Determination of pancreatic and gut Glucagon-Like Immunoreactivity (GLI) in normal and diabetic subjects

Diabetologia, Dec 1972

Summary Oral glucose tolerance tests (1.75 g glucose/ kg ideal body weight) were performed in 29 normal subjects, 17 maturity-onset diabetics (D I) and 8 juvenile type diabetics (D II). Glucose, IRI, pancreatic and gut glucagon-like immunoreactivity (GLI) were determined at various intervals. The basal levels of pancreatic GLI in the three groups were as follows: normals: 0.33 ±0.03; D I: 0.43±0.06; and D II: 0.50±0.05 ng equiv./ml (mean±s.e.m.). After glucose, a significant decrease in pancreatic GLI was observed in the normals, whereas the diabetics showed no significant change. An increase in gut GLI was observed in all persons. The importance of the GLI levels and variations is discussed.

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Determination of pancreatic and gut Glucagon-Like Immunoreactivity (GLI) in normal and diabetic subjects

Determination of Pancreatic and Gut Glucagon-Like Immunoreactivity (GLI) in Normal and Diabetic Subjects L.G. Heding 0 S. Munkgaard Rasmussen 0 0 Novo Research Institute , tIvidore ttospital, Copenhagen Summary. Oral glucose tolerance tests (1.75 g glucose/ kg ideal body weight) were performed in 29 normal subjects, 17 maturity-onset diabetics (D I) and 8 juvenile type diabetics (D II). Glucose, I R I , pancreatic and gut ghcagon-like immunoreactivity (GLI) were determined at various intervals. The basal levels of pancreatic GLI in the three groups were as follows: normals: 0.33 :~ 0.03; D I: 0.43-u0.06; and D I I : 0.50-t-0.05 ng equiv./m1 (mean  s.e.m.). After glucose, a significant decrease in pancreatic GLI was observed in the normals, whereas the diabetics showed no significant change. An increase in gut GLI was observed in all persons. The importance of the GLI levels and variations is discussed. glucagon; insulin; glucose; pancreatic GLI; gut GLI; oral glucose tolerance test; normals; diabetics Introduction The interest in determining glucagon-like immunoreactivity (GLI) in animal and h u m a n plasma has been greatly stimulated since the possibility of determining specifically pancreatic G L I was described (Keding, 1968; Aguilar-Parada et al., 1969a) . Several papers have been published during the past few years, chiefly b y Unger's group in Dallas, Texas, describing the glucagon was found as in normals despite the fact t h a t the diabetics had hyperglycaemia (Unger et al., 1970). A possible explanation for the lack of suppression of the diabetic ~-cell was given b y Mfiller et al. (1971), who showed t h a t the normal suppression of glucagon release did not occur when severe insulin deficiency was produced. This paper is a follow-up of the previous publication on glucose tolerance tests performed in 7 normal Group Sex Number Age Normals range 22--69 15--67 22--75 22--72 51 19--70 mean variations in pancreatic G L I dmdng a series of tests. I t has been shown t h a t infusions of amino acids and pancreozymin increased the level of pancreatic G L I in normal subjects (Unger and Eisentraut, 1969) , t h a t glucose decreased the level of pancreatic G L I in norreals (Heding, 1971) but not in diabetics (Mfiller et al., 1970), t h a t 3 days' starvation increased the level of pancreatic G L I (Aguilar-Parada et al., 1969b) and so did insulin-induced hypoglycaemia (Persson et al., 1971) . I n diabetic subjects, the same level of pancreatic subjects (Heding, 1971). The I R I (immunoreactive in sulin), glucose, pancreatic and gut G L I were determined in 29 normal subjects and 25 diabetics during an oral glucose load. Materials and Methods The GLI estimates were done by radioimmunoassay according to I-Ieding (1971). The two anti-glucagon sera were raised in rabbits (Heding, 1969) . One of these antiL.G. Heding and S.M. 1%asmussen: Determination of Pancreatic and Gut GLI scra (K 47) showed hardly any reaction with a crude gut GLI extract whereas the other antiserum (K 36) reacted with gut GLI giving parallel dilution curves for gut and pancreatic GLI. Twice crystallized pork glueagon (NOVO) was used as a standard. Pork glueagon was iodinated using a modification of the Hunter and Greenwood procedure (1962). The modification and purification was developed by Jorgensen and Larsen (1972 ). I R I was determined according to Heding (1972) and glucose was determined using a glucose oxidase method. The oral glucose loads were performed at the Hvidcre Diabetes Hospital in the following manner: after an overnight fast, a catheter was placed in the antecubital veins of the trial subjects and they ingested a cold solution of glucose containing 1.75 g of glucose per kg of ideal body weight. The glucose rng % 400 blood was collected into heparinized glass tubes containing 1 ml of trasylol (5000 KIE/ml, Bayer) per 10 ml of blood. The plasma was pipetted into plastic tubes and stored at --18~ until used. The diabetic patients were all newly diagnosed and untreated. The 25 diabetics were divided into two groups as described by l~asmussen et al. {1969): group II, juvenile-type diabetics, had I R I values below 30 ~zU/inl before and during the test, and group I, maturity-onset-type diabetics, comprised all the remaining diabetic subjects. Four of the eight juvenile-type diabetics had ketosis while none of the 17 maturity-onset diabetics showed ketosis during the two weeks of observation. Table 1 shows the age and sex of normals and diabetics as well as the excretion of urine glucose during the OGTT. Results The levels of pancreatic GLI reported here are higher than those obtained by Unger's group. The reason for this difference is unknown, but it might be methodological, or due to the differences in the antibodies used. For instance, the antibody K 47 used in this study does react with fragments of pancreatic glucagon. On the other hand, the results agree reasonably well with the findings of M/iller et al. (1970), who showed t h a t a dietary carbohydrate load of approximately 200 g resulted in an approximately 0.04 ng/ml decrease in the pancreatic GLI level in the normals whereas no decrease was observed in the juvenile and maturity-onset diabetics. I n the diabetic group -- all newly diagnosed, yet untreated cases -- there was a clear tendency to higher plasma pancreatic G L I levels t h a n in the normal subjects, despite their hyperglycaemia. This lends further support to the theory put forward by Unger to the effect t h a t the function of the ~-cell is augmented in Diabetes mellitus. But one should bear in mind t h a t the specificity as claimed for the various anti-glucagon sera stands only for their not reacting with a crude gut extract. The term specificity m a y lead to the false assumption t h a t the antibodies react only with the intact glueagon molecule. This is very unlikely considering the fact t h a t different antibodies react in a practically identical manner both with glucagon and with deshistidine glucagon (no biological activity) (Sundby, 1970) and mono-desamidoglucagon (about 60% biological activity) (tteding, 1972). L.G. t I e d i n g and S.M. Rasmussen: Determination of Pancreatic and Gut G L I Diabetologia E l e v a t e d g u t G L I levels were f o u n d a f t e r an oral glucose l o a d in all t h e t r i a l s u b j e c t s , b o t h d i a b e t i c a n d n o r m a l . T h r e e j u v e n i l e - t y p e d i a b e t i c s showed an exc e p t i o n a l l y m a r k e d increase. The significance of g u t G L I in b l o o d r e m a i n s obscure. Crude g u t e x t r a c t s c o n t a i n i n g less t h a n 1~o of G L I h a v e been s h o w n to h a v e insulin-releasing effect in t h e i s o l a t e d r a t islet s y s t e m , b u t no c o r r e l a t i o n was f o u n d b e t w e e n t h e G L I a n d t h e insulin-releasing a c t i v i t y (Moody et al., 1970). Marco et al. (1971) s h o w e d t h a t an e n h a n c e d secretion of endogenous g u t tion for the S t u d y of Diabetes, Louvain, Belgium, 1968. Heding, L. G. : RadioimmunologieM determination of panereatie and gut glucagon in plasma. Diabetologia 7, 10--19 (1971). Heding, L.G. : Irmmmological properties of pancreatic glueagon: Antigenicity and a n t i b o d y characteristics. Monograph on Glueagon. Ed. R. Unger and P. Lefebvre. Pergamon Press (1972). Heding, L. G. : Determination of t o t a l serum insulin (IRI) in insulin-treated diabetic patients. Diabetologia 8, 260--266 (1972). Hunter, W.M., Greenwood, F.C. : P r e p a r a t i o n of iodine8 Diabetics I I (all I R I values < 30 ~zU/ml) basal level ( - - 5 rain) increase after glucose sign. of increase (pairwise t-test) et al., 1970). T h e possible insulin-releasing or glycogenolygic a c t i v i t y of t h i s s u b s t a n c e c a n n o t be s t u d i e d p r o p e r l y u n t i l a p u r e g u t G L I p r e p a r a t i o n has b e e n made. Aclcnowledgements. I wish to t h a n k Mrs. Ulla Dahl Larsen for preparing the 12aI-glucagon, Mr. Erling P a r b s t for performing insulin determinations, Mrs. Majken P e t r i Petersen and Mrs. Connie Eriksen for excellent technical assistance. L.G. Heding and S.M. l~asmussen: Determination of Pancreatic and Gut GLI Aguilar-Parada , E. , Eisentraut , A.M. , Unger , I%.I-I. : Pancreatic glucagon secretion in normal and diabetic subjects . Amer. J. med. Sei . 257 , 415 -- 419 ( 1969a ). Aguilar-Parada , E. , Eisentraut , A. M. , Unger, i%.H. : Effects of starvation on plasma pancreatic glucagon in normal man . Diabetes 18 , 717 -- 723 ( 1969b ). Hedino' , L.G. : Glucagon antibodies for the radioimmunoassay of pancreatic and gut glucagon. Paper presented at the fourth annual meeting of the European Assoeia131 labelled h u m a n growth hormone of high specific activity . Nature 194 , 495 -- 496 ( 1962 ). J~rgensen , K . H . , Larsen , U . D . : Purification of ~25I-glucagon in anion exchange chromatography . Norm. Metab. Res . 4 , 223 -- 224 ( 1972 ). Marco , J. , Faloona , G.R. , Unger , R . H . : Effect of endogenous intestinal glucagon-like i m m u n o r e a c t i v i t y (GLI) on insulin secretion and glucose concentration in dogs . J. clin. Endocr . 33 , 318 -- 375 ( 1971 ). Miiller , W.A. , Faloona , G.R. , Aguilar-Parada , E. , Unger , R. H. : Abnormal alpha-cell function in diabetes . New Engl. J. Med . 283 , 109 -- 115 ( 1970 ). Miiller , W.A. , Faloona , G.R. , Linger , R.I-I. : The effect of experimental insulin deficiency on glucagon secretion . J. clin. Invest . 50 , 1992 -- 1999 ( 1971 ). Persson , I. , Gyntelberg , F. , Heding , L.G. , Boss-Nielsen , J. : Pancreatic glucagon-like immunoreactivity after intravenous insulin in normals and chronic-pancreatitis patients . Acta endocrin . 67 , 401 -- 404 ( 1971 ). Rasmussen , S. Munkgaard , Heding, L.G. , Parbst , E. , Volund , An. : Glucose and insulin responses to OGTT in newly detected diabetics and their subsequent treatment . Ahs. in Diabetologia 5 , 428 -- 429 ( 1969 ). P a p e r presented at the fifth annual meeting el the Scandinavian Society for the Study of Diabetes , Copenhagen 1969 . Sundby , F. : Abs . Seventh Congress of the InternationaI Diabetes Federation , Buenos Aires, Exerpta Medica No. 2 @ 9 , Abstract No . 179 . Unger , l~.H., Aguilar-Parada , E. , Miiller , W.A. , Eisentraut , A.M. : Studies of pancreatic alpha-cell function in normal and diabetic subjects . J. elin. Invest . 49 , 837 -- 848 ( 1970 ). Valverde , J. , Rigopoulou , D. , Marco , J. , Faloona , G.l~., Unger , R.H. : Characterization oi' glueagon-like immunoreaetivity (GLI) . Diabetes 19 , 614 -- 623 ( 1970 ). 215 Nordre Fasanvej DK-2200 Copenhagen N


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L. G. Heding, S. Munkgaard Rasmussen. Determination of pancreatic and gut Glucagon-Like Immunoreactivity (GLI) in normal and diabetic subjects, Diabetologia, 1972, 408-411, DOI: 10.1007/BF01212168