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

EUROPEAN ASSOCIATION FOR THE STUDY OF

DIABETES

ASSOCIATION E U R O P ] ~ E N N E P O U R L ' ]~TUDE

D U D I A B E T E

E U R O P A I S C H E G E S E L L S C H A F T F U R

D I A B E T O L O G I E

ORGANIZATION SECTION President: W. CREUTZFELDT, GSttingen Past President: K. LUNDBAEK, Aarhus

(retires 1974) Secretary: K. SC]~SFFLING, Frankfur t (retires 1972) Vice-Presidents: B. I-IEL~,~AN, Umea (retires 1973) Editor-in-Chief of Diabetologia

M. I)F~RO~C, Paris (retires 1972) K. OBERDISSE, I)iisseldorf (retires 1972) The administrative offices of the Association are located with the Executive Director, Mr. J.G.J. Jackson, 3/6 Alfred Place, London WC 1E TEE, England.

Term expiring 1972 R. Koa~e, Kosice I. MAGYAR, Budapest P . J . RANDLE, Bristol J. SCHLICItT]~RULL, Copenhagen

COUNCIL Term exp~rin.g 1973 J .G. AI~VIS~.Tos, Athen A. BERIlVGEI~, Vienna G. PozzA, Milan J.L. ROD~I(~Ez-MI~o~, Madrid

HONORARY MEMBERS

Term expiring 1974 K.D. ItEPP, Munich P. LEF]~BVRE, Li6ge D.A. PYKE, London W. STAUF~'ACH:ER, Geneva

C.H. BEST, Toronto - t I . C . HAGEDO~, Copenhagen

SUPPORTING AND ASSOCIATE MEMBERS Ames Europe, Slough, Aron of Suresnes, France, Buckinghamshire - Boehringer, Mannheim, Germany - British Insulin Manufacturers, Great Bri tain - Chemic G~nenthal, Stolberg - Hoechst, Frankfur t - Hoffmann-La Roche, Basel - Ho~wwn Chemic, Miinchen - Novo Foundation, Copenhagen - Pfizer Europe, Bruxetles - Phartee, F r a n c e . Rona, Hitchin, Hert- fordshire - Sandoz A.G., Basel - Servier, Neuilly-sur-Seine - Upjohn Co., Kalamazoo

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

Southampton , England , September 15-17, 1971

Abstracts, Part 1

Reproducibility of Borderline OGTT Data on 163 Subjects. J .P . Aboulker, A.J. Valleron, L. Papoz, M. Rathery. In- s t i tut National de La Sant~ et de la Rgcherche M@dicale, Unit6 de Reeherches Statistiques U 21, Villejuif. France. Quanti tat ive data relative to the variat ion of individual blood glucose levels during repeated OGTT are few and concern normal subjects. -- 163 men, 20--55 years old, with suspect 0- -2 h OGTT or normal tests bu t positive glycosuria have been re-tested 2--25 weeks later by 0-- 5 Hrs OGTT. Both tests used a 75 g glucose load and identical procedures. Obvious diabetics and normal subjects were excluded at first OGTT. Analysis of variance for replicate OGTT, allowing for systematic and between- individual variation give, for fasting and 2 h blood glucose levels, residual standard deviations which measure mainly physiological imprecision of blood glucose levels and laboratory error. According to data in the literature, the ratio of residual variance to total variance is found to be constant during the 0--2 h OGTT; the greater these residual variances are, the poorer the reproducibility of OGTT is. Thus the changes observed in the allocation of the subjects into the different categories of diagnostic criteria between the first and second tests are important . Correlations between individual variations of blood glucose and other parameters show that the labili ty of OGTT depends on post-glucose OGTT levels and not significantly on age, obesity, history of diabetes in the family. -- Diag-

nostic implications of these results are discussed, particularly in relation to the problem of early detection of diabetes.

The Effect of Oral Contraceptives and Glucocorticoids on Insulin Response to intensive Islet-Cell Stimulation. P.~V. Adams, M.J. Munday, N.~V. Oakley, V. ~Vynn. Alexander Simpson Laboratory, St. l~[ary's Hospital IVied. School, London W. 2. Intravenous tolbutaraide 0.5 g and glueagon 1 mg given together 2 h after a 100 g oral glucose load stimulate massive insulin release in normal subjects (Ryan, Bibbe & Schwartz 1967). This test has been carried out on 11 young women both before and after prednisone 40 mg given in divided oral doses over the preceding 24 h; (N) & (N~-P). Both tests were repeated after at least three months on a combined oral contraceptive; (O/C) & (O/C ~-P). Compared with (N), mean glucose tolerance was slightly impaired for (O/C) and ( N + P ) tests and grossly abnormal for the (O/C§ P) test. Immunoreact ive insulin levels during the first 2 h followed the same pat te rn as plasma glucose, poorest glucose ltolerance being associated with the highest insulin vMu~s. Peak insulin response to glucagon and tolbutamide occurred at about 5 rain after injection, and was usually biphasie, with a second peak at 11 -- 16 min; peak insulin values QzU/ml =[= 1 SEM) were: N = 4 4 5 • 0 / C = 4 1 2 : ~ 8 1 , N - ~ P = 5 8 4 ~ : 6 2 ,

468 Organization Section. Abstracts Diabetologia

O/C + P : 765 -4- 94. There were marked differences between individual subjects, poor insulin responses following O/C ~- P being associated with impaired glucose tolerance for the (N + P) test. Implications of these findings, with particular reference to the detection of patients unsuited to oral contraceptive use will be discussed. -- Ref. : Ryan, W.G., Bibbe, A.F. & Schwartz, T.B. Lancet 1967 I, 1255.

Blood Glucose and Insulin in Levels in Neurogenic Diabetes Insipidns Treated with Chlorpropamide. D. Andreani, F. Fallucca, G. Stirati, G. Tamburrano, G.A. Cinotti. Seconda Clinica Mediea -- Universi ty of Rome -- I taly. Blood glucose and plasma insulin levels during OGTT were determined in 8 patients suffering from neurogenic diabetes insipidus (NDI) and in 6 control subjects. -- In the patients OGTT was repeated 15 days after t reatment with chlorpropamide (CP). -- Before t reatment 2 patients showed a diabetic type of glucose curve. The values of insulin levels, fasting and after glucose, were higher than in control subjects with differences which were statistically significant at 60i. The levels of plasma insulin grossly paralleled the daily water loss. -- During treatment with CP, when normal diuresis was reached, blood glucose and insulin levels were found within normal limits. -- On the basis of these results researches have been extended to the effects of t reatment with pitressin on glucose and insulin levels in such patients. Preliminary data are discussed.

The Effect of Various Blood Sugar Levels upon the Tol- butamidc-Stimulated Insulin Secretion in Diabetic Patients. D. Andreev, :N. Tarkolev. Diabetes Research Group, Bulg. Acad. of Sci., Sofia, Bulgaria. The effect of various blood sugar levels upon tolbutamide- stimulated insulin secretion in 30 maturity-onset diabetic patients was studied by intravenous injection of combina- tions of tolbutamide and glucose (25 and 50 rag glucose per kg body weight applied consecutively). The changes in blood sugar, I R I and F F A were followed up to one hour. The glucose-assimilation coefficient was calculated and compared with the insulin-secretion curves. -- I t was established that in diabetic patients who are tolbutamide- responders, the insulin secretion increases with the increasing initial rise of blood sugar after increasing doses of glucose. In the light of the Luft-Cerasi hypothesis it is presumed that in these cases the B-cells glucose-receptors have a diminished but not completely lost sensitivity to glucose. A high blood glucose level potentiates the action of tolbutamide in such patients. Overweight diabetic patients respond to tolbutamide-stimulation with hyper- secretion of insulin.

Immunohistological, Ultrastructural and Biochemical In- vestigations of Human Insulinomas. R. Arnold, C. Creutzfeldt, U. Deuticke, H. Frerichs, N.S. Track, W. Creutzfeldt. Department of Medicine, Univer- sity of GSttingen. Ten insulinomas were examined by routine immunohistological, ultrastructural and biochemical techniques. Using the peroxidase~labelled antibody method five insulinomas studied immediately after operation gave a positive reaction for both insulin and C-peptide. Positive reactions could not be obtained either in the same tissues after one year or in the other five adenomas studied at least nine months after the operation. Normal pancreas from these tumor patients gave a positive reaction regardless of the t ime interval following the operation. Ultrastructurally, seven adenomas showed normal beta-granules, three adenomas contained atypical granules similar to small As-cell granules or to pale granules with no discernible limiting

membrane. The immunoreactive insulin content of the adenomas was in the range from 3.5--58 U/g wet weight compared to 0.9--4.3 in the adjacent normal pancreas. Column chromatography demonstrated the presence of higher percentages of big insulin in the adenomas examined compared with those found in the normal pancreas. The immunohistological, ul trastructural and biochemical findings will be discussed.

The Relative Importance of Arterial Concentrations and Tissue Uptakes (of Hormones and Substrates) in the Re- gulation of Peripheral Metabolism in Normal, Obese and Diabetic Subjects. A.C. Asmal, W . J . H . Butterfield, B. Karamanos, M.J. Whichelow. Department of Medicine, Guy's Hospital Me- dical School, London. Measurement of blood levels and tissue uptakes of glucose, insulin, free fa t ty acids, triglycerides, cortisol and oxygen have been made in the steady state and after glucose load, and the data examined for evidence about: 1. The general influence of arterial concentration on tisssue uptake (tissue substrate thresholds). -- 2. The interaction between tissue uptake of different substances. -- 3. The relationships of hormone and substrate levels in the arterial plasma. -- In normal subjects in the basal state there are significant positive correlations between the arterial concentrations and tissue uptakes of insulin, triglycerides and glucose, and in obese subjects between insulin concentra- t ion and insulin uptake. Also in the basal state there are positive correlations between the arterial concentrations of triglycerides, F F A and glucose in the obese, but not the normal group. In both groups there is an inverse correla- t ion between the uptake of insulin and that of I~FA and triglyceride, and none between insulin and glucose uptake. -- There are considerable alterations in these relationships in the diabetics. Since arterial concentrations of substances represent the direct signals from central regulatory mechanisms (nervous, hormonal, hepatic, etc.) and tissue uptake reflects metabolic events there, this study represents initial a t tempts at discrimination between the effects of central events, and of tissue processes in peripheral metabolism.

Insulin Secretion and Glucose Utilization by Human Pan- creatic Islets in Vitro. S.J .H . Ashcroft, J .M. Bassett, P . J . Randle. Dept. of Biochemistry, Universi ty of Bristol, U .K. Human pancreatic tissue was obtained immediately post- operatively from a patient undergoing surgical pan- createetomy for idiopathic hypoglycemia with evidence of hyperinsulinism but without islet cell adenoma. Islets were prepared by a collagenase method and incubated in bicarbonate medium. Insulin released into the medium was measured by radioimmunoassay and glucose utilization by the rate of formation of [3H] water from [5-all] glucose. -- The human islets had a mean insulin content of 1.3 mU/ islet: dilution curves of the islet extract were identical to those obtained with MRC standard human insulin. In the presence of caffeine, the rate of insulin release was increased from 10 to 67 microunits/islet/90 min on raising the medium glucose concentration from 60 to 300 rag%. Paralleling the increase in secretion rate was an increase in islet glucose utilization rate from 15 to 85 pmol/islet/ 90 min. In the absence of caffeine, glucose-stimulated insulin release was reduced by about 50 per cent. -- The rates of insulin release and glucose utilization were similar to those obtained in parallel studies with normal mouse islets.

Glucose Metabolism and the Secretion of Insulin from the B-Cell of Neonatal Rats. K. Asplund, C. Hellerstr6m. Histological Department, Universi ty of Uppsala, Uppsala, Sweden.

VoL 7, No. 6, 1971 Organization Section. Abstracts 469

The fetal and neonatal rat has been used as a model for studying the functional maturat ion of the pancreatic B- cell and, as previously reported, in this species glucose was found to be a poor stimulus for insulin secretion unti l the second postnatal day. In the present s tudy we have investigated whether this lack of insulin response to a glucose challenge might be due to an immature glucose metabolism of the B-cell. -- At a low level of extracellular glucose (0.6 mg/ml) the oxygen consumption rate was higher in isolated islets from one day old than from six days old rats. However, at a glucose concentration of 3 mg/ml similar respiratory rates were recorded in the two age groups. The rate of anaerobic glycolysis was of the same magnitude in islets from one and six days old animals in the absence of glucose. When the glucose concentration was raised to 3 mg/ml the anaerobic glycolytic rate increased by five times in islets from one day old rats, and was significantly higher than in the older age group. The breakdown of uniformly labelled radioactive glucose to 14CO2 was investigated at low and high glucose concentr- tions in the incubation medium and was found not to differ in islets from one and six days old rats. -- I t appears that the failure of glucose to promote insulin release during the fetal and immediate neonatal period in the rat should not be ascribed to a deficient glycolysis and oxida- t ion of glucose.

Effects on Rat Plasma Glueagon of @ Sugars, 8 Amino Acids, Lactate Load and Induced Hyper-Lactacidemia. I%. Assan, J. Hanoune, J. 1%. Attali. H6tel-Dieu de Paris and H6pital Cochin, Paris, France. Groups of adult rats were submitted to intra-gastrie loads (i g/animal) of 6 sugars after a 48 h fast; to intra-gastrie and intra-peritoneal loads (0.45mmol/100g) of 8 amino- acids after a 8 h fast; to lqa-Lactate and lactic acid (2.5 mmol/animal) intra-peritoneally; to hyperlaetaeidemia induced by hypothermia, or muscular exercise. Panereato- glueagon (P.G.) and total glucagon-like immunoreaetivity (GLI) were assayed by 2 immunological systems; GLI from extra-pancreatic origin (NP-GLI) was calculated (total BLI minus PG). Initial values, for total GLI and PG, were higher in 48 h fasting controls (GLI 1310 pg/ ml• 200, PG = 966 • 245) than in 8 h fasting controls (GLI 664 :k 33; PG 226 :k 184). NP-GLI rose only after intra- gastric loads of actively absorbed sugars with an early peak (30 rrm) for glucose ( x 3), a delayed one (60 ran) for fructose (• 1.90), galaetose (• 1.25), glycerol (• 1.40). Iqo rise occurred after mannose and ribose, nor after non- glucidic loads. -- P.G. decreased (• 0.6) 30 rain after all sugars, and rose later only for ribose load (during ribose- induced hypoglycemia). PG electively rose after amino- acids, intra-gastrie (i.g.) as well as intra-peritoneal (i.p.) TRY (ip • 9; ig • 5); LEU (ip • 5 ; ig • 5); TYR (ip • 5; ig • 5); ALik (ip • 4; ig • 4); ARG (ip • 4; ig • 2); VAL (ip x3 ; ig • 2.5); GLU (ip • 3; ig • 2); GLY (ip • 3; ig • 1.2). PG rose after i.p. lactate (• 2.5) and lactic acid

( • 3.5), hypothermia-induced hyperlaetatemia ( • 4) and muscnlar exercise (• 3). A strong rise for PG was found in human lactic acidosis. I t is suggested that while active intestinal absorption of sugars is the only known factor associated with lqP-GLI secretion, amino-acids and lactate play (besides glucose supply) a prominent role on PG secretion.

Umbilical Catheterization and Portal Blood Sampling for Sensitive, Physiological Exploration of Insulin and Glu- cagon Secretions in Man. R. Assan, G. Tehobroutsky, G. Gross. Hotel-Dieu, Paris, F r a n c e .

Humeral venous blood (H) and, through a catheter in- serted for radiological purposes in umbilical vein, portal blood samples (P) were collected from 6 subjects (2 normals, 2 diabetics, 2 cirrhotics), during arginine and

pancreozymin infusions, and during a protidoglucidie meal, then assayed for insulin and glueagon (pancreatic and total GLI). While glucagon basal levels were not significantly different in P and H plasmas, the glucagon peaks measured after stimulation by arginine and panereozymin were 2-- 5 times higher in P than in H, with an earlier occurrence (15 rain versus 30 rain) for 4/6 subjects; no P/I-I gradient was found in eirrhotics with large patho- logical porto-eaval shunts. The slight variations induced by meals were constant in P, weaker or absent in H plasmas. Acute insulin-induced hypoglycaemia did not raise glucagon in H; only a slight, insignificant rise occurred in P plasma. -- Insulin secretion after meal was sluggish and delayed in a mild maturi ty-onset diabetic (P as well as H plasmas), but after arginine stimulation, the insulin peak occurred at the 15th rain. A constant P/I-I gradient was observed for insulin, except in the 2 eirrhot- ies. Intra-portal administrat ion of glucagon or insulin induced greater variations of glycaemia than peripheral i.v. injections. Disappearance t ime for exogenous injected glueagon was significantly shorter after intra-portal than after peripheral injections. -- Conclusion: Umbilical vein catheterization is technically simple and well tolerated, allowing precise physiological measurements to be made.

Biochemical and Eleetronmieroscopic Studies on Perfused Livers from Normal and ObOb Mice. F. Assimaeopoulos, L. Orei, Ch. Rouiller, B. Jeaurenaud. Ins t i tu t de Biochimie clinique et Ins t i tu t d'Histologie et d'Embryologie, Geneva, Switzerland. The present experiments have been carried out with livers from normal Swiss (nS), O57 Black (C57) and obese hyperglycemic (ObOb) mice. The perfusion medium consisted of Krebs-Ringer bicarbonate buffer containing 2% albumin and washed bovine red blood cells. In perfused livers from fed nS mice, glycolysis was markedly stimulated by adrenaline (10-~ cyclic AMP (10-5M), and glucagon (10-1~ Gluconeogenesis in livers from fasted nS mice was increased by the addition of pyruvate, lactate, flue- tose, glycerol and alanine. Under the conditions used, however, glueoneogenesis was little influenced by glucagon or cyclic AMP, suggesting that this process might be re- gulated mostly by substrate levels. ObOb mice had considerably more liver lipids than the C57 controls. Glu- eoneogenesis, expressed as a function of delipidated liver weight, was 3--4 times higher in livers from fasted ObOb mice than in those from C57. This was true whether alanine, lactate or no substrate was added to the perfusion medium. In addition, preliminary experiments in livers from both ObOb and C57 mice indicate that gluconeogenesis was reduced upon addition of insulin. Electron- microscopic studies showed that both nS and C57 livers perfused for 1 h were morphologically normal and had an excellent state of conservation. On the contrary, livers from ObOb mice were characterized by a very marked in- filtration of lipid droplets which greatly varied in size, and by the frequent finding of lipid droplets within the nucleus. Thus, both lipid metabolism and gluconeogenesis appear to be markedly altered in the ObOb mice, but the causes of these changes are not yet determined.

Oral Glucose Tolerance Test and Insulin Response to Glu- cagon in Pheochromoeytoma. M. Austoni, G. Federspil, D. Casara, N. Sieolo, C. Scan- dellari, I. Mastrogiaeomo. Is t i tuto di Semeiotica Mediea. Padua University. I taly. In pheochromocytoma it has been claimed that the insulin secretion should be inhibited by catecholamines. In a series of 8 pheochromocytoma cases tested with OGTT, the average glyeemic curves showed a very typical pattern; that is a sharp rise with a maximal value at 60 min followed by a rapid drop to normal levels. In three cases which were restudied after surgery the response became

Diabetologia, Vol. 7 33

470 Organization Section. Abstracts Diabeto[ogia

normal. Three of these patients were tested for insulin secretion during a OGTT, and two of this latter group were subjected to a rapid i.v. glucagon test and insulin production measured. During the OGTT insulin response was found (A ~o -~ 223) to exceed that in normal subjects; however in one of these subjects the maximal response was delayed to the 180 th rain. Glueagon injection, on the contrary, brought only a very modest increase in plasma insulin level, perhaps related to the excessive catecholamine output induced by glucagon. These data suggest that in pheochromocytoma 1 �9 there may not be a constant inhibition of insulin secretion in the OGTT; 2. there is no evidence of peripheral resistance to endogenous insulin; 3. during a glucagon infusion, the catecholamine excess seems to inhibit insulin secretion even in eases with abun- dant insulin reserve, as shown by the OGTT.

The Effects of Furosemide and Amiloride (MK 870) on Glucose Tolerance and Insulin Secretion in the Rat. A. Aynsley-Green, K.G.M.M. Alberti. Nuffield Depart- ment of Clinical Medicine, The Radcliffe Infirmary, Ox- ford. Diuretic therapy may be associated with glucose intolerance but the mechanism and the effect on insulin secretion are not clear. -- This communication reports the effect of two structurally different diuretics, furosemide and amiloride, on insulin secretion and blood glucose in the conscious rat when given intravenously alone or with glucose. -- Furosemide alone at doses of 1 mg and 2 mg/kg caused a decrease in plasma insulin of 26% and 54% respectively one minute after administration and a rise in blood glucose of 19~ and 27% twenty minutes later. The same doses given with or th i r ty minutes before intravenous glucose (0.5 g/kg) caused a 40% decrease (p < 0.01 for all doses) in glucose disappearance rate with decreased initial insulin secretion. Chronic administration did not affect glucose tolerance or insulin secretion although total body potassium and sodium were decreased. -- On the other hand, amiloride alone (5 mg or 10 mg/kg) caused an up to 20 fold increase in plasma insulin after one minute. When given with glucose, marked hyperinsutinaemia occurred without altering the glucose disappearance rate. The effects of the two drugs are thus markedly different. Their effects on the isolated perfused rat pancreas will also be presented.

Can Tolbutamide Treatment be Harmful in Diabetes Melli- tus ? Gh. Baeanu, L. Stoichescu, F. Nistor, V. Turcanu, L. Anghelescu. Antidiabetic Centre, Timisoara, Rumania.

The findings of the Universi ty Group Diabetes Program, suggesting that Tolbutamide t reatment is likely to increase the mortal i ty due to cardiovascular disease, has astonished quite a number of diabetologists. -- Among the 4500 diabetic patients controlled at our Centre, we divided selected patients into 3 groups. The patients were aged between 51 and 90 years and had had corresponding antidiabetic therapy over 7--13 years. The 3 groups consisted of: 260 subjects whose diabetes was controlled by diet (1 st group), 200 insulin-treated patients (2rid group), and 266 Tolbutamide-treated diabetics. 100 patients of each group have already died. -- The cause of death was cardiovascular disease in 68% of the first group, 56% of the

o second, and 78 ~o of the third group. The difference was due to the age group ranging between 71 and 80 years included in the study. The mean age at death was 67 years in the first, 60.3 years in the second, and 70.3 years in the third group. -- Our evaluation, overall and of the results observed in diabetics still being treated, does not warrant our stating that the judicious administration of Tolbutamide is harmful in the t reatment of diabetes.

The Effects of Sympathetic Blockade, Adrenaleetomy and 5-Hydroxytryptamine on Arginine-Induced Insulin Secre- tion in the Rat. R.A. Bacchus, L.G. Meade, D.R. London. Department of Chemical Pathology, St. Thomas' Hospital, London. Various agents involved in the action of biogenie amines have been studied in rats. Their effects on blood insulin, arginine and glucose have been investigated. Guanethi- dine, reserpine, desipramine and phentolamine all enhanced the insulin response to arginine and, contrary to the effect previously reported in man, reduced the rise in blood glucose. Adrenalectomized animals showed a similar enhancement of insulin levels and reduction of rise in blood glucose after arginine. All these results can be inter- preted as showing that both the sympathetic nervous system and adrenal medullary hormones are involved in the modulation of insulin secretion in the rat. The seeming- ly anomalous behaviour of desipramine can be explained by its known action as a blocker of 5-hydroxytryptamine. In rats in whom catecholamine stores had been depleted by prior administration of reserpine, and therefore in whom the effect could not have been ascribed to secondary catecholamine release, 5-hydroxytryptamine reduced the secretion of insulin after arginine. Thus the insulin response to arginine may be determined by a com- ple.x control system involving both groups of biogenie amlnes.

Effect of Ketone Bodies and Free Fatty Acids (FFA) on Glucose Oxidation in the Dog. E. O. Balasse. Laboratory of Experimental Medicine, Uni- vers i ty of Brussels. Nineteen anesthetized dogs were infused at a constant rate with glucose (10--13 mg/kg rain), glueose-UJ4C and insulin (1.2 -- 2.1 mU/kg min) for 5 h. Arterial concentrations of glucose, aceto-aeetate, 3-hydroxy-butyrate and FFA, and the specific act ivi ty of expired C02 were followed throughout the study. Experiments were divided into 3 groups. -- In the first group (control experiments), no other exogenous substrate was provided to the animals. Glycemia remained in the normal range and was steady, indicating that glucose uptake was equal to the rate of infusion. The specific act ivi ty of expired CO 2 rose exponen- tially with time, reaching an asymptotic value at the end of the study. The CO2 derived from prompt oxidation of glucose represented about 50% of the total C02 production. -- In the second group of studies, a constant infusion of sodium aeeto-aeetate (64--110 ~moles/kg rain) was superimposed on the glucose-insulin infusion during the last 2 h of the experiment. The hyperketonemia thus obtained averaged 5 ~moles/ml. Ketone bodies inhibited glucose oxidation by 40 ~o without altering significantly the rate of glucose uptake. -- In the third group of studies, a similar experimental model was used to test the influence of increased blood F F A levels on glucose oxidation. High F F A levels (1.1 ~moles/ml) were obtained by infusing a triglyeeride emulsion and heparin. Under these conditions, glucose oxidation was inhibited by 22% when compared with the control data. A slight inhibition (5O/o) of glucose uptake by tissues was also observed. -- I t is concluded that ketone bodies and, to a smaller extent FFA, can inhibit glucose oxidation i n vivo .

The Diagnostic Role of Fluorescein Angiography in Juve- nile Diabetes. L. Barfs, G. Brooser, Maria Moln~r. I st Department of Pediatrics of the "Semmehveis" University Medical School and Ophthalmological Department of the Medical Post- graduate School, Budapest. Ophthalmological examinations were performed in 201 diabetic patients whose first symptom had appeared before

VoI. 7, No. 6, 1971 Organization Section. Abstracts 471

their 14th year. In 25 of them, ret inopathy was estimated by ophthalmoscopy. In 102 patients -- whose ophthalmoscopic findings were repeatedly negative, -- fiuorescein angiography was carried out, and in more than the half of the observed cases, microaneurysms were detectable. Fluorescein angiography was done in 1/3 of the patients between 8--14 years. There was no correlation between the duration of diabetes and the retinal lesion in those patients who were negative on ophthalmoscopic examina- tion and positive on fluorescein angiography. There was a close correlation between the duration of diabetes and the occurrence of ret inopathy in those presumably advanced cases, where a retinal change was found even by ophthalmoscopy. The frequency of the microaneurysms estimated by fluoreseein angiography was the same in the poorly controlled as in the well controlled groups. According to our observations, retinal microaneurysms are detectable in the early phase of diabetes, as the asymptomatic phase is of short duration in iehildhood. The early ophthalmological changes are regarded as part of the diabetic syndrome.

Serum Beta-Glucuronidase Activity in Diabetic Patients, and its Relationship to Vascular Complications and Ke- toacidosis. F. Belfiore, L. Lo Veeehio, E. Napoli. Ins t i tu te of Medical Pathology and Clinical Methodology, Universi ty of Ca- tania, Italy.

In a group of 120 diabetics, a statistically significant increase (about 70%) of serum beta-glucuronidase was found, in relation to normal subjects. The increase was more pronounced in the diabetics with vascular complications (at renal, cardiac or ocular level), where it reached a level of almost 100%, as well as in the ones with high blood sugar, and was very striking in the patients with ketoacidosis and coma, where it ranged from 300 to 900%. In diabetics without complications the elevation was less accentuated but still statistically significant. These findings seem to indicate tha t the increased level of serum beta-glucuronidase is correlated with both the derange- ment of glucose metabolism and the vascular lesions which take place in diabetes. -- As beta-glucuronidase, together with other enzymes, contributes to the degradation of mucopolysaccharides (which accumulate in diabetics and are implicated in the genesis of vascular lesions), our findings are in agreement with the hypothesis that the elevated glucuronidase act ivi ty in diabetes could be connected with the increased synthesis of these eom- potmds, which occurs in this disease as a result of the high act ivi ty of the glucuronic acid pathway, which is not sensitive to insulin.

The Insulin Potentiating Activity of Different Sulfonyl- ureas. J. Bayer, U. Cordes, G. Sell, N. Krall, K. SchSffling. De- par tment of Endocrinology, Center of Internal Medicine, Johann Wolfgang Goethe University, Frankfurt /M, Ger- many. Results obtained on the insulin potentiat ing effect of tolbutamide and Glibornuride (Ro 6-4563) in total ly de- pancreatized, chronically insulin-treated Beagle dogs have shown, using equimolar doses (0.37 mmol/kg i.v.), different effects on blood glucose and non esterified fat ty acids. Whereas tolbutamide leads to a significant, pronounced decrease of the blood glucose when administered with insulin, in contrast to insulin alone, glibornuride has shown the most marked decrease of NEFA. Whereas both of the main metabolites of tolbutamide, hydroxy- mcthxl tolbutamide and earboxytolbutamide, given with insulin, are ineffective in additional blood glucose lowering activity, they have a more marked antilipolytie effect than tolbutamide. Carboxydiaboral, a metabolite of glibornuride, leads under the same conditions to a similar bchaviour to glibornuride. Fur ther investigations with

doses in the therapeutic range may provide evidence on different effects, which may also exist under therapeutic conditions.

Effects of Halothane on Blood Metabolites and Serum In- sulin in the Rat. J . F . Biebuyck, K.G.M.M. Alberti. Metabolic Research Laboratory and Nuffield Departments of Clinical Medicine and Anaesthetics, The Radcliffe Infirmary, Oxford. tIalothane, a commonly used anaesthetic, causes a marked rise in blood glucose and serum insulin in rats. To elucidate the mechanisms involved the effects of the drug have been studied on the isolated perfused liver, fed and starved normal rats, and adrenalectomized, and diabetic animals. The effects of repeated exposure to halothane are also reported. -- Halothane caused an immediate rise in lactate production by the perfused liver but there was no change in glucose release, contrasting markedly with the in vivo situation in fed rats where no change in blood lactate was seer~ but a marked rise in blood glucose concentration occurred within 5 rain persisting throughout anaesthesia and accompanied by a progressive rise in serum insulin. This hyperglycaemia was not found in starved rats although a rise in serum I R I still occurred. In adrcnalectomized fed rats blood glucose rose slightly with no change in insulin. Diabetic fed rats showed no change in glucose with halothane although there was a hyperglycaemic effect in starved diabetic animals. Re- peated intermit tent exposure to halothane of fed normal rats resulted in a progressively decreasing hyperglycaemic response with some diminution of liver glycogen. The significance of the findings in relation to glucose homeos- tasis during anaesthesia, and anaesthesia in the diabetic state will be discussed.

Early Glucose Intolerance and Impaired Insulin Secretion in Idiopathic Hemochromatosis. ]3. Bierens de Haan, J . R . Scherrer, W. Stauffacher, D. Pometta. D6partement de M6decine, H6pital Cantonal, Gen6ve. The relationship between iron storage and glucose metabolism was studied in 21 relatives of 4 patients with idiopathic hemoehromatosis and in 10 healthy control subjects. In all individuals, plasma iron and iron binding capacity were measured and liver function assessed. In addition, intravenous and oral glucose tolerance tests (IVGTT, OGTT) as well as tolbutamide (TT) and insulin tolerance tests (IT) were performed. Liver biopsies were performed on the 21 relatives only. In the relatives of patients with hemochromatosis, glucose tolerance was impaired and insulin secretion in response to hyperglycemia diminished and/or was delayed. Glucose intolerance increased with age but did not depend on abnormal liver function or excessive iron storage. Insulin release in response to tolbutamide was normal and failed to reveal insulin resistance. The results suggest that glucose intolerance in hemochromatosis results from a defect in insulin secretion in response to glucose, similar to tha t observed in diabetes, and that this anomaly is not directly related to measurable anomalies in iron metabolism.

The Associations between the Degree of Filling of Adipose Tissue and Metabolic Disturbances in Obesity. P. Bj6rntorp, L. Sj6strSm. First Medical Service, Sahl- gren's Hospital, Gothenburg. The reason for the increased frequency of cardiovascular disease and diabetes among obese patients is not completely known. Statistical analyses of the associations between the two factors responsible for adipose tissue size, fat cell number and fat cell size, on the one hand and metabolic variables on the other, provide a means of examining this question more closely. A simple, non-

33"


t raumatic technique for fat celt size determination has made possible comparisons between randomly selected middle.aged persons (n ---- 123) and obese patients (n = 145), demonstrating a group of moderate obesity characterized by enlarged fat ceils (hypertrophic obesity) while severely obese patients had an increased number of fat cells (hyper- plastic obesity). -- Statistical correlations between fat cell size and insulin were stronger than between body fat and insulin in mlddle-aged and young men, in obesity, endogenous hy2oertriglyeeridemia and in myotonic dystrophy. Fa t cell number showed no positive correlation with insulin. Plasma triglyeerides correlated with insulin and glucose tolerance. The well-known moderate obesity of endogenous hypertriglyeeridemia and adult-onset diabetes was found to be characterized by enlarged fat cells. The statistical associations were broken by caloric restriction or increased physical activity. I t was concluded that the degree of filling of adipose tissue is the factor in obesity that is associated with disturbed metabolism.

The Evaluation of Photocoagulation in Diabetic Retlno- pathy: A Report on Behalf of the National Multi-Centre Photocoagulation Trial by R .K. Blaeh, (secretary) and Hung Cheng. Seratiny of published claims of the value of photoeoagula- t ion in diabetic ret inopathy does not provide proof of its effectiveness. A controlled clinical trial has therefore been established under the aegis of the British Diabetic Asso- ciation which is open to European centres. The protocoI of this trial together with tiie methods of assessment are briefly described and the techniques of treating the various features and types of diabetic ret inopathy are indicated.

A Specific and Sensitive Glucagon Immunoassay. S.R. Bloom. The Ins t i tu te of Clinical Research, The Middlesex Hospital, London. An assay for enteroglucagon and pancreatic glucagon has been set up without the need for plasma extraction. A sensitive antiserum is used to measure changes of 30 pg within 95% confidence limits. Samples are also assayed with an antiserum relatively specific for pancreatic glucagon, which enables estimations to be made of the pro- portions of pancreatic and enteroglucagon present. Standard curves are set up in pooled human plasma, initially stored at 4°C for some weeks to allow proteolysis of endogenous glucagon. Separation of ant ibody-bound from free glucagon is achieved with dextran-eoated charcoal. -- An antibody has been raised to an extract from a human enteroglueagon tumour. This has enabled a better estimation to be made of the true level of circulating enteroglucagon. -- Following a s tandard 100 gram oral glucose load, enteroglucagon levels rise by 450 pg and pancreatic glucagon falls by 65 pg per ml. The level of both glucagons obtained in reactive bypoglycaemia and after gastric surgery will be discussed.

The Studies on the Behaviour of Glucose in Plasma a n d Erythrocytes, and Especially of their Levels after Ingestion of 50 g Glucose in Diabetes Mellitus and Hyperinsulin- ism. K. Bojanowiez. Gastroenterological Clinic, Medical Aca- demy, Lodz, Poland. Determination of glucose levels in plasma and erythrocytes were conducted by the enzymatic (hexokinase) method in 46 adults with diabetes, 4 with hyperinsulinism and 20 healthy subjects. I t has been found that the g lu- cose level was highest in the plasma, intermediate in the capillary blood and lowest in the erythrocytes. The curves drawn from the differences between glucose levels in the plasma and the erytbroeytes depended on the kind of disease. Such differences have been smaller in the hyper-

insulinlsm, and higher in chemical diabetes, than in the healthy subjects; in clinical diabetes treated with tolbutamide or insulin they were higher than in chemical diabetes, they were highest in severe diabetes. The above curves of differences of glucose levels in the plasma and the erythrocytes have been in agreement with the degree of glucose tolerance in various states of intrinsic secretory activity of the pancreas. The studies strongly suggest tha t determination of glucose level curves in plasma and erythrocytes, and especially of their difference, enrich the methods of investigation of glucose tolerance. They also provide a better insight into the meehanisms conditioning penetration of glucose from the plasma to the erythro- eytes, as well as into their relation with insulin secretion.

Obesity and Pancreatic Islet Hyperplasia in the Mongol- ian Gerbil. L. Boquist. Inst i tute of Pathology, University of Ume&, Ume&, Sweden. Syndromes associated with hyperglycemia and/or obesity are known to occur in about a dozen different laboratory rodent species. I n the colony of Mongolian gerbils which has been bred in our laboratory since 1968, some of the animals have been found to develop obesity and morpho- logic alterations in the pancreatic islets. Such changes have not been reported in this species previously. Some gerbils show transitory obesity, whiIe others are perma- nent ly obese. Only a few cases of gineosnria have been found among the obese animals. The blood glucose level has shown individual variations bu t only a few cases of slight hyperglycemia have been recorded. The glucose tolerance has either been decreased or normal in the obese gerbils. Preliminary investigations of immunoreactive insulin have disclosed occasional eases of hyperinsulin- emia. The light and electron microscopic appearance of thepancreaticislets in the obese animals varies. Occasion- ally there is hyperplasia of the endocrine pancreas, in- eluding the occurrence of a few "giant islets" with rich vascular supply. The t-cells show decreased granulation, glycogen accumulation, cytoplasmic vacuoles and some. times mitochondrial alterations. The significance of these findings is discussed.

Sorbitol Pathway in the Human Umbilical Cord. E. Brachet. Laboratory of Physiology, University of Brus- sels, Brussels. Some oedematous changes which occur in tissues have been correlated with the local conversion of glucose into sorbitol, which might promote water retention. The same reaction has been identified in some embryonic and vascular tissues. The normal umbilical cord is a vascular structure of emb~sronic origin, which contains as much as 90 ~o water. For these reasons, this tissue was investigated for a possible sorbitol pathway. -- The umbilical cord contains fructose (0.6 ~xmol/g) and sorbitol (2 yxnol/g). The chopped tissue shows oxygen consumption and uptake of glucose, which is accompanied by production of sorbitol. The consumption of exogenous sorbitol is not constant and rarely leads to an increase of fructose. However, this reaction cannot be ruled out, since the umbilical cord is able to take up fructose from the medium without pro- ducing az W sorbitol. -- I n conclusion, the first step of the sorbitol pathway occurs in the umbilical cord, where i t ace0lmts for 1/6 of the glucose uptake. This reaction might thus have some importance in maintaining this tissue in a state of "physiological oedema".

Photokinetic Micro Assay of Metabolites and Cofactors in the Pancreatic Islets. S.E. Brolin, C. Berne. tIistologieal Department, Univer- sity of Uppsala, Uppsala, Sweden. Application of photokinetie teetmiques has made it possible to measure metabolic intermediates with light-

Vol. 7, No. 6, 1971 Organization Section �9 Abstracts 473

yielding reactions, at and below the picomole level. Bac- terial luciferase has proved to be of particular value since reduced pyridine nucleotides which are formed in many dehydrogenase reactions can be transferred into light reactions. By coupling to dehydrogenase steps the analytical applicability has been much extended. The extraction of metabolites to be measured has to be combined with destruction of reduced pyridine nucleotides by acidification. These nucleotides would otherwise interfere with the assay. In islet research it is also often desirable to determine the cellular content of both reduced and oxidized pyridine nucleotides. After extraction combined with acidification both NAD + and NADP + can be selectively reduced and assayed. -- By alkaline extraction the reduced pyridine nucleotides are preserved whereas the oxidized forms are destroyed. I n photokinetic assay differences in the light response induced with NADH and NADPH reveM the presence of each nucleotide. A quanti ta t ive estimation of the separate nueleotides, however, requires enzymatic oxidation of NADPH before measurements of NADI-I and vice versa. Analytical procedures for the measurements of NADH and NADPH have been developed and applied in various states of islet function.

Changes in Blood Glucose, IRI and FFA During Thera- peutic Use of Glucagon in Cardiology. B. Bruni, E. Capra. Maria Vittoria Hospital, Torino, Italy. The insulinogenic, glycogenolytic-hyperglycemie and lipolytie effects of glucagon must be considered during glueagon t reatment of severe eardiologic conditions, such as resistant heart failure and cardiogenie shock, both in non-diabetic and in diabetic or pre-diabetic subjects. In particular, an increased insulin secretion might be of clinical relevance in these eases. -- The concentrations of glucose, I R I and F F A in plasma was studied at 0, 30, 60, 90, 120, 180, 240 min during an intravenous infusion of 5% glucose solution with 10 mg Glueagon Novo (2 mg per hour) in 10 heart-normal subjects (2 diabetics) and in 7 cardiac patients (2 diabetics). The most important finding was an hyperinsulinemic peak at 60 (90) min, mainly in obese subjects; the insulinemie response was delayed in diabetics. Hyperglycemia of various degrees was observed for about 90 min ; mild reactive hypoglycemia was present in some eases. FFA showed a marked fall within 2 h, followed by a slow rise almost to the basal values. The hyperinsulinemie peak coincides with the distressing nausea occurring in all patients. -- The same parameters wore investigated at 1, 2, 3, 4, 5 h after subcutaneous administration of 5 mg Zinc Protamine Glucagon Novo in 8 subjects with refractory heart failure (2 diabetics). I R I and glucose curves demonstrated a small elevation above the initial values, maintained for about 4 h. F F A had a biphasic pattern, with a decline within 2-- 3 h and a gra- dual re turn to baseline at about 5 h. No side-effects during this type of t reatment are fotmd.

Effect of Sulfonylurea Treatment on Starvation Diabetes. V. Biiber, J .P . Felber. D@artement de Biochimie Clini- que, Clinique Mddicale Universitaire, Lausanne. 6 normal subjects were put on a 48 h fast in order to pro- duce starvation diabetes. During an OGTT, before and after the fast, glucose, insulin HIGH and GLI (glueagon- like immunoreactivity) were measured. There was a net deterioration of the glucose tolerance after the fast, with a concomitant increase of the insulin values after oral glucose stimulation, in spite of lower fasting glucose and insulin values. No significant changes in ~IGI.I and GLI levels could be found. -- The same procedure was repeated, bu t the subjects were treated with sulfonylureas during the period of fast, there was no influence of the drug on the development of starvation diabetes, even though the insulin levels after oral glucose load were rather higher than without SU treatment. No influence of

the t reatment on HGH and GLI levels could be observed. -- I n conclusion, sulfonylureas cannot prevent the development of starvation diabetes in spite of increased sensitivity of the pancreas to glycemic stimulus. This suggests a dissociation between the therapeutic and fi- cytotropic actions of the drug.

Insulin and Glucose Control of Pancreatic Glueagon Re- lease. K.D. Buchanan. Department of Medicine, The Queen's Universi ty of Belfast. Isolated islets (eollagenase digestion) were prepared from severely diabetic (streptozotocin treated) and control rats and were incubated in groups of 10 in Krebs Ringer bicarbonate buffer with albumin for 30 min at 37~ Immuno- reactive glucagon (IRG) was measured by radioimmunoassay and expressed as m~xg/ml/30 rain. Islets from control rats showed a slight bu t insignificant increase in IRG release at 30 rag% glucose (1.18) compared with tha t of 300 mg% glucose (1.02). However, diabetic islets reversed this ratio (1.68 at 300 rag% glucose and 1.28 at 30 rag% glucose). Addition of insulin to the incubation media of the diabetic islets resulted in significantly more IRG being released at 30 rag% glucose (2.15, 1000 ~ units/ml insulin addition; 1.28, no insulin addition, p < 0.025). How- ever no effect of added insulin was seen on IRG release at 300 rag% glucose and this therefore resulted in significantly more IRG being released at low glucose concentrations than at high when insulin was added (1000 ~z units/ml insulin addi t ion: 30 rag% glucose 2.15 m~g IRG, 300 rag% glucose 1.57 m~g IRG, p<0.05). I t is concluded that both insulin and glucose exert regulatory control over glucagon release, insulin deficiency abolishing the usual suppression of glucagon release at high glucose concentrations, and insulin excess increasing the need for glucagon at low glucose concentrations.

The Effect of Beta-Adrenergie Blockade in Thyrotoxic Patients. R.M. Buckle, Department of Endocrinology, The General Hospital, Southampton. A group of five patients with coincident diabetes mellitus and thyrotoxicosis have been studied. Short term treatment with Propranalol led to a decreasing glycosuria and an increase in glucose tolerance. The concentrations of free fat ty acids were reduced, but inconstant effects on plasma immuno-reactive insulin were seen. -- These results suggest that the effect of thyrotoxicosis in worsening of the diabetic state is part ly due to the increased mobili- sation of free fat ty acids which thereby impair glucose tolerance.

In Vivo Insulin Secretion Dynamics in Rodents with Spon- taneous or Acquired Obesity or Diabetes. D.P. Cameron, M. Amherdt, F. Mira, L. Orci, W. Stauffa- cher. Ins t i tu t de Biochimie Clinique and Ins t i tu t d'Histo- logie et d'Embryologie, Geneva, Switzerland. Insulin secretory dynamics were studied in vivo in rodents with spontaneous or induced obesity and/or diabetes. Plasma glucose was measured with glucose oxidase, plasma immunoreaetive insulin (IRI) by radioimmunoassay, on sequential samples from individual animals. Fasting I R I concentrations of o5o5 mutan t mice and goldthioglucose- obese (GTG) mice were elevated. After glucose 0.5 g/kg intraperitoneally (IP), a marked and sustained elevation of plasma I R I was seen in both types of obese mice -- peak values being 25 m~g/ml in obob and 8.5 m~zg/ml in GTG respectively. Following intravenous glucose (0.5 g/kg) the peak of plasma I R I was reached in 1 min in both types of mice. I n the spiny mouse, IP glucose (0.5 g/kg or 1 g/kg) elicited a much smaller though variable I R I release than in obob and GTG mice despite greater pancreatic I R I


content. Tolbutamide (250 mg/kg) failed to stimulate significant I R I release in this species. Food restriction of obob mice until they reached the weight of controls, decreased but failed to normalise insulin release. The differences in insulin-secretory patterns between obob and GTG mice on one hand and spiny mice on the other may be related to established differences in the ultrastructure of B-cells of these animals.

Primary Dysproteinaemia in Diabetes Mellitus. S. Campeanu, L. Campeanu, M. Ionescu. Department of Nutrit ional Diseases, "Dr. I. Cantacuzino" Hospital, Bucharest, Romania. The suggestion of a primary dysproteinaemia in Diabetes 3/[ellitus is made by the authors on the basis of liver, kidney and skeletal muscle ultrastructural examinations durin~ prediabetes, latent and chemical diabetes mellitus, as well as in clinical diabetes. -- The authors t ry to dif- ferentiate these patterns, which are very similar to those recorded in experimental prolonged protein deprivation, from those previously found in diabetics with late complications. These they regard as due to a secondary dysproteinaemia. -- The importance of a metabolically ade- quate protein balance is emphasized as being as essential for the diabetic as the good control of carbohydrate and lipid metabolism. In this respect the importance of ade- quate insulin and of generous dietary protein is stressed. The indications for oral antidiabetic t reatment and for the use of anabolic agents are discussed.

Dose-Response Relationships of the Glucose-Induced In- sulin Release in Normal and Prediabetie Subjects. E. Cerasi, R. Luft S. Efendic. Department of Endocrino- logy and Metabolism, Karolinska Hospital, Stockholm, Sweden. The dose-response curve for glucose-induced insulin secretion from animal pancreas in vivo and in vitro is sine-sha- ped, the secretion starting at a glucose concentration of 100-- 120 rag% and reaching a plateau of 300--400 rag%. Similar curves were obtained in healthy volunteers in whom 4 to 5 glucose infusion tests with varying glucose levels were performed. The type of the dose-response curves were similar both for the initial and the late insulin responses to glucose, although the magnitude of response was greater during the later phase of insulin release. Prediabetic subjects have been characterized by us as individuals with a decreased and delayed insulin response to glucose. This statement was based on tests performed at a glucose level of 300--400 mg~ . When complete dose-response studies were performed in prediabetie subjects it was found that both the initial and late insulin responses could reach normal values when blood glucose reached 600-- 900 mg%. On a semilogarithmie scale, the dose-response curve of the prcdiabeties showed a parallel shift to the right of the normal curve. -- These findings support strongly our hyloothesis that the defective insulin release in prediabetes is due to a decrease in the sensitivity of the d-cell glucose receptor which transmits the glucose signal for insulin release.

Disaupearance of the Peak of "Big" Insulin in Plasma Following Neutral Fat Infusion in Man. V. Chabot, F. Gomez, J .P . Felber. D@artment de Bio- chimie, Clinique !VI6dieMe Universitaire, Lausanne. 5 h fat infusions (Lipofundine, 10~/o solution) were performed in 5 normal subjects. After 2 h an oral glucose tolerance test (OGTT) was performed ~nd blood drawn before and 30, 60, 120 and 180 rain after the glucose load. Control tests were performed in the same subjects, isoto- nic saline replacing the fat infusion. -- Decreased glucose tolerance with high insulin levels was shown during fat infusion. Whereas the two peaks of "big" and "lit t le" insulin were observed in samples taken after 60 and 120

min in the absence of fat infusion, only the peak of "li t t le" insulin could be demonstrated at the same times during the fat infusion. This peak is higher while the peak of "big" insulin is absent or at the limit of the sensitivity of the radioimmunoassay. -- This s tudy demonstrates that fat might interfere not only with the process of insulin release, bu t also with the quality of the insulin produced.

The Role of Growth Hormone in the Pathogenesis of Dia- betes Mellitus in Childhood. G. Chiumello, M. J. del Guereio, M. Carnelutti. Department of Pediatrics and Child Health, Milano, Italy. The behaviour of growth hormone (GH) in basal conditions and after pharmacological stimulations (insulin induced hypoglycemia and arginine infusion) was investigated in diabetic children with or without ketoacidosis, in normal weight children with chemical diabetes, and in obese children with reduced glucose tolerance. The GH secretion in obese children with reduced glucose tolerance did not differ from that observed in obese children with normal glucose tolerance. In children with chemical diabetes the GH levels are normal; in newly diagnosed diabetics without ketoaeidosis the GI-I levels were usually normal under basal conditions, but in some cases they were definitely increased in spite of high blood sugar levels; in these groups the changes in GIrI levels after insulin and arginine infusion were quite similar to the controls. In diabetic children with severe ketoaeidosis there was usually a significant increase of Gt t values notwith- standing the marked hyperglycemia; t reatment normal- ized the GH values, even though there was no correlation between this phenomenon and either the severity of ketoacidosis or the degree of hyperglycemia.

Plasma Cateeholamincs in Suvenile Diabetics. Niels Juel Christensen. The Second Clinic of In ternal Medicine, Kommunehospitalet , Aarhus, Denmark. Plasma adrenaline (A), plasma noradrenaline (NA) and total plasma catecholamine concentration (TPCA) were measured in juvenile diabetics employing a precise and sensitive radio-enzymatic assay. -- The following groups of subjects were examined in the recumbent position and 5 and i0 min after assuming the standing position: 1. 7 non-diabetic control subjects: TPCA (mainly HA) averaged 0.26 ng/ml in the recumbent position rising to 0.69 ng/ml (5 min) and 0.72 ng/ml (10 mln) after assuming the standing position. -- 2. 9 long-term diabetics with neuropathy: TPCA averaged 0.11 ng/ml in the recumbent position rising to 0.30 ng/ml (5 rain) and to 0.37 ng/ml (10 rain) in the standing position. These mean values were all significantly reduced compared with the non-diabetic control subjects. -- 3. 6 long-term diabetics without neuropathy: normal values. -- 4. 8 long-term diabetics with neuropathy hypophysectomizcd for diabetic retino- pathy. TPCA (mainly HA) averaged 0.28 ng/ml in the recumbent position rising to 0.74 ng/ml (5 rain and 0.73 ng/ml (10 min) in the standing position. The mean values are highly significantly increased compared with the diabetic control subjects (group 2). The significance of elevated plasma noradrenaline levels (sympathetic vaso- constriction) for the increased capillary resistance in hy- pophysectomized patients is discussed. -- A number of diabetics have also been studied during keto-acidosis. Both A and NA increases considerably. I t is possible tha t circulatory changes in keto-acidosis are partially caused by elevated plasma adrenaline levels.

Levels of Insulin-Binding Immunoglobulins in Diabetics Compared with Clinical Data. An. Hein Christiansen, S. Munkgaard Rasmussen, An. VMund. Novo Research Inst i tute and Hvidore Hospital, C o p e n h a g e n . . . . . . . .

Vol. 7, No. 6, 1971 Organization Section �9 Abstracts 475

The binding of insulin to IgG and IgM was measured by immunoelectrophoresis in sera obtained from 280 diabetics t reated with insulin. The total I g I was assayed on acid ethanol extracts of the sera. The mean level of insulin- binding IgG was was 2 mU/ml, range 0--12 mU/ ml. For IgM, the mean was 0.5 mU/ml, range 0--5 mU/ ml. The mean total Ii~I was 0.5 mU/ml, range 0--6 mU/ ml. Insulin-binding immunoglobulins could be demonstrated in almost all the patients tha t had been treated with insulin for more than one year. There was a high and significant positive correlation between the total I R I and the level of insulin-binding IgG and IgM. There was no significant correlation between the daily insulin dose and the levels of insulin-binding immunoglobulins or the total whereas there was a slight but significant positive correlation between these parameters and the duration of insulin t reatment . The degree of control of the diabetes has been compared with the levels of insulin-binding IgG and IgM. The significance of these results will be discussed.

Studies of Insulin-Binding Serum Proteins in Normals and Diaber Applying Immunoelectrophoresis. Aa. Hein Christiansen, Aa. Volund. Novo l~esearch In- stitute, Copenhagen. Crossed electrophoresis has shown that added 125I-insulin is bound by albumin, e2-macroglobulin and fl-lipoprotein in normal sera. -- In sera from insulin-treated diabetics, insulin is bound to the same proteins and, furthermore, in most eases to IgG and IgM. The binding of insulin to each of these proteins has been quant i ta ted by a special immunoelectrophoresis. The binding of insulin to albumin, e2-maeroglobulin and fl-lipoprotein was about 10 times higher in the diabetics than in the normals. In the diabetics, there was a close correlation between the binding to immunoglobulins and to the other proteins. By following the binding in diabetics from the start of the insulin treatment, it was found that increases in the binding to immunoglobulins and to the other proteins occurred simultaneously. During treatment, some patients later showed a decrease in the binding to immunoglobu- fins which was accompanied by a decrease in the binding to e2-maeroglobulin. The abnormally high levels of insulin binding to albumin, a2-macroglobulin and fl-lipoprotein in the insulin-treated diabetics developing insulin-binding immunoglobulins show that the changes in serum proteins are more profound than has been realized before.

Membrane Stabilizers and Sugar Transport. T. Clausen. Inst i tute of Physiology, Universi ty of Aarhus, 8000 Aarhus C, Denmark. Membrane stabilizors inhibit the excitabili ty of and the propagation of impulses in the plasma membrane -- perhaps by decreasing the mobili ty of ions or macromolecules. The significance of these changes for the function of the glucose transport system was investigated by assessing the effect of local anesthetics (tetracaine, lidocaine, co- caine) and other membrane stabilizers (ehlorpromazine, thiomebumal) on the transport of glucose and 3-0-methyl- glucose (3-0-MG) in the isolated rat soleus muscle. Tetra- caine (0.1 --2.0 raM) produced no changes in the basal rate of glucose uptake or 3-0-MG transport. At higher concentrations, the permeabili ty to 3-0-MG and cations was markedly increased, indicating lysis of the plasma membrane. When 3-0-MG transport was st imulated by insulin (1 mU/ml), trypsin (1 rag/m1), dinitrophenol (0.05 mM), electrical stimulation or hyperosmolari ty (200 mM mannitol), tetracaine (0.5--2.0 mM) produced a marked and prompt inhibition. When present at concentrations from 0.1 to 1.0 raM, all of the above mentioned membrane stabilizers diminished the rate of 3-0-MG transport considerably, but only when this process was accelerated by insulin or other stimuli. -- The data suggest tha t mobili ty

for ions or charged groups in the plasma membrane is of importance for the act ivation of the glucose transport system, not only by insulin, but also by a var ie ty of other factors.

Differential Effect of Phenformin on Gluconeogenis by Normal and Diabetic Rat Livers. J . J . Connon. Department of Medicine, The Queen's Uni- versi ty of Belfast, N. Ireland. The hypoglycaemie action of phenformin is not clearly understood. Using the isolated perfused rat liver, we have previously noted that it inhibits gluconeogenesis from a number of substrates. -- Although phenformin is an effective hypoglycaemie agent in maturi ty-onset diabetics, it does not lower the blood sugar of normal subjects unless they have been fasted for prolonged periods. The differential effect of phenformin on hepatic gluconeogenesis by normal and diabetic rats was therefore studied. -- Wistar rats were made diabetic by the intravenous administration of streptozotoein (12.5 mg/kg body wt.) 7--10 days before the experiment. Using a modification of the Millar technique, control and diabetic livers were then perfused with Krebs/Ringer bicarbonate solution containing 4 gO/o human serum albumin. Glueo- neogenesis was assessed by measuring glucose production from 10 mM pyruvate, in the presence of various phenformin concentrations. -- The rate of glueoneogenesis was 30% greater in diabetic livers. 4-0 mg% phenformin produced complete inhibition of gluconeogenesis in both normal and diabetic livers. However, when only 12~ mg phenformin was used, there was almost total cessation of gluconeogenesis in the diabetic livers and only a minimal effect on the normal ones. -- Thus a parallel between the action of phenformin in normal and diabetic man and normal and diabetic rats can be demonstrated.

Influence of an Adrenergic Alpha- and Beta-Blockade on Insulin Secretion and Plasma Catecholamines Following Increasing Doses of Different Sulfonylureas in the Dog. U. Cordes, J . Beyer, G. Sell, 1~2. SchSffling. Centre of Internal Medicine, Johann Wolfgang Goethe Universi ty Frankfur t /M, Department of Endocrinology (Director: Prof. Dr. K. SchSffling). Increasing doses of glibenclamide, glibornuride and tolbutamide have been administered intravenously to a group of fasting beagle dogs. A second group was pre- t reated with the alpha-blocking agent phentolamine (1 mg/kg), a third group with the beta-blocker K6 592 (2 mg/kg). -- The results demonstrate tha t insulin secretion is further stimulated by sulfonylureas even at doses which have no further hypoglycemie potency. The pat tern of insulin secretion becomes biphasie under these conditions. The first insulin peak with its maximum at 5 min is followed by a second one, which is characterized by its sluggish and prolonged increase of insulin concentration with a maximum between 30 and 60 rain. Pre t reatment with the alpha-blocker phentolamine causes a potentia- t ion of insulin secretion, particularly that of the second phase. The blockade of the beta-receptors leads to a marked depletion of insulin levels, especially the second peak. The open question, whether the modified dynamics of insulin secretion following the different adrenergic blockades are caused by a synchronous release of eate- eholamines or by direct effect of the sulfonylureas upon the adrenergic receptors, should be answered by measurement of plasma cateeholamines. These determinations are in progress and will be discussed.

The Effect of Diguanides on Exocrine Function of the Pan- creas and Insulin Secretion Induced by ttCL and Secretin. A. Czyzyk, M. Szadkowski, I t . Rogala, J. Lawecki. 3rd Depar tment of Internal Diseases, Medical Academy, Waxszawa.


I n 13 heMthy subjects stimulation of the pancreatic exoerine secretion by intraduodenal drip infusion of 100 mt 0.1 N HC1, followed after 50 min by i.v. injection of secretin in a dose of i U per kg body weight, was performed. The volume of duodenal juice, its amylase and bicarbonate content, as well as its p i t were determined. In six subjects serum insulin (LgI) was simultaneously measured. The test was carried out twice, firstly as control and secondly after administration of phenformin in a daffy dose of 150 mg for 4 days. -- After phenformin, the basal secretion of pancreatic juice was markedly lower, and the increase in this secretion due to stimulation with I-IC1 and secretin was smaller. In the control tests, IIC1 infusion resulted in a much smaller increase in insulinemia induced by HC1 was no longer detectable, whereas that due to secretin failed to show any dear-cut changes. -- The present studies suggest that biguanides exert an effect on the biosynthesis and/or action of some proteins of the intestinM wall, which control the exocrine function of the pancreas and insulin secretion.

Influence of Biguanides on Glucose Turnover in the Rat and Dog. W.It . Davies, L.E. Martin, 5.G. Mills, C.J. Vardey. Bio- chemistry Department, Research Division, Allen & Han- burys Ltd., Ware, England. Glucose turnover has been studied in the 5 h fasted ra t (140 g weight) using intravenous 6-3H and 6-z4C glucose. Pretreatment orally with either phenfermin or buformin (80 mg/kg) caused all-glucose to be cleared from the plasma more rapidly than l~C-glucose. Metformin, Fen- fluramine and ?-Gnanidinobutyramide had no significant effect at. this dose level. Whilst phenformin had no effect at this dose level. Whilst phenformin had no effect on the total glycogen content or glucose incorporation into liver and hind limb muscle glycogen in wvo it produced a significant lowering in the 3HfldC ratio of liver glycogen. This ratio in muscle glycogen did not differ from that. of control animals. The hind limb muscle glucose-6 phos- phate levels were significantly elevated from 0.t i 6 ~ moles/g tissue to 0.209 ~z moles/g tissue. No differences were observed on the total CO~ output in the expired air. These results suggest tha t phenformin increases Cori cycle activity of rats as measured by the rate of disappearance of sH label relative to the 1~C label of plasma glucose. No evidence was found to substantiate inhibition of gluconeogenesis. Similar studies with dogs have shown that phenformin (20 mg/kg) orally per day for 7 days significantly increased glucose turnover and glucose recycling.

Serum Insulin after Glucose, Isoprenaline and Phentola- mine in Non-Obese Maturity-Onset Diabetics. T. Deckert, U. Birk Lauridsen, J. Linde & S. Nistrup Madsen. Frederiksberg Itospital, Copenhagen, Denmark. I t is well-known that, as well as d-glucose, stimulation of beta-receptors results in insulin secretion in man. In non- obese diabetics d-glucose is kno~na to give only a small and delayed insulin response, if any. We investigated the question of whether beta-receptor st imulation with isoprenaline or blockade of endogenous alpha receptor stimulation resulted in a normal insulin response in diabetics. -- Twelve non-obese non-diabetics and i0 non-obese maturity-onset diabetics were given a continuous infusion of isoprenaline 2 ~g/min before and during i.v. GTT. Con- trol i.v. GTT was carried out during infusion of 0.9% NaCI the day before. Serm~ insulin (IRI) and glucose was measured. -- I R I increased sig~aifieantly in the diabetic group and no difference between I R I rise in diabetics and non-diabetics could be shown. During phentolamine infusion I R I increase was delayed in normals as well as in diabetics,

Onset of Diabetes after Acute Telone Intoxication. I. De Leeuw. A.Z. Middelheim, Antwerp, Belgium. Following aecidentM intoxication due to dichloropropene (Tetone) inhalation, a man of 43 years developed manifest diabetes, besides pulmonary and renal complications. The diabetes could be well controlled by dieting and oral administration of antidiahetic agents. After 3 months, when the pulmonary and renal complications were cured, the diabetic state was improved and controlled by diet alone. There was no antecedent of diabetes nor was there evidence of mieroangiopathy. Fasting plasma insulin levels became normal, but the enhancement observed after glucose st imulation is clearly insufficient. No manifest liver disease was observed. -- The etiology of this sort of diabetes, and the possible relationship between pesticide intoxication and diabetes, wilt be discussed on the basis of the biological and anatomo-pathologicM data.

Comparative Requirements of Bovine and Porcine In- sulins. R.R. de Mowbray, J . J . Turner, S.D. Garner, E. Bruek, L. Nye, S. Triggs. Queen Mary's Hospital for the East End and St. Bartholomew's Hospital, London. The mean insulin requirement of diabetics increased rapidly during the first year on commercial bovine IZS but. less rapidly on ten times reerystMlized bovine IZS; while in patients receiving ten times recrystallized porcine IZS the mean requirement declined during the first six months and not unt i l the end of the third year did it approach the level at tained by the reerystallized bovine group after one year. -- Insul in binding antibodies were measured a t intervals in the sere of patients receiving ten times recrystMlized IZS, by radioimmunoassay following adsorption of free insulin on to dextran-coated charcoal. The percentage insulin binding rose more rapidly and reached higher levels in the bovine than in the porcine group. There appeared to be some correlation between binding and initial insulin requirements bu t this was not maintained and in some cases binding subsequently declined, regardless of the t rend in insulin requirement. -- I n patients transferred from commercial bovine to 70% bovine/30~/o porcine IZS the mean requirement fell by 28%. No difference could however be demonstrated in the plasma glucose response to a single intravenous injection of soluble insulin or to a single intramuscular injection of amorphous fraction, whether as commercial or as ten times recrystalllzed preparations.

"Endocrine Function in Medical and Surgical Stress". J.G. Devlin, M. Varma, J. Kuti . Endocrine/Metabolic Unit , St. Laurence's Hospital, Dublin. Intravenous ghmose tolerance tests have been performed sequentially on 30 patients with systemic stress, i.e., surgery or myocardial infarction, and 26 patients with acute head injury. Assessment of K value, growth hormone secretion, insulin response to the glucose load, free fat ty acid levels, and cateeholamine excretion has been carried out. -- The results indicate, to date: 1. Growth hormone respose falls into a number of defined patterns. I n 19 patients there was suppression followed by a rise after 15 rain. -- A smaller group showed a delayed rise in growth hormone with very severe head injury and high cateeholamine excretion growth hormone secretion was suppressed in two patients. The severe decerebrate head injury failed to suppress growth hormone output- with a glucose load, and recovery was associated with re turn of growth hormone secretion to a normal pattern. -- Insul in followed to a certain extent, the expected published patterns. Lower levels of insulin in the first, compared with subsequent studies, were found in 18 patients, and in a further group, an abnormal pat tern of insulin secretion associated with raised fasting values and poor response

Vol. 7, No. 6, 1971 Organization Section. Abstracts 477

to glucose loading was observed. -- Catecholamine levels were elevated in all studies. -- Cortisols were elevated as expected, and suppressed with glucose loading. The findings with reference to pi tui tary function in diabetes and control of growth hormone secretion in acromegaly will be discussed.

Insulin-Binding Antibodies in Insulin-Treated and Un- treated Diabetic Patients. S. Ditzov, I. Pencev, D. Ancreev. L. Sirskov, N. Tarkolev. Diabetes Research Group, Bulg. Acad. of Sci., Sofia, Bul- garia. Some years ago the authors demonstrated the presence of insulin-precipitating antibodies in insulin-treated as well as in insulin-untreated diabetic patients. Following some critical remarks about the method used, they have re- cently tried to prove or disprove the existence of autoantibodies with a new method. The serum was mixed with labelled insulin-1125 and then disc electrophoresis in acry- ]amide gel was performed. A separate portion of the same serum before mixing with the tracer was treated with cellulose-fixed insulin and therefore was cleared of insulin antibodies. After removing the cellulose a disc electrophoresis of the supernatant portion of the serum was performed. The method allowed the insulin antibodies to be demonstrated. -- The authors have studied 50 insulin- t reated and 88 insulin-untreated diabetic patients as well as 200 control persons with the method described above. A positive test for insulin-binding antibodies was found in 58% of the insulin-treated and in 18.7% of the un- t reated diabetics, and never found in controls. The results indicate that production of insulin autoantibodies in diabetics is possible and may play a substantial role in the pathogenesis of the disease.

Investigations Concerning Females with Fetal Gigantism. C. Dumitrescu, lYl. Bolea-Feldman. Clinic of Metabolic and Nutr i t ion Diseases, Hospital "Cantacuzino", Bucharest, Rumania. The paper deals with glucose tolerance tests (GTT) carried out on a group of 219 young females with a history of fetal gigantism and on two groups (111 each) of obese females with, or without, fetal gigantism in their obstetric history. -- Disturbance of glucose tolerance in females with a history of fetal gigantism was unrelated to the degree or duration of obesity and did not differ pro- portionately from glucose tolerance responses in obese females with children of normal bir th weight. -- Fetal gigantism thus appears to depend upon other diabetes- related factors and these should be investigated. If identified, they offer the hope of effective prophylaxis.

Oxidation of Glucose and Fatty Acids in the A2-Cells of Isolated Guinea-Pig Islets of Langerhans. John C. Edwards, C. HellerstrSm, B. Petersson, K.W. Taylor. Histological Department, Universi ty of Uppsala, Uppsala, Sweden and School of Biological Sciences, Uni- versi ty of Sussex, Falmer, Brighton, England. The rates of oxidation of ltC-glucose, 14C-oetanoate and liC-palmitate have been measured in normal guinea-plg islets and in A2-cell rich islets isolated from guinea-pigs t reated with streptozotocin. The rates of oxidation of these compounds in B-cells and in A2-cells can therefore be estimated. -- Raising the concentration of ~4C-glucose from 1.7 ~ to 16.7 mM caused a 450% increase in glucose oxidation in B-cells but only a 150% increase in A 2- cells. When the laC-octanoate concentration was raised from 0.5 mM to 5 mlg, octanoate oxidation in B-cells increased 400% but in Aa-cells there was a 600% increase. 14C-palmitate did not appear to be oxidised by the B-cells, although the rate of oxidation of l~C-palmitate in the A 2- cells was again very sensitive to changes in the extracellular fatty acid concentration. -- It appears that in the A~-

cells, fatty acids are oxidised at a faster rate than glucose, and that fatty acid oxidation is very sensitive to changes in extracellular fatty acid levels. -- The results will be discussed in relation to the known effects of glucose and fat ty acids on insulin and glucagon release. (J. C. E. is in receipt of a Wellcome-Swedish Travelling Re- search Fellowship).

Arginine-Induced Insulin Release in Relation to the Cyclic AMP System in Man. S. Efendic, E. Cerasi, 1%. Luft. Department of Endocrino- logy and Metabolism, Karolinska Hospital, Stockholm, Sweden. The amino acid arginine for its insulin releasing action in man depends on a normal blood glucose concentration. Furthermore, arginine potentiates glucose-induced insulin release, probably by amplifying in the beta-cell the insu- linogenie signal evoked by glucose. The beta-adrenergic blocking agent, propranolol, in doses known to suppress glucose-induced insulin release, did not alter insulin response to arginine infusion. Arginine, under experimental conditions in which it stimulates markedly insulin release on glucose administration, had no enhancing effect on the action of glucagon on insulin secretion. Finally, pretreatment with aminophylline potentiated the insulinogenic potency of the amino acid. -- We have previously postulated tha t glucose elicits insulin release by acting directly on a specific cell membrane receptor which acti- vates adenyl cyelase, cyclic AMP itself then discharging insulin. In the light of this hypothesis and the above results we suggest tha t arginine acts on the beta-cell by amplifying the transmission of the glucose-induced signal at a locus between the glucose receptor and adenyl cyclase.

Comparison of Normal Subjects with or without a Family History of Diabetes Examined in a Systematic Survey. Re- sults on 1828 Men in their Fifties. E. Esehwege, A.J . Valleron, G. Rosselin, J . R . Claude, J . M. Warner, L. Papoz, J .L . Richard. Ins t i tu t National de la Sant@ et de la Recherche M@dicale, Unit@ de Reeherches Statistiques U 21 94, Villejuif, France. In a prospective survey of the "Groupe d 'Etudes sur l 'Epid6miologie de l'Atherosel@rose" (Paris) whose aim is to determine the risk factors of atherosclerotic cardiovascular diseases, a systematic oral glucose tolerance test 0--2 h (75 g) was carried out in 1828 men in their fifties. In each blood sample, glucose, free fa t ty acids, triglycerides, cholesterol, insulin, H G H and cortisol levels were determined. At the same time, family history of diabetes (father, mother, brothers, sisters), cardiovascular status and degree of obesity were recorded. The correlations between all the parameters were studied within each group of diabetic and non-diabetic subjects, whether they have a family history of diabetes or not. Amongst the 1725 sub- jeers whose diabetes was not previously diagnosed, 8% have a family history of diabetes; the only difference between those with a family history of diabetes and those without concerns the plasma glucose level, which is significantly higher in the former group. No significant difference is noticed in blood pressure, obesity, F F A (0--2 h), triglyeerides, cholesterol, cortisol, I-IGH, I R I values. -- These results and the relationship between all the biological variables shown above make it possible to give a bet ter definition of early diabetes.

Effects of Pancreozymin and Caerulein in Patients with Insulinoma. F. Fallucca, G. Menzinger, G. Tamburrano, M. Javicoli, D. Andreani. Second Cliniea Medica, Universi ty of Rome, Italy. In 5 patients with islet-cell tumours and in 6 normal subjects blood glucose and plasma insulin levels have been studied after intravenous administration of panereozymin


(75 IDU) and of caerulein (10 ng/kg of body weight). Caerutein is a decapeptide, resembling panereozymin, extracted from amphibians. -- In the normal subjects panereozymin was followed by a slight increase of blood glucose, as well as of plasma insulin; on the other hand in the group of patients with insulinoma, three eases did not show any change of blood glucose and plasma insulin where~s two other patients showed a marked increase of plasma insulin with ~ fall of blood glucose levels. -- The injection of caerulein in normal subjects did not bring about any variation either in blood glucose or in insulin levels ; no change was also observed in three patients with insulinoma, bug the other two who had responded to pancreozymin showed a significant fall in blood glucose and a marked increase in plasma insulin. -- The authors suggest that caerulein shares a similar mechanism of action with panereozymin; they discuss the value of these substances for the diagnosis of islet cell turnouts.

New Possibilities to Avoid the Formation of Insulin-Anti- bodies in Diabetic Patients. S. Fankhauser, J . l~Iich]. Medical Polielinie, Universi ty of Berne. Previous investigations showed tha t t reatment with commercial Lente insulin, or highly purified ox Lente insulin, is regularly followed by formation of insulin antibodies. The antigenicity of highly purified pig Lente insulin is significantly less, but, nontheless, present. 12 diabetic patients, previously untreated with insulin, received a new monocomponent pig Lente insulin, which was purified by chromatography and did not contain a or b components (including proinsulin). Insulin antibodies were determined qum~titatively at monthly intervals by 2 different methods. After t reatment for 3 to 12 mont, hs none of the 12 patients showed a rise of the antibody titer. These results indicate that monocomponent insulin is not antigenic. The antigenicity of the insulin preparations used unti l now would not seem to be caused by the insulin molecule itself, but rather by the a or b components. Additional experiments in 10 psychiatric patients with a short acting pig insulin, containing 2 to 3 times as much b component as commercial preparations, did not show an increase of the antibody titer. These findings indicate that the a component might be more antigenic than the b component.

Study of 125I-Glucagon Transport in Rabbit Plasma. G. Federspil, G. Piemonte, 1~. Sicolo, S. Frezzato, C. Sean- dellari. Inst i tute of Medical Semeiology, Universi ty of Padova, Italy. The transport of 1~SI-glucagon in rabbit plasma was studied by means of gel column chromatography. 125I-glu- eaton was administered by intravenous injection to the rabbit, and ~ rain later blood was drawn and the plasma examined by chromatography on Sephadex G-75. The radioactivity of the eluate showed two peaks: the first corresponded to the elution volume of the proteins whereas the bigger second peak corresponded to the elution of unbound glucagon. Exclusion of the liver from the cir- culation by ligature of vessels did not cause a decrease in the size of the first pea.k. A temporal study of TCA pre- eipitable radioactivi ty showed tha t the radioactivi ty of the first peak has a slower turnover than unbound radio- act ivi ty does. -- A hyperglycemic effect was noted when the eluate of the firs~ peak was injected intraperitoneally to the rat. -- Injection of progressively increased doses of glncagon to the same rabbit was followed by an ab- solute augmentation of protein-bmmd radioactivity.

Muscular Exercise and Lipolysis. In r i v e Studies of Endo- crine Regulation in the Rat. G. Federspil, G. C. Reffo, A. Luyekx, P. Lefebvre. Ins t i tu t of Semeiotica lYIedica. Padua University. I taly. Ins t i tu t de Medecine. Universite de Liege. Belgique.

A study has been undertaken of the effects of prolonged muscular exercise (swimming) on the blood concentration of energy substrates (glucose, N E F A and glycerol) in the rat. -- An analysis has been made of the role of various hormones in this type of exercise. -- A slight but statistically significant decrease in blood glucose, and a marked rise in plasma N E F A and glycerol have been observed. These changes are accompanied by a significant decline in plasma insulin and clearcut increase in plasma eorti- costerone. -- The blockade of the beta-adrenergie receptors (mediators of the lipolytie effect of cateeholamines) by two different pharmacological agents (propranolol and D(-)IIqPEA) was shown to be incapable of preventing the rise in plasma ~TEFA and glycerol. -- Similar results have been obtained with the administration of .Methy- rapon (an inhibitor of cortieosterone synthesis). -- In h~qoophyseetomized rats, on the contrary, the rise in- plasma N E F A and glycerol was minimal and not statisti- tal ly significant. -- These experiments suggest tha t the hormones of the adrenal medulla and cortex are not essential in determining the fat mobilization tha t occurs in rats during muscular exercise and indicate tha t the hypophysis plays a major role in these metabolic phenomena �9

Acute Metabolic Effects of Neutral Red in Rats. G. Federspil, ~f. Zaccaria, G.C. l~effo, C. De Pale. Insti- tu te of Semeiotica l~Iedica. Padua University. I taly. Acute metabolic effects induced by intraperitoneal administration of Neutral l~ed have been studied in rats. -- Neutral Red produces a definite rise in blood glucose, which first appears after 15 min and reaches maximum values 60--120 rain after the injection. 7 h later blood glucose returns to normal. Plasma glycerol shows a moderate increase, but the rise only becomes significant 60 rain after administration of the dye. -- Lactic acid in the blood increases very sharply after 60 min. -- To establish whether hyperglycemia induced by Neutral l~ed is due exclusively to a release of cateeholamines, the effects of the dye were studied after bloekade of the adrenergie receptors by ergotamine. -- This substance effectively blocks epinerphrine-induced hepatic glyeogenolysis in rats. In rats treated with ergotamine, Neutral l~ed produces a less marked rise in blood glucose than in untreated rats; the rise is however statistically significant (p < 0.05). -- These results are compatible with the idea tha t the metabolic effects of Neutral Red are in par t due to the release of glucagon by the alpha-cells of the pancreas.

The Pancreatic Function Related to Lipo-Metabo|ic Vari- ables in Treated Diabetics. A. Fernandes-Cruz (3u C. Lopiz-Quijada. Patologia General y C.S.I.C., l:[ospitat Clinica. Ciudad Universitaria, Madrid. After the cessation of the therapy (24 h for insulin and 72 for the oral antidiabetie agents) an oral test (260 Cal.) was performed and total cholesterol, total triglycerides, phospholipids, serum I R I and blood glucose were studied in 30 treated diabetic patients of both sexes without specific mieroangiopathy or other vascular disease. -- Blood samples after t5 h of fasting and before the oral test, and 2 h after the oral test were obtained. -- No significant correlations between blood glucose, serum insulin release, serum phospholipids, total cholesterol and total triglyeerides, were obtained after the oral test. -- After cessation of therapy the oral test was performed in 17 treated diabetic patients in similar conditions, samples for blood glucose, total triglycerides, total cholesterol, phospholipids, serum I R I and urine Ii%I being obtained both before and 2 h after the oral test. -- A significant correlation between urine I R I 2 h after the oral test and phospholipid levels after 15 h fast, was obtained. -- A mathematical rela-

Vol. 7, No. 6, 1971 Organiza t ion Sec t ion . Abs t rac t s 479

t ionship was also deduced. -- The Sta t is t ica l analysis was pe r fo rmed b y computer .

The Effect of Various Factors in the Presence of Insulin in Bile and Urine. A. Fernandez-Cruz , J r . , M. L u q u e Crete . Pa to log ia Ge- neral. Hosp i t a l Clinico. Ciudad Univers i ta r ia . Madrid, Spain. I n order to s t udy the presence of insul in in bile and urine, and to define t e n t a t i v e l y the effect of var ious gu t factors (secretin, pancreozymin (Jorpes) and oral glucose load) on islet cell hormone release, three experimental situations were used to collect bile samples : 1. Spontaneous bi le fistula. - - 2. Af t e r a cholecys tee tomy, a T - tube eholed- ocho tomy. - - 3. F r o m b o t h a control group and gastrec- tomized pa t ien ts , t h rough a Mil ler -Abbot tube . -- The bile and ur ine were collected in ice-surrounded containers. Blood samples were ob ta ined v ia an indwell ing po lye thy- lene ca the te r inser ted in an an tecub i ta l vein. Blood glucose was measured by the au toana lyzer , and serum, ur ine and bile I R I by rad io immunoassay . - - Samples were t aken a t 0,3, 5, 10, 15, 30, 60, 120, 180 rain and a t 24 h. A compar ison of effects was carr ied ou t fol lowing infusion of glucose and Hb-419 admin is te red in t r avenous ly in the subjects . The excre t ion per m i n u t e and the clearance of I R I in bile and ur ine showed an increase in response to an oral glucose load. The resul ts will be dis- CUssed.

Do Different Mechanisms of Insulin Release Depend on the Feeding States ? H. Fiedler , H . J . H a h n , Br ig i t t e Ziegler, M. Ziegler, E . Ju tz i , 1%. Michael. Zen t ra l ins t i tu t ffir Diabetes "G. K a t s c h " Kar l sburg-Garz , D D R . Different resul ts concerning var ious agents s t imula t ing insulin secret ion in in rive and in vitro exper iments were expla ined by the differences in basal metabo l ic act ions of t he islets and in p r e t r e a t m e n t of animals . - - Us ing 4 male ra t siblings we inves t iga ted the influence of the feeding states (72 h and 24 h s ta rva t ion , feeding ad l ib i tum and three weeks ' admin i s t r a t ion of 10% sucrose solution) on the secret ion ra te of insulin on the isolated islets. Ten islets f rom one pancreas were incuba ted under t he following condi t ions : 2.5 m M glucose, 15 m M glucose, 2.5 mM glucose and 50 ~g/ml glybenclamide, 1 m M nicot inic acid_, 10 mM caffeine, 15 mM glucose and 50 fzg/ml diazoxide. The I R I con ten t of t he m e d i u m was inves t iga ted a f te r a 30 rain incubat ion. The s ta t is t ical eva lua t ion was b y analysis of the var iance. - - Af te r 72 h s t a rva t ion only caffeine s t imula ted the I R I ou tpu t . Af te r feeding ad l ib i tum or 24 h s t a rva t ion all used substances were effective. Af t e r 3 weeks sucrose feeding the increase of insulin secret ion seen in exper iments wi th nicot inic acid was no t produced. Our resul ts also give references for var ious responses of isolated islets in vitro.

Reflex Insulin-Mobilization Triggered via the Oral Cavity of the Conscious Dog and its Inhibition by the Anesthesia of the Mucosa. U. Fischer, H. H o m m e l , M. Ziegler, H . Bibergeil . Central I n s t i t u t e for Diabetes " G e r h a r d t K a t s c h " , Kar lsburg , DDR. Oral administration of 1.0 g glueose/kg body weight to trained conscious German shepherd dogs was followed by a mul t iphas ic increase in I R I levels in the per iphera l venous blood. A definite increase was observed wi th in 5 min a t which t i m e blood glucose was still unchanged. The ear ly phase of insulin mobi l iza t ion af te r glucose corre- sponds in t ime to an increase of the ho rmone concentra- t ion which the au thors found dur ing the first 2 .5-- 15 rain af ter oral admin i s t r a t ion of dr inking wa te r or af ter sham- feeding wi th glucose in animals w i th a double-ended oesophageal f istulae (while b lood glucose r emained un-

changed). U p o n t e t r aca in anesthes ia of t he mucosa of muzzle and pharynx , the first peak of I R I in t he in t ac t an imal does no t appear af ter an oral in take of glucose nor is there any rise af ter sham-feeding. I t is suggested t h a t when food is t aken a first phase of insulin mobi l iza t ion is t r iggered v ia reflex mechan isms in t he oral cav i ty . The afferent pa r t of t he arc p robab ly begins in the ne rve endings of the mucosa of the oral c av i ty or in the pha rynx . -- Different ial assessment of th is ear ly phase of I R I release in the course of t he oral glucose to lerance tes t should get more considerat ion as regards the possible par t ic ipa- t ion of t he vagus ne rve and /or the en te rohormones in the efferent section.

The Effect of Starvation at Various Time Intervals after Streptozotoein Application to Rats. O. F5rster , W. Rippel , B. Rudas . I n s t i t u t e for General and E x p e r i m e n t a l Pa tho logy of t he Un ive r s i t y of Vienna. There are controvers ia l repor ts concerning the develop- m e n t of ketosis and the increase of se rum F F A levels in ra ts w i th s t rep tozotoc in (STR) diabetes. I n mos t studies the ra ts were in jec ted wi th S T R af ter a per iod of fas t ing and the s t a rva t ion is l ikely to interfere wi th the subse- quen t metabol ic changes. We induced diabetes in fed rats by in jec t ing 65 mg S T R / k g i. v. Blood sugar, se rum F F A and l iver glycogen were de te rmined 2 - - 3 days and 6 weeks af ter S T R admin i s t r a t ion bo th in fed ra ts and in ra ts which had fasted for 24 h. I n t he acu te ly diabet ic fed ra ts b lood glucose and serum F F A levels were e leva ted and l iver glycogen marked ly decreased especial ly in ke- re t i e rats. A 24 h fast effeeted a lowering of se rum F F A and a fur ther decrease of l iver glycogen, b u t b lood sugar remained high. F e d ra ts w i th long t e r m S T R diabetes differed f rom acute ly diabet ic ra ts only in demons t ra t ing near to normal FFA-leve ls . Af te r the 24 h fast low blood sugar levels and a fur ther decrease of F F A was observed. I n a few rats which had higher fast ing blood sugar serum FFA was also elevated. In this respect starvation effects in STI~ diabetes differ from those observed in alloxanized or normal rats. A possible explanation of this differenc e is discussed.

The Metabolism of Insulin during Prolonged Muscular Exercise in Man. J . R . M . Franckson, R. Vam'oux, R. Leclereq, H. Brunen- graber, H. Corns. D e p a r t m e n t of Medical Chemistry , Uni- ve r s i ty of Brussels. Most studies concerning the influence of exercise on insulin secret ion only deal w i th t he level of p lasma I R I . Our a im was to measure b o t h insulin ca tabol i sm and pancreat ic responsiveness. -- Muscular exercise (bicycle ergo- mete r , Power 80 W), ma in ta ined a t a s t eady s ta te of ven t i l a t ion dur ing 40 rain, was per formed by several groups of normal subjects. The following resul ts were ob- ta ined. 1. I n normoglycemia or in hyperg lycemia caused by glucose infusion, exercise induced no decrease in p lasma I R I level. 2. The metabo l ic c learance ra te of a cons tan t infusion of 125I-insulin (0.2 a t o m I /mol) showed a reduc t ion averag ing 20% whereas no change in ext ra- cellular space could be detected. This decrease in insul in ca tabol i sm was associated wi th a s imilar decrease in the clearance ra tes of labelled h ippuran and BSP. 3. The insulin response to g lueagon in jec t ion was m a r k e d l y reduced. -- I n conclusion, the absence of changes in p lasma I R I levels of ten descr ibed in non-s t renuous exercise results f rom antagonis t ic phenomena : a decreased secret ion ra te p robab ly re la ted to the release of endogenous catecholamines, and a decreased catabol ic ra te appa ren t ly due to changes in l iver and k idney haemodynamics .

Contribution to the Immnnopathology of Experimental Insulitis. G. F rey tag , G. K15ppel. I n s t i t u t e of Pa tho logy , Un ive r s i t y of H a m b u r g , Germany.


In experimental medicine, the tansmissible insulin antibodies (passive immunisation) can lead to three different forms of inflammation in the isolated islets of Langerhans, namely: 1. acute insulitis showing predominantly eosino- phile cell infiltrates; 2. chronic-recurrent insulitis with mainly monocy%e and histiocyte infiltrations; 3. primary chronic insulitis with purely lymphocyte infiltrations. The result is reproducibly predictable and, at equivalent quantities of serum injected, it depends on the following factors: a) the insulin antibody titre, b) the kind of insulin used to form antibodies, c) the mode of injection (the frequency of injections depends upon blood sugar levels). The first two forms are reversible and represent an im- munopathological reaction in the antigen-antibody com- plex in the area of the elution of insulin secreted. Chronic lymphocytic insulitis is irreversible and progresses even after injection of the antibodies is discontinued. This form is of special interest : its cytologic aspects correspond to the type of insulitis due to active immunization in the rabbit. Furthermore, it complies with the findings observed in the early phase of juvenile diabetes. The authors consider that this third form of insulitis is a mechanism of auto-immunisation induced by the insulin antibodies transmitted. -- The results of the histological, histochemical and autoradiographic studies are correlated with the biochemical and immunological observations and will be discussed in this light.

Abnormally High Glucagon Secretion of the Streptozotezin Diabetic Isolated Perfused Rat Pancreas. Evidence for a Lack of Suppression Due to the Deprivation of Endogenous Insulin Release. 1~. D. Fussg/inger, 1%. Goberna, K.E. SchrSder, H. Laube, E.F. Pfeiffer. Department of Endocrinology and Meta- bolism, Center of Internal Medicine and Pediatrics, Uni- versity of Ulm, Germany. The possibility of a mutual interaction of glucose, insulin and glucagon within the islets of Langerhans was studied by measuring the release of both hormones of the isolated perfused rat pancreas. Normal and diabetic glands, de- prived of insulin with streptozotozin 48 h before were used. Mean secretion rates of insulin and glueagon following stimulation with glucose and cholecystkinin-pancreozymin (CCK-PZ) were evaluated radioimmunologi- cally. -- Concomitant increase of both hormones in response to CCK-PZ was recorded at low glucose concentrations. With increasing glucose concentrations, with or without CCK-PZ, glucagon responses were progressively abolished in the normal gland. Additional release of insulin in response to CCK-PZ was also diminished. I n contrast, glucagon secretion of the streptozotozin diabetic gland was characterized by a paradoxical dose-response- relationship. With increasing glucose, glucagon secretion rose. At 5.5 mM glucose no difference of glucagon response between normal and diabetic pancreas was observed. CCK-PZ potentiated glucagon release of normal glands with glucose below 5.5 raM, bu t did not potentiate glucagon secretion in the diabetic gland, at low or high glucose concentrations. Perhaps endogenous insulin supports glucagon suppression in the presence of high glucose levels, bu t enhances release at low glucose. Following CCK-PZ endogenous insulin seems to control glucagon release.

Insulin Action on Amino Acid Transport in Chick Embryo Heart. Relationship to Protein Synthesis. G.C. Gazzola, 1%. Franchi, P. l%onehi, E. Saibene, G.G. Guidotti. Is t i tut i di Patologia Generale, Universita di Mi- lano e di Cagliari, I taly. Insul in has been found to enhance the uptake of analogue and natural ly occurring amino acids by intact chick embryo hearts and by isolated cardiac cells (Guidotte et al., Biochem. J. 1t)7, 565 (1968) ; 114, 97 (1969). The action

of the hormone was consistent with an acceleration of the maximal velocity of saturable transport systems (Gui- dottiet a[., Bioehem. J. 122, (1971) in the Press), suggesting increased 'capacity' or enhanced operational rate of the agencies involved. To explore this situation further, the initial velocity of ~-aminoisobutyrie acid uptake by isolated eardiae cells has been studied in 5 rain experiments at sequential intervals during prolonged incubation periods (up to 5 h), in the presence and absence of insulin, under conditions of active and inhibited protein synthesis. Puromyein and eycloheximide (at sufficient concentrations to stop protein synthesis) depressed the rate of c~- aminoisobutyrate uptake progressively during the first 3 h and abolished active transport completely thereafter. These effects did not arise from energetic failure. Under conditions of impaired transport, the stimulation by insulin of ~-aminoisobutyrate accumulation, expressed as percentage of basal uptake, was little affected. Similar results were obtained when actinomycon D was used to inhibit transcription. The results presented suggest that the insulin-mediated acceleration of ~-aminoisobutyrate uptake by chick embryo heart cells does not involve an increase of the transport system 'capacity' that requires active protein synthesis (Aided by N.I.H. Grant AM- 05290).

The Effect of Phenformin in Patients with Diabetes Melll- tus under Strict Balance Conditions. C. Geldermans, J. Terpstra. H.M.J . Krans. Dept. of Endocrinology and Metabolism, University Hospital, Lei- deI).. According to some reports phenformin induces a lowering of the blood sugar level combined with weight reduction in obese maturi ty-onset diabetics. -- As yet no studies under real balance conditions have been published. We studied patients under strict conditions of food intake (constant diet at fixed times during the whole study) and daily blood, urine and faeces analysis. -- In all patients phenformin induced a decrease in blood glucose levels and concomitantly a reduction in glycosuria. Contradictory to most statements the patients showed a (slight) increase in body weight under phenformin. No change in caloric value of the faeces (bomb calorimetry) could be demonstrated. The B.M.l~., l%.Q. and nitrogen balance were not significantly altered during phenformin therapy. Blood cholesterol levels were decreased, whereas triglyceride levels became slightly increased. The adrenal function was not affected. -- Our balance studies do not confirm that phenformin lowers the body weight when food intake is constant.

The Influence of Adrenalectomy on Glucose and Insulin Levels after Injection of Glucose, Ghicagon, and Arginine into the Pancreas of Stressed Rats. C.A. Geser, E. Rattenhuber, J.P. Felber. Department of Clinical Biochemistry, University of Lausanne. An intrapancreatic injection technique was used to study insulin secretion in rats undergoing a surgical stress which consisted of laparatomy. Adrenalectomy was performed to investigate the influence of adrenal hormones on gly- comic and insulinemia. Pulses of glucose (0.5 g/kg), glu- eaton (15 ~g/kg) and arginine (35 mg/kg) were injected. -- In stressed rats without adrenalectomy the plasma insulin was 47 ~U/ml after glucose, i15 after glucagon, and 96 after arginine administration. The blood glucose was between 180 and 220 rag/100 ml. -- After adrenalectomy insulin respose to glucose and glueagon was higher than in non-adrenalectomized rats. Insulin peaks were now I12 ~U/ml after glucose and 185 after ghicagon. Arginine did not modify insulin response. Blood glucose reached 255 rag/100 ml after glucagon and 85 rag/100 ml after arginine. -- Stress increases epinephrine secretion. We assume that the changes in plasma insulin and blood glu-


cose after adrenMectomy are mainly due to the lack of epinephrine, because they were reversible in adrenalectomized rats by epinephrine infusion. These data show a decreased insulin response to glucose in stress, mediated by the presence of the adrenals. Insul in response to glucagon is depressed to a smaller extent and arginine-in- dueed insulin release seems to be independent of the adrenals. I t is suggested that in stress epinephrine has no additive effect upon glueagon action on glyeogenolysis.

Rat Plasma Glucagon during the Perinatal Period. J. Girard, D. Bal, R. Assan. Laboratoire de Physio]ogie eompar6e, Facult6 des Sciences, Paris and H6tel Dieu, Paris, France. Panereatoglueagon (PG) and total glucagon-like immune- reactivity (GLI) were assayed by radioimmunoassay in plasma of rat fetuses (18 ~ to 21 �89 days of gestation) and in 211~ day-old fetuses delivered by uterine section and maintained, after umbilical section in an Humidierib at + 37~ PG and GLI were detectable in fetal plasma from 181~ days (earliest stage studied) at levels lower than in maternal plasmas. Maximal fetal values were found at 20 ~ days (GLI 580 • 80 pg/ml ; PG 500 • 40 pg/ml). The demonstrated absence of transplaeental passage for exogenous glucagon in the rat indicated the fetal origin of cir- cnlating PG in the fetus. -- ThirW mill after separation from mother a significant increase occured for PG (430 =k 60 to 1140-_t= 80 pg/ml). PG remained elevated after 60 and 90 mill (1070 • 170 and 965 :k 150 pg/ml), then progressively decreased to a steady level, from 120 to 360 mill. Assay of total GLI demonstrated an exclusive contribu- t ion of PG to these perinatal variations. -- A fall of Mood glucose (beginning at detachment with a minimal value 60 mill later) accompanied the rise of PG. Liver phos- phorylase activity rose after 60 rain, followed by a sharp decrease in liver glycogen content (60 to 360 mill). This t ime relationship makes conceivable the suggested se- quence : neonatal fuel need (and/or hypoglycemia) triggers glucagon secretion, which in tu rn inc[uces liver glycogeno- lysis.

Diabetic Cystopathy: CHnical and Functional Aspects. The Importance of its Diagnosis in the Prevention of Ascending Nephritis. V. Gligore, N. Mosora, T. Fekete, T. Beraru. Second Medi- eM Clinic and Clinic of Urology, Cluj, 1%umania. 24 diabetic patients underwent eystography (micturating, ascending, and radiologieal) in order to assess residual content of the bladder, as well as cystovolumetry. 8 of these patients then underwent cystomanometry, and graphical recording of the endovesieal pressure was performed in 5 patients. The result of these studies were correlated with age, sex, duration of the diabetes, and the presence or absence of ur inary infections, proteinuria, or diabetic microangiopathy. -- The high incidence of diabetic eystopathy and its relation to the duration of the diabetic state have to be emphasized. Both cystography and cystovoIumetry were also performed in 3 obese non-diabetic women. -- The authors will discuss diabetic cystopathy on the basis of the results published in the literature and on their own findings. They insist on the need for early detection of diabetic cystopathy, both from the point of view of prevention and treatment , as well as in the aim to l imit further its incidence and, above all, to prevent ascending nephritis.

The Importance of Cutaneous Biopsy and the Risah Test in Evaluation of Diabetic Microangiopathy. V. Gligore, N. Mosora, V.V. Pipilian, L. Olteanu, A. Ser- ban, I. Calusera, T. Holan, M. Mielutia. Second Medical Clinic, Proseeture, Ins t i tu te of Nuclear Medicine, Cluj, Rumania.

The authors have shown the value, of cutaneous biopsy by demonstrating the presence of diabetic mieroangiopathy (MAD). Correlation between the histological and histochemical results of cutaneous MAD and the modification of capillary permeability obtained by the RISAH test has been established. All subjects studied underwent the RISAH test. 89 biopsies were performed. Signs of the presence of MAD have been found in 40 diabetics (44.89% of the patients studied). -- The RISAI-I test demonstrated the abnormalities in capillary permeability. A surprising fact was the incidence of MAD in a large number of patients, aged between 0 and 5, and also between 5 and 10 years. The percentage totalled by these young patients is close to the total cases with MAD (42.80/0 and 40.7~o, respectively, versus 44.8%). The percentage of cases with MAD detected by cutaneous biopsy was higher than that of ret inopathy (diagnosed by ophthalmoscopy) and also higher than that of albuminuria, which once more under- scores the importance and technical superiority of cutaneous biopsy.

The Effects of Allopurinol Administration on Carbohy- drate and Lipid Metabolism in Normal and Hyperuricemic Patients. A. Gnudi, C. Coscelli, G. Ballerio, V. Palmari, O. Alpi, G. Cavazzini. Is t i tuto di Semeiotica Medico, Universi ty of Parma, Parma, I taly. The effects of allopurinol administrat ion on glycolipid metabolism of normal and hyperuricemic subjects were examined. Allopurinol was administered at the dosage of 200 and 400 mg/day for a period of one or two weeks. IVGTT, Tolbutamide test, blood lipids, cholesterol, beta/ alpha lipoprotein ratio, free glycerol were measured at the beginning and at the end of the t reatment with allopurinol; uric acid levels and urieuria were measured every second day during the administrat ion of the drug. -- The predictable decrease of uric acid levels and of uricuria were observed in all patients. The authors also noticed changes in lipid profile and a diminished effect of tolbutamide both in control and hyperurieemie groups. Peripheral glucose utilization was impaired, but not significantly. -- Some possible explanations of the obtained data (action of allopurinol through the hyperlipemia, action on enzymes) are being considered.

The Effect of Carbohydrate and Lipid Oxidation on Glucose Tolerance and Insulin Sensitivity in Man. F. Gomez, E. J6quier, V. Chabot, V. Briber & J .P . Felber. Clinique M6dieale Universitaire, Lausanne, Switzerland. Several types of glucose tolerance tests (OGTT, rapid IVGTT, 90 min glucose infusions) were carried out on 41 normal subjects in whom free fat ty acid (FFA) levels were either raised by neutral fat infusion or lowered by ~-pyridyl earbinol infusion. -- With continuous measurement of O 2 consumption and of non-protein respiratory quotient (RQ) during these tests it could be demonstrated that the fat infusion before glucose load was followed by an increased lipid oxidation whereas carbohydrate oxida- t ion was diminished. The glucose load (without fat infusion) induced a rapid increase of carbohydrate oxidation and a decrease of lipid oxidation. However, when F F A were raised a glucose lead was followed by a smMler increase of carbohydrate oxidation than was observed in the eases where glucose only was given. In addition, glucose tolerance was found to be depressed by the simultaneous administrat ion of lipids, while insulin (IRI) levels after glucose load were significantly increased. -- These observations fmd support in the known inhibit ion of glucose oxidation induced by fat ty acid oxidation. Glucose intolerance and insulin insensitivity appear to be a consequence of this mechanism. -- The reverse, i.e. decreased lipid oxidation, increased carbohydrate oxidation, improved glucose tolerance and lower insulin response to glucose


load with apparent "insulin hypersensi t ivi ty" was observed while lowering F F A levels by a fl-pyridyl carbinol infusion.

The Effect of Carbohydrate-Intake on the Fat Mobilizing Activity of Urine Extracts. E. Goth, J . FSvenyi, A. Hegedtis. Dept. of Medicine II . Janos Hospital, Budapest,. Urinary fat mobilizing substance (FMS) was extracted quanti tat ively by the method of Chalmers et al. The urine was collected for 24 h, the precipitate was purified by chromatography and 5% of the extract was injected into mice. The blood ketenes were determined by the method of Pawan before and six hours after injection. The control animals received saline, which caused slight increase of kctones, presumably due to stress. 30 obese persons were put on diets containing daily 300, 200 and 50 g carbohydrate and finally they fasted. The extracts of urines collected on a low carbohydrate diet and during fasting caused a significant increase of ketches in mice. There was no difference in FMS act ivi ty between patients with normal and decreased glucose tolerance. Some data of the chemical analysis of the extracts will be presented.

Immunoassay of Insulin in the Aqueous Humour of Dia- betics and Non-Diabetics with or without Cataract. A.V. Greco, G. Ghirlmlda, G. Fedeli, R. Feniei, G. Gem- bassi. Department of internal Medicine, Catholic Univer- sity of S. Heart , Rome. The immunoassay of insulin has revealed the presence of the hormone not only in the plasma but also in the other extracelhdar fluids: urine, bile, cerebrospinal fluid, aqueous humour (Daniel and Henderson). In order to check the importance of insulin in the carbohydrate metabolism of the lens, our studies have been extended to a group of diabetics with ret inopathy and to a group of diabetics and non-diabetics with cataract. The aqueous humour was sampled by piercing the anterior chamber of the eye according to Amsler st al. The average value of insulin in the aqueous humour of normal subjects is 11.3 4- 0.94 aU/mt ; in the other groups the concentration of the hormone is decreased. The fact tha t the insulin levels are low in the aqueous humour of hyperglyeaemic patients with cataract leads to the thought tha t insulin may have some influence in the onset of opacity of the lens. The observation of a similar decrease in the aqueous humour of diabetics with ret inopathy but without cataract weakens this hypothesis. The fall of insulin is probably related to a modification of the blood-ophthalmic barrier with regard to insulin as a result of vascular damage in diabetics and non-diabetics.

Insulin Resistance withou~ Circulating Antibodies, Evi- dence for an Antagonist ? D. Griineklee, J . Hessing, H. Daweke, L. Herberg, F .A. Gries. 2nd Med. Clinic and Diabetes Research Institute, University Diisseldorf (Director: Prof. K. Oberdisse). Insulin resistance (IR) usually is caused by circulating antibodies to insulin (AB). However, in some eases no AB could be demonstrated. 3 of these patients, 1 male and 2 females from 57 to 68 years of age, with a maximal need of 360, 560 and 650 U of insulin per day were investigated. Other causes of increased insulin requirement, such as infection or increased levels of insulin-antagonistic hormones, were excluded. -- The following results were obtained: The plasma disappearance rate of i .v. injected insulin is delayed in patients with AB; however, it was normal in I R without AB. Using acetate cellulose electrophoresis or immune electrophoresis, lslI-insulin added to serum migrates in the alpha-globulin fraction. Glucose metabolism of subcutaneous adipose tissue of IR patients with AB is not stimulated by insulin. In 2 patients without AB a dose-dependent response to insulin was observed.

When incubated in autologous serum, the tissue of one pat ient responded to insulin. The effect was suppressed by incubation in serum with AB. -- I n ob ob mice and in ob+ ob+ mice the blood sugar lowering effect of i.p.- injected insulin was inhibited by the addition of serum of patients with AB and, to a lesser extent, also by a serum of a pa- t ient without AB. -- Serums of patients both with and without AB injection into mice caused a (significant) increase of blood sugar within 1 h. This increase was more distinct with serum of an I R patient without AB than with AB. -- The existence of an insulin-antagonistic factor in serum of such patients will be discussed.

Comparative Studies of the Blood Sugar Lowering Effect of Insulin from Different Species in Insulin Resistant Pa- tients. D. Griineklee, J. Hessing, H. Daweke. 2nd Med. Clinic and Diabetes Research Inst i tute, Universi ty Diisseldorf (Director: Prof. K. Oberdisse). 9 patients with insulin resistance (IR) were investigated after receiving 27 U of various insulins intravenously in successive tests, namely, bovine (B), porcine (P), human (H) and bonito (fish) (F). In 7 of these patients with insulin resistance high titres of specific antibodies (AB) were observed. -- Patients resistant to insulin as caused by antibodies exhibited marked differences in the drop of blood sugar after injection of the various insulin preparations. P was found to be more effective than B in 3 out of 7 cases. In 3 out of 8 cases H showed a more marked effect than B and in 4 cases also more than P ; however, in one case it proved the most ineffective. -- In 7 cases F was found to be more effective than B or P and in 5 out of 9 cases also more effective than H. -- In 2 cases circulating antibodies could not be determined in spite of an increased insLflin requirement; other causes for I R could not be found. In these cases the different responses described above were not apparent. All insulins showed a reduced effect, the blood sugar curves being similar. -- I t may be concluded that. differences in the response to insulin of va~'ious species may be the resut~ of different binding capacities of antibodies for insulin and on the other hand a decreased effect of insulin in I R without AB may be due to a different kind of insulin antagonism. -- Therapeutic trials with bonito insulin are suggested when insulin resistance related to antibodies is present.

The Regulation of Renal Gluconeogenesis by Cyclic AMP and Free Fatty Acids. W. Guder, O. Wieland. Inst. Clin. Chem., Schwabing City Hosp., MLmich, Germany. Renal gluconeogenesis was found to be increased in starvation and diabetes. Since glueagon and insulin are meta- bolicMIy ineffective in the kidney, other mechanisms of hormonal and metabolic regulation have been discussed. For instance, free fa t ty acids and parathyroid hormone were shown to stimulate glucose formation from various precursors in vitro. -- In order to study the mechanisms of action of these effectors, the st imulatory action of free fa t ty acids was compared with tha t of cyclic AMP, the intracetlular mediator of parathyroid hormone action. Glucose formation was measured in isolated rat kidney tubules in vitro. Cyclic AMP stimulated glucose formation from all precursors that enter gluconeogenesis via phosphoenolpyruvate carboxykinase. -- This increase was paralleled by a stimultaneous increase in substrate uptake. -- In contrast, the increase of glucose formation after addition of fa t ty acids was coupled with a decrease in substrate uptake "sparing" substrate. Both effeetors, when given simultaneously, resulted in additive stimulations. These results suggested different mechanisms for both stimulators. Whereas the effect of free fa t ty acids can be explained by inactivation of the pyruvate dehydrogenase, cyclic A h ~ seems to act by providing more


phosphoenolpyruvate. The importance of both regulatory mechanisms in s tarvat ion and diabetes will be discussed.

Long-Term Evolution of Diabetes in Familial Idiopathic Hemochromatosis. 22 cases studied over more than 5 years. J . Guillon, L. 13odic, B. Charbonnel. Medical Clinic ]3, HStel-Dieu, Nantes, Trance.

The evolution of diabetes was studied for 5--11 years in 22 patients with familial idiopathic hemochromatosis and is presented on the slides shown. 14 of these subjects were treated by veneseetion. -- 6 cases of diabetes were well controlled over several years without insulin therapy; -- 4 subjects treated by venesection have been well controlled for more than 6 years; 1 patient had to be given insulin; -- in the 17 insulin-dependent diabetics, the dose of the hormone varies between 20 and 80 units, depending on the individual case. In many cases, the initial dose could be lowered after some months; in 7 subjects the dose was even further diminished, and in only 1 case did the insulin requirements have to be increased over the years. -- There is late-onset angiopathy; 2 cases of ret inopathy were observed after 9 and 10 years respectively. -- Dia- betes is more stable and more easily controlled in the subjects undergoing venesection; after some years of t reatment with venesection, the diabetes in hemochromatosis does not appear to be different from the habitual diabetes mellitus.

Myocardial Infarction during Treatment with Oral Anti- diabetic Agents. D.R. Hadden, D.A.D. Montgomery, Elizabeth Mayne, J .A . Weaver. Sir George E. Clark Metabolic Uni t and I-Iaematology Department, Royal Victoria ~ospi ta l , 13el- fast, Northern Ireland. A retrospective study has been made of 550 diabetic patients who commenced t rea tment with oral antidiabetic agents between 1957 and 1965. The occurrence of myocardial infarction during their subsequent follow-up until 1971 at a diabetes outpat ient clinic has been analysed and compared with the occurrence of myocardial infarction in diabetic patients t reated by conventional carbohydrate diet restriction. -- A prospective study of 180 new diabetic patients was commenced in 1965. These patients had initial cardiovascular assessment, including blood pressure levels, electrocardiogram, serum cholesterol and presence of tibial pulsation, and these tests were repeated at three years (1968) and six years (1971). Effect of various t reatments on cardiovascular status is evaluated. Data on changes in platelet adhesiveness and fibrinogen levels during t rea tment with oral agents, diet and insulin, are also considered.

Influence of Isolated Insulin Antibodies on the Insulin Secretion in Vitro. H . J . Hahn, M. Ziegler. Zentralinsti tut fiir Diabetes "G. Katsch" Karlsburg-Garz, DDR. Having found an inhibitory effect of mouse insulin and insulin fractions on IRI-release of isolated mice islets, we studied the influence of insulin antibodies (IAB) on IRI - secretion in vitro, because such antibodies diminish the free insulin content of the medium. The isolated antibodies (binding capacity 2.3 mU/mg IAB) were prepared from goat anti-bovine-insulin-serum by means of immuno- precipitation. 6 isolated islets of normal mice were incubated with increasing concentrations (0--32 rag/incubation) of pure insulin antibodies or IgG-immunoglobulins (controls without addition of proteins). After incubation the insulin content of the medium was assayed and the islets were reincubated with glucose or glybenclamide. -- The IRI-secret ion was strongly st imulated by antibodies, independent of the glucose concentration in the

buffer. The greatest effect was already reached by a concentration of 8 mg IAB. IgG slighly inhibited the IRI- output. The reincubation results show that the stimulators used are effective. The described influence of IAB does not seem to be based on unspecific effects of proteins or on immunological extraction.

Comments on the Antigenic Role of the Beef-Diinsulin- Contamination in Commerical Insulins*. J. Hahn, O. Richter, S. Steinhilber, L. Kerp**. Medizini- sehe Univ. Klinik, Freiburg i. Br. Summary. -- Sere from 38 insulin-treated diabetics con- rained the wellknown main components of antibody- combining sites, Ak I with higher and Ak 2 with lower affinity, both forming complexes with beef-monocomponent-insulin and beef-diinsulin. The combining parameters (concentrations of antibody binding sites Ak 1 and Ak 2 and association constants kl) for beef-monocomponent-insulin were significantly higher than those of beef- diinsulin. The association constant k s showed no sigzfifi- cant difference between antigens. I t is suggested that the beef-monoeomponent-insulin molecule is better adapted to antibodies from diabetics against insulin than the beef- diinsulin molecule. After adsorption of the antisera by beef-monoeomponent-insulin coupled to cellulose, there was no evidence for antibodies specifically directed against antigenic sites only represented by beef-diinsulin. The beef-diinsulin contamination of commercial insulins does not seem to play an important role as antigenic st imulant for the production of antibodies against beef insulin in m a n .

Hyperlactaemla Associated with Phenformin Therapy. S.J. Heaney, S.K. Varma, W.G. ~VVhyte, R.S. Walker. Law Hospital, Carluke, Scotland.

To explore the relationship between Phenformin therapy and hyperlactaemia in diabetes mellitus, treated patients have been compared with untreated diabetics and normal subjects using the intravenous glucose tolerance test (IVGTT). At the higher pharmacological dose levels hyperlactaemia was found in the fasting state and was exaggerated by the glucose load. At lower dose levels this effect was less pronounced. Parallel measurements of pyruvate showed that a consistent excess of lactate over pyruvate was obtained and that the ratio was further increased in the treated group. This change is demonstrated in several patients who were tested before and after treatment. -- The results point to the mode of action of Phen- formin being associated with a block of tissue respiration at pharmacological levels of dose.

Biphasic ATP Drop during Lipolysis in Mouse Adipocytes. J . J . Heindel, S.W. Cuslnnan, B. Jeanrenaud. Ins t i tu t de 13iochimie Clinique, Geneva, Switzerland. Isolated mouse adipose cells incubated with a var ie ty of lipolytic agents release non-esterified fat ty acids (FFA) into the medium. As a consequence of F F A release, marked increases in intracellular F F A (referred to as CAFA for cell-associated fa t ty acids) together with decreased cellular ATP content have been observed. Since several energy processes have been shown to be altered during lipolysis, a detailed analysis of CAFA and ATP changes has been carried out. In all experiments, when saturating F F A / Albumin ratios of about 5 are reached, a rapid fall in ATP level is observed that coincides with the cessation of F F A release and a rapid and sizeable accumulation of CAFA (10--20 ~moles/g lipids). This ATP drop is not prevented

* We are very indebted to Dr. H. Seh6ne, Farbwerke Hoechst A.G. for the gift of beef-monocomponent and beef-diinsulin.

** Supported by the Deutsche Forschungsgemeinsehaft.


b y addi t ion of substrates. As C.~FA continues to rise, the fall I n A T P appears to approach bu t never reaches a new s teady state. ATP values as low as 10% of control have been measured. When the approach to sa tura t ing FFA/Alb ratios is lengthened by increasing the albumin concentra- t ion in the medium from 3 to 7~/o, by lowering the epinephrine concentration, or b y bra ldng the act ion of epinephrine x~dgh insulin, a small drop in ATP prel iminary to t ha t just described occurs. This drop remains constant

~ O " " " at about 1~ ~ of control, and is associated with relat ively constant CAFA levels (4--6 ~moles/g lipids) and F F A / Alb ratios from 2--4. - - These two dist inct drops in cellular ~kTP produced b y lipoly~ie agents may be of quite different significance in the control of energy-requiring processes and lipotysis in fa t ceils.

Increased Glueagon Sensitivity of Mouse Liver Adenyl Cyclase in Streptozotoein Diabetes. K.D. Hepp. Diabetes Research Unit , ]~Iunieh. A number of studies have presented evidence for a key role of 3 ~, 5'-A~'~{P in the regulat ion of l iver metabolism. Recent experiments from this labora tory have demon- s t ra ted a aireet inhibi tory effect of insulin and NSILA upon glucagon-stimulated adenyl cyclase ac t iv i ty in mouse liver. I n contrast , the effect of fluoride was not inhibited b y these substances. This would suggest t ha t interact ion of glueagon and insulin occurs a t some step ~long the act ivat ion process betwee-n hormone receptors and eatal?Nie malt of this enzyme. I n view of these findings we have invest igated the control of liver adenyl cyclase after induction of diabetes by i.p. injection of 200 mg/kg streptozotocin in mice. I n three diabetic groups the maximal glueagon effect, was significantly elevated (40, 55, and 90%) over the controls without any eha~ge in the fluoride- stimulated enzyme act ivi ty. Insulin treatment of the diabetic animMs again reversed this effect. These results in~ dieate tha t in insulin-deficient livers the adenyl eyelase system exhibits an increased sensit ivity towards glucagon.

Is Insulin Release Triggered by Glucose Translmr$ Across the t6-Cell N[embrane ? B. Hellman, A. Lernmark, J. Sehlin, I .-B. T~ljedal. De. pa r tment of Histology, Universi ty of Umes Ume&, Swe- den. The effects of phlorizin and phloret in on several parameters of fl-eeIt ~ c t i o n were s tudied with microdisseeted islets of obese-hyperglycemic mice. At a concentration of 10 m?r phlorizin signifieaxitly depressed glucose-stimulated insulin release, glucose t ransport , glucose oxidation and the level of fructose 1.6-diphosphate. Whereas 1 mM phlorizin inhibited glucose t ranspor t b y about 50 %, the glucose- s t imulated insulin release remained unagfeeted b y 1- -5 m2v[ phtorizin. I n the absence of glucose, 10 mM phlorizin significantly s t imulated insulin release, Similar effects, al though less pronounced, were obtained with phloretin. -- The results corroborate our previous hypothesis of active glucose t ranspor t into the pancreat ic fl-cell. Insul in release does not seem to be governed b y the glucose trans- p o ~ in tote. Stimulat ion of insulin release might ra ther be elicited b y the binding of glucose to a dist inct membrane receptor, which accounts for but a minor fraction of the to ta l glucose uptake.

~-Cell Function in Sireptozotocin Diabetes. M. HSnz, N. Katsi lambros, Y. Abdel Rahman, H. Sehatz, V. Maier, E. SchrSder, E . F . Pfeiffer. Dep. Endocrinology and YIetabolism, Center Internal Medicine and Pediatrics, Univers i ty of Ulm, Germany. As previously shown, p lasma glucagon levels are increased in severe streptozotocin diabetic rats. For clarification of this rise in p lasma glue~gon ~-cett function has been

e x a m ~ e d in streptozotoein diabetic r~ts, bo th under in. vivo and in ~qtro conditions. - - 48 h after i.v. inject ion of streptozotoein (80 mg/kg b.w.) the plasma glueagon levels were significantly increased in ketoacidotic diabetic rats but were only slightly higher in non-ketoacidotic diabetic rats as compared with normM animals. I .v. injection of insulin failed to depress the elevated glueagon levels in t he ketoaeidotie diabetic animals while decreases rapidly followed insulin injection in the non-ketoaeidotie diabetic rats. Increases in glueagon release have also been demon- s t ra ted in incubation and perfusion experiments employing islets of Langerhans isolated from ketoacidotie diabetic rats . The glucagoa release from isolated islets of non-ketoaeidotie diabetic animals and of control rats was of similar magnitude. On the other hand, addition of streptozo~ocin to the incubation medium had no significant effect on glueagon secretion from isolated islets of normal rats. -- I t is concluded tha t the increased ~-cell function is due to the ketoaeidotie s ta te as such, and not to the direct effect of streptozotocin.

The Role of SkeletM ~Iuscle in Post-Insulln Hyperlae~a- temia. C.R.S, Houghton, I .D. Caterson, D,H. Williamson. Me- tabolic Research Laboratory , Radcliffe Infirmary, Oxford. Adminis t ra t ion of insulin to hyperglyeacmie pat ients can lead to a considerably htcreased blood lac ta te concentra- t ion and this raises the question of the source of the lactate. The isolated, perfused ra t hind-quarter has been used to evaluate the role of skeletal muscle in lactate production in this si tuation. When the hindquarter from normal ra ts was perfused with 5 m3/i glucose the addi t ion of insulin (25 mU/ml) did not increase laeta.te release, At. a high glucose concentration (20 rn~) there was a slight increase in lacta te release (from 2.0 to 3.0 ~mol/min/30 g muscle), when insulin was added lac ta te production increased to 7.2 lxmol/mln/30 g muscle. Experiments wi th 20 toni fructose showed no comparable increase. The results for diabetic muscle from stveptozotocin-treated ra t s perfused with 5 mM glucose and insulin segregated into two groups. One had tow blood glucose (7 raM) and showed a 3-fold increase in lacta te production with a slight increase in glucose uptake compared with normal muscle. The other had high blood glucose (16 mM), a lower than normal glucose and an approximate ly 2-fold increase in lac ta te release. - - I t is suggested tha t ha the absence of l iver disease, the severi ty of the hyper laet~temia af ter insulin- adminis t ra t ion will depend on the existing hyperglyeaemia and the sensit ivity of skeletal muscle to insulin.

A Study of Diabetes in a Relatively Atheroma-Free West African (Gambian) Population. D.P.M. Howells. ]~I.R.C. The Gambia aa~d -Unk, ers i ty Hospi ta l of W'ates, Cardiff. The prevalence of disease varies great ly in different Com- munities and the s tudy of these differences may help in the bet ter understanding of disease. Diabetes is wor thy of s tudy not only for i ts own sake bu t also because of i ts association with atheroselerotio vascular disease. - - 70% of the adul t populat ion of a rural Gambian village were examined, numbering 272 persons, and using the 2 h blood sugar after 50 grams of ghieose taken orally, the prevalence of hyperglyeaemia was defined. I t w?~ found to be low when compared with Bri t ish da ta and da ta about genetically similar to New World negroes in Trinidad. The influence of pa r i ty was examined. Close ties of kinship amongst the subject made i t possible to s tudy the influence of heredi ty as the 272 subjects also provided 375 first degree relatives. ~ Hyperglycaemia, hypereholes- terolaemia, and obesi ty are all factors which are associated with the development of atheroselerotie vascular disease. I n a Bri t ish pop~flation Abrams e~ al. (1969) have shown a

Vol. 7, N o . 6, 1971 Organization Section. Abstracts 485

correlation between these three factors. I t was not possible to show such a correlation in the Gambian subjects, and it is postulated that this might be a characteristic of a relatively atheroma-free population.

Adrenergie Receptors for the Secretion of Immunoreactive Glucagon and Insulin from the Isolated Perfused Canine Pancreas. J. Iversen. The Second Clinic of Internal Medicine, Kom- munehospitalet, Aarhus, Denmark. The effect on secretion of pancreatic glucagon and insulin of infusions of L-epinephrine at physiological concentrations from 0.5--2 ng/ml for 8 min was studied during perfusion with low (25 mg/100 ml) as well as with high (150 rag/t00 ml) concentrations of glucose. -- During perfusion with high concentrations of glucose, L-epinephrine stimulates secretion of glucagon in a dose-dependent manner in a non-biphasic response pattern, contrasting with the biphasic response pat tern normally seen after glucagon- releasing stimuli, as previously reported from our laboratory. Glucagon is stimulated in spite of a glucose concen- t rat ion which by itself effectively inhibits glucagon release. Release of insulin exhibits the well-known inhibition even at these low concentrations of L-epinephrine. At low concentrations of glucose secretion of glucagon is greatly accentuated while insulin remains unchanged. The epinephrine-induced glucagon release is further accentuated when the alpha-adrenergic blocking agent phentolamine is simultaneously infused at a concentration of 1 ~M, while the inhibit ion of insulin is converted to a stimulation. -- The epinephrine-induced glucagon release is suppressed or abolished when the beta-blocking agent pro- panolol is simultaneously infused at a concentration of 1 fxlVI while the inhibit ion of insulin becomes further accentuated. -- There was no effect upon the release of glucagon or insulin by the adrenergie blocking agents themselves, in the concentrations used in this study. -- Evidence is thus presented that both the alpha cells and the beta cells are under the influence of adrenergic substances, the stimulation of glucagon release being mediated through a beta receptor and the inhibition of insulin being mediated through an alpha receptor.

The Relationship between Peripheral Glucose Uptake and Glucose Tolerance in Maturity-Onset Diabetes. R.A. Jackson, G. Perry, J. Rogers, U. Advani, T .R .E . Pilkington. Medical Unit , St. George's Hospital, London S.W. 1. Forearm glucose uptake (FGU) was studied during i00 g oral glucose tolerance tests in normal subjects and maturity-onset diabetics following a normal carbohydrate intake and again in the latter after carbohydrate restriction. -- Following a normal diet, the increment in FGU after glucose loading was reduced in the diabetics and glucose tolerance (assessed as the incremental area under the curve) was grossly impaired. Calculations of glucose disposition in the body as a whole based on these observations suggest, however, tha t the reduction in peripheral uptake can account to only a minor extent for the concomitant impairment in glucose tolerance. Following carbohydrate restriction basal glucose concentrations were reduced and the increment in FGU after glucose loading was increased to equal the mean normal response, bu t despite this glucose tolerance was decreased even further. -- I t is concluded that after a normal diet decreased peripheral glucose uptake is NOT the major cause of the impairment in glucose tolerance in the diabetics. I t is suggested that diminished hepatic glucose conservation is the major underlying disturbance, and that the further deterioration in glucose tolerance following carbohydrate restriction is due to the total ablation of the already limited hepatic capacity for glucose utilization.

Incidence of Different Therapeutic Antidiabetic Agents on Iodine Metabolism and Metabolism of Thyroid Hormones. C. Jaffiol, L. Baldet, Y. Vierne, J. Mirouze. Dept. of Me- tabolism and Endocrinology, St. Eloi Hospital, Mont- pellier. The cycle of inorganic and organic iodine, the daily thyroid hormone production, the tissue requirements for tri- and tetra-iodothyronine as well as for the vector pro- reins for these hormones have been assessed in two groups of insulin-treated diabetics and in orally treated diabetics. The results are compared within themselves, by statistical methods, and compared with those of a non-diabetic control population. Sulfonamide therapy has no effect whatsoever on inorganic iodine metabolism and thyroid hormonogenesis. Insul in therapy increases the daily requirement for T4 and above all for T3 and diminishes the transport capacity of TBPA. Insul in and oral hypogly- comic agents show no competitive action as regards the protein fixation sites of thyroxine, and this has been con- firmed in in vitro experiments.

The Ability of MC-Insulin or t X Crystallized Insulin in the Development of Immunological Tolerance in Mice. F.K. Jansen. Diabetes Research Inst i tute, Diisseldorf, Germany. Immunological tolerance to insulin prevents the forma- t ion of antibodies against insulin. We therefore tried 1be induce tolerance by immunizat ion with high and low doses of insulin. 600 NMRI mice had been immunized i.p. by 6 doses of MC-pork-insulin or 1 • crystallized pork insulin ranging from 100 ~g to i ng (diluted 1:10) 3 times per week up to 3 months. Both insulins had been kindly supplied by Nero, Copenhagen. Insul in shock by 100 ~g or 10 ~g had been -avoided by the s.c. injection of 2 ml 20% glucose. 14 groups of 10 mice each had been treated for �89 1, 2, or 3 months by immunizations. 13 days after the last injection all animals got a single dose of 100 ~g insulin in complete Freunds adjuvant to test the tolerance. 3 weeks and 5 weeks thereafter blood samples had been taken from all animals for analysis of ant ibody content. Immunizat ion with 1 • crystallized insulin led to two zones of tolerance after three months of t reatment , one zone by high doses of 100 ~g or 10 ~g, and the other by a low dose of 100 ng of insulin. The intermediate dose of 1 ~g was followed by a high antibody formation. I t seems as though immunization with MC-insulin leads to similar results: to ant ibody formation and tolerance, bu t it takes about 4 times longer. So the curves of ant ibody content 2 months and 3 months after MC-insulin treatment are similar to the ones of �89 and 1 month after t reatment with crystalline insulin.

Urinary Albumin Excretion in Diabetics, Borderline Dia- betics and Potential Diabetics. R.J . Jarrett , H. Keen. Department of Medicine, Guy's Hospital, London. Urinary albumin excretion has been measured, using a sensitive, double ant ibody immunoassay method, in several groups of people. These include diabetics and borderline diabetics, together with a control, normogly- caemie group, derived from the population of Bedford, England, cooperating in the 1962 Diabetes Survey and diabetics, potential diabetics and a control group under s tudy by the Boston investigators (Balodimos, Soeldner et al.). - - In the Bedford series, the mean albumin excretion was greatest in the newly diagnosed diabetics, least in the controls and intermediate in the borderline diabetics in each of the three age categories (20--39 ; 40-- 59; 60 -}- ). -- In the Boston series, the diabetics also had higher mean albumin excretion rates than the controls. -- However, potential diabetics (offspring of con- jugal diabetics), with or without abnormalities of glucose

Diabetologla, u 7 34

486 Orgaa~ization Section. Abstracts

tolerance, did not differ from the controls. -- I t is con- eluded that increased alburain excretion is associated with diabetes or impaired glucose tolerance, bu t is not associated with genetic predisposition to diabetes.

Epidemiology of Diabetes Mellitus in a Country District. A. Kacding and coworkers. Medical Polielinie, The Uni- versity of Rostock, Rostock, DDR. I n a country district with a population of 38 000 prevalence of the diabetes has been investigated by carrying out two screening surveys in 1960 and 1970, using the same methods. The influence of the two surveys on the prevalence of spontaneous diabetes is reported for the period between the two screening surveys. I~esulting from the first screening in 1960 the diabetes "prevalence" doubled in this district. Ten years later, in 1970, the second screening revealed an increase of only 15% in the apparent prevalence of diabetes. -- Clinical data, including secondary diabetic complications of the newly discovered diabetics from the two surveys are discussed and compared. -- The possible reasons for the significant differences between the two surveys are discussed, and therapeutic aa~d epi- demiologieal conclusions are drawn.

Comparison of the Results of an Analysis of the i.v. Tolbu- tamide Test According to Lange and Krick as well as by the ~vlethod of Unger and Madison. I-I. Kaffarnik, U. Heink, W.D. Gassel, P. Z6fel, K. 1Viy. larch. Medical Policlinie, University of Marburg/Lahn, W.-Germany. I n order to improve the assessment of the diabetic metabolic state, Lange and Krick have applied a modification of the method of Unger and Madison, suggesting extension of the Tolbutamide test from 30 to 60 rain. I n routine diagnosis a I h test, including additional blood sugar measurements, means considerable additional workload and material. The authors have therefore tested the reliability of the two methods in 150 subjects. A close correlation was observed between the T~ value and the blood sugar assessment 20 and 30 rain after the injection of Tolbuta- mide. Only slight correlations were found between the T 3 value and the increase in blood sugar 40 and 60 rain after the injection. -- In comparing the above values with the glucose assimilation constant K (according to Conard), we found sufficient correlation between the values at 20 and 30 rain, bu t no correlation between the 40 and 60 min values. -- These results demonstrate that the Totbutamide test according to Unger and )~[adison is really sufficient in routine diagnosis.

Peripheral Insulin and Glucose Utilization in Tolbutamide- Induced Hypeglyeaemia. ]3. Karamanos, W.J.I-L Butterfield, A.C. Asmal, M.J. Whiehelow, B.D. Cox. Deparment of Medicine, Guy's Hospital Medical School, London. This study was designed to investigate (a) the early ar.

terial insulin response to i.v. tolbutamide, (b) the relationship between insulin and glucose uptake in the peri- phery, (c) the role of peripheral insulin uptake and glu. cose metabolism in the development of tolbutamide in. duced hypoglyeaemia. -- Five normal subjects were studied before and for 60 min after 1 g i.v. tolbutamide. Continuous half minute arterial and venous samples were taken for glucose and insulin estimations over the first. 10 rain and thereafter a t regular intervals for the rest of the test. Tissue uptake was measured using the forearm technique. -- Our results indicate tha t there is a well marked insulinaemie response in the arterial plasma within half a minute of commencing the tolbutamide injection and this reaches a peak by the second minute. The arterial response is reflected in the venous plasma with a time lag of less than half a minute bu t the venous insulin levels are lower. -- The arterial insulin response is paralleled by changes in the peripheral insulin uptake. The correlation between the two is highly significant (p < 0.001). -- The increase in insulin uptake is accompanied by a rise in glucose uptake bu t the changes are not concomitant and there is no correlation between the two. :However, there is a good correlation between the total one hour glucose uptake and the total one hour insulin uptake. -- The results suggest that, the magnitude of the peripheral glucose uptake after tolbutamide cannot in itself account for the degree of fall in the blood sugar.

Utilization and Exhalation as lalJO~ of I.V. Administered Loads of laC Labelled Glucose, Xylitel, Fructose and Sorbi- tol in the Fasted and in the Streptozotocin-Diabetic Rat. U. Keller, E.I~. Froeseh. Metabolic Unit , Department of Medicine, Universi ty of Zurich, Switzerland. 35--37% of equivalent loads of glucose, xyIitol and fructose and 20% of sorbitol were exhaled as ~4CO2 by 24 h fasted rats within six hours. The peak of laCO~ was 20--30 rain after administration. Streptozotocin-diabetic rats exhaIed li--18~/o of the administered dose of each substrate. Maximal l~COe-production was registered 20 rain after fructose and 40--60 rain after glucose, xylitol and sorbitol. After six hours, the amounts of carbon-14 re- covered in serum, serum osazones, glycogen and total lipids of liver and diaphragm glycogen were similar after each of the four substrates in either of the two groups of animals. -- Diabetic rats incorporated only minimal quantities of carbon-J4 into diaphragm glycogen. 40-- 55% of the given dose of any one of the substrates was lost in the urine, mostly as glucose-laC. Only one clear cut difference between glucose and the other substrates was apparent. Much less laC-glueose was incorporated into glyceride-glycerol of liver total lipids, particularly in diabetic rats. The results suggest ~hat alI three glucose substitutes are rapidly converted to glucose and that. their util ization is, therefore, highly dependent on insulin. -- Thus, the value of the application of these glucose substitutes to diabetics is presently reassessed in our laboratory.

Corrigendum Notice Diabetologia, 7, 349-356 (1971) U. Keller and E .R . Froesch: Metabolism of U-~C-Glucose, Xylitol, Fructose aaad Sorbitol in the Fasted and in the Streptozotocin-Diabetic Rat . Unfortunately the figures in this article have been changed around by mistake. The following order would have been right: Fig. 4 should be Fig. 1 ; Fig. 5 should be Fig. 3 ; Fig. 2 should be Fig. 5; Fig. 6 should be Fig. 2; Fig. 1 should be Fig. 4; Fig. 3 should be Fig. 6. Legends are correct as printed, l~equests for correct reprints to : Professor E. R. Froesch

Kantonsspi tal Ziirieh Medizinische Universit/~tstdinik Stoffwechsellabor CH-8006 Ziirich, R/imistrat?e 11

l~esponsible for the tex~: Prof. Dr. K. O~E~)tSSE, II. Med. Klinik u. t?olikli~ik der Universit/~t, D-4000 Dfisseldorf, Moorenstr. 5. Responsible for advertisements: EDGAI~ SEID]SER, D-1 Berlin 16, Kurfiirstendaxam 237. Springer-Veriag, Berlin, Heidelberg, New York.

Prhu~ed in Germany by Druekerei Georg AppI, V~'emding/Schwaben. Copyright. �9 by Springer-u Berlin - Heidelberg 1971

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

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