Upsala J Med Sci 87: 127-134, 1982 Release of Insulin and Pancreatic Somatostatin in Response to Increased Circulating Growth Hormone (GH) Levels Sven Gustavsson' and Gudmar Lundqvist' Departments of Surgery' and Clinical Chemistry2, University Hospital, Uppsala, Sweden ABSTRACT In an experimental study on 5 anaesthetized pigs the effect of increased circulating levels of growth hormone (GH) on the release of insulin and somato- statin from the pancreas was investigated. Blood was sampled simultaneously from the pancreatic venous effluent and from mixed venous blood. As measured by radioimmunoassay infusion of GH (20 pg x kg-I x h-') resulted in a significant (p < 0.01 ) increase both in insulin and somatostatin concentration in pancreatic venous blood. In mixed venous blood no significant changes in hormone levels were found. The results illustrate one possible mechanism for the close relation between circulating GH and endocrine pancreas. INTRODUCTION Considerable evidence is now available showing that growth hormone (GH) is capable of inhibiting its own secretion (12, 15, 8). involves a "short feed-back loop" operating on the hypothalamus /14/ is not finally proven. Indirect evidences have, however, been obtained for such a hypothesis, where hypothalamic somatostatin seems to contribute to the GH release regulation (1 2). excess and hypothalamic somatostatin concentration in rats ( 1 3 ) , which might indicate an increase in somatostatin secretion during GH treatment. Whether this mechanism A positive correlation is also demonstrated between GH So far no physiological significance has been attributed neither to the well-known extrapituitary actions of somatostatin, n o r to the localization of large amounts of somatostatin in the pancreatic islets and in the gastric mucosa. However, numerous reports on somatostatin as inhibitor of gastro- intestinal (GI) hormone release points to a specific function for somatostatin as a modulator of GI hormone response, especially within the pancreatic islets but also in the stomach (7, 3 ) . The effect of GH o n pancreatic hormonal release is controversial. Thus, GH does not appear to have any immediate effect on insulin secretion of the &cell 127 i n t h e i n v i t r o s t u d y of M a l a i s s e e t a l . ( l o ) , b u t t h e r e s u l t s of T a i & Pek ( 1 6 ) i n d i c a t e t h a t G H may h a v e a t o n i c e f f e c t b o t h on i n s u l i n and g l u c a g o n r e l e a s e . C o n s t a n t l y e l e v a t e d l e v e l s of G H i n t u m o u r - b e a r i n g rats r e s u l t e d i n an i n c r e a s e i n t h e f a s t i n g i n s u l i n l e v e l s a s w e l l a s i n t h e e x t r a c t a b l e p a n c r e a t i c i n s u l i n ( 1 1 ) . i n h i b i t o r y e f f e c t on i n s u l i n r e l e a s e ( 1 ) . m i g h t i n d u c e a change i n p a n c r e a t i c s o m a t o s t a t i n s e c r e t i o n o r n o t h a s n o t been s t u d i e d p r e v i o u s l y . However, j u d g i n g from c l i n i c a l s t u d i e s a d m i n i s t r a t i o n of G H h a s an Whether c i r c u l a t i n g l e v e l s o f GH In t h e p r e s e n t p a p e r we a r e e v a l u a t i n g t h e h y p o t h e s i s t h a t p a n c r e a t i c somato- s t a t i n r e l e a s e i s a f f e c t e d by t h e c i r c u l a t i n g l e v e l s o f GH i n t h e same way a s h y p o t h a l a m i c s o m a t o s t a t i n i s supposed t o be r e g u l a t e d . F o r t h e s e s t u d i e s we h a v e u s e d a n e x p e r i m e n t a l model which p e r m i t s s e l e c t i v e blood s a m p l i n g from t h e p a n c r e a t i c v e i n s d u r i n g and a f t e r GH i n f u s i o n . MATERIAL AND METHODS Animals F i v e p i g s of Swedish l a n d - r a c e weighing 20-28 kg were used. A n a e s t h e s i a A n a e s t h e s i a was i n d u c e d by a n i n t r a m u s c u l a r i n j e c t i o n of k e t a m i n e ( K e t a l a g ) 250-500 mg and m a i n t a i n e d by r e p e a t e d i n t r a v e n o u s i n j e c t i o n s of p h e n o p e r i d i n ( L e a l g i n R , 4 mg e v e r y 30-45 min) and pancuron bromide ( P a v u l o n R , 4 mg e v e r y 30-45 min). A f t e r e n d o - t r a c h e a l i n t u b a t i o n p o s i t i v e p r e s s u r e v e n t i l a t i o n was g i v e n w i t h a m i x t u r e of N20 and O2 ( 4 : l ) by means of a r e s p i r a t o r . A r t e r i a l blood p r e s s u r e was m o n i t o r e d t h r o u g h a c a t h e t e r ( i n f a n t f e e d i n g t u b e No 5 ) i n t r o d u c e d i n t o t h e common c a r o t i d a r t e r y of t h e l e f t s i d e . An i n f a n t f e e d i n g t u b e No 8 was i n s e r t e d i n t o t h e r i g h t atrium v i a t h e i n t e r n a l j u g u l a r v e i n o f t h e l e f t s i d e . T h i s c a t h e t e r was u s e d f o r measurement of t h e c e n t r a l venous p r e s s u r e and f o r blood s a m p l i n g (mixed venous b l o o d ) . S u r g i c a l p r o c e d u r e An u p p e r l a p a r o t o m y was performed u s i n g a m i d l i n e i n c i s i o n . A c a t h e t e r ( i n f a n t f e e d i n g t u b e No 5 ) was i n t r o d u e d i n t o t h e s u p e r i o r p a n c r e a t i c o - d u o d e n a l v e i n i m m e d i a t e l y b e f o r e i t s e n t r a n c e i n t o t h e p o r t a l v e i n . T h i s c a t h e t e r was u s e d f o r blood s a m p l i n g from t h e p a n c r e a t i c venous e f f l u e n t . The c a t h e t e r was e x t e r i o r i z e d t h r o u g h a s t a b wound i n t h e abdominal w a l l and t h e laparotomy wound was c l o s e d . E x p e r i m e n t a l p r o t o c o l A f t e r a n i n i t i a l c o n t r o l p e r i o d of s a l i n e i n f u s i o n v i a an e a r v e i n and blood s a m p l i n g from b o t h c a t h e t e r s ( - 4 5 , -30, -15 and 0 ) human growth hormone 128 (Crescormone, KABI AB, Sweden) was infused for 30 min via an ear vein in the dose 20 pgxkg-lxh-l. to the results of Adamsson and Efendic ( 1 ) to achieve an increase in circulating GH simulating the highest levels seen during a 24 h period in man and well within the range of circulating GH in acromegalic patients. Blood was then sampled at 15, 30, 45, 60, 75 and 90 min after the beginning of the growth hormone administration. In 2 of the pigs a repeated infusion of growth hormone was given in the same dose starting 90 min after the beginning of the first infusion (Fig. 1 ). protocol. The dose of administered GH was chosen according Blood was sampled according to an identic experimental In different experiments the drip rate of blood from the catheter in the superior pancreatico-duodenal vein varied between 20-60/min. experiment the constancy of the drip rate (+ 10 $ ) was taken as evidence that the venous blood flow did not change. Within each Blood samples (5 ml each) were collected in chilled tubes with the addition After centrifugation at of Heparin (143 USP-units) and Trasylol (400 KIE/ml). -4°C plasma was decanted and frozen at -20°C until radioimmunoassay. Radioimmunoassae Before radioimmunoassay of somatostatin plasma samples were extracted with acetone-petroleum-ether as suggested by Arimura et al. (2). The radioimmuno- assay was performed with a solid phase technique (9) with somatostatin anti- bodies coupled to microcrystalline cellulose. The antiserum used, R 141, is well characterized with respect to reactivity against different parts of the somatostatin molecule as well as to the lack of reactivity against other GI peptides (2). Tyr-1-somatostatin (Beckman, Geneva) was used for iodination with the lactoperoxidase method and synthetic somatostatin (Beckman, Geneva) was used for preparation of standards. Insulin and GH were determined with solid phase radioimmunoassays (Phadebas, Pharmacia, Sweden). Statistical analysis The degree of significance for the difference between hormone levels before and after the beginning of the GH infusion was tested by analysis of variance. RESULTS The GH infusion in the selected dose resulted in a 10-fold increase in GH levels as measured in one of the pigs (Fig. 1). The insulin concentration in pancreatic venous blood was significantly (p < 0.01 ) increased after the beginning of the GH infusion (Table 1 ) . There was also noted a tendency to an increase in insulin in mixed venous blood although this increase did not reach 9-822x57 129 T a b l e 1 L e v e l s o f immunoreac b e f o r e and a f t e r t h e A n a l y s i s o f v a r i a n c e t i v e i n s u l i n (mU/l) i n p a n c r e a t i c venous blood b e g i n n i n g of t h e G H i n f u s i o n . Mean + SEM. r e v e a l e d F = 10.08, p = 0.004. Animal No. B e f o r e G H i n f u s i o n A f t e r G H i n f u s i o n 10.7 + 2.7 23.6 + 6.0 121 + 28.0 7.8 + 1.8 9.3 + 1.8 20.5 + 8.6 114 + 13.9 741 + 188 21 + 15 18.0 + 2.9 T a b l e 2 L e v e l s of immunoreactive i n s u l i n (mU/l) i n mixed venous blood b e f o r e and a f t e r t h e b e g i n n i n g of t h e G H i n f u s i o n . Mean + SEM. A n a l y s i s of v a r i a n c e r e v e a l e d F = 3.86, p = 0.058. Animal No. B e f o r e G H i n f u s i o n A f t e r G H i n f u s i o n 1 9.6 + 0.7 2 3.7 + 0.6 3 4.2 + 1.9 4 7.0 + 0.5 5 7.8 + 1.3 32.4 + 21 6.2 + 0.5 11.9 + 1.3 9.8 + 0.6 19.4 + 3.0 T a b l e 3 L e v e l s o f immunoreactive s o m a t o s t a t i n (pg/ml) i n p a n c r e a t i c venous blood b e f o r e and a f t e r t h e b e g i n n i n g of t h e G H i n f u s i o n . A n a l y s i s of v a r i a n c e r e v e a l e d F = 10.21, p = 0.007. Mean + SEM. Animal No. B e f o r e GH i n f u s i o n A f t e r G H i n f u s i o n 799 + 47 458 + 44 391 + 56 331 + 106 429 + 56 1755 + 265 500 + 62 487 + 72 628 + 55 537 + 87 I30 T a b l e 4 INSUUN nw' 150- KO- 50- 0 - L e v e l s o f immunoreactive s o m a t o s t a t i n (pg/ml) i n mixed venous blood b e f o r e and a f t e r t h e b e g i n n i n g of t h e GH i n f u s i o n . Mean + SEM. A n a l y s i s o f v a r i a n c e r e v e a l e d F = 0.73, p = 0.400. . . . . . . . . . . 'T \ ', ** Y' +-+-. *--+ c-+ Animal No. B e f o r e G H i n f u s i o n A f t e r G H i n f u s i o n 567 + 103 284 + 64 275 + 31 244 + 18 232 L 29 611 + 161 240 + 37 295 + 48 250 + 1 1 329 L 23 F i g . 1 . A r e p r e s e n t a t i v e example of hormone l e v e l s i n one i g b e f o r e , d u r i n g and a f t e r GH i n f u s i o n i n t h e d o s e 20 pgxkg-'xh-l ( h a t c h e d a r e a ) 131 s t a t i s t i c a l s i g n i f i c a n c e ( T a b l e 2 ) . measured i n t h e p a n c r e a t i c venous e f f l u e n t i n c r e a s e d s i g n i f i c a n t l y ( p < 0.01). I n mixed venous blood no s i g n i f i c a n t change was observed i n t h e s o m a t o s t a t i n c o n c e n t r a t i o n ( T a b l e 4). venous blood o c c u r r e d s i m u l t a n e o u s l y . In 2 a n i m a l s a r e p e a t e d GH i n f u s i o n was a d m i n i s t e r e d , i n b o t h c a s e s f o l l o w e d by a r e p e a t e d i n c r e m e n t i n t h e i n s u l i n and s o m a t o s t a t i n l e v e l i n p a n c r e a t i c venous blood ( F i g . 1 ) - A c c o r d i n g t o T a b l e 3 t h e s o m a t o s t a t i n l e v e l The i n c r e a s e i n i n s u l i n and s o m a t o s t a t i n i n p a n c r e a t i c DISCUSSION The i n c r e a s e i n i n s u l i n and s o m a t o s t a t i n found i n t h e p r e s e n t e x p e r i m e n t s as a r e s p o n s e t o GH i n f u s i o n was s t a t i s t i c a l l y s i g n i f i c a n t i n p a n c r e a t i c venous b l o o d b u t n o t i n mixed venous blood. Thus t h e p r e s e n t r e s u l t s i l l u s t r a t e t h e b e n e f i c i a l v a l u e o f blood s a m p l i n g a l s o from p a n c r e a t i c v e i n s i n s t u d i e s of hormonal i n t e r p l a y , a s a l s o p o i n t e d o u t b e f o r e ( 5 , 6 ) . is e s p e c i a l l y n e c e s s a r y i n s t u d i e s of p o l y p e p t i d e s w i t h a r a p i d metabolism o r e l i m i n a t i o n , l i k e s o m a t o s t a t i n . T h i s e x p e r i m e n t a l d e s i g n The u s e o f human GH m i g h t be e r r o n e o u s due t o t h e well-known s p e c i e s d i f f e r - e n c e s known f o r G H ( 4 ) . n o t e d between human and p o r c i n e GH (18) which j u s t i f i e s t h e p r e s e n t e x p e r i m e n t a l p e r f o r m a n c e . The p o s s i b i l i t i e s of d i r e c t measurement of t h e G H l e v e l s reached by G H i n f u s i o n is a n o t h e r r e a s o n f o r t h e u s e of human G H i n t h e s e e x p e r i m e n t s . The mechanism whereby G H a f f e c t s i s l e t f u n c t i o n i s n o t known and t h e e f f e c t of GH o n i n s u l i n s e c r e t i o n i s c o n t r o v e r s i a l . The p r e s e n t r e s u l t s w i t h an i n s u l i n r e s p o n s e d e t e c t e d i n p a n c r e a t i c venous b l o o d a f t e r GH i n f u s i o n a r e i n a c c o r d a n c e w i t h r e p o r t s of a s t i m u l a n t e f f e c t of G H o n i n s u l i n s e c r e t i o n ( 1 1 , 19). On t h e o t h e r hand a small b u t s i g n i f i c a n t d e c r e a s e of i n s u l i n r e l e a s e h a s a l s o been r e p o r t e d a f t e r GH i n f u s i o n i n man ( 1 ) . v a r i o u s d o s e s o f GH is u n c l e a r . However, m o l e c u l a r and immunological s i m i l a r i t i e s a r e Whether t h i s d i s c r e p a n c y is due t o I n t h e p r e s e n t s t u d y t h e l e v e l of immunoreactive s o m a t o s t a t i n i n t h e pan- c r e a t i c venous e f f l u e n t i n c r e a s e d s i g n i f i c a n t l y d u r i n g i n f u s i o n o f GH. T h i s i n c r e a s e was e v i d e n t i n a l l a n i m a l s b u t w i t h c o n s i d e r a b l e i n t e r - i n d i v i d u a l v a r i a t i o n i n m a g n i t u d e , which j u s t i f i e s t h e p r e s e n t s t a t i s t i c a l e v a l u a t i o n . Although l i t t l e i s known a b o u t t h e r o l e of s o m a t o s t a t i n - p r o d u c i n g c e l l s w i t h i n t h e p a n c r e a t i c i s l e t s , it seems t o be r e a s o n a b l e t o s u g g e s t t h a t t h e p a n c r e a t i c D c e l l s r e a c t i n t h e same manner as h y p o t h a l a m i c s o m a t o s t a t i n c e l l s upon b l o o d GH a l t e r a t i o n s . The unchanged s o m a t o s t a t i n l e v e l i n mixed venous b l o o d , found by u s , do n o t s u p p o r t t h e h y p o t h e s i s t h a t p a n c r e a t i c s o m a t o s t a t i n d u r i n g p h y s i o l o g i c a l c o n d i t i o n s p a r t i c i p a t e s i n a feed-back c o n t r o l o f GH s e c r e - t i o n from t h e p i t u i t a r y g l a n d . I t i s d i f f i c u l t t o f u r t h e r s p e c u l a t e on t h e complex i n t e r p l a y between GH o n one hand and i n s u l i n and p a n c r e a t i c s o m a t o s t a t i n on t h e o t h e r . 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