I -- 65 pa- t ients with atherosclerosis. The polarographic method was compared with digital photoplethysmography and, for the diabetics, with ophthalmoscopic examinations. -- The mean values for "oxygen tension recovery t ime" were: Group 1--27 see, I I - - 4 5 sec, and IYI--85 sec. -- These findings point to high sensitivity of postischaemic polarography, which indirectly reflects changes in muscle blood flow, and may prove useful for examination of pe- ripheral vascular changes in diabetes.

Effects of 2-Aminonorbornane-2-Carboxylic Acid on Insu- lln Secretion and Metabolism of Isolated Pancreatic Islets. U. Panten, J. Christians. Ins t i tu te of Pharmacology and Toxicology, University of GSttingen, Germany.

The stimulation of insulih relem~c by the non-metabolized leucine analogue 2-aminonorbornane-2-carbexylic acid (BCH) has been used as an argument against the view that L-leucine induces insulin secretion only after being metabolized. When perifusing pancreatic islets of obese- hyperglycaemie mice in the absence of substrate, the addition of BCH (20 raM) triggered insulin release with an initial overshoot. An accompanying increase of fluores- cence of reduced pyridine nucleotides (PNH) began 10-- 15 see after BCH reached the islets and rose with a two- phase kinetics. In the presence of glucose (5 raM), only a slight insulin release wa.q noted after addition of BCH (20 raM) and the kinetics of PNH-:fluorescence were also

Diat,etologia

changed. -- These rapid metabolic effects of BCH strik- ingly resemble those induced by L-leucine. ] t seems thus possible that BCH triggers insulin secretion indirectly by metabolic event, s in the beta cell.

Increased Capillary Permeability to Albumin in Long- Term Juvenile Diabetes Mellitus. H.-tt . Parving, S. Munkgaard Rasmussen. Dept. of Clini- cal Chemistry A, Rigshospitalet, University of Copenha- gen, and Nero Research Inst i tute, Hvidore Hospital, Co- penhagen, Danmark.

Transcapillary escape rate of albumin (fraction of intra- vascular mass per uni t time) was determined during the first hour after an intravenous injection of 131I human serum albumin in eleven non-diabetic, ten short-term diabetic (mean durat ion 2.3 years) and eleven long-term diabetic subjects (mean duration 16.8 years). The inves- t igation was preceded by at least 12 h fasting and 30 min rest in supine position. -- Transcapillary escape rate of a lbumin was found significantly increased in the long- term diabetic group, average 8.0 (SD -F i.0) per cent/hour, compared to the short-term group, average 5.7 (SD • 1.0) per cent/hour and to the non-diabetic group, average 5.8 (SD :t: 0.9) per cent/hour (p < 0.0001). Similar results were obtained for the efflux of a lbumin (mass per unit time), (p < 0.05). -- I t is assumed that the present findings re- flect an increased capillary permeabili ty to a lbumin due to the presence of diabetic microangiopathy, which prob- ably makes the capillaries abnormally leaky to albumin.

The Possible Role of a Glycolytic Metabolite as a Signal for Glucose-Induced Insulin Biosynthesis and Release. D.G. Pipelcers, W.J . Malaisse. Laboratory of Experimen- tal Medicine, Brussels University, Brussels.

By combining measurements of insulin synthesis, calcium uptake and insulin release by isolated islets, we have at tempted to localize the metabolic sequence which could mediate the s t imulant action of glucose on biosynthesis and secretion of insulin. Iodoacetate (I.0 raM) markedly inhibited glucose-induced insulin synthesis, calcium up- take and insulin release. At a lower concentration (0.1 raM) a lesser but significant inhibition of all three processes could still be detected. Moreover, even at low concentra- tion, iodoacetate altered the ratio of (pro)-insulin to total protein synthesis. Except for this latter abnormality, addition of a mixture of pyruvate, glutamate and fumar- ate (each 5.0 mM) to the incubation medium failed to modify the iodoacetate-induced inhibitions. Sodium fluoride, in the 2.0 to 0.2 mM range, caused a dose-related inhibition of insulin synthesis, bu t had little if any effect on ghmose-induced calcium uptake and subsequent insu- lin release. Sodium fluoride caused the same abnormali ty in the ratio of (pro)-insulin to total protein synthesis as tha t evoked by iodoacetate. Addition of the pyruvate- glutamate-fumarate mixture to the incubation medium reduced or abolished all the effects of sodium fluoride. -- These findings are compatible with the hypothesis that (i) insulin biosynthesis is ve~" sensitive to partial depletion of ATP; and (ii) tha t a glycolytic metabolite in the 1-3- diphosphoglycerate to 2-phosphoglycerate sequence serv- es as a signal in both ghmose-induced insulin synthesis, on one hand, and glucose-induced calcium uptake, and subsequent insulin release, on the other hand.

Metabolic, Cationic and Pharmacological Influences on In- sulin Biosynthesis. D.G. Pipeleers, M. Marichal, ~u Malaisse. Laboratory of Experimental Medicine, Universi ty of Brussels, Brus- sels, Bolginm.

Ghmose and mannose, unlike galactose, fruciose, ribose, and xylitol st imulated (pro)-insulin biosynthesis in

Vol. 9, No. 1, 1973 Organization Section �9 Abstracts 87

isolated islets. Glucose-induced insulin synthesis was in- hibited by mannoheptulose, 2-deoxyglucose, dinitrophe- nol, and cycloheximide. Neither dinitrophenol nor cyclo- heximide, in contrast with other inhibitors, affected the specific s t imulant action of glucose on insulin biosynthesis as judged by the ratio of insulin to total protein synthesis. Glucose-induced synthesis was unaffected in the absence of extracellular calcium, at high magnesium concentra- tion, o r i n the presence of agents known to influence cal- cium handling by the beta cell, such as theophylline, epinephrine, diazoxide and sulphonylureas. In contrast, glucose-induced insulin synthesis was markedly inhibited when large amounts of extracellular sodium were re- placed by choline, potassium, l i thium or tris-(hydroxy- methyl)- aminomethane. When compared with the effects of cations upon glucose-induced insulin release, these data suggest tha t the s t imulant action of glucose upon both insulin synthesis and release is mediated through a sodium- dependent step of glucose handling by the beta cell.

Epidemiological Survey of Serum Lipoproteins in Diabetic Subjects. H. Platilov~, ]~I. Dufek, E. Konophsek. Clinical Depart- ment and Department of Clinical Biochemistry, Regional Ins t i tu te of National Health, Prague, CSSR.

Over a period of 18 months, serum lipoproteins were examined in 1122 diabetics. Lipoprotein levels were as- eessed by a modification of the electrophoresis technique on agarose. Values in 110 controls with normal cholesterol levels were determined in different age groups. The values of beta- and pre-beta lipoproteins are higher in obese con- trols than in subjects with normal weight. Pathological increases of beta- and pre-beta lipoproteins and changes of these values during t reatment were assessed. The fac- tors of age, sex, body weight, heredity, hypercholester- olaemia, and t reatment were investigated in manifest dia- betics and in the group of borderline diabetics. -- Obese diabetic subjects show significantly higher beta- or pre- beta lipoproteins than subjects of normal weight . .The incidence of organ arteriosclerosis in diabetics is higher in the obese regardless of pathological changes of the lipide- gramme. -- The importsalce of lipoprotein levels for epidemiological surveys of diabetes is evaluated.

Action of Insulin on Human Serum Serotonin. V. Pozuelo, J. Tamarit , A. Suffer, C. Castell. Department of Endocrinology, Ins t i tu te Nacional de Prevision, 45 Doctor Esquerdo Street, Madrid, Spain.

The inhibitory action of serotonin on insulin secretion and release is at present subject to discussion. The behaviour of human serum serotonin was studied by us in 11 patients after intravenous injection of 0.05 units of insulin per kg of pat ient 's ideal weight, The serum serotonin level was measured by the method of Vane, modified by Tamarit . Serum samples were obtained before the injection of in- sulin and 3, 30, 60, 90 and 120 min thereafter. -- The means in every serum sample were 156.99, 149.18, 143.90, 135.18, 134.0 and 134.63, respectively. A fall in serotonin levels was seen in nine out of eleven patients, and a slow rise began at 120 mi~. I t should be noted that besides the discussed action of serotonin on the pancreatic insulin secretion, there exists an action of insulin on serum serotonin.

Energy Metabolism and Physical Activity in Juvenile Dia- betics. E. D. R. Pruet t and S. Maehlum. Ins t i tu te of Work Phys. iology, Oslo, Norway.

Results of a previous investigation of the effect of exercise on metabolism in healthy yotmg men living on three different diets (E.D.R. Pruett , Fat and Carbohydrate

Metabolism in Exercise and Recovery, and its Dependence Upon Work Load Severity, Universitetsforlagets Try- kningssentral, Oslo) have been compared with the results of a similar s tudy of young male i~sulin treated juvenile- onset diabetics, living on their prescribed diet (ca. 130 g carbohydrate per day). The diabetic subjects came to the laboratory in the post-absorptive state and without insu- lin on the day of the experiment. Each subject performed five experiments, one at rest and one each at 20%, 50~o, 70% and 90% of his maximal oxygen utilization capacity (max Vo2) exercising on a bicycle ergometer for three hours or unt i l exhaustion, whichever came first. Blood samples taken before, during and after the exercise were analyzed for glucose and free fat ty acids (FFA) among other parameters, and expired air taken during the exer- cise was analyzed for oxygen and the respiratory quotient (RQ) in order to determine the exact severity of the ex- orcise and the percentage contr ibution of fat and car- bohydrate to the energy utilized. I t was found that the contribution of carbohydrate in grams per minute was less in diabetic subjects than in non-diabetic subjects even living on a high fat diet containing 180 g carbohydrate per day when the subjects exercised at 50% max ~7o2. At 70% and 90% max ~ro 2, however, the percentage con- t r ibut ion of carbohydrate to the energy utilized was similar for diabetic subjects and for non-diabetic subjects living both on a high fat and on a standard diet (420 g carbohydrate). The util ization of fat in g[min was similar at all work loads in both groups of subjects.

Influence of Dimethylxanthine (BL 191) on Insulin Secre- tion Induced by Tolbutamlde, GHbenclamide, and Glucose in Man. S. Raptis, B. Grebe, M. Chrissiku, G. Rothenbuchner, R. Miiller, H.J . Hinze, E .F . Pfeiffer. Department of In ternal Medicine, Endocrinology and Metabolism, University of Ulm, Germany.

Potent ia t ion of experimental st imulation of insulin secre- t ion by phosphodiesterase inhibitors was shown both in r ive and in vitro. Inhibi t ion of phospbodiesterase and increases of cAMP have been established following BL 191. The combined actions of various sulphonylureas (tolbutamide iT) 1 g i.e., glibenclamide (GL) 25 ~/kg body weight i.e.) as well as glucose (G) and BL 191 lave been examined in 10 metabolically healthy subjects and 10 diabetics with matur i ty onset diabetes, respectively. The serum concentration of BL 191 was also determined. No change was observed in immunologically measurable in- sulin (IMI) and blood glucose (BG)levels after infusion of BL 191, whereas a significant increase in free fa t ty acids (FFA) and free glycerol (FG) was found. After the ad- ministrat ion of G (0.33 g/kg b.w.) and BL 191 (4.5 mg/kg/ 60 min), more important increases in both insulin secre- t ion and K values were noted than with glucose alone. On the other hand, BL 191 did not potentiate T actions. (IMI following T: 10Q:t: 19 izU/ml, IMI following T and BL 1 9 1 : 1 0 8 4 - 1 2 iz/ml). In contrast, BL 191 clearly potentiated GL effected insulin secretion (by 48%) and caused greater BG falls than after single GL administra- tion. These results indicate tha t only the effect of GL on the beta cell seems to be mediated by the same mechanism as that .of glucose.

Regulation of the Pyruvate Dehydrogenase Complex in the Perfnsed Heart of Normal and Diabetic Guinea Pigs. H. Reinauer, E .R. Miiller-Ruchholtz. Ins t i tu te for Dia- betes Research, Ins t i tu te of Physiology, University of Diisseldorf, Germany.

The activity of the pyruvate dehydrogenaee complex has been studied in isolated perfused guinea pig hearts using either the Langendorff technique or a preparation per- forming a defined stroke work, with pyruvate-l-14C, as

88 Organization Section �9 Abstracts 19iabetologia

substrate. The following parameters were measured: heart rate, coronary flow, left intraventricular pressure, ventricular enddiastolie pressure, t he rate of change of left intraventricular pressure (dp/dt), arterial and venous PO~. At the end of the experiments, the hearts were: fixed by a freeze stpp clamp, previously cooled in liquid nitro- gen. The actual and total act ivi ty of the pyruvate dehy- drogenase complex, creatine phosphate, ATP, ADP, AMP, glycogen, glucose-6-phosphate, pyruvate, lactate were determined in the heart tissue. The effects of the following experimental conditions on the activity of the pyruvate dehydrogenase complex were examined: anoxia, change of heart work, potassium-chloride arrest, suprarenin, fat ty acids, diabetes. From these experiments the con- clusion is drawn that regulation of the enzymes of the pyruvate dehydrogenase complex is dependent on the energy charge, i.e. on the availabil i ty of ATP. The results �9 of these experiments are compared with studies on the purified dehydrogenase complex from pig heart:

Early Recognition of Diabetic Retinopathy byFluorescein Angiography, X. Rietzler, P. Passa, J. Canivet. Clinic of Endocrinology, Htp i ta l Saint-Louis, Paris, France.

The first signs indicating the presence of diabetic retin0- pathy are the microaneurysms detectable by eye fundus ophthalmoscopy. The aim of this study was to determine whether such retinal vascular changes would already be present a t a stage preceding the appearance of the early signs. 58 diabetics were subjected to fluorescein angiogra- phy, although standard ophthalmoscopic examinations including retinography and biomieroseopie control of the eye fundus did not give positive results as to the existence of a visible retinopathy. -- Angiography in 23 (40~/o) of these patients indicated: (a) the presence of microaneury- sins not detectable by ophthalmoscopic or biomicroscopic investigation of the eye fundus; (b) the existence of cir- culatory disorders consisting of areas with delayed cap- illary blood inflow characterized by too early arterio- venous t ime and substantiated by real "gaps" on the photographs. Their significance will be discussed. -- These angiographie abnormalities suggestive of infra- clinical ret inopathy are analyzed with respect to the age of the patient, both durat ion and type of diabetes, the degree of control of the metabolic disturbance and its association with possible arterial hypertension.

st imulated insulin secretion and did not affect conversion of P to I per se. ,Glucagon, theophylline, and cAMP pc. tentiated both glucose st imulated L-incorporation and insulin secretion; at 0 glucose no significant st imulation occurred. The effects on synthesis and secretion were not?

�9 always correlated. The potentiat ing action of glucagon on L-incorporation was strongest at high glucose eoncentra-

�9 tion. No major effects were observed with other hormones. The size of the intracellular leucine pool was estimated in the range of 10 -1~ mol/100 islets and, unlike the radio- activity, was found to remain unchanged under different conditions. -- Conclusions: 1. Secretion and synthesis of insulin appear not always to be correlated. 2. Glucagon and/or the adenyl cyclase system seen to be involved in the regulation of insulin biosynthesis. 3. Glucose is of ut- most importance for these regulatory actions.

Sulfation Activity of NSILA-S and Relation of NSILA-S to Growth. U. Sehlumpf, B. Morell, A. Zingg, E. R. Froesch. Metabolic Unit , Department of Medicine, University of Zurich, Zurich, Switzerland.

NSILA-S mimicks all the in vitro biological effects of in- sulin on adipose tissue and diaphragm. I t is 50 times less active than insulin on these tissues, Mole for Mole. Clearly I~SILA-S, unlike insulin, is not concerned with blood sugar regulation. The following study was undertaken in order to gain better insight in the possible physiological role of NSILA-S. Undifferentiated growing cells need One or several serum factors in order to grow. Insul in in phar- macologic doses can replace these serum factors to a cer- ta in extent. NSILA-S also stimulates glucose uptake, lactate production and growth of chicken embryo fibre- blasts, but it is 50 times more active, Mole for Mote, than insulin. Since g rowth was stimulated by NSILA-S in undifferentiated fibroblasts, it was of interest to find out whether differentiated ceils concerned with growth would also respond to NSILA-S. Therefore, NSILA-S was tested for sulfation activity. NSILA-S was found to be 50 times more active than insulin in promoting 85S incor- poration into rat and chicken cartilage in vitro. -- With the hypothesis tha t NSILA-S may be a physiological growth factor in mind, its act ivi ty is presently b e i n g tested in urine and serum of normal subjects, patients with growth hormone deficiency, off and on hormone re- placement and in subjects with aeromegaly.

Influence of Insulin Secretagogues on 8H-Leucine Incor- poration into Proinsulin (P) and Insulin ( I ) a n d on Intra- cellular Pools of Amino Acids in Isolated Pancreatic Islets of Mice. H. Schatz, Ji Otto*, M. tt inz, V. Major, C. Nierle, G. Behrens*, T. Biicher*, E .F . Pfeiffer. Dept of Endocrino- logy and Metabolism, Center of Internal Medicine and Pediatrics, Universi ty of Ulm, and Dept. of Biochemistry, University of Munich*, Germany.

This study was designed to investigate the regulation of insulin biosynthesis and its relation to insulin secretion. Isolated pancreatic islets of mice were incubated with 3I-I-

' leueine (L) for 3 h at 0, 100 and 300 mg~o glucose in the presence of tolbutamide, glibenelamide, arginine, gluca- gon, theophylline, cAMP, and gastrointestinal as well as pi tui tary hormones. Insul in secretion has been determined hourly, incorporation of L into P and I after fractionation of the islet proteins on Sephadex G-50 fine. Conversion of P to I has also been studied by pulse labelling. Specific radioactivity of free intracellular leueine has been esti- mated by chromatography on an ultrasensitive amino acid analyzer. -- Tolbutamide, glibenclamide, and argin- ine inhibited L-incorporation into P and I, whereas they

Insulin Release from the Isolated Perfused Rat Pancreas in the Presence of L-Leucine, 2-Aminonorbornane-2-Car- boxylie Acid, L-NorvaHne, and ~-Ketoisocaproie Acid. J. Schtnborn, P. Westphal, U. Panten. Ins t i tu te of Phar- macology and Toxicology, University of GSttingen, Ger- many.

The kinetics of insulin release were studied to test the hypothesis that L-leueine, the non-metabolized leucine analogue 2.aminonorbornane-2-earboxylic acid (BCH), and ~-ketoisoeaproie acid (KIC), the t ransaminat ion pro- duct of L-leueine, stimulate insulin release by the same mechanism. In rat pancreas perfused without sub- strafe, L-leucine (10 mM) and BCH (20 raM) induced a steep increase of insulin release, which fell to basal levels after 2 min. In the presence of glucose (5 mM) these two amino acids caused only a slight augmentat ion of basal insulin secretion. L-norvaline (10 mM) which should comply with a hypothetical common receptor site of L- leueine and (-)b-BCH, did not stimulate insulin release. KIC (10 raM) induced a marked insulin release for the whole t ime of its administration. These kinetics were not changed by addition of 5 mM glucose. -- I t is suggested that L-leucine, (-)b-BCH and KIC. do not react directly with the same receptor site for insulin release.

Vol. 9, No. 1, 1973 Organization Section �9 Abstracts 89

A Possible Role of Membrane Thiols in Insulin Release. J. Sehlin, G.D. Bloom, B. Hellman, L.-A. Idahl, A. Lern- mark, M. S6derberg, I.-B. Tir Department of Histo- logy, Universi ty of Umea, Umea, Sweden.

The effects of p-chloromercuribenzoic acid (PCMB) and chloromercuribcnzene-p-sulphonic acid (PCMBS) on mic- rodissected pancreatic islets were studied. Both mercurials stimulated insulin release at concentrations of 0.01 to 1.0 raM, the highest concentration tested. The st imulation was significantly inhibited by the omission of Ca 2+ ions or the addition of adrenaline, diazoxide or 2.4-dinitrophe- nol. Neither PCMB nor PCMBS interfered with the insulin releasing abil i ty of glucose. Micro-perifusion experiments revealed that insulin release in response to organic mer- curials occurrccl almost instantantaneously, was revers- ible, and was biphasic. The two mercurials inhibited glu- cose transport as well as glucose oxidation, and increased the mannitol and sucrose spaces of isolated islets. Com- pared with the effects on insulin release, those on glucose transport and membrane permeabili ty were characte- rized by a longer latency and/or required higher concern trations of organic mercurial. The uptake of 2~ labelled FCMBS showed complex kinetics, which is in agreement with strong binding to islet tissue and a slow membrane penetration. Apart from a seemingly higher

of fl-cells which exhibited certain degenerative tures in islets exposed to 0.1 mM PCMBS for 60 rain,

there were no significant changes of the fl-ccll ultrastruc- ture. I t is suggested that relatively superficial thiol groups in the fl-cell p lasma membrane may be involved in the regulation of insulin release.

Insulin Secretion in Klinefeiter's Syndrome. The Effect of Testosterone. M. Serrano Rios, F.G. Hawkins, F. Eseobar, J.M. Mate, L. Larrodcra, M. de Oya, J .L. Rodriguez-Mifion. Funda- cion Jim~nez Diaz, Madrid, Spain.

For unknown reasons diabetes mellitus is a rather com- mon metabolic disorder in patients with Klinefelter 's syndrome (XXY males). In this study 10 nonobese pa- t ients with cytogenetically proven XXY syndrome have been selected. A control group of 20 nonobese nondiabetics subjects and 10 nonobose patients recently diagnosed as juvenile-type D.M. were studied under the same condi- tions. OGTT (100 gin), IVGTT (.33 grn/kg body weight) and i.e. tolbutamide were performed in the three groups. Plasma glucose (glucose-oxidase) and Inmunorcact ive In- sulin (IRI, Heding's method) were assayed at different intervals during each test. Also insulin secretion under the continuous infusion (2 ml/min) of different doses of test- osterone (2.5 and 5 mgs) administered alone or in com- binat ion with glucose infusion was studied in Klinefelter's patients. Glucose tolerance was normal in 6 and abnormal (chemical diabetes) in 4" Klinefelter 's patients. Fast ing I R I and its release pat tern after OGTT, IVGTT and i.v. tolbutamide in non-diabetics Klinefelter's patients did not significantly differ from t h a t in the normal group. Chemi- cal diabetic Klinefelter's patients had a typically dimin- ished insulin release after any test. In this group insulin, ogenic reserve was even less than in the subjects with clinical diabetes. Testosterone at any dose improved K indexes, bu t had no significant effect on glucose-induced insulin release. I t seems, therefore that the high incidence of D.M. in Klinefelter 's syndrome is much more directly connected with the chromosomal aberrat ion than with endocrine factors.

The Effect of Cortisol Treatment on Insulin Secretion in Addison's Disease. M. Serrano Rios, F. G. Hawkins, L. Larrodera, J.M. Mate, F. Escobar, M. de Oya, J .L. Rodri~lez-Mifion. Fundacion Jimdnez Diaz, Madrid, Espagne.

Carbohydrate tolerance and insulin secretion in 10 Ad- dison's patients (8 m and 2 f aged 30--45 yr) were studicd, with 18 nonobese nondiabetic subjects (10 m and 8 f aged 18--35 yr) as controls. Oral glucose tolerance tests (OGTT, 100 g), and intravenous gh cose tolerance tests (IVGTT, 0.33 g/kg body weight) as wel l as intravenous tolhutamide tests were carried out in both groups. Plasma glucose and IRI (method of Hcding) were assayed at fixed intervals during each test. The results showed : Nor- mal glucose tolerance was found in 9 patients and overt diabetes in 1 subject. The insulin secretion after OGTT and IVGTT in the group with normal carbohydrate toler- ance was not significantly different from that in the con- trol group. Tolbutamide elicited a marked hypoglycaemic response, but insulin release was not different from that in normal subjects. The pat ient with clinical diabetes had a diminished insulin response to the three tests, similar to tha t seen in matur i ty onset diabetes. Cortisol t reatment (20 mg hydrocortisol/day for 15 days) did not significantly modify the glucose tolerance to OGTT and K-indexes after IVGTT. Insul in secretion after OGTT and IVGTT remained also essentially unchanged a f a r cortisol ther- apy. However, a striking potentiat ing effect of cortisol on tolbutamide-induced insulin release was observed.

Effect of Fasting and Triamcinolone on the Activity of Glucogenic and Amino Acid Metabolizing Enzymes in Spiny Mice (Acomys catdrinus). E. Shafrir, A. Gutman. Department of Biochemistry, Heb- rew University-Hadassah Medical School, Jerusalem, Israel.

Spiny mice, trapped in the Negev and kept on standard chow, had high activity of hepatic enzymes of gluconeo- genesis in relation to those of glycolysis (Gutman et al. Isr. J. Med. Sci. 8:364, 1972). No hyperglycemia, glu- cosuria, hyperinsulinemia or obesity were evident. On 48 h fast, the activity of glucokinase decreased but other enzymes of glycolysis did not change appreciably. The act ivi ty of G6Pase, pyruvate carboxylase (PC) and phos- phoenolpyruvate carboxykinase (PEPCK) increased by 30 to 50O/o. Among enzymes of amino acid metabolism, the activity of tyrosine and aspartate aminotransferases and of serine dehydratase rose by less than 50O/o . In albino mice, which served as reference, the changes in activity of glycolytic and of glucogenic enzymes were larger. -- After 3 injections of 0.75 mg of triamcinolone, 12 h apart, the aet!vity of PC, PEPCK, tyrosine and aspartate trans- ammase and of t ryptophan oxygenase pronouncedly in- creased in albino mice. In spiny mice only the activity of aspartate transaminase increased markedly, and of PEPCK moderately. Liver glycogen and blood glucose rose only in albino mice. -- The small or absent response of enzymes associated with gluconcogenesis to conditions promoting augmented glucose production, suggests that !n spiny mice these enzymes are induced to capacity. This Is supported by our findings that synthetic carbohydrate diets markedly induce glycolytic enzymes and suppress glucogenie enzymes in spiny mice. Glucose supply during fast appears to be mainly regulated by the provision of precursors rather than by ari increase in the activity of enzymes.

Glucagon-Insulin (G/I) Plasma Ratio in Normal and Pan- createctomized Geese infused with Insulin and Glucagon. G. Sitbon, P. Mialhe*. Ins t i tu te of Physiology, University Louis Pasteur, Strasbourg, France.

Measurements of plasma glucagon and insulin in normal fasting geese before and during constant i.e. "infusion of saline gave G/I ratios of 2 .20• 0.12 and 2.25 _-t: 0.05 (cal- culated on a molar basis). As total pancreatectomy induces

* With the help of the C.N.R.S. and the I.N.S.E.R.M.

90 Organization Section �9 Abstracts Diabetologia

hypoglycacmia and a decreased glucose tolerance, -- showing that both glueagon and insulin play an important role in this species -- , it was at tempted to mainta in blood glucose within the normal range by constant i.e. infusion of glucagon and insulin in operated animals. The results are as follows: 1. Blood glucose levels can be maintained within the normal range during experiments lasting 6 or more hours with a constant G/I ratio. 2. The G/I ratio obtained (1.71 -b 0.23) is near to, but slightly lower than that found in infused normal animals; this small difference may be due to a reduced efficacy of infused insulin, a con- sequence of the diabetic state.

New Diagnostic Methods for Better Classification of Bor- derline Diabetes. Z. Skrabalo, N. Panajatovi~, Z. Pupil , J. Posinovec, A. StavljeniS, V. Lipovac, I. Aganovid. Ins t i tu te for Diabetes "Vuk Vrhovac", Zagreb, Yugoslavia.

The standard tests for detection of the early carbohydrate metabolic disorders today are not sufficient enough to permit early classification of every borderline case of diabetes, according to prognosis of its development to- wards clinical manifestation of diabetes, or towards its normalising (eventually as the result of therapy). As dia- betes is a complex disorder involving different metabolic processes in the human organism, the endeavours of the "Zagreb group" over the last three years were directed toward finding .new, additional methods which would provide us with more rapid, better and earlier establishing the prognosis concerning the development in each partic- ular case of diabetes. -- In a group of diabetics, group of 50 borderline cases, and a control group of 50 normal, healthy subjects, the changes in lipid metabolism (chol- esterol, free fat ty acids (FFA), triglycerides, lipidogram) were examined as well as changes that appear in the phase of borderline diabetic disorders of the metabolism in the skin (accumulation of glycogen), in adipose tissue (changes in esterases and aldolases), in muscle tissue (changes in transaminases) and in peripheral blood lymphocytes (ac- cumulation of PAS positive substances). Based on dis- covery and statistically proved findings of changes in lipid metabolism and changes in the biochemistry of cells and tissues during the early phase of diabetes, those tests can be used as new, additional diagnostic methods for better classification of borderline cases of diabetes.

A Study of Potassium Balance During the Treatment of Diabetic Ketoacidosis with Special Reference to the Use of Bicarbonate. N.G. Soler, M.A. Bennett , K. Dixon, M.G. Fitzgerald, J.M. Malins. Diabetic Clinic, The General Hospital, Bir- mingham, England.

Twenty-five patients with severe diabetic ketoacidosis were studied during the initial 24 h of treatment. In the early stages insulin was given intravenously every 2 h and later when the blood sugar had fallen it was given as required by the subcutaneous route. Saline or sodium bicarbonate was infused rapidly to correct dehydration and acidosis. All patients were given saline and in 18 cases sodium bicarbonate was also used: 11 patients were given up to 250 m-equiv of bicarbonate and 7 pa t ien ts were given over 250 m-equiv, of bicarbonate. Potassium re- placements were guided by continuous E.C.G. monitoring and frequent serum potassium estimations. -- In the pa- tients treated with saline alone and in the group given up to 250 m-equiv, of Na bicarbonate a mean of 30 m-equiv. of potassium per litre of intravenous fluid was required to keep the serum potassium normal. The high potass ium requirements of these patients were par t ly explained by the considerable leases of potassium in the urine. When more than 250 m-equiv, of sodium bicarbonate were used in t reatment the potassium requirements increased. The

increase in the amount of potassium infused was not ac- counted for by urinary loss of potassium.

On the Mechanism of Inhibition of a Monoamino Oxidase Inhibitor (Phenelzin) on Hepatic Gluconeogenesis. H.D. S51ing, H. Peters, H. Peters, G. Jansen. Department of Clinical Biochemistry, Medical Clinic of the University, GSttingen, Germany.

Hypoglycaemic episodes have been reported after treat- ment of psychotic patients with phenelzin. Since an in- hibition of hepatic t ransamination had been thought to account for this effect, the mechanism of action of this drug on isolated perfused rat livers and isolated rat liver mitochondria was studied in this laboratory. Phenelzin (1 • 10aM) completely blocks gluconeogenesis from L- lactate, bu t only partially from pyruvate. Gluconeogene- sis from fructose is not affectecl by phenelzin. -- Crossover plots revealed a site of interaction a t the site of pyruvate decarboxylase in the absence of st imulated fat ty acid oxidation, in accordance with a severe fall in acetyl-CoA, whereas the site of interaction was seen mainly at the site of PEP-earboxykinase in the presence of exogenous fa t ty acids. Phenelzin does not directly inhibit this enzyme but competes with it as well as with glutamate oxaloacetatc transaminase for oxaloacetate. In isolated ra t liver mitochondria, phonelzin exerts a rotenone-like action. The possible therapeutic use of this hypoglycaemic principle will be discussed.

Plasma Glucose, Serum Growth Hormone and Cortisol Responses to Graded Infusions of Monocomponent Human Insulin in Normal Subjects and Patients with Diabetes Mellltus. P.H. SSnksen, M.C. Srivastava, C.V. Tompkins, J .D:N. l~'abarro. St. Thomas' and The Middlesex Hospital, Lon- don.

Insul in was infused into 9 normal subjects, 9 patients with mild-moderato and 4 patients with severe diabetes melli- tus by the priming dose -- constant infusion technique. Steady state insulin concentrations were obtained at four separate concentrations ranging from 15--200 ~U/ml and were maintained for 30 rain at each level. On average 13 of insulin was infused over a two-hour period at rates ranging from 22 mU/min in the first period, to 176 mU/ rain in the fourth. In normal subjects, plasma glucose fell to 50% of fasting value at 50 rain and in mild-moderate diabetics a t I00 rain, while in severe diabetics it fell only 30% in 120 min. In normal subjects serum growth hor- mone and cortisol rose as anticipated in response to hypo- glycacmia (mean rise of 27 ng]ml and 14 ~g/100 ml re- spectively). Fasting growth hormone was lower than nor- mal in the diabetics (normal 6.1 ng/ml, mild-moderate diabetics 1.9 ng/ml and severe diabetics 2.5 ng/ml) while cortiscl values were elevated in the severe diabetics (nor- mal 7.27, mild-moderate diabetics 7.33 and severe dia- betics 15.25 i~g/100 ml). Unexpectedly, serum growth hormone rose within 10 rain of starting the insulin infusion in both groups of diabetics. These rises occurred before there had been an appreciable fall in plasma glucose in either group and were accompanied by a parallel rise in cortisol. The results are compatible with the hypothesis tha t in uncontrolled diabetes, release of growth hormone and ACTH by the pi tui tary may be limited by impaired glucose metabolism within the gland and that t reatment with insulin restores energy metabolism towards normal allowing the release of growth hormone and ACTH in response to hypothalamic stimulation.

Changes Induced by Fasting in Blood Patterns of Aceto- acetate and 3-Hydroxybutyrate after Infusion of Insulin or Glucose in Obese Persons. N. Schwartz Seronsen, K. Ladefoged and K.E. Wilden-

Vol. 9, No. 1, 1973 Organization Section �9 Abstracts 91

heft. Medical Depar tment I I I and the Central Labora tory , Aarhus County Hospi ta l , Aarhus, Denmark.

The blood pa t te rns of actoacetate, 3 -hydroxybutyra te , free f a t ty acids and glycerol were registered 60 rain before and 90 rain after a rap id intravenous infusion of insulin (0.05 i.u./kg body weight) or glucose (0.4 g/kg body weight) in 6 obese persons. In each individual s tudied the experiment wi th insulin was carried out first, and the ex- per iment with glucose two days later. Both sets of experi- ments were repeated af ter 8 and 10 days of absolute fasting. -- Before fasting, infusion of insulin or glucose resulted in an immedia te and significant decrease in all parameters which lasted a t least 30 rain. -- During fasting, infusion with insulin did not resul t in a significant change in the blood concentrat ion of acetoacetate in spite of a significant decrease in all other parameters during the first 30--45 min af ter the infusion. Infusion with glucose resulted in a significant increasi~ in the blood concentra- t ion of acetoacetate which s ta r ted immedia te ly after the

�9 infusion and lasted for the rest of the experimental period. The concentrations of 3 -hydroxybutyra te , free f a t ty acids and glycerol showed a significant fall during the same period.

Insulin Response to Oral Glucose in Patients after Myo- cardial Infarction and in Patients with Peripheral Vascular Disease. F. Serge, H. : J . Diehl, H. Hoffmann and *vV. Schwartz- kopff. Kl iniktun Westend, Free Univers i ty , Berlin, W- Germany.

I n 69 pat ients 6 months to 1 year after myocardia l infarc- t ion (MI) and 44 pa t ien ts with per ipheral vascular disease (PVD) an OGTT was done. Glucose tolerance was a b - normal in 43% of PVD and 61% of MI, t r iglycer ide levels (TG) were raised (above 150 mg~o) in 5 7 % of PXCD and 64 % of MI, Insulin basal values correlated s t r ic t ly with ~o ideal weigh t (~/oIW) in bo th groups (PVD: % I W 117~ :t: 14, r = 0.48, p < 0.001; MI: % I W 120% q- 14, r - 0,53, p < 0.001) and with TG values only in PVD group (r = 0.44, p < 0.005). Insul in response to OGTT, calculated as the sum of the values on 60 a n d 120 rain (Z 60 ' -k 120'), showed no difference in both groups (PVD : 384 -4- 224 ~U/ ml; MI : 399 :]: 220 ~U/ml, means 4- SD) bu t correlated with % I W only in MI group (r ---- 0.35, p < 0.005) and not in PVD group ( r ----- 0.18, NS). On the contrary, insulin Z (60 '+ 120 ) correlated very well wi th TG values (r----- 0.59, p < 0.001) only in pat ients wish PVD and not in those af ter MI (r----0.13, NS). - - These results and their�9 pos- sible implicat ions in pathogenesis of PVD and MI are discussed.

The Effect o f Diabetes on Erythrocyte 2,8-Diphosphogly- cerate. E. Standl , H. Kolb, A. Standl , }I. Mehnert. Diabetes Re- search Unit , Munich, Germany.

2,3-diphosphoglyeerate (2,3-DPG) modifies the oxygen binding of hemoglobin. I t play~ thus an impor tan t r01e for the adequat.e oxygen supply in per ipheral tissues. Invest i- gat ions have so far not been conducted as to the aspects of if and how the ac tual glucose concentrat ion in blood affects the levels of the glyeolyt ie in termediate 2,3-DPG in erythrocytes . -- I n normal subjects, 2,3-DPG re- mained constant for several weeks. Randomly selected diabetics (n = 38) wi th incidental ly high and low blood sugar levels showed only an insignificant decrease of 2,3- DPG in comparison with normal subjects in ~ 14). When considering the respective blood glucose concentrations, however, there were significant differences. M a r k e d l y hyperglycacmic, non-ketot ic subjects developed a signifi- cant decrease of 2,3-DPG (p < 0.001) immedia te ly after

the blood glucose level fell tO normal. This fall in 2,3-DPG was also significant as compared to normal subjects (p < 0.01). I t Could be demonstra ted in ini t ia l ly hypergly- cacmic diabetics tha t , while the blood glucose concentra- t ion was brought to normal, an a k l o s t dai ly assessment of the 2,3-DPG levels indicated their decrease to go paral lel to the fall in blood glucose. After a few days, 2,3-DPG levels increased again in spite of persisting nor- mal blood sugar levels. - - I t is concluded tha t certain metabolic si tuations in diabetes might impair peripheral oxygen supply through changes in 2,3-DPG.

Clues in the Detection of Glucose Intolerance in Human Pregnancy. H . W . Sutherland, J .M. Stowers, J . C. G. Whetham. Aber- deen Materni ty Hospi ta l , Aberdeen, Scotland.

845 pregnant women had 25 gm rapid I.V.G.T.T.s because of various s t r ic t ly defined features (indicators) suggestive of diabetes. - - The indicators- previous del ivery of a ~ "large-for-dates" baby, fasting glycosuria, family h is tory in a first degree relat ive and materna l obesi ty had, alone, predict ive values for abnormal glucose tolerance of 16, 12, 11, and 7% and, in association with other indicators, 26, 19, 23, and 22% respectively. Polyhydramnios , previous st i l lbir th, or radiol0gical evidence of excessive fetal sub- cutaneous fat in utero in late pregnancy occurred in a few bu t these indicators also had significant predict ive values individual ly i.e. " P r i m a r y indicators" . I n association with other indicators their values were increased to 38, 13, and 50~/o respectively. - - "Secondary indicators" which have no individual predict ive values bu t which enhance those of the p r imary indicators were found to be a family hist- ory of diabetes exclusive of first degree relatives, re- current abort ions, f e t a l congenital anomaly, and pre- vious nee-natal death. - - Compared to paternal , materna l h is tory of diabetes is more significant, and high bir th- weight of obese women may be a useful diseriminant . =- Increasing numbers of indicators correlate w i t h an in- creased incidence of glucose intolerance and also an in- creased per inatal mor t a l i t y even in those with normal glucose tolerance.

Dynamic Studies of and Effect of Physiological Doses of Epinephrine on the Insulin Secretion in Vitro. B. Ch. ft. Surfer, B. Billaudel. Laboratoiro de Physiologic et Centre de Biologic et de Biochimie du Dtveloppement , Facul td des Sciences, Reims, France.

The biphasic response of insulin secretion to glucose in vi t ro depends on the r ap id i ty and the importance of the var ia t ion of the glucose concentrat ion and on the per- fusion ra te : 1. Perfusion ra te : 1.2 mg/g/min: when glucose varies between 0 and 1 mg/ml the two typical phases of insulin secretion are seen and the first peak corresponds exact ly to the moment ' when the glucose pla teau is reached. I f the perfusion ra te is 0:4 mg/gfinin with a glu- cose var ia t ion from 0.8 to 1.4 mg]ml, the two r~esp0nses are observed when the p la teau is reached in 6 min, if i t takes 12 min to reach it, the first peak is absent. This means (i) tha t the first phase of insulin secretion is due to g!ucorecepters sensitive only to the rap id i ty of the glu- cose var ia t ion and, t ha t the second phase depends on the t ranspor t of glucose into the cell, and (ii) t ha t a t both physiological perfusion rates and glucose variations, only the second secretion phases can be obtained. 2. A phy- siological concentrat ion of epinephrine of 0.5 ng/ml in- hibi ts nei ther of the two phases, bu t ra ther increases the second response. At physiological conditions and especial- ly in vivo, the inhibi tory effect of epinephrine is very l imited, b u t i t may be possible tha t this hormone exerts a s t imula tory effect on the insulin secretion in the non stressed animals.

92 Organization Section �9 Abstracts Diabetologia

Inhibition of Insulin Action by Progesterone in the Rat. B.Ch.J. Sutter, M.T. Sutter-Dub, R. Leclercq, R. Jac- quot. Laboratoire de Physiologic animale et Centre de Biologic et de Biochimie du D6veloppement, Facultd des Sciences, (F) 51-Reims and Laboratoire de Chimie Clini- que, Universit~ Libre, (B) Bruxellcs.

We have shown recently tha t serum I R I is increased dur- ing pregnancy even if glycemia and ILA are at normal levels. New results are as followcs : 1. -- Serum I R I varies as progesteronemia during pregnancy and lactation, but during lactation the correlation between progesteronemia and serum I R I is not as good as during pregnancy. 2. -- Castrated female rats injected during 20 days by proges- terone (5 mg/kg/day) have elevated serum IRI and their progesteronemia is highly comparable to tha t of pregnant rats. Treatment of progesterone and oestradiol gives the same results. 3. -- A 6 days t reatment of progesterone (5 mg/kg/day) reduces the hypoglycemic response to insulin in castrated streptozotocin treated female rats ; the same reduction is observed when insulin and progeste- rone are injected simultaneously in castrated streptozoto- cin treated females without prior progesterone treatment. Progesterone also inhibits the insulin effect on glucose uptake of rat diaphragms in vitro. We can conclude 1. that progesterone inhibits insulin action in vitro and in wee and 2. tha t this hormone is responsible for the high I R I levels during pregnancy and perhaps during lactation.

The Pancreatic ~-Cell Recognition of Glucose and Phlo- rizin. I .B . T/fljedal, B. Hellman, A. Lernmark, J. Sehlin. De- par tment of Histology, University of Ume~, UmeA, Swe- den.

In the absence of glucose, microdissected islets of obob. mice released insulin in response to phlorizin. This effect was not due to the glucose residue of phlorizin, since phloretin also st imulated insulin release. Mannoheptulose inhibited the effects of phlorizin and phloretin, suggesting that both stimuli were sensed by the system which re- cognizes glucose as an insulin secretagogue. However, the mechanism sensitive to phlorizin does not possess the full competence of the glucose-recognizing system, since phlorizin did no t potentiate the insulin-releasing actions of arginine or theophylline. Leucine potentiated the effect of phlorizin. Glucose and leucine but not arginine are rapidly oxidized in the islets. The synergism between phlorizin and leucine may therefore suggest tha t glucose recognition involves a metabolic component as well. However, pyruvate or succinate did not potentiate the effect of phlorizin. Radioactive phlorizin accumulated in amounts exceeding the urea space of the islets. PCMBS and ant imycin A, which increase the islet uptake of extra- cellular space markers, stimulated the uptake of phlorizin, suggesting that phlorizin normally hinds predominantly to the fl-eell plasma membranes. Although the binding was not specific for glucose-sites, it is conceivable that phlorizin and phloretin stimulate insulin release by re- acting with such sites.

Changes in Insulin Secretion Induced by Serotonin. J. Tamarit , J. Tamarit-Rodriguez, R. Goberna. Depart- ment of Physiology and Biochemistry, Facul ty of Medi- cine, Universidad Complutense, Madrid-3, Spain.

The influence of serotonin (5-HT) on carbohydrate meta- bolism has not yet been elucidated and the results of Various authors are contradictory. In earlier experiments, glucose levels in rabbits given 0.5 g/kg glucose i.e. plus 5-HT at a dose of 250 ~tg/kg i.v. were much higher than those in control animals. Furthermore, serum 5-HT levels 240.6 ~ 38.36 ng/ml (method of Vane) were found in dia- betic patients vs. 115.6~ 10.35 ng/ml in controls, and

this difference was statistically significant (p < 0.01). Serum insulin levels of animals treated with glucose plus 5-HT were lower than those observed in animals who had received glucose alone. In the isolated perfused rat pan- creas (100 rag% glucose), a slight increase in insulin re- lease resulted after a pulse of serotonin. Perfusion with 300 mg~o of glucose plus 5-HT (100 ~Lg/lnl) showed a normal first peak of insulin release, while the second peak was absent.

Effect of Glucose-I-Phosphate on Insulin Release in the Isolated Perfused Rat Pancreas. J. Tamarit-Rodriguez, R. Goberna, J. Tamarit . Depart- ment of Physiology a n d Biochemistry, Facul ty of Medi- cine, Complutense University, Madrid 3, Spain.

Matschinsky (1971) suggested that glucose itself triggers insulin release and Rand]e (1970) felt this would be sup- ported by 'the discovery of a sugar which may inhibit the insulin secretory response specifically, without inhibiting the metabolism of glucose'. We consider that glucose-I- phosphate (G-X-P) and glucose-6-phosphate (G-6-P) (as analogues of glucose itself and possibly not as inhibitors of its metabolism) could compete with glucose at the level of the postulated glucoreceptor. Neither G-I-P nor G-6-P, each perfused alone, st imulated insulin release. After perfusion with G-6-P (300 mg%) for 30 rain, a per- fusion of glucose (300 mg%) plus G-6-P (300 rag%) in- hibited the insulin secretion that would result from glu- cose alone. After perfusion with G-I-P (300 rag%) for 30 rain, the administrat ion of glucose (300 mg%) plus G-I- P (300 rag%) produced a disordered increase in insfilin re- lease, resulting in the disappearance of its characteristic dynamics. The perfusion of G-6-P (300 mg%) plus glucose (300 mg~/o) plus glucose (300 mg%), without previous perfusion of G-6-P, produced an increase in insulin secre- tion, bu t the corresponding levels of the first and second peaks were significantly lower than those observed after perfusion with 300 mg% glucose alone. After a 15 rain period with glucose (50 rag%) the perfusion of glucose (300 rag%) again provoked a normal insulin release with its inherent characteristic dynamics. I n the same manner, arginine produced an increase in insulin release after a short periled of perfusion with 50 rag% glucose.

Computerized Evaluation of Intravenous Glibenclamide- Tests as a Diagnostic Test. Gy. Tam~s, Jr., ]~va Baranyi, A. Baranyi, A. Radvanyi . First Dept. of Medicine, Semelweis Universi ty Medical School, Budapest, and Research Ins t i tu te of Telecom- munications, Budapest, Hungary. A standard intravenous glibenclamide test (1 or 2 mg of glibenclamide) was performed in approximately 60 healthy subjects, latent diabetics, and diabetics. The changes in blood sugar levels and, in some cases, in serum insulin levels were recorded before and after the adminis- t rat ion of glibenclamide at 15, then respectively 30min intervals for a period of 180min. The results of the gliben- clamido test were compared with those of the oral glucose tolerance test and, in some cases, with those of the tolbuta- mide test. -- The results of the glibenclamide tests were evaluated and analyzed by computer. The shape of the curves (time and per cent maximal decrease in blood sugar) is characteristic of the different types of carbohy- drate intolerance. -- The importance of the glibenclamide test as a diagnostic aid will be discussed.

The Effects of Insulin and Biguanides on the Glucose and FFA Metabolism of Isolated Coronary Artery Cells. J. Tatofi, A. Hinek, A. Wi~miewska. Medial Academy, I I I rd Department of Internal Medicine, Warsaw, Poland. The aim of this s tudy was to investigate the metabolic regulatory effects of insulin and biguanides in the system

Vol. 9, No. 1, 1973 Organization Section �9 Abstracts 93

of isolated coronary artery ceils. The original enzymatic method of cell isolation from fragments of the porcine coronary artery provides free cells with well preserved morphological and functional integrity. In five types of experiments, glucose and F F A util ization per cell were determined under the following conditions: (a) physio- logical, (b) low concentration of substrates, (c) phy- siological concentration of glucose and very high concen- trat ion of FFA, (d) addit ion of increasing concentrations of insulin, and (e) of biguanides to the systems a, b, c. -- Results: (i) The kinetics of glucose and F F A utiliza- t ion per cell are not related to the concentration of these substrates, within the range studied. (ii) Glucose utiliza- t ion is not inhibited by the very high concentration of FFA. (iii) Util ization of glucose and F F A by the cells is positively related to the insulin concentration within the range 20--100 tzu/ml. (iv) Biguanides exert no effect on glucose and F F A metabolism of isolated coronary artery cells.

Diabetic Retinopathy in Identical Twins. R.B. Tattersall and D.A. Pyke. King's College Hospital, London, England.

I t is not known whether ret inopathy and the other com- plications of diabetes are related to "control" of the condition or consti tutional factors. We have studied the prevalence and features of ret inopathy in identical twins who have been diabetic for 15 years or more -- 10 dis- cordant twins and 12 concordant pairs (24 individuals). Four of the 12 concordant pairs, bu t none of the 10 dis- cordant pairs, had a diabetic parent. -- We have found 1. no abnormali ty in the retinae .of the 10 non-diabetic twins in the discordant pairs. 2. In the concordant twins, 21 of the 24 showed some degree of ret inopathy, l I had proliferative changes and 4 were blind. In the 10 dis- cordant twins, 5 showed some ret inopathy but only one had proliferative changes. I t appears tha t ret inopathy may be commoner and more severe in "genetic" than in "non-genetic" diabetes. 3. In the 12 concordant pairs, the development and pat tern of ret inopathy was strikingly similar in both twins, with one exception -- a pair becom- ing diabetic 20 years ago, of whom one has normal eyes and the other is nearly blind from proliferative retino- pathy. 4. Within the l imitations of this study, we have not been able to relate the manifestations of diabetic re- t inopathy to the "control" of the diabetes.

Outcome of Pregnancies in Women wi th Clinical and Latent Diabetes and Factors Influencing the Results. J. Terpstra, J. Bruins Slot, P . L . M . v . d . Sande, J .K . Radder, K. J. J. Waldeck, R. C. P . A . v . Muijden. Diabetes Uni t of the Dept. of Metabolism and Endocrinology, and Dept. of Obstetrics and Gynaecology, University Hospital, Leiden, The Netherlands.

This report analyses, over a period from 1959 till 1966, the outcome of 75 pregnancies in 53 women with clinical dia- betes mellitus m relation to both the severity of diabetes and complicating factors. In 67 pregnancies of more than 28 weeks duration, there were 7 perinatal deaths. Ac- cording to White 's classification (Am. J. Med. 7 : 609, 1949) no difference between groups was observed. Using the classification of Pederson and Pedersen (Acta Endocrin. 50:70, 1965), a significantly higher perinatal morta l i ty was found in the group with "prognostically bad signs during pregnancy". The series studied gave no indication of a relationship between perinatal mortal i ty and durat ion of diabetes, but our data suggest the existence of a re- lationship between perinatal mortal i ty and vascular com- plications. -- The effect of the adequate t reatment of dia- betes on fetal mortal i ty was studied in a series of 25 wom- en with latent diabetes (as defined by the Brit. Diabetes

Association). 31 pregnancies during which the diabetes was under strict control, showed a significantly bettor outcome compared to the 79 earlier pregnancies in the same women before they had been diagnosed as latent diabetics. -- The comparatively small series of pre- gnancies in diabetic women al~ further extended over the next five years (1966-- 1971). The data are being prepared also to be presented during the meeting.

Hypoglucagonemia in Pancreatic Human Diabetes: In- fluence on Blood Glucose and Sensitivity to Insulin. A. Tiengo*, R. As~an**. *Univ. of Padova, Italy. **H6- tel-Dieu of Paris, France.

Glucagon responses were measured during arginine and insulin tests in 27 patients with pancreatic diabetes: cal- cifying pancreatitis (CP) 12; partial pancreatectomy (P. P.): 4; hemochromatosis (H): 11 (regularly bled). They were compared with 10 insulin-dependent genetic dia- betic patients (GD), and 7 normal subjects (N) with com- parable age range, nutr i t ional status absence of over- weight. -- A significant glucagon increment occurred at 30th mn of arginine test in N (-f- 100 pg/ml; p < 0.01) and in GD (q- 160 pg/ml; p < 0.005) ; not in 2 of the pancreatic diabetic groups (q- 50 pg/ml for CP, and q- 40 pg/ml for PP) ; at l imit of significance for H (q- 100 pg; p < 0.02). A significant difference was found between GD and CP, and between GD and PP. -- Insul in test induced a signifi- cant glucagon increment at 60 min in GD (-b 130 pg/ml; p < 0.005) and in treated hemochromatosis (+ 90 pg/ml; p < 0.005), not in the 2 other pancreatic diabetic groups (-{- 20 pg/ml; p < 0.05). A highly significant intergroup difference was found between GD and CP. -- Glucose variations during both tests were significantly larger in GD than in the pancreatic diabetic groups. This supports the concepts of: 1. an efficient contribution of glueagon to glucose homeostasis; and 2. of dampened or abeen% hy- perglucagonemia in pancreatic diabetes as a cause of hyper-sensitivity to insulin.

Release of Newly Synthesised Proinsulln and Insulin from Granulated and Degranulated Isolated Rat Islets. N.S. Track, H. Frerichs, W. Creutzfeldt. Division of Gastroenterology and Metabolism, Department of Medi- cine, University 5f Goettingen, GFR.

Collagenase isolated islets from control and tolbutamide- treated (500 mg/kg twice daily for 3 days) rats were in- cubated in Krebs-Ringer bicarbonate buffer supplemented with glucose (3.0 mg/ml), tritiated-leucine (20 ~C/ml) and an amino acid mixture (20 tzg]ml). Radioactivi ty was recovered in proinsulin (PI) and insulin (Ins) in both the islets and the media. This incorporation was inhibited by cycloheximide (250 ~g/ml). Glucose (3.0 mg].ml) enhanced the release of PI a n d Ins from the islets; release was arrested by the presence of cyanide (65 [~g/ml). -- Islets were pulsed for 60 min with tritiated-leucino plus glucose (3.0 mg/ml) and then chased with L-leucine (375 ~g/ml) for an additional 120 rain. After the 60 min pulse period, radioactivity was recovered in P I and Ins in a 4 : 1 ratio ; no newly synthesised material was released into the me- dium at this time. After the 120 rain chase period, no radioactivity was found in the islet PI and Ins fraction; in the medium, radioactive PI and Ins were found again in a 4:1 ratio. Similar labelling and release pe, t terns were found after shorter chase periods. Comparable results wore obtained with both granulated and degranulated islet preparations which contained 9% and o/ 18/o endogen- ous PI respectively. -- The fact that 80% of the newly synthesieed and released I RI material was PI which differs considerably from the endogenous PI levels sug- gests tha t the newly synthesieed material may follow a

94 Organization Section �9 Abstracts Diabetologia

different release route out of the B-cells. Since the newly synthesised I R I material amounts to less than 1% of the total I R I release, it �9 not play a major physiological role.

The Preparation of Partially Purified Intestinal Insulin Re- leasing Polypeptide and its Effect in Vitro and in Vivo in the Pat. D.S. Turner, R.W. Baker, W.G.L. Gent, A. Shabaan, V. Marks. Biochemistry Department University of Surrey and Guy's Hospital Medical School London, England.

A basic polypeptide fraction, Intest inal Insulin Releasing Polypeptide, (IRP), has been isolated from porcine duo- denojejunal mucoea by acid ethanol extraction and par- tially purified by dialysis and chromatography on Sepha- dex G50 and carboxymethyl cellulose. IRP shows a probable molecular weight of 3--4.000 and has a basic isoelectric point. I R P contains negligable contamination of secretin, pancreozymin or glucagon-like immunore- activity (GLI) and no insulin. -- IRP enhances glucose and leucine st imulated insulin release in rabbit pancreas pieces in vitro and has no effect in the absence of stimula- tory glucose or leucine concentrations. -- IRP greatly enhances the insulin secretory response of the rat to intra- venous glucose and produces a marked acceleration of glucose disappearance. I t is without effect when adminis- tered to fasting animals without glucose. -- I t is suggested that I R P contains the intestinal hormone responsible for the greater insulin secretory response to oral as compared to intravenously administered glucose.

The Effect of Fasting and Selective Re-Feeding on Insulin Release in the Pat. D.S. Turner, D.A.B. Young. Biochemistry Department University of Surrey Guildford, England and The Well- come Foundat ion Beekenham, England.

The insulin secretory response to intravenous glucose in the rat was found to be greatly impaired by fasting for three days, whereas tha t to orally administered glucose was not significantly affected. Eighteen hours before the intravenous administrat ion of glucose to three-day fasted" rats, ghmose, protein hydrolyzate or both in combination were given to them orally. This pretreatmcnt failed to affect the insulin secretory response to intravenous glu- cose. F(trther, rats fasted for two days were given either protein or starch pellets for six hours, and tnhen fasted for a further eighteen hours before the intravenous glucose test. ' l~e protein pre-feoding failed to affect significantly the subsequent insulin secretory response to intravenous (~lucoso, whereas starch pre-feeding greatly enhanced it. I t is suggested that intestinal hormones released by. glu- cose ingestion may exert not only an acute effect on insu- lin release, but also a 'priming' effect on the insulin re- lease mechanism of the fl-cell which enables it to respond to the subsequent stimulus of glucose alone.

Hormonal and Metabolic 'Variations alter Moderate Ex- ercise in the Afternoon in Normal, non Diabetic Obese and Insulin-Treated Diabetic Subjects. Ph. Vague, H. Heim, C. Martin Laval, M. Vegezzi, C.Di Campo. Hepital de la Conception, Marseillo, France.

Does a moderate exercise in every-day lifo conditions induce appreciable changes in the plasma concentrations of pancreatic glucagon, insulin, HGH, ACTH, and glyce- rol and NEFA=? These substances have been measured in brachial vein plasma in 7 nmTnal subjects, 11 non diabetic obese and 7 insulin-treated diabetics (luring and after a moderate leg muscular exercise (80 W--10 rain) 3 �89 h a f a r eating hmch. -- Plasma glycerol levels increase during exorcise and return to basal levels at 60 rain in nor- real and obese subjects, while they do not change signifi-

cantly in treated diabetics. Plasma NEFA do not decrease significantly during exercise in the normal subjects and rise by up to 30% at 30 rain. In the obese they diminish 34% during exercise and return to basal values at 60 rain. I n the diabetics, the drop is not corrected at 60 min. ACTH increases moderately but significantly during exercise in all groups. HGH levels increase moderately after exercise in normal, insignificantly in obese subjects, and quite variably in diabetics. Pancreatic glucagon assayed by a specific antiserum increases insignificantly from 50 to 94 pg/ml after exercise in normal, bu t from 90 to 300 pg/ml in obese subjects, the rise being mmtained till the 40th rain. -- These results show that this type of exercise induces an immediate lipolysis which overshoots the mark in normal but not in obese subjects. I t does not enhance lipolysis in diabetics with correct plasma levels of exogen- ous insulin. The hypersecretion of glucagon observed in the obese subjects may be related at least in part, to the accompanying fall in plasma NEFA.

Differential Insulin Response to Glucose and Tolbutamide. A Genetic Marker of Diabetes Mellitus ? Ph. Vague, G. Rahamandridona, D. Garron, B. Hcyraud, J. Vague. Clinique Endocrinologique, Hepital do la Con- ception, Marseille, FR.

After an intravenous injection of glucose (0.3 g/kg) followed 90 rain later by one of tolbutamide (20 mg/kg), the early phase of insulin response to glucose (0 to 10' plasma insulin area: iG = 372 micro U/ml r a n + 63) in nonna l subjects is similar to the one to tolbutamide (iT = 345 =[= 65) with a iG/iT ratio of 1.30 • 0.26 always'higher than 0.70. -- In normal weight subjects with chemical diabetes (K < 1.2.1~ iT is in the normal range (392 116). This suggests that the insulin excretory mechanism is not in itself involved. However the response to glucose, while not delayed, is of lower magnitude (iG := 179 ~ 59) and iG/iT=0.447=t= 0.04 always lower than 0.60. The same l~henomenon is observed in obese subjects classified according to their glucose tolerance. -- The individual iG/iT ratio is of "diabetic" type in ahnost all genetic prediabetics (offsprings of two diabetics or twin of a dia- betic) with normal K value, evidencing in individual cases a B cell insensitivity to glucose. -- In 42 normal weight 6 - 50 yr old subjects with high genetic predisposition to diabetes (at least one direct diabetic parent) 23 of them having a "diabetic" type iG/iT ratio, this latter is in- dependen$ of the age, whereas the K value shows an inverse correlation with age. -- These results suggest that the decreased sensitivity to glucose of the insulin excre- tory mechanism established in individual cases by the iG/iT ratio may be used as a genetic marker for diabetes mellitus.

Effect of Diazoxide and Sulphonylureas on Glucagon Secre- tion in Normal Subjects. I. Valverde, M.L. Villanueva, I. Lozano, M. Diaz-Fierros, J. Marco. Fundacion Jim(inez Dlaz, and Clinica P,mrta de Hierro, Univorsidad Autonoma, Madrid, Spain.

The secretion pat tern of glucagon and insulin in the so- called diazoxide (DZ) diabetes and under the influence of sulphonylureas, the chemical cogeners of DZ, were studied in three different groups of normal volunteers. An oral dose of 600 mg DZ was given over three days (group 1, n = 5). Two highly active sulphonylureas, namely glibizide (group 2, n = 7), and glibenclamide (group 3, n = 6) each 10 mg daily were administered for 7 days. Arginine (468 mg]kg over 10 rain) was used to stimulate the endocrine pancreas. Glucagon w a s determined by radioimmunoassay using a highly specific antiserum (donated by Dr. R.H. Unger). Both insulin (IRI) and blood sugar (BS) levels were also assessed. After DZ treat.- ment, basal BZ increased 11.6~ (p < 0.02) and a frank

Vol. 9; No. 1, 1973 Organization Section �9 Abstracts 95

impairment of the insulin response to glucose was found, although thearg in ine- induced insulin secretion was not, significantly affected. Glueagon values, post- treatment were significantly lower both at the baseline points (dif- ference ~ 37 pg]ml; p < 0.02) and after the arginino load (differences from control osc i l la t ing between 85 and 137 pg/mt; p < 0.02). Neither the fasting gluea.gon con- centrations nor the glueagon response to axglnme were modified by glipizide or glibenelamide. Conclusions: a) in normal subjects, DZ administrat ion is followed by a de- pression of fasting plasma glucagon levels as well as by a diminished gtueagon reponse to arginine. "DZ diabetes" offers an experimental model of impaired function of both alpha and beta cells; b) contrary to other reports, sul- phonylureas failed to significantly affect the secretion of glucagon.

Quantitative Histology of the Pancreas in Human Anon- cephalic Fetuses and in Decapitated Rat Fetuses. F. A / V a n Assche and W. Gepts. Lab. of Gynecol. physic- path. Univ. of Leuven. Lab. of Path. Univ. of Brussel. A hypothalamo-hypophyseal system is not necessary f o r the basal development and histological differentiation of the fetal pancreas. However a functional hypothalamo- hypophyseal system is needed for the complete morpholo- gical and functional maturat ion of the pancreatic endo- crine tissue. The same morphological and functional chan- ges are present in Anencephalics with a functional hypo- thalamo-hypophyseal system born to mothers with re- duced carbohydrate tolerance as in infants from diabetic mothers, namely islet hypertrophy, B cell hyperplasia and an increased number of Argyrophilie ceils. However these changes are completely lacking in aneneephalics without a functional hypothalamo-hypophyseal system to mothers with reduced glucose tolerance. -- The experimental s tudy on decapitated rat fetuses confirms our data from hmnans. The percentage endocrine tissue is unchanged in decapit- ated fetuses comparing with the control fetuses. The per- centage of exoerine tissue however is slightly reduced. Prel iminary results will be showed on decapitated rat fetuses from diabetic mothers.

Participation of the Microfilamentous Cell Web in the Initial Secretory Response of the Beta Cell. E. Van Obberghen, G. Seiners, G. Devis, G.D. Vaughan, F. Malaisse-Lagae, L. Orei, W.J . Malaisse. Laboratory of Experimental Medicine, Brussels University, Brussels, and Inst i tutes of Histology and Biochimie Clinique, Ge- neva. The microfilamentous cell web is thought to be involved in the mechanism of insulin release during prolonged s t imulat ion of the beta cell. In order to assess the role of the cell web in the initial insulin secretory response, the effect of eytochalasin B, on both glucose- and sulfonyl- urea-induced insulin release was investigated in the per- fused isolated pancreas. Cyt, ochalasin enhanced both the initial and later release of insulin induced by either glucose or gliclazido. At the ul trastructural level, in control islets, the cell web appeared as a band of mierofilaments located just beneath the plasma membrane. Cytochalasin provok- ed an alteration Of the ceil web, with the appearance of large masses of micro filamentous material which often contained clumps of densely packed microfilaments hav- ing lost their prevailing polygonal arrangement. Granule cores extruded in the extracellular space were also often seen, spatially related with these micro filamentous mas- ses. -- I t is concluded that the cell web plays an equally important role in both the initial and later secretory response of the beta cell to various insulinotropic agents.

Effect of Butylbiguanide on Glucose and Fat Metabolism and Plasma Insulin Concentrations in Obese non-Diabetic and Diabetic Subjects after an Oral Glucose Load.

J. Veleminsky, E. Spirova. Ins t i tu te of Endocrinology, Lubochna, Czechoslovakia.

I n 10 obese non-diabetic persons and 10 obese maturi ty- onset diabetics the blood or plasma levels of glucose, pyruvate, citrate, FFA, glycerol, triglyeerides and im- munoreaetive insulin (IRI) were examined during 3 h after oral administrat ion of 100 g of glucose. Butylbiguan- ide (Silubin-Retard) was then given 900 mg daily for 6 days. O n t h e 7th day the examination was repeated 2 h after administrat ion of 600 mg of butylbiguanide. The butylbiguanide t reatment induced lower fasting glucose and I R I concentrations and a lower rise of glucose and I R I levels during the oral GTT in both observed groups. The fasting levels of pyruvato and citrate remained tin- changed and the partial metabolic block between pyru- r a t e a n d citrate, characterized by a higher rise of pyru- r a t e levels and an inhibit ion of the rise of citrate levels in obese non-diabetic and diabetic subjects, was Uninfluenc- ed by butylbiguanide. There was no significant change of the fasting FFA, glycerol and triglyceride levels and the response of the FFA and glycerol levels to glucose administrat ion was not altered by butylbiguanide treat- ment. -- The significance of these findings in relation to the mode of action of biguanido drugs will be discussed.

Insulin Antibodies in Diabetics: Incidence and Treatment by Monoeomponent Insulin, W. Waldhgusl, H. Frisch and H. Haydl. 1. Medizinische Universitgtsklinik, Wien, Austria.

Antibodies against insulin are regarded as a cause of in- sulin resistance. This s tudy investigated the incidence of antibodies against insulin (IA) in 299 diabetics treated both with boYine and/or hog insulin. IA were estimated by a radioimmunological double ant ibody technique and results expressed as ug insulin bound per litre serum. The pat tern of distr ibution of the insulin binding capaci ty (IBC) was as follows: < 10 ug:51~/o; 1 1 : 5 0 ug :23%; 51--100 ug:13~/o; 101-- 150 ug:6.34~o; 151--400 u g : 5 % ; 401--1000:1% and < 1000 ug:0.66~ None of the pa- t ients studied required more than i50 traits of insulin. In 9 patients with an IBC greater than 150 ug/1 t reatment with commercial insulin was terminated and subst i tuted by administrat ion of monocomponent insulin (semilente). This change was accompanied by an initial rise of the IBC in 3 out of 9 patients. All patients showed a significant decrease of IBC within 5 months of treatment. This effect however was no t consistently paralleled by a better con- trol of the diabet ics i tuat ion. I t appears, tha t the use of highly purified insulin may prevent the formation of IA vs. heterologous insulin.

Control of Fat Cell Pyruvate Dehydrogenase (PDH) by Insulin. L. Weiss, G. L6ffler, O. Wieland. Ins t i tu te of Clinical Chemistry, Schwabing City HOspital, and Diabetes Re- search Unit, Munich.

Mammalian PDH is an interconvertible enzyme, which exists in an active (dephospho-) form (PDH a) and in an inactive (phospho0 form (PDtt b). -- In rat fat cells insulin concentrations from 50--500 ~U/ml give rise to formation of PDH a within 5 min of incubation. The effect of insulin is mimicked by antilipolytic drugs such as nicotinate, 4-(aminobutyl)-guanidine and isopentyl-guan- idine. In contrast, prostaglandin El, which markedly inhibits lipolysis, has no effect on PDH-interconversion. Insul in is effective on .the PDH-system even in the presence of sufficient epinephrine to stimulate lipolyms. The effect of insulin is not abolished by NaF (5 raM), a blocker of glyeolysis. These results give evidence for an .insulin effect in adipose tissue independent of its action on lipolysis or glucose metabolism.

96 Organization Section �9 Abstracts

Alcohol Tolerance with Different Sulphonylureas. B. Willms, U. Deuticke. Medical Clinic of the University, G6ttingen, Germany.

Alcohol intolerance is a well kno~m side effect of the sui- phonylureas earbutamide, tolbutamide, and chlorpropa- mide. The biochemical basis of this so-called "antabuse syndrome" is not clear especially not as regards its possible correlation with an increased acetaldehyde concentration in the blood. Furthermore, it is not known whether with the more recent sulphonylureas of high potency an alcohol intolerance also occurs. -- Alcohol tolerance tests w e r e carried out with and without equi-hypoglycaemic doses of chlorpropamide (500 mg/day), glisoxepide (4 rag/day) and glibenclamide (2.5 mg/day). -- No differences were observed in the lactate]pyruvate ratios or ethanol disappearance rates with different sulphonylureas. An "antabuse syndrome" was found only with chlorpropamide and it correlated well with an increased concentration of acetaldehyde in blood. I t is thought to be due to a direct inhibition of the aldehyde dehydrogenase by the sul- phonylurea compound. -- The absence of the alcohol intolerance with glisoxeiMde and glibenclamide may re- sult from the lower dosage of these sulphonylureas.

A-Cell Function in Monolayer Cultures of Newborn Rat Pancreas. C.B. Wollheim, E.B. Marliss, B. Blondcl, L. Orei, A. Like, and A.E. Renold. Inst i tuts de Biochimie Clinique et d' Histologie, Universi ty of Geneva, Switzerland.

A-cell morphology and glueagon (IRG) content, and re- gulation of I R G release have been studied in monolayer cell cultures. Persistence of normal structure and function of cultured B-cells from enzymatieally-dissociated new, born rat pancreas has been demonstrated previously. -- Pancreas IRG content of 2-day old rats was 550 ng, and that of insulin (IRG) 6400 ng. Similar proportions of hormones were present, after 3 days of culture ( IRG: 25 ng, I R I : 240 ng/culture dish). I R G was present and A-cell ultrastructure was normal during culture periods up to 8 days. The influence of individual agents on IRG release was defined during 4-hour periods of i~cubation in Krebs-Ringer bicarbonate buffer. An inverse relation be- tween glucose concentration and IRG release existed be- tween 1.38--16.5 raM. Release was increased by 10 mM lactate (180% of control), pyrnvate (250%), alanine (210%), and arginine (250~ whereas DL-beta-hydroxy- butyrate I0 ~ inhibited to 60O/o (all with 2.75 mM glu- cose). Epinephrine 0.5 [zg/ml stimulated release to 155%o of control (11 mM glucose). Addition of propranolol or phentolamine suggested that epinephrine stimulation is

fl-adrenergic. Diazoxide did not alter IRG output. Cy- tochalasin B (5 and 10 ~g/ml) augmented IRG release with glucose 2.75, 5.5 and 16.5 raM, but increased I R I release ozfly with 16.5 raM. -- Conclusions: 1. A-cell structure and ftmction are preserved in monolayer culture.

2 . Methylnicotinamide increased I R G content and re- lease. 3. Several gluconeogenic substrates and epinephrine st imulated IRG release. 4. An agent affecting the micro- filamentous system augmented IRG release.

Protein Kinase Activities in Various Tissues of Normal and Streptozotocin-Diabetic Rats. J. Zapf, E .R . Froeseh. Metabolic Unit, Department of Medicine, Universi ty of Zurich, Switzerland.

Cyclic AMP (cAMP)-stimulated protein kinases may be third messengers in the hormonal regulation of lipolysis, gtyeogenolysis, glycogen synthesis and protein synthesis. Since lipid, glycogen, and protein metabolism are dis- turbed in insulin deficiency, we examined 1. whether protein kinase act ivi t ies of various tissues are quantita- t ively affected by the diabetic state, 2. if the enzyme pat tern in normal skeletal muscle of the rat which we found to consist of six different fractions on elution from a DEAE-cellulose column undergoes any quali tat ive changes. - : Our results indicate tha t bo.th basal and cAMP-stimulated specific activities of protein kinases in adipose tissue are reduced corresponding to the duration of diabetes. Basal protein kinase activities of rat liver and heart are not significantly/different in normal and diabetic animals, whereas cAMP-stimulated activities are de- creased in diabetic liver and diabetic heart. In skeletal muscle, cAMP-stimulated activities from tissue of normal and diabetic rats are identical; basal activities seem to be diminished in diabetic muscle resulting in a greater Stimulation by cAMP compared to normal muscle. The enzyme elution pat tern was the same in both normal and diabetic animals. It, is concluded that insulin deficiency effects protein kinase levels and their response to cAMP in a different manner in different tissues.

Causes of Death in Diabetics and Nondiabetics. M. Zrhstov~, J. Ro~,tlapil. Facul ty of Hygiene, Charles University, Prague.

A report of the causes of death in 16 033 cases covering the years 1957-- 1970 is presented -- autopsies of 14465 non- diabetics and 1568 diabetics. The most considerable differences are seen in vascular lesions, especially myo- cardial infarcts, and renal diseases. The low incidence of cancer among diabetics is striking~

Responsible for the text: Prof. Dr. W. CI~]r Med. Universit/~tsklinik, Humboldtallee 1, D-34 G6ttingen/F.R.G. Prof. Dr. K. SC~6FFLI-'~(~. Zentrum der Inneren Medizin, Theodor-Stern-Kai 7, D-6 Frankfurt 70IF. R. G. Responsible for advertisements : L. S~EGEL,

G. MARTIN, D-1000 Berlin 15, Kurfiirstendamm 237. Springer-Vorlag, Berlin, l=Ieidelberg, New York. Printed in Germany by Druekerei Georg Appl, Wemding]Sehwaben.

Copyright �9 by Springer-Verlag Berlin �9 Heidelberg 1973