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Studies on the in vitro effects of insulin on exocrine pancreatic function Sinanan, Mika Narad 1991

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S t u d i e s on t h e In Vitro E f f e c t s o f I n s u l i n on E x o c r i n e P a n c r e a t i c F u n c t i o n MIKA NARAD SINANAN B.A., Johns Hopkins U n i v e r s i t y , 1977 M.D., Johns Hopkins U n i v e r s i t y , 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY THE FACULTY OF GRADUATE STUDIES (Department o f P h y s i o l o g y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J a n u a r y 1991 ' \PJ M i k a Narad S i n a n a n , 1991 i n In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date DE-6 (2/88) i i A b s t r a c t E n d o c r i n e and e x o c r i n e components of t h e p a n c r e a s c o e x i s t a n a t o m i c a l l y b u t have been c o n s i d e r e d t o f u n c t i o n i n d e p e n d e n t l y . A v a s c u l a r p o r t a l system l e a d i n g from i s l e t s t o t h e s u r r o u n d i n g a c i n a r t i s s u e o f t h e p a n c r e a s has been d e s c r i b e d b u t t h e p h y s i o l o g i c a l s i g n i f i c a n c e o f t h i s " i n s u l o a c i n a r " p o r t a l system i s u n c l e a r . The u n d e r l y i n g h y p o t h e s i s o f t h i s work was t h a t i s l e t hormones, p a r t i c u l a r l y i n s u l i n , a r e c a r r i e d i n h i g h c o n c e n t r a t i o n t o s u r r o u n d i n g e x o c r i n e a c i n a r t i s s u e and modulate p a n c r e a t i c s e c r e t i o n . The s e c r e t a g o g u e dependency, magnitude, t i m e c o u r s e , and n a t u r e o f i n s u l i n e f f e c t s on t h e e x o c r i n e p a n c r e a s were i n v e s t i g a t e d i n two in v i t r o model systems, an i s o l a t e d , p e r f u s e d r a t p a n c r e a s , and a p r e p a r a t i o n o f d i s p e r s e d r a t p a n c r e a t i c a c i n i . The e x o c r i n e i n t e g r i t y o f t h e model systems was i n v e s t i g a t e d and v e r i f i e d i n b o t h systems f o r CCK, s e c r e t i n , and V I P , and i n a c i n i , a d d i t i o n a l l y , f o r m e t h a c h o l i n e dose r e s p o n s i v e e f f e c t s . I n b o t h s y s t e m s , CCK p r o v e d t o t h e most p o t e n t s e c r e t o r y s t i m u l u s f o r p a n c r e a t i c e x o c r i n e s e c r e t i o n though maximal s t i m u l a t i o n dosages o f s e c r e t i n and VIP r e s u l t e d i n g r e a t e r e x o c r i n e s e c r e t i o n t h a n CCK a t any dose. Normal a d u l t a n i m a l s , a n i m a l s o f d i f f e r e n t ages, and a n i m a l s t r e a t e d t o i n c r e a s e ( h y p e r i n s u l i n i s m ) o r d e c r e a s e ( e x p e r i m e n t a l d i a b e t e s ) c i r c u l a t i n g i n s u l i n l e v e l s were t e s t e d under a v a r i e t y o f a c u t e i n s u l i n and s e c r e t a g o g u e c o n d i t i o n s f o r p a rameters d e t e r m i n i n g t h e r e s p o n s i v e n e s s t o i n s u l i n . i i i Endogenous and exogenous i n s u l i n c l e a r l y p o t e n t i a t e d s t i m u l a t e d s e c r e t i o n i n t h e i s o l a t e d p e r f u s e d p a n c r e a s though t h e p o t e n t i a t i n g e f f e c t was e v i d e n t w i t h some s e c r e t a g o g u e s ( s e c r e t i n and CCK) b u t n o t o t h e r s ( V I P ) . I n c o n t r a s t , i n s u l i n e f f e c t s i n i s o l a t e d p a n c r e a t i c a c i n i were much l e s s r e p r o d u c i b l e . Of g r e a t e s t s i g n i f i c a n c e was t h e l o s s i n a b s o l u t e amylase c e l l c o n t e n t a f t e r development o f d i a b e t e s w i t h r e c o v e r y towards normal amylase c o n t e n t and r e l e a s e a f t e r i n s u l i n t r e a t m e n t . T h i s o b s e r v a t i o n , however, was i n r e s p o n s e t o c h r o n i c i n s u l i n t r e a t m e n t o v e r s e v e r a l d a y s . I n t h e s e t t i n g o f more a c u t e i n s u l i n t r e a t m e n t , h i g h dosages o f i n s u l i n seemed t o a c t u a l l y i n h i b i t C C K - s t i m u l a t e d s e c r e t i o n w h i l e i n some b u t n o t a l l s t u d i e s , a c i n i from d i a b e t i c a n i m a l s g a i n e d i n c r e a s e d s e n s i t i v i t y t o i n s u l i n . C o n d i t i o n s o f i n c r e a s e d c i r c u l a t i n g i n s u l i n n e g ated any a c u t e r e s p o n s e t o i n s u l i n . A c i n i from younger, more i n s u l i n s e n s i t i v e a n i m a l s , a c i n i t r e a t e d ^o d e p l e t e zymogen and i n d u c e p r o t e i n s y n t h e s i s , and a c i n i t r e a t e d w i t h m i c r o t u b u l e o r p r o t e i n s y n t h e s i s i n h i b i t o r s a l l f a i l e d t o show any a c u t e p o t e n t i a t i o n o f s t i m u l a t e d s e c r e t i o n t o i n s u l i n . However, a c i n i h a r v e s t e d s e p a r a t e l y from v e n t r a l and d o r s a l p a n c r e a s , d e v e l o p e d under t h e i n f l u e n c e o f i s l e t s h a v i n g d i f f e r e n t c o m p o s i t i o n s , d i d d i f f e r i n s e c r e t o r y r e s p o n s i v e n e s s . A l t h o u g h t h e y seemed o n l y a s m a l l c o n t r i b u t i n g f a c t o r t o t h e v a r i a b i l i t y i n i n s u l i n r e s p o n s i v e n e s s o f a c i n i , i s l e t f r agments c o n t a m i n a t i n g t h e p u r i f i e d a c i n i p r e p a r a t i o n were a s s e s s e d and appeared t o be i v n o n f u n c t i o n a l , r e l e a s i n g low c o n c e n t r a t i o n s o f i n s u l i n o n l y . Immunocytochemical s t u d i e s p r o v i d e d a ^ g r a p h i c c o r r e l a t i o n t o t h e e n d o c r i n e and e x o c r i n e changes i n d u c e s by t h e v a r i o u s a n i m a l t r e a t m e n t s and a p i l o t a u t o r a d i o g r a p h i c s t u d y o f 1 2 5 I - i n s u l i n b i n d i n g t o a c i n i i n t h e p e r f u s e d p a n c r e a s s u g g e s t e d t h a t i n s u l i n from i s l e t s w i t h i n t h e p a n c r e a s d i d r e a c h a t l e a s t t h e f i r s t f o u r a c i n i deep around t h e i s l e t . These d a t a s u g g e s t t h a t i n s u l i n does r e a c h s u r r o u n d i n g e x o c r i n e t i s s u e i n h i g h c o n c e n t r a t i o n and i s c a p a b l e of a c u t e l y m o d u l a t i n g e x o c r i n e f u n c t i o n i n t h e i n t a c t g l a n d . A t t h e a c i n a r l e v e l , e i t h e r t h i s r e s p o n s e i s i n p a r t i n d i r e c t , o r t e c h n i c a l f a c t o r s i n t h e p r e p a r a t i o n o f a c i n i v a r i a b l y reduce r e s p o n s i v e n e s s . I n s u l i n i n most c a s e s p o t e n t i a t e d s e c r e t i o n i n s t i m u l a t e d normal and d i a b e t i c a c i n i b u t a t v e r y h i g h dosages d u r i n g a p r o l o n g e d p r e i n c u b a t i o n , became i n h i b i t o r y . I t would appear t h a t a n i m a l age, s e c r e t a g o g u e dosage, c a l c i u m c o n c e n t r a t i o n , and a s t a t e o f zymogen d e p l e t i o n were n o t i m p o r t a n t p arameters i n d e t e r m i n i n g a c i n a r r e s p o n s i v e n e s s t o i n s u l i n . TABLE OF CONTENTS A b s t r a c t i i T a b l e o f C o n t e n t s v T a b l e s x i F i g u r e s x i i I n t r o d u c t i o n 1 Statement of t h e Q u e s t i o n 1 H i s t o r i c a l Background 1 Embryology and Anatomy of t h e P a n c r e a s 3 I n s u l o a c i n a r " P o r t a l " System 6 P h y s i o l o g i c a l F u n c t i o n s o f t h e Pancreas 9 E x o c r i n e F u n c t i o n 9 E n d o c r i n e F u n c t i o n 13 R e g u l a t i o n o f E x o c r i n e P a n c r e a t i c F u n c t i o n 16 R e g u l a t i o n o f I n s u l i n S e c r e t i o n 19 E x t r a p a n c r e a t i c E f f e c t s o f I n s u l i n 22 I n s u l o a c i n a r A x i s 24 S p e c i f i c Aims 27 Methods 28 E x p e r i m e n t a l P r e p a r a t i o n s 28 I s o l a t e d P e r f u s e d P a n c r e a s 28 A n i m a l s 28 A p p a r a t u s 2 8 P e r f u s i o n s o l u t i o n s 29 P e r f u s i o n b u f f e r c o n c e n t r a t e ( S t o c k ) 29 G l u c o s e c o n c e n t r a t e ( S t o c k ) 30 P e r f u s a t e c o m p o s i t i o n ( f o r m u l a t i o n ) 30 H e p a r i n 31 v i I s o l a t i o n o f p a n c r e a s 31 P e r f u s i o n p r o c e d u r e 3 3 S a m p l e c o l l e c t i o n 34 P a n c r e a t i c A c i n i P r e p a r a t i o n 34 B u f f e r p r e p a r a t i o n 3 5 R e a g e n t s a n d e q u i p m e n t 3 5 A c i n i i s o l a t i o n b u f f e r 3 5 I n c u b a t i o n b u f f e r 36 I s o l a t i o n p r o c e d u r e 3 7 I n c u b a t i o n p r o c e d u r e 3 9 S a m p l e c o l l e c t i o n 4 0 A n i m a l T r e a t m e n t s 4 1 E x p e r i m e n t a l s t r e p t o z o t o c i n d i a b e t e s 4 1 E x o g e n o u s i n s u l i n 4 2 C h l o r p r o p a m i d e 4 3 G l u c o s e T o l e r a n c e T e s t s 4 3 A s s a y s 4 3 H o r m o n e A s s a y s 4 4 I n s u l i n 4 4 R e a g e n t s o u r c e s 4 4 A s s a y b u f f e r 4 4 H o r m o n e - f r e e p l a s m a 4 4 A n t i b o d y 44 1 2 5 I - I n s u l i n 4 5 S a m p l e p r e p a r a t i o n 4 6 S t a n d a r d s a n d C o n t r o l s 4 7 C h a r c o a l p r e p a r a t i o n 4 7 v i i Technique o f a s s a y 48 Data c a l c u l a t i o n 48 S e n s i t i v i t y and s p e c i f i c i t y 49 S o m a t o s t a t i n 50 Reagent s o u r c e s 50 Assay b u f f e r 50 A n t i b o d y 50 C h a r c o a l s o l u t i o n 50 1 2 5 I - S o m a t o s t a t i n 51 S t a n d a r d s 52 Sample p r e p a r a t i o n and s t o r a g e 52 Assay P r o c e d u r e s 5 3 S e n s i t i v i t y and s p e c i f i c i t y 53 E x o c r i n e A s s a y s 54 Amylase 54 S t a r c h - I o d i n e 54 P r o c i o n y e l l o w - s t a r c h 55 L i p a s e 56 T o t a l p r o t e i n 58 G l u c o s e measurement 59 O s m o l a r i t y 59 T i s s u e S t a i n i n g and Immunocytochemistry 59 A u t o r a d i o g r a p h y ( I n s u l i n R e c e p t o r D i s t r i b u t i o n ) 61 R e a g e n t s / P e p t i d e s 63 S t a t i s t i c a l A n a l y s i s 63 R e s u l t s 2 87 S t u d i e s i n t h e I s o l a t e d P e r f u s e d P ancreas 287 E x o c r i n e Response t o S e c r e t a g o g u e s 287 P a n c r e a s J u i c e Volume Response 287 Amylase S e c r e t i o n i n P a n c r e a t i c J u i c e 288 T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e 289 E n d o c r i n e Response t o E x o c r i n e S e c r e t a g o g u e s 290 I n s u l i n R e l e a s e i n P e r f u s a t e 290 S o m a t o s t a t i n R e l e a s e i n P e r f u s a t e 291 E f f e c t s o f G l u c o s e and Exogenous I n s u l i n on S t i m u l a t e d E x o c r i n e S e c r e t i o n 292 C C K - S t i m u l a t e d E x o c r i n e S e c r e t i o n 292 S e c r e t i n - S t i m u l a t e d E x o c r i n e S e c r e t i o n 294 V I P - S t i m u l a t e d E x o c r i n e S e c r e t i o n 295 E f f e c t s o f A n o x i a on C C K - S t i m u l a t e d E x o c r i n e S e c r e t i o n 297 S t u d i e s i n P u r i f i e d , D i s p e r s e d P a n c r e a t i c A c i n i 299 E x o c r i n e Response t o S e c r e t a g o g u e s 299 S i g n i f i c a n c e o f I s l e t Fragments i n t h e D i s p e r s e d P a n c r e a t i c A c i n i P r e p a r a t i o n 300 E f f e c t o f I n s u l i n on C C K - S t i m u l a t e d A c i n a r S e c r e t i o n 304 E f f e c t o f I n s u l i n on S e c r e t i n - S t i m u l a t e d A c i n a r S e c r e t i o n 304 E f f e c t o f I n s u l i n on M e t h a c h o l i n e - S t i m u l a t e d A c i n a r S e c r e t i o n 305 E f f e c t o f I n s u l i n Dosage on B a s a l and S t i m u l a t e d S e c r e t i o n 305 E f f e c t o f A l t e r e d C i r c u l a t i n g I n s u l i n C o n c e n t r a t i o n s i n t h e Rat on Subsequent A c i n a r R e s p o n s i v e n e s s t o I n s u l i n in v i t r o 307 E f f e c t o f S t r e p t o z o t o c i n - D i a b e t e s and Subsequent Exogenous I n s u l i n Replacement on t h e A c i n a r Response t o I n s u l i n in v i t r o 309 i x E f f e c t o f I n s u l i n on A c i n i from Weanling R a t s 311 E f f e c t o f C a l c i u m C o n c e n t r a t i o n i n I n c u b a t i o n Medium on t h e A c i n a r Response t o I n s u l i n 312 E f f e c t s o f M i c r o t u b u l e ( c o l c h i c i n e ) and P r o t e i n -S y n t h e s i s ( c y c l o h e x i m i d e ) I n h i b i t o r s on t h e A c i n a r Response t o I n s u l i n 313 E f f e c t o f I n s u l i n on S t i m u l a t e d S e c r e t i o n o f A c i n i d e r i v e d from D o r s a l and V e n t r a l P a n c r e a s 313 H i s t o l o g i c and Immunocytochemical S t u d i e s 314 Normal A d u l t R a t Pa n c r e a s 314 S t r e p t o z o t o c i n - D i a b e t i c Rat Pancreas 316 I n s u l i n - T r e a t e d , D i a b e t i c Rat Pancreas 316 I n s u l i n - T r e a t e d , N o n d i a b e t i c Rat Pa n c r e a s 317 C h l o r p r o p a m i d e - T r e a t e d R a t Pa n c r e a s 318 A u t o r a d i o g r a p h i c S t u d i e s 318 D i s c u s s i o n 3 20 Model Systems 323 E x o c r i n e and e n d o c r i n e f u n c t i o n i n t h e i s o l a t e d , p e r f u s e d p a n c r e a s 324 P a n c r e a t i c a c i n i p r e p a r a t i o n 3 29 D i s t r i b u t i o n o f endogenous i n s u l i n t o a c i n a r t i s s u e : a u t o r a d i o g r a p h i c s t u d y 331 A c t i o n s o f i n s u l i n 333 A c u t e e f f e c t s o f i n s u l i n 33 3 F a c t o r s m o d u l a t i n g i n s u l i n r e s p o n s i v e n e s s o f t h e e x o c r i n e p a n c r e a s 3 39 E f f e c t o f a l t e r a t i o n s i n t h e r a t e o f p r o t e i n s y n t h e s i s 339 E f f e c t o f C a + 2 c o n c e n t r a t i o n i n t h e medium 342 E f f e c t o f s h o r t - t e r m d i a b e t e s 342 X E f f e c t o f a g e - r e l a t e d , i n s u l i n s e n s i t i v i t y 343 E f f e c t s o f a l t e r e d c i r c u l a t i n g i n s u l i n c o n c e n t r a t i o n s o n i n s u l i n r e s p o n s i v e n e s s 345 E f f e c t s o f i n s u l i n - t r e a t e d and u n t r e a t e d d i a b e t e s 349 B i b l i o g r a p h y 352 Appendix 368 TABLES T a b l e 1 308 Pan c r e a s Weights from A n i m a l s T r e a t e d t o A l t e r C i r c u l a t i n g I n s u l i n C o n c e n t r a t i o n s T a b l e 2 310 Pa n c r e a s Weights from C o n t r o l , D i a b e t i c , and I n s u l i n - t r e a t e d D i a b e t i c A n i m a l s a t t h e Time o f A c i n i P r e p a r a t i o n x i i FIGURES F i g u r e 1 P a n c r e a t i c J u i c e Volume Response t o I n c r e a s i n g Doses o f CCK-8 i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 65 F i g u r e 2 P a n c r e a t i c J u i c e Volume Response t o I n c r e a s i n g Doses o f S e c r e t i n i n t h e I s o l a t e d P e r f u s e d Pancreas 67 F i g u r e 3 P a n c r e a t i c J u i c e Volume Response t o I n c r e a s i n g Doses o f VIP i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 69 F i g u r e 4 Amylase S e c r e t i o n Due t o I n c r e a s i n g Doses o f CCK-8 i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 71 F i g u r e 5 Amylase S e c r e t i o n Due t o I n c r e a s i n g Doses o f S e c r e t i n i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 73 F i g u r e 6 Amylase S e c r e t i o n Due t o I n c r e a s i n g Doses o f VIP i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 75 F i g u r e 7 T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n Response t o I n c r e a s i n g Doses o f CCK-8 i n t h e I s o l a t e d P e r f u s e d Pancreas 77 F i g u r e 8 T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n Response t o I n c r e a s i n g Doses o f S e c r e t i n i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 79 F i g u r e 9 T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n Response t o I n c r e a s i n g Doses o f VIP i n th e I s o l a t e d P e r f u s e d Pancreas 81 x i i i F i g u r e 10 I n s u l i n R e l e a s e i n t o P e r f u s a t e ( e x p r e s s e d per ml o f p e r f u s a t e ) i n Response t o I n c r e a s i n g Doses o f CCK-8 i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 83 F i g u r e 11 I n s u l i n R e l e a s e i n t o P e r f u s a t e ( e x p r e s s e d p e r min) i n Response t o I n c r e a s i n g Doses o f CCK-8 i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 85 F i g u r e 12 I n s u l i n R e l e a s e i n t o P e r f u s a t e ( e x p r e s s e d p e r min) i n Response t o I n c r e a s i n g Doses of S e c r e t i n i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 87 F i g u r e 13 I n s u l i n R e l e a s e i n t o P e r f u s a t e ( e x p r e s s e d per min) i n Response t o I n c r e a s i n g Doses o f VIP i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 89 F i g u r e 14 S o m a t o s t a t i n R e l e a s e i n t o P e r f u s a t e ( e x p r e s s e d p e r ml o f p e r f u s a t e ) i n Response t o I n c r e a s i n g Doses o f CCK-8 i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 91 F i g u r e 15 S o m a t o s t a t i n R e l e a s e i n t o P e r f u s a t e ( e x p r e s s e d p e r min) i n Response t o I n c r e a s i n g Doses o f CCK-8 i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 93 F i g u r e 16 S o m a t o s t a t i n R e l e a s e i n t o P e r f u s a t e ( e x p r e s s e d p e r min) i n Response t o I n c r e a s i n g Doses o f S e c r e t i n i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 95 F i g u r e 17 S o m a t o s t a t i n R e l e a s e i n t o P e r f u s a t e ( e x p r e s s e d p e r min) i n Response t o I n c r e a s i n g Doses o f VIP i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 97 x i v F i g u r e 18 P a n c r e a t i c J u i c e Volume Response t o I n c r e a s e d G l u c o s e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 99 F i g u r e 19 E f f e c t o f I n c r e a s e d G l u c o s e on Amylase S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e CCK-S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 101 F i g u r e 20 E f f e c t o f I n c r e a s e d G l u c o s e on T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e CCK-S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 103 F i g u r e 21 E f f e c t o f I n c r e a s e d G l u c o s e on I n s u l i n R e l e a s e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 105 F i g u r e 22 E f f e c t o f I n c r e a s e d G l u c o s e on S o m a t o s t a t i n R e l e a s e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 107 F i g u r e 23 E f f e c t o f I n s u l i n on P a n c r e a t i c J u i c e Volume i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 109 F i g u r e 24 E f f e c t o f I n s u l i n on Amylase S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 111 F i g u r e 25 E f f e c t o f I n s u l i n on T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 113 F i g u r e 26 E f f e c t o f Added I n s u l i n on Measured I n s u l i n R e l e a s e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 115 XV F i g u r e 27 E f f e c t o f I n s u l i n on S o m a t o s t a t i n R e l e a s e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 117 F i g u r e 28 P a n c r e a t i c J u i c e Volume Response t o I n c r e a s e d G l u c o s e i n t h e S e c r e t i n -S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 119 F i g u r e 29 E f f e c t o f I n c r e a s e d G l u c o s e on Amylase S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a ncreas 121 F i g u r e 30 E f f e c t o f I n c r e a s e d G l u c o s e on T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 123 F i g u r e 31 E f f e c t o f I n c r e a s e d G l u c o s e on I n s u l i n R e l e a s e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 125 F i g u r e 32 E f f e c t o f I n c r e a s e d G l u c o s e on S o m a t o s t a t i n R e l e a s e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 127 F i g u r e 3 3 E f f e c t o f I n s u l i n on P a n c r e a t i c J u i c e Volume i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 129 F i g u r e 34 E f f e c t o f I n s u l i n on Amylase S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s x v i F i g u r e 35 E f f e c t o f I n s u l i n on T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e S e c r e t i n -S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 133 F i g u r e 36 E f f e c t o f Added I n s u l i n on Measured I n s u l i n R e l e a s e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 135 F i g u r e 37 E f f e c t o f I n s u l i n on S o m a t o s t a t i n R e l e a s e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 137 F i g u r e 38 P a n c r e a t i c J u i c e Volume Response t o I n c r e a s e d G l u c o s e i n t h e V I P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 139 F i g u r e 39 E f f e c t o f I n c r e a s e d G l u c o s e on Amylase S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e V I P -S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 141 F i g u r e 40 E f f e c t o f I n c r e a s e d G l u c o s e on T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e V I P -S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 143 F i g u r e 41 E f f e c t o f I n c r e a s e d G l u c o s e on I n s u l i n R e l e a s e i n t h e V I P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 145 F i g u r e 42 E f f e c t o f I n c r e a s e d G l u c o s e on S o m a t o s t a t i n R e l e a s e i n t h e V I P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 147 F i g u r e 4 3 E f f e c t o f I n s u l i n on P a n c r e a t i c J u i c e Volume i n t h e V I P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s x v i i F i g u r e 44 E f f e c t o f I n s u l i n on Amylase S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e V l P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 151 F i g u r e 45 E f f e c t o f I n s u l i n on T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e V I P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 153 F i g u r e 46 E f f e c t o f Added I n s u l i n on Measured I n s u l i n R e l e a s e i n t h e V I P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 155 F i g u r e 47 E f f e c t o f I n s u l i n on S o m a t o s t a t i n R e l e a s e i n t h e V I P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s 157 F i g u r e 48 E f f e c t s o f A n o x i a on P a n c r e a t i c J u i c e Volume i n t h e I s o l a t e d P e r f u s e d P a n c r e a s : Lack of E v i d e n c e f o r a P r o t e c t i v e E f f e c t from G l u c o s e o r I n s u l i n P r e t r e a t m e n t 159 F i g u r e 49 E f f e c t s o f A n o x i a on Amylase S e c r e t i o n i n t h e I s o l a t e d P e r f u s e d P ancreas 161 F i g u r e 50 E f f e c t s o f A n o x i a on T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 163 F i g u r e 51 E f f e c t s o f A n o x i a on I n s u l i n R e l e a s e i n t o P e r f u s a t e i n t h e I s o l a t e d P e r f u s e d P ancreas 165 F i g u r e 52 E f f e c t s o f A n o x i a on S o m a t o s t a t i n R e l e a s e i n t o P e r f u s a t e i n t h e I s o l a t e d P e r f u s e d P a n c r e a s 167 x v i i i F i g u r e s 53 and 54 Amylase R e l e a s e from A c i n i i n Response t o I n c r e a s i n g Doses o f CCK-8 169-70 F i g u r e s 55 and 56 Amylase R e l e a s e from A c i n i i n Response t o I n c r e a s i n g Doses o f S e c r e t i n 172- 3 F i g u r e s 57 and 58 Amylase R e l e a s e from A c i n i i n Response t o I n c r e a s i n g Doses o f VIP 173- 6 F i g u r e s 59 and 60 Amylase R e l e a s e from A c i n i i n Response t o I n c r e a s i n g Doses o f M e t h a c h o l i n e 178-9 F i g u r e s 61 and 62 S i g n i f i c a n c e o f I s l e t Fragments i n t h e D i s p e r s e d P a n c r e a t i c A c i n i P r e p a r a t i o n : The Effect of Increasing Glucose Concentration on I n s u l i n and Amylase Release into the Incubation Medium 181-2 F i g u r e 63 S i g n i f i c a n c e o f I s l e t Fragments i n t h e D i s p e r s e d P a n c r e a t i c A c i n i P r e p a r a t i o n : The Effect of Increasing CCK-8 Doses on I n s u l i n Release into the Medium 184 F i g u r e 64 S i g n i f i c a n c e o f I s l e t Fragments i n t h e D i s p e r s e d P a n c r e a t i c A c i n i P r e p a r a t i o n : The Effect of GIP and/or Glucose on I n s u l i n Release into the Medium 186 F i g u r e s 65 and 66 E f f e c t o f I n s u l i n P r e s e n t Only D u r i n g t h e I n c u b a t i o n P e r i o d on C C K - S t i m u l a t e d Amylase S e c r e t i o n 188-9 F i g u r e s 67 and 68 E f f e c t o f I n s u l i n P r e s e n t Only D u r i n g t h e P R E i n c u b a t i o n P e r i o d on C C K - S t i m u l a t e d Amylase S e c r e t i o n 191-2 x i x F i g u r e s 69 and 70 E f f e c t o f I n s u l i n P r e s e n t D u r i n g Both t h e P R E i n c u b a t i o n and I n c u b a t i o n P e r i o d s on CCK-S t i m u l a t e d Amylase S e c r e t i o n 194-5 F i g u r e s 71 and 72 E f f e c t o f I n s u l i n P r e s e n t Only D u r i n g t h e P R E i n c u b a t i o n P e r i o d on S e c r e t i n - S t i m u l a t e d Amylase S e c r e t i o n 197-8 F i g u r e s 73 and 74 E f f e c t o f I n s u l i n P r e s e n t D u r i n g B o t h t h e P R E i n c u b a t i o n and I n c u b a t i o n P e r i o d s on S e c r e t i n - S t i m u l a t e d Amylase S e c r e t i o n 200-1 F i g u r e s 75 and 76 E f f e c t o f I n s u l i n P r e s e n t D u r i n g B o t h t h e P R E i n c u b a t i o n and I n c u b a t i o n P e r i o d s on S e c r e t i n - and t h e C o m b i n a t i o n o f S e c r e t i n -and C C K - S t i m u l a t e d Amylase S e c r e t i o n 203-4 F i g u r e s 77 and 78 E f f e c t of I n s u l i n P r e s e n t D u r i n g P R E i n c u b a t i o n and I n c u b a t i o n P e r i o d s on M e t h a c h o l i n e - S t i m u l a t e d Amylase S e c r e t i o n 206-7 F i g u r e s 79 and 80 E f f e c t o f Zymogen D e p l e t i o n and I n s u l i n Dosage on P o t e n t i a t i o n o f B a s a l and S t i m u l a t e d Amylase S e c r e t i o n 209-10 F i g u r e s 81 and 82 E f f e c t o f S h o r t - t e r m D i a b e t e s and I n s u l i n Dosage on P o t e n t i a t i o n o f B a s a l and S t i m u l a t e d Amylase S e c r e t i o n 212-3 F i g u r e 8 3 Body Weight Changes i n Response t o C h r o n i c a l l y A l t e r e d C i r c u l a t i n g I n s u l i n C o n c e n t r a t i o n s 215 F i g u r e s 84 and 85 F a s t e d Plasma G l u c o s e and I n s u l i n i n Response t o C h r o n i c a l l y A l t e r e d C i r c u l a t i n g I n s u l i n C o n c e n t r a t i o n s XX F i g u r e s 86 and 87 E f f e c t o f I n s u l i n in v i t r o on B a s a l and S t i m u l a t e d Amylase S e c r e t i o n i n C o n t r o l , N o r m o i n s u l i n e m i c R a t s 220-1 F i g u r e s 88 and 89 E f f e c t o f I n s u l i n in v i t r o on B a s a l and S t i m u l a t e d Amylase S e c r e t i o n i n S T Z - D i a b e t i c R a t s 223-4 F i g u r e s 90 and 91 E f f e c t o f I n s u l i n in v i t r o on B a s a l and S t i m u l a t e d Amylase S e c r e t i o n i n H y p e r i n s u l i n e m i c , C h l o r p r o p a m i d e - t r e a t e d R a t s 226-7 F i g u r e s 92 and 93 E f f e c t o f I n s u l i n in v i t r o on B a s a l and S t i m u l a t e d Amylase S e c r e t i o n i n H y p e r i n s u l i n e m i c , NPH I n s u l i n - t r e a t e d R a t s 229-30 F i g u r e 94 Body Weight Changes i n Response t o STZ-D i a b e t e s W i t h and W i t h o u t I n s u l i n Treatment 232 F i g u r e 95 E f f e c t o f t h e D u r a t i o n o f U n t r e a t e d D i a b e t e s on T o t a l , C C K - S t i m u l a t e d Amylase R e l e a s e 234 F i g u r e 96 E f f e c t o f t h e D u r a t i o n o f U n t r e a t e d D i a b e t e s on T o t a l , S e c r e t i n - S t i m u l a t e d Amylase R e l e a s e 236 F i g u r e 97 E f f e c t o f t h e D u r a t i o n o f U n t r e a t e d D i a b e t e s on S e c r e t i n - and t h e C o m b i n a t i o n o f CCK- and S e c r e t i n - S t i m u l a t e d Amylase R e l e a s e 238 F i g u r e 98 E f f e c t o f I n s u l i n Treatment on T o t a l , CCK-S t i m u l a t e d Amylase R e l e a s e from D i a b e t i c A n i m a l A c i n i 240 x x i F i g u r e 99 E f f e c t o f I n s u l i n Treatment on T o t a l , S e c r e t i n - S t i m u l a t e d Amylase R e l e a s e from D i a b e t i c A n i m a l A c i n i 242 F i g u r e 100 E f f e c t o f I n s u l i n Treatment on S e c r e t i n - and t h e C o m b i n a t i o n o f CCK- and S e c r e t i n -S t i m u l a t e d Amylase R e l e a s e From D i a b e t i c A n i m a l A c i n i 244 F i g u r e 101 I n f l u e n c e o f A n i m a l Age on t h e Plasma G l u c o s e Response t o a G l u c o s e C h a l l e n g e : GTT 246 F i g u r e 102 I n f l u e n c e o f A n i m a l Age on t h e Plasma I n s u l i n Response t o a G l u c o s e C h a l l e n g e : GTT 248 F i g u r e s 103 and 104 E f f e c t o f I n s u l i n on C C K - S t i m u l a t e d Amylase S e c r e t i o n from A c i n i D e r i v e d from Weanling R a t s 250-1 F i g u r e s 105 and 106 E f f e c t o f I n s u l i n on S e c r e t i n - S t i m u l a t e d Amylase S e c r e t i o n from A c i n i D e r i v e d from Weanling R a t s 253-4 F i g u r e s 107 and 108 E f f e c t o f I n s u l i n on S e c r e t i n - and V I P -S t i m u l a t e d Amylase S e c r e t i o n from A c i n i D e r i v e d from W e a n l i n g R a t s 256-7 F i g u r e s 109 and 110 I n s u l i n E f f e c t s on S e c r e t i n - S t i m u l a t e d Amylase R e l e a s e : Effect of the Calcium Concentration of the Medium During Incubation 259-60 F i g u r e s 111 and 112 I n s u l i n E f f e c t s on S t i m u l a t e d Amylase R e l e a s e : Effect of Colchicine Treatment During Acini Preincubation 262-3 x x i i F i g u r e s 113 and 114 I n s u l i n E f f e c t s on S t i m u l a t e d Amylase R e l e a s e : Effect of Cycloheximide Treatment During Acini Incubation 265-6 F i g u r e s 115 and 116 D i f f e r e n t i a l R e s p o n s i v e n e s s o f The V e n t r a l and D o r s a l P a n c r e a s t o C C K - S t i m u l a t e d Amylase S e c r e t i o n 268-9 F i g u r e s 117 and 118 M i c r o s c o p i c Appearance o f a Normal Rat P a n c r e a t i c I s l e t S t a i n e d w i t h H e m a t o x y l i n and E o s i n o r Immunostained f o r I n s u l i n (600x) 271-2 F i g u r e s 119 and 120 M i c r o s c o p i c Appearance o f an S T Z - D i a b e t i c Rat P a n c r e a t i c I s l e t (a) d i s p l a y e d b e s i d e Normal I s l e t s ( b ) , b o t h S t a i n e d w i t h e i t h e r H e m a t o x y l i n and E o s i n o r Immunostained f o r I n s u l i n (600x) 274-5 F i g u r e s 121 and 122 M i c r o s c o p i c Appearance o f an I n s u l i n -T r e a t e d , D i a b e t i c Rat P a n c r e a t i c I s l e t (a) d i s p l a y e d b e s i d e a Normal I s l e t ( b ) , S t a i n e d w i t h e i t h e r H e m a t o x y l i n and E o s i n o r Immunostained f o r I n s u l i n (600x) 277-8 F i g u r e s 123 and 124 M i c r o s c o p i c Appearance o f a N o n d i a b e t i c , NPH I n s u l i n - T r e a t e d Rat P a n c r e a t i c I s l e t (a) d i s p l a y e d b e s i d e a Normal I s l e t ( b ) , b o t h S t a i n e d w i t h e i t h e r H e m a t o x y l i n and E o s i n o r Immunostained f o r I n s u l i n (600x) 280-1 F i g u r e s 125 and 126 M i c r o s c o p i c Appearance o f a C h l o r p r o p a m i d e -T r e a t e d Rat P a n c r e a t i c I s l e t (a) d i s p l a y e d b e s i d e a Normal I s l e t ( b ) , b o t h S t a i n e d w i t h e i t h e r H e m a t o x y l i n and E o s i n o r Immunostained f o r I n s u l i n (600x) F i g u r e 127 The E f f e c t o f I n c r e a s e d Endogenous I n s u l i n R e l e a s e and D i s t a n c e from t h e I s l e t on B i n d i n g o f 1 2 5 I - I n s u l i n t o A c i n i : R e s u l t s o f an A u t o r a d i o g r a p h i c Study A f t e r 1 2 5 i -I n s u l i n I n f u s i o n o f t h e I s o l a t e d P e r f u s e d P a n c r e a s 286 1 INTRODUCTION Statement o f t h e Q u e s t i o n The p a n c r e a s i s a c o m p o s i t e g l a n d . E n d o c r i n e components r e l e a s e hormones w i t h i m p o r t a n t e x t r a p a n c r e a t i c e f f e c t s and e x o c r i n e components produce s e c r e t i o n s c r i t i c a l f o r t h e i n t e s t i n a l phase o f d i g e s t i o n . I n t h e p a s t , t h e two components o f t h e p a n c r e a s have been c o n s i d e r e d t o f u n c t i o n i n d e p e n d e n t l y . However, i n c r e a s i n g e v i d e n c e s u g g e s t s t h a t an i n t e r a c t i o n between t h e e n d o c r i n e and d i g e s t i v e f u n c t i o n s o f t h e p a n c r e a s may e x i s t . The p r i m a r y q u e s t i o n a d d r e s s e d i n t h i s s t u d y was whether e n d o c r i n e p a n c r e a t i c hormones a c u t e l y c o n t r o l e x o c r i n e s e c r e t i o n . The p r e v a l e n t hormone o f t h e e n d o c r i n e p a n c r e a s , i n s u l i n , was chosen f o r s t u d y . A proposed p h y s i o l o g i c a l pathway f o r t h e e x o c r i n e a c t i o n s o f i n s u l i n has been termed t h e i n s u l o a c i n a r a x i s (Kanno 1976a). The n a t u r e , t i m e c o u r s e , and a c t i o n s o f i n s u l i n on b a s a l and s t i m u l a t e d s e c r e t i o n o f t h e e x o c r i n e p a n c r e a s form t h e e x p e r i m e n t a l b a s i s o f t h i s t h e s i s . H i s t o r i c a l Background The c o n c e p t t h a t t h e e n d o c r i n e and e x o c r i n e p a n c r e a s might be f u n c t i o n a l l y l i n k e d i s n o t new. Postmortem s t u d i e s o f t h e human d i a b e t i c p a n c r e a s r e p o r t e d i n 1909 ( C e c i l 1909) showed t h a t marked a t r o p h y and f i b r o s i s o c c u r r e d i n t h e e x o c r i n e g l a n d . An e n d o c r i n e - e x o c r i n e r e l a t i o n s h i p was a l s o s u g g e s t e d by d i f f e r e n c e s i n a c i n a r c e l l s i z e and c o n t e n t depending on d i s t a n c e o f t h e c e l l from t h e i s l e t s o f Langerhans (Kramer 2 1968). These o b s e r v a t i o n s s u g g e s t e d t h a t i s l e t hormones might a f f e c t a c i n a r c e l l growth and s t r u c t u r e . W i t h t h e a d d i t i o n a l f i n d i n g o f d i r e c t v a s c u l a r c o n n e c t i o n s between i s l e t s and s u r r o u n d i n g e x o c r i n e t i s s u e , Henderson (1969) h y p o t h e s i z e d t h a t t h e e n d o c r i n e p a n c r e a s , v i a l o c a l passage o f i s l e t hormones, had d i r e c t c o n t r o l o f t h e e x o c r i n e p a n c r e a s . He proposed t h a t such an i n t e r a c t i o n b e s t e x p l a i n e d why t h e e n d o c r i n e p a n c r e a s i n mammals e x i s t s as m u l t i p l e hormone-s e c r e t i n g i s l a n d s s c a t t e r e d t h r o u g h t h e mass of e x o c r i n e t i s s u e , r a t h e r t h a n as t h e s e p a r a t e organ found i n e v o l u t i o n a r i l y l e s s d e v e l o p e d c r e a t u r e s . O t h e r w o r k e r s have expanded t h i s argument i n p r o p o s i n g t h a t e n d o c r i n e - e x o c r i n e i n t e r a c t i o n s might a l s o p r o v i d e a means f o r a d a p t i n g t h e p a n c r e a s t o changes i n e a t i n g p a t t e r n o r d i e t (Grossman 1944, Ben A b d e l j l i l 1963, Mossner 1987). The p r e s e n c e o f i n s u l i n -l i k e m a t e r i a l i n t h e p a r o t i d and s a l i v a r y g l a n d s o f mice ( P a t e l 1986) s u g g e s t s t h a t i n s u l i n may have a more g e n e r a l r o l e i n s u p p o r t o f e x o c r i n e g l a n d s . A d d i t i o n a l e v i d e n c e f o r i s l e t e f f e c t s on e x o c r i n e p a n c r e a t i c f u n c t i o n has been r e p o r t e d ( v i d e i n f r a ) , b u t p h y s i o l o g i c a l r e l e v a n c e has o n l y been shown f o r one hormone, i n s u l i n , i n t h e l o n g - t e r m r e g u l a t i o n o f a c i n a r enzyme c o n t e n t ( W i l l i a m s 1986). R a p i d o r s h o r t - t e r m e f f e c t s o f i n s u l i n and o t h e r i s l e t hormones on e x o c r i n e f u n c t i o n have n o t been c h a r a c t e r i z e d . 3 Embryology and Anatomy o f t h e Pan c r e a s D u r i n g e m b r y o l o g i c development, o u t p o u c h i n g s o f t h e f o r e g u t form d o r s a l and v e n t r a l p a n c r e a t i c buds. The d e v e l o p i n g v e n t r a l bud, a s s o c i a t e d w i t h t h e b i l e d u c t , r o t a t e s p o s t e r i o r l y and f u s e s t o t h e d o r s a l p a n c r e a s w i t h , i n most c a s e s i n t h e human f e t u s , f u s i o n o f t h e d i s t a l v e n t r a l d u c t t o t h e m i d - p r o x i m a l d o r s a l d u c t (Langman 1975). A l t h o u g h s i m i l a r i n e m b r y o l o g i c p r o g r e s s i o n t o t h e human, t h e d e v e l o p e d r a t pancr e a s shows a d i f f e r e n t d u c t p a t t e r n as a r e s u l t o f t h e common p a n c r e a t i c o - b i l i a r y d u c t ( O r c i 1976a). F u s i o n o f t h e two e m b r y o l o g i c buds o b l i t e r a t e s a c l e a r t i s s u e p l a n e between d o r s a l and v e n t r a l p a n c r e a s . The two p a r t s may, however, be i d e n t i f i e d by t h e i r d u c t a l d r a i n a g e p a t t e r n ( L l o y d - J o n e s 1972, C o t t o n 1985), b l o o d s u p p l y (Bonner-Weir 1982), and d i s t i n c t p a t t e r n s o f i s l e t c e l l u l a r c o m p o s i t i o n (Bencosme 1955, Baetens 1979). The e x o c r i n e p a n c r e a s d e v e l o p s as a s e r i e s o f anastomosing t u b u l e s r a t h e r t h a n a t r u e a l v e o l a r g l a n d (Bockman 1978). Enzyme-producing a c i n a r c e l l s s e c r e t e i n t o a b r a n c h i n g d u c t system w h i c h m o d i f i e s t h e s e c r e t i o n d u r i n g t r a n s p o r t from t h e s m a l l e s t i n t e r c a l a t e d d u c t s t o t h e major d o r s a l and v e n t r a l d u c t s . E x o c r i n e c e l l t y p e s i n c l u d e t h e a c i n a r c e l l s , c e n t r o a c i n a r c e l l s , and d u c t c e l l s , t o g e t h e r w i t h t h e v a s c u l a r e l e m e n t s , f i b r o b l a s t s , and i n f l a m m a t o r y c e l l s t h a t make up t h e i n t e r s t i t i u m . The a c i n a r c e l l s , d e r i v e d from t h e e m b r y o l o g i c d u c t system, form t u f t e d c e l l masses a t t h e ends o f i n t r a l o b u l a r d u c t s . I n d i v i d u a l a c i n a r c e l l s have a s p e c i f i c 4 o r i e n t a t i o n . B a s o l a t e r a l b o r d e r s a r e o r g a n i z e d f o r c e l l - c e l l i n t e r a c t i o n ( W i l l i a m s 1984a) o r i n t e r s t i t i a l - c a p i l l a r y n u t r i e n t t r a n s f e r , w h i l e t h e a p i c a l p o r t i o n i s t h e s p e c i a l i z e d s e c r e t o r y s u r f a c e . A group o f a c i n a r c e l l s d e v e l o p t o g e t h e r t o form an a c i n u s w h i c h i s t h e p r i m a r y enzyme s e c r e t o r y u n i t o f t h e p a n c r e a s . C e n t r o a c i n a r c e l l s form t h e i n i t i a l l i n i n g o f i n t r a l o b u l a r d u c t s a t t h e o u t l e t o f each a c i n u s . They a r e r e s p o n s i b l e , a l o n g w i t h d u c t c e l l s , f o r t h e b i c a r b o n a t e , w a t e r , and some o f t h e e l e c t r o l y t e components o f t h e p a n c r e a t i c j u i c e ( H o l l a n d e r 1952). Members o f each c e l l t y p e a r e f o u n d i n p r o x i m i t y t o i s l e t s and may t h e r e f o r e , t h e o r e t i c a l l y , be exposed t o h i g h c o n c e n t r a t i o n s o f i s l e t hormones. The i s l e t s o f Langerhans a r e a l s o d e r i v e d from t h e e m b r y o l o g i c d u c t a l system (Rasbach 1984). I n g e n e r a l , a l l i s l e t s c o n t a i n t h e same b a s i c c e l l t y p e s a l t h o u g h i n v a r y i n g p r o p o r t i o n s . F our d i f f e r e n t t y p e s o f i s l e t c e l l s a r e g e n e r a l l y i d e n t i f i e d by immunocytochemical and u l t r a s t r u c t u r a l c r i t e r i a ( O r c i 1982). The B o r b e t a - c e l l s e c r e t e s i n s u l i n and, i n most mammals, forms t h e l a r g e s t mass o f t h e c e l l s c o n s t i t u t i n g t h e c o r e o f t h e i s l e t . The mantle o r o u t e r l a y e r o f c e l l s i s made up o f t h e A o r g l u c a g o n - s e c r e t i n g c e l l s , t h e D o r s o m a t o s t a t i n - c o n t a i n i n g c e l l s , and t h e PP o r p a n c r e a t i c p o l y p e p t i d e - s e c r e t i n g c e l l s . C o - l o c a l i z a t i o n o f o t h e r p e p t i d e hormones w i t h i n i s l e t c e l l s , f o r example b e t a - e n d o r p h i n i n t h e D c e l l ( B r u n i 1979), t h e c l a s s i c i s l e t hormones and v a r i o u s 5 p e p t i d e p r o d u c t s o f i s l e t p e p t i d e r g i c i n n e r v a t i o n ( i e . v a s o a c t i v e i n t e s t i n a l p o l y p e p t i d e ( V I P ) , c a l c i t o n i n gene r e l a t e d p e p t i d e (CGRP)) a l l i n c r e a s e t h e number o f p o t e n t i a l l y a c t i v e hormonal p r o d u c t s r e a c h i n g t h e i s l e t c a p i l l a r y system. U l t r a s t r u c t u r a l and f u n c t i o n a l s t u d i e s o f i s o l a t e d i s l e t s and r e a g g r e g a t e d i s l e t c e l l s show c e l l t o c e l l i n t e r a c t i o n s t h a t may modulate i s l e t s e c r e t i o n . An i n t r a i s l e t c a p i l l a r y system forms a complex " g l o m e r u l a r " network ( D a n i e l 1978) w i t h b l o o d f l o w i n g from t h e B - c e l l c o r e outward (Bonner-Weir 1982). I f i n d e e d t h e p r i n c i p l e p a t t e r n o f f l o w i s c e n t r i f u g a l , i n s u l i n may have i m p o r t a n t e f f e c t s on A, D, o r PP c e l l s ( L e c l e r c q -Meyer 1983). D i f f e r e n c e s i n i s l e t c o m p o s i t i o n t h r o u g h t h e p a n c r e a s may be a cause f o r r e g i o n a l h e t e r o g e n e i t y o f e x o c r i n e f u n c t i o n w i t h i n t h e p a n c r e a s . Immunocytbchemical s t u d y o f i s l e t s from t h e v e n t r a l l o b e o f t h e dog, r a t , and human has shown a g r e a t e r p r o p o r t i o n o f PP c e l l s , w h i l e t h o s e from t h e d o r s a l l o b e have i n c r e a s e d numbers o f A - c e l l s ( O r c i 1976b, Baetens 1979). B-and D - c e l l numbers have n o t been found t o d i f f e r between l o b e s ( L e c l e r c q - M e y e r 1985) b u t appear t o f u n c t i o n d i f f e r e n t l y . A l t h o u g h i s l e t i n s u l i n c o n t e n t i s s i m i l a r i n b o t h l o b e s o f t h e r a t p a n c r e a s , d o r s a l i s l e t s r e l e a s e s i g n i f i c a n t l y more i n s u l i n and g l u c a g o n i n r e s p o n s e t o g l u c o s e t h a n v e n t r a l i s l e t s ( T r i m b l e 1981). L o c a l e f f e c t s o f g l u c a g o n on B - c e l l s may be r e s p o n s i b l e f o r t h i s e f f e c t . E v i d e n c e f o r i n c r e a s e d amylase c o n t e n t i n t h e d o r s a l l o b e ( M a l a i s s e - L a g a e 1983) may be a 6 consequence o f d i f f e r e n c e s i n i s l e t f u n c t i o n , p r o v i d i n g f u r t h e r s u p p o r t f o r an i s l e t - a c i n a r e f f e c t (Bruzzone 1986). I n s u l o a c i n a r " P o r t a l " System One o f t h e s t r o n g e s t p i e c e s o f e v i d e n c e f o r a d i r e c t e f f e c t o f t h e e n d o c r i n e system on e x o c r i n e f u n c t i o n has been t h e f i n d i n g o f l a r g e , unbranched, d i r e c t v a s c u l a r c o n n e c t i o n s between i s l e t s and t h e s u r r o u n d i n g a c i n a r c a p i l l a r y bed ( F u j i t a 1973a, Kanno 1976b). M i c r o s c o p i s t s o f t h e l a t e 19th c e n t u r y , i n e x a m i n i n g t h e c a p i l l a r y p a t t e r n o f t h e in vivo p a n c r e a s , d e t e c t e d many s m a l l r e g i o n s o f i n c r e a s e d c a p i l l a r y d e n s i t y w h i c h appeared s i m i l a r t o t h e v a s c u l a r t u f t s o f t h e g l o m e r u l a r c a p i l l a r y system (Henderson 1981). Indeed, t h e s e m i c r o v a s c u l a r f e a t u r e s were r a p i d l y a t t r i b u t e d t o t h e d i s t i n c t p a n c r e a t i c c e l l u l a r i s l a n d s d e s c r i b e d by Langerhans (Langerhans 1869) and a f u n c t i o n a l r o l e i n d i a b e t e s s u g g e s t e d . More r e c e n t l y , d e t a i l e d dye p e r f u s i o n and v a s c u l a r c o r r o s i o n c a s t s t u d i e s ( F r a s e r 1979) o f t h e pancreas^ have shown d i r e c t i s l e t p e r f u s i o n by s h o r t , wide a r t e r i o l e s from i n t r a l o b u l a r a r t e r i e s (Henderson 1981). Q u a n t i t a t i v e b l o o d f l o w s t u d i e s showed t h a t i s l e t b l o o d f l o w was p r o p o r t i o n a t e l y up t o e i g h t e e n t i m e s g r e a t e r p e r gram o f t i s s u e t h a n e x o c r i n e p a n c r e a s ( J a n s s o n 1982, L i f s o n 1985). Thus, t h e i s l e t mass i s r i c h l y p e r f u s e d i n p a r a l l e l w i t h t h e e x o c r i n e p a n c r e a s ( F r a s e r 1979). B l o o d l e a v i n g t h e i s l e t p a s s e s t h r o u g h v a s a e f f e r e n t i a c o n n e c t i n g t o t h e c a p i l l a r y s y stem o f t h e s u r r o u n d i n g e x o c r i n e 7 t i s s u e ( F u j i t a 1976, F r a s e r 1980). C o n s i d e r a b l e v a r i a b i l i t y i s e v i d e n t i n t h e p a t t e r n o f c a p i l l a r y i s l e t d r a i n a g e . L i f s o n e t a l (1985) n o t e d i n t h e i r r a t s t u d i e s t h a t w h i l e a range of i s l e t volumes a r e p r e s e n t i n t h e normal p a n c r e a s , most i s l e t s a r e s m a l l ( d i a m e t e r 30 t o 90 nm). These s m a l l i s l e t s r e c e i v e p r o p o r t i o n a t e l y more b l o o d f l o w p e r gram o f t i s s u e t h a n l a r g e r i s l e t s and o n l y b l o o d from t h i s group o f s m a l l e r i s l e t s seems t o f l o w c e n t r i f u g a l l y t o t h e a c i n a r c a p i l l a r y system. L a r g e r i s l e t s d r a i n d i r e c t l y i n t o i s l e t - s u r f a c e v e n u l e s (Bonner-Weir 1982). I f t h e i n s u l o a c i n a r h y p o t h e s i s i s c o r r e c t , a c i n i i m m e d i a t e l y around i s l e t s i n r e g i o n s most d i r e c t l y r e c e i v i n g c a p i l l a r y o u t f l o w from t h e i s l e t , s h o u l d be most a f f e c t e d by i s l e t e n d o c r i n e hormones. H i s t o l o g i c e x a m i n a t i o n o f h e m a t o x y l i n -and e o s i n - s t a i n e d normal p a n c r e a s shows a " h a l o " appearance around i s l e t s t h a t i s due t o an a l t e r e d s t a i n i n g i n t h e s e p e r i - i s l e t ( near i s l e t ) a c i n a r c e l l s . Morphometric s t u d i e s have shown i n c r e a s e d c e l l and n u c l e u s s i z e , and c e l l u l a r zymogen c o n t e n t (Hellman 1962, J a r o t s k y 1899) i n t h e s e same c e l l s . C o r r e s p o n d i n g f u n c t i o n a l changes have a l s o been shown. Amino a c i d u p t a k e and p r o t e i n s y n t h e s i s a r e i n c r e a s e d , and s e c r e t a g o g u e ( p i l o c a r i n e ) s e n s i t i v i t y i s a l t e r e d i n p e r i - i s l e t a c i n i as compared t o a c i n i l o c a t e d a t a d i s t a n c e from i s l e t s ( t e l e - i s l e t ) (Kramer 1968). Study o f t h e h a l o phenomenon i n duck p a n c r e a s has p r o v i d e d c l u e s as t o w h i c h o f t h e i s l e t hormones may be a c t i n g on t h e e x o c r i n e p a n c r e a s . Duck i s l e t s 8 a r e o f two k i n d s , " l i g h t " and " d a r k " , t h e f o r m e r s e c r e t i n g o n l y i n s u l i n and t h e l a t t e r g l u c a g o n and s o m a t o s t a t i n . H a l o e s a r e o n l y e v i d e n t i n t h e duck p a n c r e a s around t h e i n s u l i n -s e c r e t i n g , " l i g h t " i s l e t s ( W a l l g r e n 1962). An i n s u l i n e f f e c t has a l s o been s u g g e s t e d by p r o m i n e n t p e r i - i s l e t h a l o e s i n o b e s e - h y p e r g l y c e m i c mice w h i c h have v e r y e n l a r g e d , h y p e r s e c r e t i n g ( i n s u l i n ) i s l e t s ( H e l lman 1962). A l t h o u g h i t i s t e m p t i n g t o a s c r i b e p e r i - i s l e t a c i n a r changes t o l o c a l e f f e c t s o f i n s u l i n , s e v e r a l p i e c e s o f e v i d e n c e f a i l t o s u p p o r t t h i s h y p o t h e s i s . F i r s t , p e r f u s i o n s t u d i e s p e r f o r m e d by Henderson a f t e r t h e i n i t i a l i d e n t i f i c a t i o n o f i s l e t - a c i n a r c a p i l l a r y b r a n c h e s , showed t h a t t h e s e c o n n e c t i o n s t r a v e r s e d a r e g i o n o f s p a r s e v a s c u l a r i t y , a r e g i o n w i t h few c a p i l l a r y b r a n c h e s t o a c i n a r t i s s u e i m m e d i a t e l y around t h e i s l e t (Henderson 1981). A l t h o u g h v a s c u l a r pathways from t h e e n d o c r i n e t o t h e e x o c r i n e p a n c r e a s seemed t o e x i s t , t h e y d i d n o t appear t o d i s t r i b u t e p r e f e r e n t i a l l y t o p e r i - i s l e t a c i n i , c a s t i n g doubt on t h e p r e v i o u s e x p l a n a t i o n f o r t h e h a l o phenomenon. Second, d i r e c t c e l l - c e l l c o n t a c t s were i d e n t i f i e d between i s l e t and a c i n a r c e l l s a t t h e i s l e t p e r i p h e r y (Bendayan 1982) p r o v i d i n g an a l t e r n a t i v e e x p l a n a t i o n f o r h a l o phenomena. T h i r d , a l t h o u g h B c e l l d e s t r u c t i o n by a l l o x a n r e s u l t e d i n d i s a p p e a r a n c e o f p e r i - i s l e t h a l o e s (Kramer 1968), when t h e e x p e r i m e n t was r e p e a t e d w i t h s t r e p t o z o t o c i n , h a l o prominence a c t u a l l y i n c r e a s e d d e s p i t e l o s s o f B - c e l l mass ( M a l a i s s e - L a g a e 1976). I f t h e h a l o e f f e c t was due t o a 9 r e g i o n a l t r o p h i c r e s p o n s e , t h e s e d a t a s u g g e s t e d t h a t hormones o t h e r t h a n i n s u l i n might have been a c t i v e . And f o u r t h , s i g n i f i c a n t l y g r e a t e r c o n c e n t r a t i o n s o f amylase, l i p a s e , and c h y r a o t r y p s i n o g e n were measured i n t e l e - i s l e t a c i n i compared t o p e r i - i s l e t a c i n i , c o n t r a d i c t i n g t h e h i s t o l o g i c e v i d e n c e f o r a t r o p h i c e f f e c t ( M a l a i s s e - L a g a e 1976). These r e s u l t s i n normal r a t s , s t r e p t o z o t o c i n - d i a b e t i c r a t s , and t h e s p i n y mouse l e d t h e a u t h o r s t o s p e c u l a t e t h a t r a t h e r t h a n an absence of i n s u l o a c i n a r e f f e c t s , hormones o t h e r t h a n i n s u l i n might a c t v a r i a b l y i n c o m p e t i t i o n as i n h i b i t o r s o f a c i n a r f u n c t i o n . R e s o l u t i o n o f t h e s e c o n f l i c t i n g r e s u l t s appeared t o r e q u i r e a more complete u n d e r s t a n d i n g o f s e c r e t i o n p a t t e r n s and i n t e r a c t i o n s between t h e v a r i o u s hormones r e l e a s e d by t h e i s l e t . A l t h o u g h t h e e x a c t m e d i a t o r ( s ) o f t h e h a l o phenomenon remains u n c l e a r , a c i n a r changes about t h e i s l e t remain p o w e r f u l e v i d e n c e f o r a p h y s i o l o g i c a l e n d o c r i n e - e x o c r i n e a x i s . P h y s i o l o g i c a l F u n c t i o n s o f t h e Pancreas W h i l e t h e i n t e n t i o n o f t h i s work was t o b r i n g t o g e t h e r t h e s e p a r a t e f u n c t i o n s o f t h e p a n c r e a s by s u g g e s t i n g i s l e t m o d u l a t i o n o f e x o c r i n e f u n c t i o n , i n s u l o a c i n a r a c t i o n s o n l y o c c u r i n t h e c o n t e x t o f major c o n t r o l mechanisms f o r p a n c r e a t i c e n d o c r i n e and e x o c r i n e f u n c t i o n . Exocrine Function The e x o c r i n e p a n c r e a s i s a c r i t i c a l o rgan o f d i g e s t i o n . I t f u n c t i o n s by s e c r e t i n g a complex j u i c e i n t o t h e lumen o f t h e p r o x i m a l d i g e s t i v e t r a c t . T h i s j u i c e i s a m i x t u r e o f enzymes 10 and o t h e r s o l u t e s d i s s o l v e d i n an e l e c t r o l y t e s o l u t i o n . The components o f p a n c r e a t i c j u i c e s u b s e r v e t h e d i g e s t i v e f u n c t i o n s o f t h e g l a n d , t h u s e x o c r i n e f a i l u r e i s measured by l o s s o f j u i c e s e c r e t i o n . F i v e t o e i g h t grams o f p r o t e i n , l a r g e l y e n z y m a t i c , a r e produced p e r day by t h e human p a n c r e a s . Over a dozen s e p a r a t e enzymes have been i d e n t i f i e d by two d i m e n s i o n a l e l e c t r o p h o r e s i s , many bands r e p r e s e n t i n g enzymes o f s i m i l a r s u b s t r a t e s p e c i f i c i t y b u t w i t h d i f f e r e n t optimum pK v a l u e s ( S c h e e l e 1975). The major groups i d e n t i f i e d a r e t h e s e r i n e p r o t e a s e s ( t r y p s i n , c h y m o t r y p s i n , c a r b o x y p e p t i d a s e A and B, e l a s t a s e , and k a l l i k r e i n ) , l i p a s e s , p h o s p h o l i p a s e A, DNA and RNA'se, and amylase ( R i n d e r k n e c h t 1986). Of t h e s e , t h e s e r i n e p r o t e a s e s and p h o s p h o l i p a s e s a r e s e c r e t e d i n an i n a c t i v e form t o g e t h e r w i t h endogenous enzyme i n h i b i t o r t o p r o t e c t t h e pa n c r e a s from a u t o d i g e s t i o n . Once i n t h e i n t e s t i n a l lumen, e n t e r o k i n a s e w i t h i n t h e g u t w a l l ( G r a n t 1976) a c t i v a t e s t r y p s i n o g e n t o t r y p s i n w h i c h t h e n a c t i v a t e s b o t h i t s e l f and t h e o t h e r i n a c t i v e p r e c u r s o r s ( S c h e e l e 1975). B i o s y n t h e s i s o f p a n c r e a t i c p r o t e i n s i n v o l v e s an o r d e r l y sequence o f t r a n s l a t i o n a t t h e en d o p l a s m i c r e t i c u l u m f o l l o w e d by p a c k a g i n g i n t h e G o l g i a p p a r a t u s , and s t o r a g e i n a p i c a l s t o r a g e g r a n u l e s (Jamieson 1971, P a l a d e 1975). A l t h o u g h most b i o c h e m i c a l s t u d i e s o f t h i s p r o c e s s s u g g e s t t h a t f i x e d p r o p o r t i o n s o f enzymes a r e packaged and r e l e a s e d t o g e t h e r i n a " p a r a l l e l " manner as zymogen g r a n u l e s ( S c h e e l e 1975), under 11 c e r t a i n n u t r i t i o n a l and s t i m u l a t o r y c o n d i t i o n s , p r o p o r t i o n a l enzyme a c t i v i t i e s i n p a n c r e a t i c s e c r e t i o n can be a l t e r e d ( A d e l s o n 1975). T h i s has been termed " n o n p a r a l l e l " s e c r e t i o n and may c o r r e l a t e w i t h m i c r o a s s a y d i f f e r e n c e s i n t h e enzyme c o n t e n t o f d i f f e r e n t p o p u l a t i o n s o f p a n c r e a t i c zymogen g r a n u l e s (Mroz 1986). A r o l e f o r i s l e t hormones has been s u g g e s t e d i n a c u t e l y m e d i a t i n g a d a p t i v e , n o n p a r a l l e l e x o c r i n e s e c r e t i o n by t h e p a n c r e a s ( A d e l s o n 1985). Nonenzymatic components o f p a n c r e a t i c j u i c e s e r v e an e s s e n t i a l b u t s u p p o r t i v e r o l e i n t h e d i g e s t i v e p r o c e s s . They d i l u t e and b u f f e r i n t e s t i n a l c o n t e n t s t o p r o t e c t t h e g u t and o p t i m i z e enzyme f u n c t i o n . F a c t o r s c o n t r o l l i n g d u c t f u n c t i o n i n c l u d e t h e i n t e s t i n a l hormone s e c r e t i n and t h e p e p t i d e r g i c n e u r o t r a n s m i t t e r v a s o a c t i v e i n t e s t i n a l p o l y p e p t i d e ( V I P ) . R e c e p t o r s f o r i n s u l i n have p r e v i o u s l y been found on p a n c r e a t i c d u c t c e l l s (Sakamoto 1984) b u t i n s u l i n d e p r i v a t i o n due t o s t r e p t o z o t o c i n d i a b e t e s f a i l e d t o a l t e r s e c r e t i n r e s p o n s i v e n e s s ( S o f r a n k o v a 1983). E f f e c t s o f o t h e r i s l e t hormones on d u c t f u n c t i o n have n o t been r e p o r t e d . P a n c r e a t i c j u i c e has f o u r major a c t i o n s . F i r s t , t h e l i q u i d volume o f t h e j u i c e s e r v e s t o d i l u t e chyme p r o p e l l e d by g a s t r i c p e r i s t a l s i s i n t o t h e p r o x i m a l duodenum. D i l u t i o n i n c r e a s e s n u t r i e n t exposure f o r d i g e s t i o n and a b s o r p t i o n ( V i d o n 1978) and r a i s e s pH. Second, t h e b i c a r b o n a t e i o n r e s p o n s i b l e f o r a l k a l i n i t y o f p a n c r e a t i c j u i c e a c t s t o n e u t r a l i z e r e s i d u a l g a s t r i c a c i d i t y w h i c h i s n e c e s s a r y t o 12 p r o t e c t t h e i n t e s t i n a l mucosa and p r o v i d e s an o p t i m a l pH f o r b o t h p a n c r e a t i c and i n t e s t i n a l enzyme f u n c t i o n (Regan 1977, M a l a g e l a d a 1980). T h i r d , t h e s o l u t e component o f p a n c r e a t i c j u i c e c o n t a i n s d i g e s t i v e enzymes d i r e c t e d a t p r o t e i n , l i p i d , and c a r b o h y d r a t e n u t r i e n t s o u r c e s and n u c l e i c a c i d s ( R i n d e r k n e c h t 1986). That p a n c r e a t i c enzymes p l a y an e s s e n t i a l d i g e s t i v e r o l e i s c l e a r g i v e n t h e m a l a b s o r p t i o n and m a l n u t r i t i o n w h i c h d e v e l o p i n c l i n i c a l s t a t e s o f p a n c r e a t i c i n s u f f i c i e n c y . S u r g i c a l e x t i r p a t i o n o f t h e g l a n d o r t h e d i s e a s e o f c h r o n i c p a n c r e a t i t i s a r e two examples o f c l i n i c a l i n s u f f i c i e n c y ( B r a a s c h 1978). L a s t l y , i n c r e a s i n g e v i d e n c e s u g g e s t s feedback e f f e c t s by components o f p a n c r e a t i c j u i c e upon s o u r c e s o f humoral s t i m u l a t i o n f o r t h e p a n c r e a s ( F u j i t a 1976). C e r t a i n l y , t h e a c t i o n s o f p a n c r e a t i c j u i c e t o d i l u t e , n e u t r a l i z e , and d i g e s t i n t e s t i n a l components would e v e n t u a l l y remove l u m i n a l s t i m u l a t i o n f o r v a g o v a g a l r e f l e x e s (Dooley 1984), and f o r c h o l e c y s t o k i n i n (CCK), and s e c r e t i n r e l e a s e . However, a d i r e c t i n h i b i t o r y f eedback e f f e c t o f enzyme on c h o l e c y s t o k i n i n r e l e a s e has a l s o been de m o n s t r a t e d ( S l a f f 1984, L o u i e 1986). S i n c e p a n c r e a t i c j u i c e f o r m a t i o n i s c e n t r a l t o e x o c r i n e f u n c t i o n , i n s u l o a c i n a r hormonal e f f e c t s , i f p r e s e n t , s h o u l d be e v i d e n t as 1) an a l t e r a t i o n i n t h e c h a r a c t e r o r volume of j u i c e p r o duced, o r 2) a l t e r e d s e n s i t i v i t y o f e x o c r i n e t i s s u e t o n e u r a l o r humoral c o n t r o l s i g n a l s . Enzyme and volume-e l e c t r o l y t e components o f p a n c r e a t i c j u i c e r e p r e s e n t , i n 13 g e n e r a l t e r m s , t h e p r o d u c t o f two d i f f e r e n t c e l l masses wh i c h may a l s o r e s p o n d d i f f e r e n t l y t o i s l e t hormones. Endocrine Function A l l i s l e t hormones have t h e p o t e n t i a l t o e n t e r t h e i n s u l o a c i n a r a x i s and a l t e r a c i n a r c e l l f u n c t i o n . Glucagon and i n s u l i n , from t h e A and B c e l l s r e s p e c t i v e l y , s e r v e complementary r o l e s i n t h e maintenance o f g l u c o s e h o m e o s t a s i s . Glucagon i s a 29 amino a c i d p e p t i d e r e l e a s e d by a complex i n t e r a c t i o n between serum n u t r i e n t s i n c l u d i n g g l u c o s e , amino a c i d s , and f r e e f a t t y a c i d s ( A s p l i n 1983), and o t h e r i s l e t hormones b e l i e v e d t o a c t i n a p a r a c r i n e manner. The most e v i d e n t e x t r a p a n c r e a t i c r e s p o n s e t o g l u c a g o n o c c u r s i n t h e l i v e r where g l u c a g o n i s a h y p e r g l y c e m i c agent. G l y c o g e n breakdown and g l u c o n e o g e n e s i s a r e b o t h i n i t i a t e d t h r o u g h s p e c i f i c r e c e p t o r systems. A number o f p h a r m a c o l o g i c a l r e s p o n s e s i n c l u d i n g i n h i b i t i o n o f g a s t r i c mucosal b l o o d f l o w and s e c r e t i o n , c h o l e r e s i s , and i n h i b i t i o n o f smooth muscle s p h i n c t e r c o n t r a c t i o n ( g a s t r i c , e s o p h a g e a l , and c h o l e d o c h a l ) have a l s o been d e s c r i b e d ( L i n 1980) b u t o n l y t h e e x o c r i n e p a n c r e a s i s n o r m a l l y exposed t o c o n c e n t r a t i o n s o f g l u c a g o n ( v i a t h e i n s u l o a c i n a r a x i s ) w h i c h , i n t h e p e r i p h e r a l c i r c u l a t i o n , produce t h e s e p h a r m a c o l o g i c a l r e s p o n s e s . Glucagon has s e v e r a l p h a r m a c o l o g i c a l e f f e c t s on e x o c r i n e f u n c t i o n . Repeated g l u c a g o n a d m i n i s t r a t i o n caused p r o g r e s s i v e d e g r a n u l a t i o n o f a c i n a r c e l l s , an e f f e c t a t t r i b u t e d t o i n h i b i t i o n o f zymogen s y n t h e s i s ( J a r e t t 1962). Dyck e t a l 14 ( 1 9 6 9 ) r e p o r t e d m a r k e d d e p r e s s i o n o f v o l u m e a n d e n z y m e s e c r e t i o n i n t h e s t i m u l a t e d d o g p a n c r e a s . S u b s e q u e n t c o m p a r i s o n s t u d i e s i n i n t a c t a n i m a l s a n d i s o l a t e d o r g a n p r e p a r a t i o n s s u g g e s t e d t h a t t h e i n h i b i t i o n w a s i n d i r e c t , p o s s i b l y m e d i a t e d b y c h a n g e s i n g a s t r i c s e c r e t i o n o r p a n c r e a t i c b l o o d f l o w . R e c e n t o b s e r v a t i o n s i n i s o l a t e d a c i n i s h o w e d t h a t g l u c a g o n i n c r e a s e d d i g e s t i v e e n z y m e s e c r e t i o n b y a d i r e c t a c t i o n v i a c A M P w h i l e d e c r e a s i n g e n z y m e s y n t h e s i s ( S i n g h 1 9 8 0 ) . T h e s e f i n d i n g s h a v e b e e n q u e s t i o n e d b y P a n d o l w h o f o u n d t h a t e l e v a t i o n o f c A M P i n g u i n e a p i g a c i n i b y n a t u r a l s o u r c e g l u c a g o n w a s d u e t o c o n t a m i n a n t s , a n d t h a t p u r e , s y n t h e t i c g l u c a g o n h a d n o a p p a r e n t e f f e c t ( P a n d o l 1 9 8 3 ) . S o m a t o s t a t i n i s a 1 4 a m i n o a c i d c y c l i c p e p t i d e ( S c h a l l y 1 9 7 6 ) f o u n d t h r o u g h o u t t h e c e n t r a l n e r v o u s s y s t e m a n d g a s t r o i n t e s t i n a l t r a c t . T h e s t o m a c h a n d p a n c r e a s h a v e t h e h i g h e s t c o n c e n t r a t i o n o f s o m a t o s t a t i n i n t h e g u t ( L u f t 1 9 7 4 ) . N e u r a l , g a s t r o i n t e s t i n a l h o r m o n e , a n d n u t r i e n t i n f l u e n c e s a l l p l a y a r o l e i n s t i m u l a t i n g s o m a t o s t a t i n r e l e a s e . I s o l a t e d i s l e t s t u d i e s s h o w t h a t g l u c o s e , l e u c i n e , a r g i n i n e , a n d g l u c a g o n a l l s t i m u l a t e s t o m a t o s t a t i n r e l e a s e . ( S c h a u d e r 1 9 8 0 ) . T h e f u n c t i o n o f s o m a t o s t a t i n i n v i r t u a l l y a l l o r g a n s y s t e m s t e s t e d h a s b e e n i n h i b i t o r y ( T h o m p s o n 1 9 8 4 ) . G r o w t h h o r m o n e r e l e a s e f r o m t h e h y p o t h a l a m u s , i n s u l i n , g l u c a g o n , a n d p a n c r e a t i c p o l y p e p t i d e r e l e a s e f r o m t h e i s l e t , a n d b o t h g a s t r i n a n d g a s t r i c s e c r e t i o n h a v e a l l b e e n i n h i b i t e d ( E f e n d i c 1 9 8 0 , S c h a u d e r 1 9 8 0 ) . 15 I n t h e e x o c r i n e p a n c r e a s , i t i s l i k e l y t h a t s o m a t o s t a t i n i s a l s o i n h i b i t o r y . D - c e l l s o f t h e i s l e t a r e o r i e n t e d f o r c a p i l l a r y r e l e a s e o f s o m a t o s t a t i n i n t o t h e i n s u l o a c i n a r p o r t a l c i r c u l a t i o n (Aponte 1985). S t u d i e s i n dogs and r a t s have shown i n h i b i t i o n o f enzyme s e c r e t i o n due t o CCK and analog u e s ( L i n 1983) and b o t h v a g a l s t i m u l a t i o n and a c e t y l c h o l i n e ( C h a r i o t 1978). I n h i b i t i o n o f enzyme and p r o t e i n s e c r e t i o n i s g r e a t e r t h a n t h e i n h i b i t i o n o f s e c r e t i n - s t i m u l a t e d volume and b i c a r b o n a t e s e c r e t i o n and v a r i e s by s p e c i e s (Boden 1975). S o m a t o s t a t i n a l s o i n h i b i t s p a n c r e a t i c growth. B o t h DNA and enzyme p r o t e i n c o n t e n t a r e d i m i n i s h e d by c h r o n i c s o m a t o s t a t i n t r e a t m e n t ( M o r i s s e t 1982) and t h e g r o w t h - s t i m u l a t i n g e f f e c t s o f c a e r u l e i n and s e c r e t i n can be b l o c k e d ( S a r f a t i 1985). A n t i c i p a t e d e f f e c t s o f s o m a t o s t a t i n w i t h i n t h e i n s u l o a c i n a r a x i s may t h e r e f o r e be b o t h d i r e c t a c t i o n s on e x o c r i n e f u n c t i o n , and i n d i r e c t e f f e c t s on o t h e r i s l e t hormones and t h e i n t e s t i n a l hormones, CCK and s e c r e t i n . Of p a r t i c u l a r i m p o r t a n c e i s e v i d e n c e f o r a t o n i c i n h i b i t i o n o f i n s u l i n r e l e a s e by s o m a t o s t a t i n ( H o p c r o f t 1985). S i n c e t h e s t i m u l i f o r s o m a t o s t a t i n and i n s u l i n a r e s i m i l a r , s o m a t o s t a t i n may be i m p o r t a n t i n m o d u l a t i n g t h e amount o f i n s u l i n r e l e a s e d and t h e e f f e c t s o f i n s u l i n i n t h e p e r i p h e r y , i n c l u d i n g t h e e x o c r i n e p a n c r e a s . O t h e r i s l e t hormones p o t e n t i a l l y r e l e a s e d i n t o t h e i n s u l o a c i n a r a x i s i n c l u d e p a n c r e a t i c p o l y p e p t i d e and b e t a -e n d o r p h i n . P a n c r e a t i c p o l y p e p t i d e i s a 36 amino a c i d hormone 16 f o u n d i n t h e i s l e t PP c e l l s and s c a t t e r e d i n v e n t r a l p a n c r e a t i c e x o c r i n e t i s s u e ( Baetens 1979). V a g a l c h o l i n e r g i c a c t i v i t y i s t h e p r i m a r y s t i m u l u s f o r p a n c r e a t i c p o l y p e p t i d e r e l e a s e ( S c h wartz 1983). A t p h a r m a c o l o g i c a l d osages, p a n c r e a t i c p o l y p e p t i d e i s a dose-dependent i n h i b i t o r o f s t i m u l a t e d e x o c r i n e s e c r e t i o n b u t no c l e a r p h y s i o l o g i c a l r o l e f o r t h i s hormone has y e t been e s t a b l i s h e d (Thompson 1984). O p i o i d p e p t i d e s have been c o - l o c a l i z e d t o i s l e t D - c e l l s and t h e p e p t i d e r g i c i n n e r v a t i o n o f t h e p a n c r e a s ( B r u n i 1979, L a r s s o n 1979a). S i m i l a r t o p a n c r e a t i c p o l y p e p t i d e , e v i d e n c e f o r p h a r m a c o l o g i c i n h i b i t i o n o f e x o c r i n e s e c r e t i o n by o p i o i d s has n o t t r a n s l a t e d i n t o any p h y s i o l o g i c a l l y r e l e v a n t r o l e . R e g u l a t i o n o f E x o c r i n e P a n c r e a t i c F u n c t i o n The e x o c r i n e p a n c r e a s i s c o n t r o l l e d by b o t h n e u r a l and hormonal mechanisms. N e u r a l r e f l e x pathways have been d e f i n e d f o r t h e c e p h a l i c , g a s t r i c , and i n t e s t i n a l phases o f d i g e s t i o n t h a t e x e r t b o t h d i r e c t p a n c r e a t i c e f f e c t s j and modulate i n t e s t i n a l hormone r e l e a s e ( S i n g e r 1980, L o u i e 1986). T r a d i t i o n a l c o n c e p t s o f a s t i m u l a t o r y p a r a s y m p a t h e t i c system f u n c t i o n i n g t h r o u g h r e l e a s e o f a c e t y l c h o l i n e , and an i n h i b i t o r y s y m p a t h e t i c system r e l e a s i n g n o r e p i n e p h r i n e ( S m i t h 1981), have been m o d i f i e d by t h e f i n d i n g o f numerous, p e p t i d e -c o n t a i n i n g neurons w i t h i n t h e g u t t h a t e f f e c t i v e l y c o n s t i t u t e a t h i r d , p e p t i d e r g i c nervous system ( P o l a k 1979). G a s t r i n and c h o l e c y s t o k i n i n (CCK) have b o t h been i d e n t i f i e d i n t h e vagus ( R e h f e l d 1983) and s u b s t a n c e P, e n k e p h a l i n , V I P , and CCK have 17 been found i n i n t r i n s i c p a n c r e a t i c n e r v e s ( L a r s s o n 1979b). A l t h o u g h changes i n e x o c r i n e s e c r e t i o n can be d e t e c t e d d u r i n g i n f u s i o n o f many o f t h e s e p e p t i d e s , i t remains l a r g e l y s p e c u l a t i v e as t o whether any r e p r e s e n t p h y s i o l o g i c a l e f f e c t s . P a v l o v ' s a c c o u n t a t t h e t u r n o f t h e c e n t u r y a s c r i b i n g p a n c r e a t i c s e c r e t i o n e x c l u s i v e l y t o n e u r a l c o n t r o l was a l m o s t i m m e d i a t e l y m o d i f i e d by B a y l i s s and S t a r l i n g . They r e p o r t e d i n 1902 t h e f i n d i n g o f a b l o o d - b o r n e p r i n c i p l e ( s e c r e t i n ) from d e n e r v a t e d i n t e s t i n e , r e l e a s e d by a c i d and c a p a b l e o f s t i m u l a t i n g p a n c r e a t i c s e c r e t i o n ( B a y l i s s 1902). S u b s e q u e n t l y , I v y and O l d b e r g (1928) and Harper and Raper (1943) r e p o r t e d what p r o v e d t o be a s i n g l e s u b s t a n c e (CCK) w i t h g a l l b l a d d e r m o t i l i t y and p a n c r e a t i c s e c r e t o r y e f f e c t s , r e s p e c t i v e l y . The work o f many i n v e s t i g a t o r s c h a r a c t e r i z i n g p h y s i o l o g i c a l a c t i o n s o f b o t h s e c r e t i n and c h o l e c y s t o k i n i n i n t h e p a n c r e a s has s i n c e d e m o n s t r a t e d t h e i r i m p o r t a n c e as t h e i two major hormonal s e c r e t a g o g u e s o f t h e p a n c r e a s . CCK i s a p o l y p e p t i d e hormone found i n b o t h t h e c e n t r a l and p e r i p h e r a l n e r v o u s sy s t e m s , and i n t h e g u t i n 39, 33, 12, 8, and 4 amino a c i d forms. The s m a l l e r s i z e s c o n s i s t o f C-t e r m i n a l s e c t i o n s o f t h e l a r g e r forms and c o n t a i n t h e s u l f a t e d t e t r a p e p t i d e . S u l f a t i o n o f t h i s fragment i s i m p o r t a n t f o r t h e p h y s i o l o g i c a l a c t i v i t y o f a l l m o l e c u l a r forms o f CCK, as w e l l as CCK a n a l o g u e s such as c a e r u l e i n ( R e h f e l d 1981). S t u d i e s on CCK r e l e a s e from t h e duodenum i n d i c a t e t h a t a l t h o u g h l a r g e r forms a r e p r e s e n t , most r e l e a s e i n t o t h e c i r c u l a t i o n i s o f 18 s m a l l e r (CCK-12, 8, and 4) p e p t i d e s ( R e h f e l d 1981). CCK has been l o c a l i z e d i m m u n o c y t o c h e m i c a l l y and by r e g i o n a l r e l e a s e s t u d i e s ( L a r s s o n 1978, Ko n t u r e k 1986) t o t h e p r o x i m a l g u t . M a j o r s t i m u l a n t s f o r CCK r e l e a s e i n t h e dog a r e f a t (Meyer 1974) and p r o t e i n d i g e s t i o n p r o d u c t s ( K o n t u r e k 1973) a c t i n g d i r e c t l y on t h e CCK c e l l o r v i a s h o r t c h o l i n e r g i c v a g o v a g a l r e f l e x e s ( L o u i e 1986). I n t e r s p e c i e s d i f f e r e n c e s a r e emphasized by t h e f i n d i n g t h a t , i n t h e r a t , o n l y i n t a c t p r o t e i n and n o t p r o t e i n h y d r o l y s a t e s , amino a c i d s , o r f a t s r a i s e d serum CCK l e v e l s ( L i d d l e 1986). I n t h e e x o c r i n e p a n c r e a s , CCK i n a l l i t s forms b u t w i t h d i f f e r e n t i a l a f f i n i t y (Szecowka 1985) b i n d s t o s p e c i f i c CCK r e c e p t o r s t o i n i t i a t e s e c r e t i o n ( W i l l i a m s 1981). A c t i o n s o f CCK i n t h e pan c r e a s may be d i v i d e d i n t o immediate r e s p o n s e s and t h o s e due t o c h r o n i c , l o n g - t e r m e x p o s u r e t o t h e hormone. CCK i s r e s p o n s i b l e f o r much o f t h e p r o t e i n - e n z y m e component o f p a n c r e a t i c j u i c e ( K o n t u r e k 1986) s e c r e t e d a c u t e l y i n r e s p o n s e t o a meal. Over l o n g e r p e r i o d s o f t i m e , b o t h in v i t r o and in vivo s t u d i e s have shown p a n c r e a t i c h y p e r t r o p h y and h y p e r p l a s i a i n response t o CCK, an e f f e c t , p o t e n t i a t e d by a d d i t i o n o f s e c r e t i n (Logsdon 1986, O t s u k i 1983). Both CCK and g a s t r i n a r e p r o b a b l y e s s e n t i a l s t i m u l i f o r n o r m a l growth o f t h e p a n c r e a s (Johnson 1976).^ S e c r e t i n i s a 27 amino a c i d member o f t h e g l u c a g o n - V I P f a m i l y o f p e p t i d e s t h a t r e q u i r e s an i n t a c t s t r u c t u r e f o r b i o l o g i c a c t i v i t y (Dockray 1979, Grossman 1969). Immunocytochemical 19 s t u d i e s have c o n f i r m e d B a y l i s s 7 and S t a r l i n g ' s o r i g i n a l f i n d i n g o f a p r o x i m a l g u t mucosal l o c a t i o n f o r s e c r e t i n c e l l s , r e l e a s i n g s e c r e t i n i n r e s p o n s e t o l u m i n a l a c i d a t a pH t h r e s h o l d o f 4.5 (Chey 1982). S e c r e t i n r e c e p t o r s on p a n c r e a t i c a c i n i can be d i s t i n g u i s h e d from s e p a r a t e VIP r e c e p t o r s (Robberecht 1982). S e c r e t i n and n e u r a l l y - d e r i v e d V I P a r e t h e major s e c r e t a g o g u e s f o r b i c a r b o n a t e and w a t e r s e c r e t i o n by t h e p a n c r e a t i c d u c t system ( K o n t u r e k 1976). R e g u l a t i o n o f I n s u l i n S e c r e t i o n C o n d i t i o n s w h i c h i n d u c e o r s u p p r e s s s e c r e t i o n by t h e e n d o c r i n e p a n c r e a s a r e n e c e s s a r i l y a l s o c o n d i t i o n s which d e t e r m i n e t h e a v a i l a b i l i t y o f i s l e t hormones, p a r t i c u l a r l y i n s u l i n , t o a c t on t h e e x o c r i n e p a n c r e a s . As w i t h t h e e x o c r i n e p a n c r e a s , n u t r i e n t , n e u r a l , and hormonal mechanisms c o n t r o l i n s u l i n r e l e a s e . I n s u l i n i s t h e p r i n c i p l e p e p t i d e hormone R e l e a s e d by t h e b e t a c e l l ( H e l l e r s t r o m 1984). C o n t a i n e d i n dense g r a n u l e s w i t h i n t h e c y t o p l a s m , i t i s r e l e a s e d by e x o c y t o s i s t h r o u g h an energy-r e q u i r i n g p r o c e s s ( H o w e l l 1984). G l u c o s e i s t h e c r i t i c a l n u t r i e n t c o n t r o l l i n g i n s u l i n r e l e a s e ( F a i n 1984). In v i t r o s t u d i e s i n t h e i s o l a t e d , p e r f u s e d p a n c r e a s p r e p a r a t i o n have de m o n s t r a t e d t h e c h a r a c t e r i s t i c b i m o d a l n a t u r e o f g l u c o s e -i n d u c e d i n s u l i n s e c r e t i o n ( C u r r y 1968). I n g e n e r a l , m e t a b o l i c f u n c t i o n s o f i n s u l i n i n t h e normal h o s t a r e based on maintenance o f b l o o d g l u c o s e w i t h i n a r e s t r i c t e d r a n g e . Thus g l u c o s e i t s e l f p r o v i d e s t i g h t f e e d b a c k c o n t r o l o f i n s u l i n 20 s e c r e t i o n . Whether t h e a c t i o n o f g l u c o s e i s a t a s u r f a c e r e c e p t o r o r i n t r a c e l l u l a r l y ( F a i n 1984), i t seems c l e a r t h a t g l u c o s e a c t s t h r o u g h a c a l m o d u l i n - d e p e n d e n t c a l c i u m second messenger system ( H o w e l l 1984). Other n u t r i e n t s s t i m u l a t i n g i s l e t i n s u l i n r e l e a s e a r e amino a c i d s such as a r g i n i n e , c a p a b l e o f a c t i n g s y n e r g i s t i c a l l y w i t h g l u c o s e . N e u r a l c o n t r o l o f i n s u l i n s e c r e t i o n a l t h o u g h c l e a r l y s u b o r d i n a t e t o g l u c o s e ( H o i s t 1983), s e r v e s t o i n t e g r a t e i s l e t f u n c t i o n w i t h o t h e r a n t i c i p a t e d o r o n g o i n g m e t a b o l i c f u n c t i o n s o f t h e h o s t . I s l e t i n n e r v a t i o n i s made up of a complex a r r a y o f c h o l i n e r g i c , a d r e n e r g i c , and p e p t i d e r g i c n e r v e s t h e s p e c i f i c f u n c t i o n s o f w h i c h a r e p o o r l y u n d e r s t o o d ( M i l l e r 1 981). V a g a l a f f e r e n t s have been shown t o m e d i a t e a weak c e p h a l i c phase o f i n s u l i n s e c r e t i o n (Simon 1986), b u t t h i s r e m a i n s c r i t i c a l l y dependent on g l u c o s e c o n c e n t r a t i o n . A d r e n e r g i c n e r v o u s i n p u t v i a t h e s p l a n c h n i c n e r v e s appears t o be l a r g e l y i n h i b i t o r y (Edward 1980). Immunohistochemical s t u d i e s have i d e n t i f i e d a v a r i e t y o f p e p t i d e r g i c f i b e r s i n b o t h i n t r a p a n c r e a t i c g a n g l i a and i s l e t s . V I P - , e n k e p h a l i n - , CCK4-, CGRP-, and G A B A - c o n t a i n i n g n e r v e s may be f o u n d i n t h e i s l e t s . P r e sumably, p e p t i d e r g i c i n n e r v a t i o n a l t e r s b e t a c e l l f u n c t i o n (Raghu 1984) b u t s p e c i f i c p h y s i o l o g i c a l c o n d i t i o n s f o r t h e i r r e l e a s e , a c t i o n , and s i g n i f i c a n c e a r e n o t d e f i n e d . Hormones s t i m u l a t i n g i n s u l i n r e l e a s e a r e termed i n c r e t i n s , s u b s t a n c e s a c t i n g s y n e r g i s t i c a l l y w i t h c i r c u l a t i n g g l u c o s e t o 21 s t i m u l a t e i n s u l i n r e l e a s e . G a s t r i c i n h i b i t o r y p o l y p e p t i d e ( G I P ) , a g u t p e p t i d e r e l e a s e d from t h e p r o x i m a l s m a l l bowel mucosa, i s t h e b e s t c h a r a c t e r i z e d " i n c r e t i n " . I t has demon s t r a t e d g l u c o s e - d e p e n d e n t , i n s u l i n o t r o p i c a c t i v i t y i n t h e i n t a c t h o s t ( P e d e r s o n 1975) as w e l l as i n t h e i s o l a t e d , p e r f u s e d p a n c r e a s model (Pederson 1979), i s o l a t e d i s l e t s ( S i e g e l 1985), and B - c e l l s ( F u j i m o t o 1981). O t h e r p o t e n t i a l i n c r e t i n s i n c l u d e CCK and s e c r e t i n ( K o f o d 1986). I f t h e y have a p h y s i o l o g i c a l r o l e i n t h e c o n t r o l o f i n s u l i n r e l e a s e , g u t i n c r e t i n s appear t o be a c t i n g t o a m p l i f y t h e d i r e c t e f f e c t s o f abs o r b e d n u t r i e n t s on i n s u l i n r e l e a s e . S i n c e t h e a v a i l a b i l i t y o f b o t h g l u c o s e and t h e v a r i o u s s t i m u l i f o r GIP, CCK, and s e c r e t i n r e l e a s e a r e dependent, i n p a r t , on b u f f e r i n g and d i g e s t i v e a c t i o n s o f p a n c r e a t i c j u i c e , one might h y p o t h e s i z e an i n d i r e c t feedback mechanism t h a t i n c o r p o r a t e s n u t r i e n t s , i n c r e t i n s , and e x o c r i n e f u n c t i o n v i a t h e i n s u l o a c i n a r a x i s i n a p o s i t i v e f eedback l o o p . D i r e c t c e l l - c e l l c o n n e c t i o n s between i s l e t c e l l s ( u s u a l l y o f t h e same t y p e ) c o m p r i s e a range o f t i g h t and gap j u n c t i o n s a f f o r d i n g t h e p o s s i b i l i t y o f c e l l - c e l l s i g n a l i n g v i a c h e m i c a l means o r e l e c t r i c a l c o u p l i n g . A u t o r a d i o g r a p h i c e v i d e n c e f o r i n s u l i n , g l u c a g o n , and s o m a t o s t a t i n - 1 4 r e c e p t o r s on each c e l l t y p e o f t h e i s l e t ( P a t e l 1982) r a i s e t h e p o s s i b i l i t y o f i n t r a -i s l e t p a r a c r i n e e f f e c t s . A p a r t from an u l t r a s h o r t feedback l o o p o f i n s u l i n i n h i b i t i n g i t s own s e c r e t i o n , ( D r a z n i n 1986), p h y s i c a l s e p a r a t i o n o f c e l l s and a c e n t r i f u g a l b l o o d f l o w 22 s u g g e s t t h a t i s o l a t e d i s l e t s t u d i e s showing g l u c a g o n s t i m u l a t i o n and s o m a t o s t a t i n s u p p r e s s i o n o f i n s u l i n r e l e a s e ( P e t e r s s o n 1985), may have l i t t l e p h y s i o l o g i c a l s i g n i f i c a n c e . E x t r a p a n c r e a t i c E f f e c t s o f I n s u l i n I n s u l i n a c t i o n s have been most e x t e n s i v e l y s t u d i e d i n l i v e r , m u s c le, and a d i p o s e t i s s u e . The e x i s t e n c e o f c e l l u l a r s u b s t r a t e s f o r i n s u l i n t h a t a r e u n i q u e t o d i f f e r e n t t i s s u e s , f o r example, i n t h e l i v e r , r a i s e s t h e p o s s i b i l i t y t h a t i n s u l i n e f f e c t s i n one t i s s u e may be s u b s t r a t e - s p e c i f i c and n o t r e a s o n a b l y e x t r a p o l a t e d t o o t h e r t i s s u e s . N e v e r t h e l e s s i n b r o a d t e r m s , d e t e c t i o n o f an i n t e r n a l l y c o n s i s t e n t r o l e f o r i n s u l i n t h r o u g h a l l r e s p o n s i v e t i s s u e s may be i n s t r u c t i v e . I n s u l i n has b o t h m e t a b o l i c and t r o p h i c e f f e c t s . As a m e t a b o l i c r e g u l a t o r , i n s u l i n i s r e s p o n s i b l e f o r d i s p e r s i o n and h a n d l i n g o f m e t a b o l i c ( p r i m a r i l y c a r b o h y d r a t e ) f u e l s , a c t i n g as t h e c r i t i c a l a n a b o l i c o r s t o r a g e hormone ( C a h i l l 1971). I n s u l i n s t i m u l a t e s g l u c o s e uptake and s t o r a g e by muscle and a d i p o s e t i s s u e s ( F a i n 1984). Hexose c a r r i e r s t r a n s l o c a t e t o t h e c e l l membrane under t h e i n f l u e n c e o f i n s u l i n , p i c k up g l u c o s e , t h e n d i s t r i b u t e i t e i t h e r as an energy s u b s t r a t e o r i n t o s t o r a g e . Depending on t h e t i s s u e , i n s u l i n c h a n n e l s e x c e s s g l u c o s e i n t o g l y c o g e n ( l i v e r and muscle) o r t r i g l y c e r i d e ( f a t ) . S i m u l t a n e o u s a c t i v a t i o n o f m u l t i p l e enzyme systems, some by p h o s p h o r y l a t i o n and o t h e r s by d e p h o s p h o r y l a t i o n , s u g g e s t s m u l t i p l e second messenger systems a r e i n v o l v e d (Denton 1981). I n s u l i n - s e n s i t i v e t i s s u e uptake 23 o f g l u c o s e a c c o u n t s f o r o n l y a s m a l l p r o p o r t i o n o f a l l g l u c o s e m e t a b o l i z e d . The c e n t r a l n ervous system (CNS), formed b l o o d e l e m e n t s , and r e n a l m e d u l l a , a l l a c c o u n t f o r t h r e e q u a r t e r s of t h e normal p o s t - a b s o r p t i v e c i r c u l a t i n g g l u c o s e uptake ( C r y e r 1985). Q u a n t i t a t i v e l y , t h e most i m p o r t a n t g l u c o - r e g u l a t o r y f u n c t i o n f o r i n s u l i n i n t h e p o s t - a b s o r p t i v e s t a t e i s i n h i b i t i o n o f h e p a t i c g l y c o g e n o l y s i s and g l u c o n e o g e n e s i s . I n s e e k i n g c e l l u l a r p r o c e s s e s i n t h e e x o c r i n e p a n c r e a s w h i c h might be i n s u l i n r e s p o n s i v e , one p o t e n t i a l r o l e f o r i n s u l i n might be p r o v i s i o n o f g l u c o s e as an energy s u b s t r a t e f o r a c i n a r b i o s y n t h e t i c f u n c t i o n , perhaps c o u n t e r b a l a n c e d by t h e u n i q u e l y h i g h c o n c e n t r a t i o n o f o t h e r i s l e t hormones, g l u c a g o n and s o m a t o s t a t i n . A n o t h e r i m p o r t a n t m e t a b o l i c r o l e f o r i n s u l i n i s r e g u l a t i o n o f p r o t e i n s y n t h e s i s . I n s u l i n has been shown t o d i r e c t l y s t i m u l a t e amino a c i d t r a n s p o r t , e s p e c i a l l y b r a n c h e d - c h a i n amino a c i d s i n t o s k e l e t a l muscle ( C a h i l l 1971). F u r t h e r , i n s k e l e t a l muscle i n s u l i n i s n e c e s s a r y f o r i n i t i a t i o n o f p r o t e i n s y n t h e s i s a t t h e ribosome ( J e f f e r s o n 1971) w h i l e i n t h e l i v e r , t r a n s c r i p t i o n o f a l b u m i n mRNA i s c r i t i c a l l y dependent on i n s u l i n ( J e f f e r s o n 1980) Thus i n s u l i n e f f e c t s i n v a r i o u s t i s s u e s i n c l u d e p r o m o t i o n o f i n t r a c e l l u l a r amino a c i d t r a n s p o r t , t r a n s c r i p t i o n , and t r a n s l a t i o n . Q u i t e a p a r t from e f f e c t s on s u b s t r a t e m e t a b o l i s m and p r o t e i n s y n t h e s i s , i n s u l i n s t i m u l a t e s DNA s y n t h e s i s and c e l l r e p l i c a t i o n i n s e v e r a l e x p e r i m e n t a l systems. S i m i l a r l y , 24 i n s u l i n p l a y s a p e r m i s s i v e b u t e s s e n t i a l r o l e i n many serum-f r e e , hormone-supplemented t i s s u e c u l t u r e systems ( S t r a u s 1984). S i g n i f i c a n t c l i n i c a l c o r r e l a t i o n f o r t h e s e e f f e c t s i s b e s t seen i n p o s t - r e s e c t i o n h e p a t i c r e g e n e r a t i o n which i s a b s o l u t e l y dependent on i n s u l i n ( S t a r z l 1983). The e x o c r i n e p a n c r e a s r e c e i v e s p o r t a l e f f l u e n t from i s l e t s j u s t as t h e l i v e r , i n d e e d a t h i g h e r i s l e t hormone c o n c e n t r a t i o n s , and may t h e r e f o r e a l s o depend on i n s u l i n as an e s s e n t i a l growth f a c t o r . I n s u l o a c i n a r A x i s I n a d d i t i o n t o t h e v a s c u l a r c o n n e c t i o n s from i s l e t t o a c i n u s w h i c h p r o v i d e a p h y s i c a l p a t h f o r t h e i n s u l o a c i n a r a x i s , s p e c i f i c s u r f a c e r e c e p t o r s must a l s o be p r e s e n t f o r s i g n a l t r a n s d u c t i o n . R a p i d and s p e c i f i c b i n d i n g o f 1 2 5 I - i n s u l i n has been shown i n i s o l a t e d mouse a c i n i , h a l f maximal a t two minutes and maximal a t t h i r t y m i n u t es ( K o r c 1978). S c a t c h a r d a n a l y s i s s u g g e s t e d t h a t two r e c e p t o r s were p r e s e n t , one o f low e r a f f i n i t y (Kd = 83 nM) and one o f h i g h e r a f f i n i t y (Kd = 1.67 nM). E l e c t r o n m i c r o s c o p i c (EM) a u t o r a d i o g r a p h y r e v e a l e d t h a t t h e i n i t i a l s i t e o f i n s u l i n b i n d i n g i n t h i s in v i t r o system was a t t h e a c i n a r c e l l b a s o l a t e r a l membrane ( G o l d f i n e 1983). I n s u l i n b i n d i n g i n t h e in vivo e x o c r i n e p ancreas a n a l y z e d by a u t o r a d i o g r a p h y and t i s s u e d i s t r i b u t i o n s t u d i e s showed p a n c r e a t i c i n s u l i n b i n d i n g p e r gram o f t i s s u e t o be second o n l y t o t h a t o f t h e l i v e r (Sakamoto 1984). I n t h i s s t u d y , b i n d i n g was s p e c i f i c f o r a c i n a r and d u c t c e l l s . 25 I n s u l i n r e c e p t o r s i n p a n c r e a t i c a c i n i appear t o have a s u b u n i t s t r u c t u r e s i m i l a r t o t h e a l p h a and b e t a s u b u n i t s d e f i n e d i n o t h e r t i s s u e s ( G o l d f i n e 1983). The i n f l u e n c e a l i g a n d - b o u n d r e c e p t o r has on s u r r o u n d i n g r e c e p t o r a f f i n i t y , termed " c o o p e r a t i v i t y " , i s a n o t h e r c h a r a c t e r i s t i c o f t h e i n s u l i n r e c e p t o r t h a t may be i m p o r t a n t i n t h e i n s u l o a c i n a r system. Most s t u d i e s o f t h e i n s u l i n r e c e p t o r i n e x t r a p a n c r e a t i c t i s s u e s show " n e g a t i v e c o o p e r a t i v i t y " , t h a t i s p a r t i a l r e c e p t o r occupancy i n h i b i t i n g f u r t h e r i n s u l i n o r o t h e r hormone b i n d i n g (Gammeltoft 1984). D o w n r e g u l a t i o n o f i n s u l i n r e c e p t o r number may a l s o o c c u r , as a consequence o f h i g h i n s u l i n c o n c e n t r a t i o n s . I s o l a t e d a c i n i s t u d i e s comparing e x p e r i m e n t a l l y d i a b e t i c and normal mouse a c i n i have shown u p r e g u l a t i o n w i t h d o u b l i n g o f i n s u l i n r e c e p t o r s i n t h e d i a b e t i c a n i m a l s , an e f f e c t r e v e r s i b l e by i n s u l i n t r e a t m e n t (M6ssner 1984). O t s u k i and W i l l i a m s (1983) s t u d i e d i s o l a t e d a c i n i o f S T Z - d i a b e t i c r a t s and found t h a t i n s u l i n p r e t r e a t m e n t d e c r e a s e d t h e a f f i n i t y o f one group and c a p a c i t y o f a n o t h e r group o f CCK r e c e p t o r s . U n t r e a t e d d i a b e t i c a n i m a l s i n c r e a s e d CCK-33 b i n d i n g o v e r normal r a t a c i n i b u t showed d e c r e a s e d s e n s i t i v i t y s u g g e s t i n g , i n a d d i t i o n , a r e v e r s i b l e p o s t - r e c e p t o r d e f e c t . E f f e c t s on t h e b i n d i n g and number o f s e c r e t a g o g u e r e c e p t o r s , and on b i n d i n g o f i n s u l i n i t s e l f , may be a n o t h e r s i t e o f i n s u l o a c i n a r a c t i v i t y . 2 6 The a n a t o m i c a l model o f a hormone b e i n g s e c r e t e d by c e l l s p h y s i c a l l y i n p r o x i m i t y t o t h e e f f e c t o r organ has s e v e r a l t e l e o l o g i c a l i m p l i c a t i o n s . F i r s t , i s l e t s may r e c e i v e feedback o f a non-hormonal n a t u r e from t h e e x o c r i n e p a n c r e a s t h a t r e q u i r e s c e l l t o c e l l p r o x i m i t y . Second, t h e e x o c r i n e t i s s u e may r e q u i r e i s l e t hormones a t v e r y h i g h c o n c e n t r a t i o n f o r p r o p e r f u n c t i o n , a c o n c e n t r a t i o n o n l y p r o v i d e d by c l o s e p r o x i m i t y . T h i r d , b o t h i s l e t and a c i n u s might need t o be i n a common v a s c u l a r o r n e u r o l o g i c w a t e r s h e d t o re s p o n d t o o t h e r s i g n a l s , hormonal, n e u r o l o g i c , o r n u t r i t i o n a l i n n a t u r e . L a s t l y , t h e y may remain i n p r o x i m i t y f o r e m b r y o l o g i c r e a s o n s , because o f a common c e l l o f o r i g i n . C h a r a c t e r i s t i c s r e q u i r e d t o demonstrate a d i r e c t e f f e c t by i n s u l i n on t h e e x o c r i n e p a n c r e a s i n c l u d e 1) i t s p r e s e n c e i n t h e g l a n d , 2) a pathway f o r passage t o t h e a c i n a r t i s s u e , 3) a p p r o p r i a t e c e l l s u r f a c e r e c e p t o r s , and 4) one o r more a c u t e l y r e s p o n s i v e b i o s y n t h e t i c o r s e c r e t o r y systems l i n k e d t o t h e r e c e p t o r . As d i s c u s s e d , p a r t s 1-3 have been s a t i s f i e d by o t h e r w o r k e r s . The i n t e n t o f t h i s work was t o s a t i s f y t h e f o u r t h c r i t e r i o n and show p h y s i o l o g i c a l r e l e v a n c e f o r t h e i n s u l o a c i n a r a x i s by d e m o n s t r a t i n g s e c r e t o r y p a r a m e t e r s i n s e v e r a l model systems f o r an e x o c r i n e r e s p o n s e t o i n s u l i n . S p e c i f i c Aims 1. To d e v e l o p an i s o l a t e d , p e r f u s e d pancreas system i n t h e r a t f o r measurement o f e x o c r i n e p a n c r e a t i c f u n c t i o n . 2 . To demonstrate t h e i n t e g r i t y o f t h e p e r f u s e d r a t p a n c r e a s model f o r measurement o f b a s a l and s t i m u l a t e d e x o c r i n e s e c r e t i o n by major s e c r e t a g o g u e s . 3. To e v a l u a t e t h e a c u t e e x o c r i n e r e s p o n s i v e n e s s o f t h e p e r u s e d r a t p a n c r e a s t o b o t h endogenous and exogenous i n s u l i n , under c o n d i t i o n s o f d i f f e r e n t e x o c r i n e s e c r e t a g o g u e s . 4. To e v a l u a t e t h e s p e c i f i c p a r a m e t e r s o f a n i m a l age, s e c r e t o r y s t a t u s , p r i o r ambient i n s u l i n e x p o s u r e , and a c i n a r c e l l d i s t r i b u t i o n about i s l e t s - f o r t h e i r r e s p e c t i v e i m p o r t a n c e i n d e t e r m i n i n g t h e i n s u l i n r e s p o n s i v e n e s s o f t h e e x o c r i n e p a n c r e a s , u t i l i z i n g an in vitro p r e p a r a t i o n o f p a n c r e a t i c a c i n i . 28 METHODS Two in v i t r o p r e p a r a t i o n s o f t h e r a t pan c r e a s were used i n t h e s e i n v e s t i g a t i o n s . E x p e r i m e n t a l P r e p a r a t i o n s Isolated Perfused Pancreas A n i m a l s A l l p e r f u s i o n e x p e r i m e n t s were c a r r i e d o u t w i t h male W i s t a r r a t s w e i g h i n g from 250-3 25 g. A n i m a l s were e i t h e r p r o v i d e d by t h e l o c a l b r e e d i n g program o f t h e U n i v e r s i t y o f B r i t i s h C olumbia o r p u r c h a s e d from C h a r l e s R i v e r , I n c . ( W i l m i n g t o n , MA) and were m a i n t a i n e d under t e m p e r a t u r e and l i g h t - c o n t r o l l e d c o n d i t i o n s i n group cages o f 6 t o 8 a n i m a l s w i t h s t a n d a r d r a t chow and wa t e r f r e e l y a v a i l a b l e . A n i m a l s were a c c l i m a t i z e d f o r a minimum o f 3 days p r i o r t o use f o r p e r f u s i o n . A p p r o x i m a t e l y 12 t o 18 h p r i o r t o a p e r f u s i o n e x p e r i m e n t , f o o d b u t n o t wa t e r was removed from t h e cages t o s i m p l i f y t h e d i s s e c t i o n p r o c e d u r e and t o reduce a d d i t i o n a l v a r i a t i o n i n p a n c r e a t i c f u n c t i o n due t o f e e d i n g . D e s p i t e f a s t i n g , b o t h e n d o c r i n e and e x o c r i n e b a s a l r e s p o n s e s were found t o o c c a s i o n a l l y v a r y c o n s i d e r a b l y from group t o group w i t h o u t c l e a r e x p l a n a t i o n . A p p a r a t u s The components o f t h e p e r f u s i o n a p p a r a t u s a r e l i s t e d . 29 1. 95% 0 2 - 5 % C 0 2 t a n k and r e g u l a t o r 2. M a g n e t i c s t i r r e r s and b a r s 3. Dual c h a n n e l a d j u s t a b l e r o l l e r pump ( C l a y Adams) 4. T h e r m o s t a t i c a l l y c o n t r o l l e d , custom h e a t i n g u n i t 5. I n - l i n e p r e s s u r e t r a n s d u c e r 6. Bubble t r a p 7. Sidearm a c c e s s p o r t 8. Heated i s o l a t e d organ p l a t f o r m 9. F r a c t i o n c o l l e c t o r (LKB) 10. F l a s k s , t u b i n g P e r f u s i o n s o l u t i o n s E l e c t r o l y t e components, a l l o f s t a n d a r d r e a g e n t g r a d e , and s o l u t e components o f t h e p e r f u s i o n s o l u t i o n ( p e r f u s a t e ) a r e l i s t e d i n T a b l e 1 o f t h e Appendix. The p e r f u s a t e c o n s i s t e d o f a b u f f e r c o n t a i n i n g b o v i n e serum a l b u m i n (BSA, 0.2%) and d e x t r a n ( 3 % ) , t h e BSA (RIA Grade) t e s t e d on a l o t by l o t b a s i s f o r a c c u r a c y w i t h t h e i n s u l i n and s o m a t o s t a t i n radioimmunoassays. D e x t r a n was added t o p r o v i d e a d d i t i o n a l c o l l o i d o s m o t i c p r e s s u r e w h i c h was p a r t i c u l a r l y i m p o r t a n t when w o r k i n g w i t h t h e e x o c r i n e p a n c r e a s . E a r l y edema f o r m a t i o n i n t h e p e r f u s e d , i s o l a t e d p a n c r e a s u s u a l l y r e s u l t e d from o u t f l o w o b s t r u c t i o n due t o t e c h n i c a l d i f f i c u l t i e s w i t h t h e p o r t a l v e i n o u t f l o w c a n n u l a t h a t c o u l d be c o r r e c t e d by ad j u s t m e n t o f t h e c a n n u l a . However, s e v e r e l y edematous specimens were g e n e r a l l y d i s c a r d e d i m m e d i a t e l y . P e r f u s i o n b u f f e r c o n c e n t r a t e ( S t o c k ) I s o l a t e d o r gan p e r f u s i o n was c a r r i e d o u t w i t h a b i c a r b o n a t e b u f f e r t h a t m a i n t a i n e d a pH o f 7.4 when s a t u r a t e d w i t h 95% 0 2 -5% C 0 2 . P e r f u s i o n b u f f e r was d e r i v e d from a f o u r t e e n - f o l d c o n c e n t r a t e d s t o c k s o l u t i o n made monthly and s t o r e d a t 4° C. Reagent-grade components made up t h e c o n s t i t u e n t s t o c k s o l u t i o n s i n t h e perfusion buffer concentrate: K C l C a C l 2 MgS0 4-7H 20 K H 2 P 0 4 285 ml 243 ml 78 ml 97 ml [STOCK] 154 mM 102.7 mM 154 mM 154 mM combined t o a t o t a l volume o f 703 m i l l i l i t e r s . The f i n a l c o n c e n t r a t i o n s o f s o l u t e s i n t h e perfusion buffer concentrate were: G l u c o s e c o n c e n t r a t e ( S t o c k ) When n e c e s s a r y f o r e x p e r i m e n t a l p u r p o s e s , t h e g l u c o s e c o n c e n t r a t i o n i n t h e p e r f u s a t e was a l t e r e d by a d d i n g v a r y i n g amounts o f a g l u c o s e s t o c k s o l u t i o n (308 mM) made from a comme r c i a l 50% (2.8 M) i n t r a v e n o u s g l u c o s e made up i n o d i s t i l l e d H 20 b i m o n t h l y and s t o r e d a t 4 C. P e r f u s a t e c o m p o s i t i o n ( f o r m u l a t i o n ) No more t h a n 12 h p r i o r t o p e r f u s i o n , d e x t r a n and BSA components o f p e r f u s a t e were d i s s o l v e d i n normal s a l i n e (154 mM i n d e i o n i z e d H 2 0 ) . Imm e d i a t e l y p r i o r t o p e r f u s i o n , f i n a l b u f f e r p r e p a r a t i o n was c a r r i e d o u t by a d d i n g , p e r l i t e r o f p e r f u s a t e : K C l C a C l 2 MgS0 4-7H 20 K H 2 P 0 4 62.4 mM 35.5 mM 17.1 mM 21.25 mM NaHC0 3 Perfusion buffer cone. 162.5 ml 70.3 ml [STOCK] 154 mM 31 t o d i s s o l v e d d e x t r a n and BSA. G l u c o s e (308 mM) and s a l i n e (154 mM) were added t o b r i n g t h e volume t o 1 1. F i n a l perfusation buffer solution c o n s t i t u e n t c o n c e n t r a t i o n s were: K C l 4.4 mM C a C l 2 2.5 mM MgS0 4-7H 20 1.2 mM K H 2 P 0 4 1.5 mM NaHC0 3 25 mM NaCI 120 mM D e x t r a n 3 % BSA 0.2 % G l u c o s e (308 mM) was added t o a c h i e v e t h e d e s i r e d g l u c o s e c o n c e n t r a t i o n (3.25 ml s t o c k / 1 p e r f u s a t e , p e r mM f i n a l g l u c o s e c o n c e n t r a t i o n ) and t h e b u f f e r was a n a l y z e d t o v e r i f y t h e g l u c o s e c o n c e n t r a t i o n t o w i t h i n 10% o f t h e i n t e n d e d v a l u e . H e p a r i n The i n f u s i o n c a n n u l a was p r e f i l l e d w i t h 2-3 ml o f h e p a r i n t o p r e v e n t c l o t t i n g p r i o r t o washout o f b l o o d . H e p a r i n s t o c k (14.3 x 1 0 2 U/ml) was s t o r e d a t 4 C and d i l u t e d 1:50 i n / normal s a l i n e t o a f i n a l c o n c e n t r a t i o n o f 285 U/ml. I s o l a t i o n o f pancreas The s u r g i c a l i s o l a t i o n o f t h e p a n c r e a s i n p r e p a r a t i o n f o r p e r f u s i o n was a c c o m p l i s h e d by s e q u e n t i a l l y r e s t r i c t i n g b l o o d f l o w t o o t h e r v i s c e r a and t o t h e l o w e r e x t r e m i t i e s . When t h e i n f u s i o n c a n n u l a was p o s i t i o n e d i n t h e i s o l a t e d a o r t i c segment o p p o s i t e t h e c e l i a c and s u p e r i o r m e s e n t e r i c a r t e r i a l o r i g i n s , i s o l a t e d p e r f u s i o n o f t h e duodenum and p a n c r e a s w i t h venous o u t f l o w v i a t h e p o r t a l v e i n was a c h i e v e d (Penhos 1969). 3 2 I n s t r u m e n t s used i n t h e d i s s e c t i o n a r e l i s t e d i n t h e Appendix, T a b l e 2. A n i m a l s were a n e s t h e t i z e d w i t h p e n t o b a r b i t a l (0.4 mg/kg body w e i g h t i n t r a p e r i t o n e a l l y ( i . p . ) ) on a he a t e d p l a t f o r m and t h e abdomen was opened from x i p h o i d t o p u b i s . The c o l o n was i s o l a t e d and l i g a t e d (3-0 s i l k ) i n t h e r e g i o n o f t h e s i g m o i d . The a o r t a was v a s c u l a r l y i s o l a t e d above t h e b i f u r c a t i o n w i t h s u s p e n s o r y t i e s , l i g a t e d d i s t a l l y , and t h e h e p a r i n i z e d c a n n u l a o f p o l y e t h y l e n e (PE-120) i n s e r t e d t o t h e l e v e l o f t h e c e l i a c o r i g i n . B o t h r e n a l v a s c u l a r p e d i c l e s i n c l u d i n g a d r e n a l v e s s e l s were l i g a t e d and t h e a o r t a , i m m e d i a t e l y below t h e diaphragm, was i s o l a t e d w i t h an u n t i e d l i g a t u r e f o r subsequent p r o x i m a l v a s c u l a r c o n t r o l . The s p l e e n was r e s e c t e d f o l l o w e d by t h e e n t i r e s m a l l bowel e x c e p t i n g t h e duodenum. The esophagus, l e f t g a s t r i c a r t e r y , and vagus n e r v e s were a l l l i g a t e d i n c o n t i n u i t y and d i v i d e d and t h e stomach was removed by t r a n s e c t i n g t h e p y l o r u s . A duodenual d r a i n was p l a c e d by l i g a t i n g t h e f i r s t p o r t i o n o f t h e duodenum around a h o l l o w t u b e . The p a n c r e a t i c d u c t was l i g a t e d a t t h e duodenum and c a n n u l a t e d (PE-50) p r o x i m a l l y i n t h e b i l e d u c t r e g i o n a f t e r l i g a t i o n o f t h e b i l e d u c t b i f u r c a t i o n . A l t h o u g h t h i s t e c h n i q u e r e q u i r e d p a n c r e a t i c j u i c e t o f l o w i n a r e t r o g r a d e manner toward t h e l i v e r , no v a l v e s e x i s t i n t h e p a n c r e a t i c o - b i l i a r y d u c t system and more r e l i a b l e e x o c r i n e s e c r e t i o n was o b t a i n e d . P e r f u s e d b u f f e r s o l u t i o n was c o l l e c t e d by i n s e r t i o n o f a c a t h e t e r i n t h e p o r t a l v e i n a f t e r l i g a t i o n o f t h e p r o x i m a l a o r t a and t r a n s e c t i o n o f t h e t o r s o a t 33 t h e l e v e l o f t h e l i v e r . A l l p e r f u s i o n s were c a r r i e d o u t on a h e a t e d p l a t f o r m . P e r f u s i o n p r o c e d u r e P e r f u s a t e s o l u t i o n was p r e p a r e d f r e s h d a i l y as p r e v i o u s l y d e s c r i b e d . One t o t h r e e hundred m i l l i l i t e r s o f p e r f u s a t e were c o n t i n u o u s l y s t i r r e d and b u b b l e s a t u r a t e d w i t h 95% 0 2 - 5% C 0 2 t h e n c h a n n e l l e d s e r i a l l y t h r o u g h a r o l l e r pump, a h e a t i n g b l o c k , and a b u b b l e t r a p . The i n t e n d e d f l o w r a t e f o r a l l p e r f u s i o n e x p e r i m e n t s was 3 ml/min and was c a l i b r a t e d by a t i m e d volume measurement. The p o r t a l venous o u t p u t was measured t o w i t h i n 0.1 ml so t h a t t o t a l hormone r e c o v e r y o v e r t i m e c o u l d be c a l c u l a t e d . P e r f u s a t e l o s s due t o l e a k a g e from t h e p r e p a r a t i o n was v a r i a b l e and t h e i n f u s i o n r a t e was a d j u s t e d t o m a i n t a i n a c o n s t a n t c o l l e c t e d o u t p u t . I f p e r f u s a t e l e a k a g e exceeded 0.5-1.0 ml/min, t h e a n i m a l p r e p a r a t i o n was d i s g a r d e d . The p e r f u s i o n a p p a r a t u s was a r r a n g e d so t h a t s i d e a r m p o r t s e n t e r e d t h e f l o w p a t h a f t e r t h e bu b b l e t r a p , p e r m i t t i n g i n - l i n e p r e s s u r e and t e m p e r a t u r e measurement f o r feedback c o n t r o l t o t h e h e a t i n g u n i t . W h i l e s e p a r a t e p e r f u s a t e volumes c o n t a i n i n g i n e x p e n s i v e r e a g e n t s such as g l u c o s e o r a r g i n i n e c o u l d be p r e p a r e d and r u n t h r o u g h t h e e n t i r e p e r f u s i o n a p p a r a t u s , most s e c r e t a g o g u e s were p r e p a r e d a t h i g h c o n c e n t r a t i o n i n p e r f u s a t e and i n f u s e d v i a a s i d e a r m (0.1 t o 0.2 ml/min). A l l s y r i n g e s and t u b i n g f o r t h e s i d e a r m were e q u i l i b r a t e d w i t h p e p t i d e t o a v o i d 34 a d s o r p t i o n l o s s and s e p a r a t e , complete s e t s o f a p p a r a t u s were k e p t f o r each p e p t i d e . Sample c o l l e c t i o n F o r most p r e p a r a t i o n s , p e r f u s i o n was c a r r i e d out f o r 15 t o 20 min p r i o r t o t h e t e s t p e r i o d t o c l e a r b l o o d and e q u i l i b r a t e t h e system. P e r f u s a t e samples were c o l l e c t e d from t h e p o r t a l v e i n c a n n u l a on i c e a t 5 min i n t e r v a l s and, a f t e r m i x i n g and volume measurement, were a l i q u o t e d i n t o p r e c o o l e d , 12 x 75 mm o b o r o s i l i c a t e t u b e s f o r immediate s t o r a g e a t -20 C. Samples of p e r f u s a t e s t o c k were a l s o f r o z e n f o r c o n t r o l and sample d i l u t i o n p u r p o s e s . A s s a y s o f i n s u l i n and s o m a t o s t a t i n i n t h e p e r f u s a t e were c a r r i e d o u t w i t h i n 2 weeks of t h e e x p e r i m e n t i n most c a s e s . P a n c r e a t i c j u i c e was c o l l e c t e d i n s i l i c o n i z e d , c a l i b r a t e d 10 til m i c r o p i p e t s p o s i t i o n e d i n a s w i v e l clamp a g a i n s t t h e e x o c r i n e o u t f l o w c a n n u l a . A f t e r e q u i l i b r a t i o n o f t h e pancreas p r e p a r a t i o n , samples were c o l l e c t e d a t 5 min i n t e r v a l s and blown i n t o 0.5 ml o f normal s a l i n e i n s i l i c o n i z e d 12 x 75 mm o g l a s s t u b e s . These v i a l s were s t o r e d f r o z e n a t -20 C f o r l a t e r p r o t e i n and enzyme d e t e r m i n a t i o n . Pancreatic Acini Preparation P a n c r e a t i c a c i n i a r e t h e f u n c t i o n a l u n i t s o f t h e e x o c r i n e p a n c r e a s . A c u t e l y h a r v e s t e d a c i n i were d i s p e r s e d i n t o s u s p e n s i o n t h r o u g h c o l l a g e n a s e d i g e s t i o n and m e c h a n i c a l d i s r u p t i o n . The a c i n i s u s p e n s i o n s r e t a i n e d t h e i r 35 r e s p o n s i v e n e s s t o s e c r e t a g o g u e s and had a normal a p i c a l -b a s o l a t e r a l o r i e n t a t i o n . B u f f e r p r e p a r a t i o n Reagents and equipment Reagents and equipment a r e l i s t e d i n T a b l e 3 o f t h e Appendix. A c i n i i s o l a t i o n b u f f e r P a n c r e a t i c a c i n i were i s o l a t e d by c o l l a g e n a s e d i g e s t i o n o f t h e d i s s e c t e d r a t p a n c r e a s . The d i g e s t i o n was c a r r i e d o u t i n a K r e b s - H e n s l e i t b u f f e r (KHB) c o n t a i n i n g g l u c o s e (200 mg%) and amino a c i d s u b s t r a t e ( W i l l i a m s 1984). The KHB was p r e p a r e d t o a f i n a l volume o f 100 ml w i t h d i s t i l l e d H 20 and was made up o f : [ S t o c k ] anhydrous D-glucose amino a c i d s o l u t i o n L - g l u t a m i n e s o l u t i o n NaHC0 3 NaCl K C l N a 2HP0 4 M g C l 2 200 mg 2 ml 2 ml 6.5 ml 4.65 ml 0.5 ml 0.5 ml 0.5 ml 0.1 M 0.5 M 2.36 M 0.94 M 0.2 M 0.48 M # # 50X c o n c e n t r a t e The f i n a l c o n s t i t u e n t c o n c e n t r a t i o n s i n t h e KHB were: anhydrous D-glucose amino a c i d s o l u t i o n L - g l u t a m i n e s o l u t i o n NaHC0 3 NaCl K C l N a 2HP0 4 M g C l 2 11.1 mM s t a n d a r d 2 mM 1 mM 0.11 M 4.6 mM 1 mM 2.4 mM 36 The KHB was e q u i l i b r a t e d w i t h 95% 0 2 - 5% C 0 2 and soybean t r y p s i n i n h i b i t o r (SBTI, 10 mg%) was added t o p r e v e n t t r y p t i c d i g e s t i o n o f c e l l s o r s e c r e t a g o g u e s by s e c r e t e d t r y p s i n . Three s e p a r a t e i s o l a t i o n b u f f e r s were t h e n p r e p a r e d u s i n g t h e KHB as a s o u r c e b u f f e r . The digestion buffer solution was made up w i t h : KHB 10 ml C a C l 2 (67.7 mM) 20 ul BSA 20 mg c o l l a g e n a s e 1.2 mg The f i n a l C a C l 2 c o n c e n t r a t i o n o f t h e d i g e s t i o n s o l u t i o n was 135.4 uVL. Nytex rinse s o l u t i o n , used t o wash d i s r u p t e d a c i n i t h r o u g h t h e n y t e x f i l t e r i n t h e f i r s t p u r i f i c a t i o n s t e p , was made up w i t h : KHB 10 ml C a C l 2 (67.7 mM) 20 /ul BSA 100 mg The f i n a l C a C l 2 c o n c e n t r a t i o n o f t h e r i n s e s o l u t i o n was 135.4 /iM. A centrifuge wash s o l u t i o n , used t o wash a c i n i t h r o u g h s e v e r a l c e n t r i f u g a t i o n c y c l e s , was made up w i t h : KHB 50 ml C a C l 2 (677 mM) 50 / i l BSA 2 g The f i n a l C a C l 2 c o n c e n t r a t i o n o f t h e wash s o l u t i o n was 677 jxM. I n c u b a t i o n b u f f e r A c i n i were i n c u b a t e d i n a HEPES b u f f e r c o n t a i n i n g v a r i o u s d i s s o l v e d s e c r e t a g o g u e s . E x o c r i n e r e s p o n s e s were measured by q u a n t i f y i n g enzyme (amylase) s e c r e t i o n i n t o t h e medium. The 37 HEPES buffer was made up t o a f i n a l volume o f 200 ml w i t h d i s t i l l e d H 20 and i n c l u d e d : [STOCK] anhydrous D-•glucose 400 mg L - g l u t a m i n e s o l u t i o n 4. 0 ml 0.1 M K C l 1. 0 ml 0.94 M Na 2HP0 4 1. 0 ml 0.2 M M g C l 2 0. 5 ml 0.48 M NaCl 10. 8 ml 2.36 M NaOH 1. 0 ml 1.0 M HEPES 20 ml 0.1 M CaCl-> 0. 5 ml 0.677 M The f i n a l c o n c e n t r a t i o n s o f c o n s t i t u e n t s i n t h e HEPES b u f f e r i n c l u d e d : 11.1 mM s t a n d a r d 2.34 mM 70 mM 0.6 mM 1.69 mM 70 mM 0.5 mM 10 mM A f t e r m i x i n g , t h e HEPES b u f f e r was e q u i l i b r a t e d w i t h 100% 0 2 and t h e pH was a d j u s t e d t o 7.6 w i t h NaOH (1 M). Incubation medium f o r t h e p u r i f i e d a c i n i was based on t h e HEPES b u f f e r and was made by a d d i n g BSA (1 g%) and SBTI (10 mg%) t o HEPES b u f f e r . I s o l a t i o n p r o c e d u r e Rat p a n c r e a s e s were r a p i d l y h a r v e s t e d a f t e r s a c r i f i c i n g t h e a n i m a l by d e c a p i t a t i o n . S e v e r a l r a t s were u s u a l l y used i n each e x p e r i m e n t , p o o l i n g p a n c r e a s e s and a c i n i i n t h e c o u r s e o f p r e p a r a t i o n . F a t was trimmed from t h e p a n c r e a s and t h e n t h e pa n c r e a s was b l o t t e d d r y and weighed. A t u b e r c u l i n s y r i n g e anhydrous D-glucose L - g l u t a m i n e s o l u t i o n K+ N a + M g + 2 Ca+2 C l ~ P 0 4 - 3 HEPES 38 and 27 gauge n e e d l e were used t o i n j e c t digestion buffer solution (5 ml) c o n t a i n i n g c o l l a g e n a s e i n t o t h e p a n c r e a t i c parenchyma u n t i l a l l i n d i v i d u a l l o b u l e s were v i s i b l y s e p a r a t e d . The d i g e s t i o n s t e p c o n t i n u e d i n a 25 ml p o l y c a r b o n a t e f l a s k c o n t a i n i n g e x c e s s d i g e s t i o n b u f f e r , gassed w i t h 95% 0 2 - 5% C 0 2 , and p l a c e d i n a s h a k i n g water b a t h (120 c y c / m i n ) . A f t e r 10 min, t h e medium was r e p l a c e d w i t h f r e s h d i g e s t i o n b u f f e r , t h e f l a s k g a s s e d a g a i n , and r e p l a c e d i n t h e b a t h f o r an a d d i t i o n a l 40 min. F o l l o w i n g t h e d i g e s t i o n phase, SBTI (5 mg) was added t o t h e f l a s k and t h e a c i n i were d i s p e r s e d m e c h a n i c a l l y by s e r i a l f o r c e f u l passage t h r o u g h p r o g r e s s i v e l y n a r r o w e r - t i p p e d , 10 ml p o l y p r o p y l e n e p i p e t s (2.4 mm t o 0.9 mm t i p d i a m e t e r ) . The r e s u l t i n g s u s p e n s i o n o f d i s p e r s e d a c i n i was washed t h r o u g h 150 n Nytex c l o t h w i t h t h e Nytex rinse solution and l a y e r e d o v e r centrifuge wash solution i n c o n i c a l l y t a p e r e d , 12 ml p l a s t i c c e n t r i f u g e t u b e s . A c i n i were p e l l e t e d by c e n t r i f u g a t i o n f o r f o u r min a t 50g on a bench t o p c e n t r i f u g e a f t e r w h i c h t h e p e l l e t s were combined and resuspended, t h e n s u b j e c t e d t o two more wash c y c l e s . F o l l o w i n g t h e f i n a l wash c y c l e , a c i n i were resuspended i n t h e p r e v i o u s l y d e s c r i b e d incubation medium f o r a p r e i n c u b a t i o n p e r i o d . Each f l a s k was g a s s e d w i t h 0 2 and p l a c e d i n a s h a k i n g o w a t e r b a t h (37 C, 60 cyc/min) f o r between 30 min and 4 h, depending on t h e e x p e r i m e n t . I n p a r t , t h e p r e i n c u b a t i o n s t e p a l l o w e d a c i n i t o r e c o v e r and s t a b i l i z e a f t e r t h e i n c u b a t i o n p r o c e d u r e . Longer p e r i o d s o f p r e i n c u b a t i o n ( w i t h p e r i o d i c 39 r e g a s s i n g ( 0 2 ) o f t h e s o l u t i o n ) were sometimes used t o expose a c i n i t o d i f f e r e n t e x p e r i m e n t a l c o n d i t i o n s f o r p r o l o n g e d p e r i o d s p r i o r t o t h e s e c r e t o r y t e s t p e r i o d . A f t e r p r e i n c u b a t i o n , clumped a c i n i were d i s p e r s e d by g e n t l e p i p e t i n g t h e n c e n t r i f u g e d a t 50g and resuspended i n f r e s h i n c u b a t i o n medium. M i c r o s c o p i c e x a m i n a t i o n f o r c e l l v i a b i l i t y and f o r a c i n i d e n s i t y was c a r r i e d o u t a t t h i s t i m e . C e l l s t a k i n g up t r y p a n b l u e i n t r a c e l l u l a r l y ( 0 . 5 % , Sigma) were c o n s i d e r e d n o n v i a b l e and u s u a l l y r e p r e s e n t e d l e s s t h a n 5% o f t h e t o t a l c e l l number. A c i n i s u s p e n s i o n s w i t h o v e r 10% n o n v i a b l e c e l l s were d i s c a r d e d . I n c u b a t i o n p r o c e d u r e D u r i n g t h e i n c u b a t i o n p r o c e d u r e , a c i n i were suspended i n i n c u b a t i o n b u f f e r t o a f i n a l t o t a l c e l l p r o t e i n d e n s i t y o f 0.2 t o 0.3 mg/ml. Two m i l l i l i t e r a l i q u o t s o f a c i n i s u s p e n s i o n were p l a c e d i n t o p o l y c a r b o n a t e (25 ml) f l a s k s and v e h i c l e o r an e q u a l volume o f a p p r o p r i a t e l y c o n c e n t r a t e d t e s t s u b s t a n c e was added t o t h e f l a s k . S e p a r a t e f l a s k s were used f o r each t e s t c o n d i t i o n i n d u p l i c a t e o r t r i p l i c a t e . F l a s k s were mixed by g e n t l e s w i r l i n g , g a s s e d w i t h 0 2 , and p l a c e d i n t h e s h a k i n g o w a t e r b a t h (37 C, 60 c y c / r a i n ) . By f o l l o w i n g a s p e c i f i c t i m e t a b l e , t h e f l a s k s were l o a d e d , i n c u b a t e d , and sampled i n a s e q u e n t i a l f a s h i o n b u t each h a v i n g an e q u a l d u r a t i o n o f i n c u b a t i o n . I n a l l a c i n i e x p e r i m e n t s , t h e i n d e x o f t h e e x o c r i n e r e s ponse was r e l e a s e o f amylase o r l i p a s e i n t o t h e i n c u b a t i o n medium. 40 A t t e m p t s a t e x c l u d i n g t h e t r y p s i n i n h i b i t o r (SBTI) and measuring t r y p s i n r e l e a s e i n t o t h e medium were u n s u c c e s s f u l ; no t r y p s i n c o u l d be d e t e c t e d . S i n c e a c i n i c o n t i n u a l l y r e l e a s e d amylase even b e f o r e a p p l i c a t i o n of t h e s e c r e t a g o g u e -due t o some c o m b i n a t i o n o f i n j u r y and b a s a l s e c r e t i o n -measurement o f p r e t r e a t m e n t background amylase c o n c e n t r a t i o n was n e c e s s a r y . T h i s was a c c o m p l i s h e d by c o l l e c t i n g samples from t h e s t o c k a c i n i s u s p e n s i o n f l a s k f o r amylase a s s a y b e f o r e , d u r i n g , and a f t e r a l i q u o t i n g i n c u b a t i o n f l a s k s . By assuming a l i n e a r s e c r e t i o n between each o f t h e measured v a l u e s , i n t e r p o l a t i o n v a l u e s f o r p r e t r e a t m e n t amylase were d e r i v e d f o r each, i n d i v i d u a l l y i n c u b a t e d , f l a s k . These v a l u e s were s u b t r a c t e d from t h e f i n a l t o t a l amylase v a l u e s t o g i v e t h e n e t amylase r e l e a s e d d u r i n g t h e t e s t p e r i o d . Sample c o l l e c t i o n Amylase r e l e a s e was e x p r e s s e d i n two forms, as a p e r c e n t a g e o f t h e t o t a l c e l l c o n t e n t o f amylase (% amylase c o n t e n t ) which a l l o w e d comparison o f s e c r e t a g o g u e e f f i c a c y , and as u n i t s o f amylase i n t h e medium p e r mass o f t o t a l c e l l p r o t e i n (uU/uq  c e l l p r o t e i n ) w h i c h a l l o w e d c o m p a r i s o n o f changes i n b o t h t o t a l c e l l amylase c o n t e n t , and amylase r e l e a s e under d i f f e r e n t c o n d i t i o n s . The background amylase c o n c e n t r a t i o n was d e t e r m i n e d by c e n t r i f u g i n g 1 ml a l i q u o t s o f s t o c k a c i n i s u s p e n s i o n i n 1 . 5 ml Eppendorf t u b e s a t i n t e r v a l s and s t o r i n g t h e s u p e r n a t a n t on i c e u n t i l a s s a y . The 4 a c i n i p e l l e t s l e f t b e h i n d a f t e r 41 removing supernatant f o r background amylase were washed i n cold s a l i n e then u l t r a s o n i c a l l y disrupted i n cold, d i s t i l l e d H 20. Amylase and t o t a l c e l l protein (by the method of Lowry (1951)) measured from these disrupted c e l l s allowed c a l c u l a t i o n of t o t a l c e l l amylase content and amylase content per ng c e l l protein. Units of amylase were calculated by converting absorbance values f o r the procion yellow-starch reagent assay through an amylase standard curve (based on a stable, crude alpha-amylase (Sigma)) polynomial regression equation that was recalculated and v e r i f i e d for each new l o t of procion yellow starch. Animal Treatments Unless otherwise indicated, most experiments were c a r r i e d out with animals i n the weight range of 280 to 350 g that had been acclimatized f o r several days i n a heated, l i g h t - c o n t r o l l e d room. A l l animals had free access to r a t chow and water i except as otherwise indicated. In most instances, rats were fasted f o r 8 to 12 h p r i o r to perfusion studies or the i n vitro a c i n i preparation. Drugs f o r animal treatment or treatment of the i s o l a t e d pancreatic a c i n i are l i s t e d i n Table 4 of the Appendix. Experimental streptozotocin diabetes Streptozotocin (STZ) i s an a n t i b i o t i c with powerful and s p e c i f i c t o x i c i t y f or the i s l e t c e l l s of the pancreas. Treatment with STZ induces B - c e l l damage or death leading to diabetes. Depending on the dosage, route of administration, 42 and c o n d i t i o n o f t h e a n i m a l , d i a b e t e s a f t e r STZ t r e a t m e n t ranged from a m i l d t r a n s i e n t c o n d i t i o n t o a p r o f o u n d d i s e a s e t h a t was l e t h a l w i t h o u t s u p p l e m e n t a l i n s u l i n t r e a t m e n t (Junod 1969). Most e x p e r i m e n t s u t i l i z e d a 55 mg/kg i . p . dose o f STZ t o i n d u c e m o d e r a t e l y s e v e r e d i a b e t e s b u t w i t h enough f u n c t i o n a l i s l e t s t o o b v i a t e t h e need f o r s u p p l e m e n t a l i n s u l i n . N e v e r t h e l e s s , r a t s t r e a t e d w i t h t h i s dose o f STZ were s e v e r e l y h y p e r g l y c e m i c , w i t h f a s t e d serum g l u c o s e l e v e l s o v e r 300 mg%. STZ f o r i n j e c t i o n was p r e p a r e d i n a sodium c i t r a t e b u f f e r (7 mM) made up i n normal s a l i n e . The b u f f e r was a d j u s t e d t o pH 4.5 and t h e STZ was d i s s o l v e d t o a c o n c e n t r a t i o n o f 110 mg/ml. A f t e r s t e r i l i z a t i o n by passage t h r o u g h a p r e w e t t e d , s t e r i l e , d i s p o s a b l e 0.22 /i f i l t e r , r a t s f a s t e d f o r 12 h were i n j e c t e d i . p . a t a dose o f 55 mg/kg and d e p r i v e d o f f o o d f o r a f u r t h e r 2 h. The d u r a t i o n o f f a s t i n g b o t h b e f o r e and a f t e r a d m i n i s t e r i n g STZ pr o v e d n e c e s s a r y t o r e l i a b l y i n d u c e d i a b e t e s , s i n c e f e d a n i m a l s w i t h h i g h NADPH l e v e l s were more r e s i s t a n t t o t h e B - c e l l t o x i c e f f e c t s o f t h e STZ. D i a b e t e s g e n e r a l l y d e v e l o p e d i n 24 h and r a t s were s u b s e q u e n t l y p r o v i d e d w i t h a d d i t i o n a l w a t e r t o compensate f o r t h e i r c h r o n i c o s m o t i c d i u r e s i s ( L i k e 1976). Exogenous i n s u l i n A l o n g - a c t i n g f o r m u l a t i o n , n e u t r a l p r o t a m i n e Hagadorn (NPH) i n s u l i n , was used f o r exogenous i n s u l i n t r e a t m e n t s . NPH i n s u l i n was a d m i n i s t e r e d on a t w i c e d a i l y s c h e d u l e a t a dose 43 o f 7 U/kg and i n d u c e d a p e r s i s t e n t h y p e r i n s u l i n i s m t h a t made t h e r a t s h y p e r p h a g i c , l e a d i n g t o s t r i k i n g w e i g h t g a i n . C h l o r p r o p a m i d e C h l o r p r o p a m i d e i s an o r a l h y p o g l y c e m i c agent t h a t s t i m u l a t e s b o t h endogenous i n s u l i n r e l e a s e and i n c r e a s e d p e r i p h e r a l i n s u l i n s e n s i t i v i t y . I t was d i s s o l v e d i n H 20 f o r a d m i n i s t r a t i o n by o r a l gavage a t a dose o f 35 mg/kg g i v e n once each day. The e f f e c t s o f t h e d r u g became e v i d e n t a f t e r s e v e r a l days o f t r e a t m e n t . G l u c o s e T o l e r a n c e T e s t s G l u c o s e t o l e r a n c e t e s t s (GTT) were c a r r i e d o ut t o a s s e s s t h e plasma g l u c o s e and i n s u l i n r e s p o n s e s i n s e v e r a l groups o f a n i m a l s . F a s t e d r a t s o f d i f f e r e n t w e i g h t ranges were i n j e c t e d w i t h g l u c o s e (1 g/kg i . p . ) a f t e r a b a s a l b l o o d sample had been o b t a i n e d by t a i l b l e e d i n g . Subsequent b l o o d samples were o b t a i n e d i n h e p a r i n i z e d c a p i l l a r y t u b e s from u n a n e s t h e t i z e d a n i m a l s a t 5, 15, 30, 60, and 90 min a f t e r g l u c o s e i n j e c t i o n . Samples were k e p t on i c e u n t i l plasma c o u l d be s e p a r a t e d f o r g l u c o s e measurement and i n s u l i n a s s a y as d e s c r i b e d below. A s s a y s A s s a y s were c a r r i e d o u t f o r t h e p e p t i d e hormones i n s u l i n and s o m a t o s t a t i n by radioimmunoassay ( R I A ) , f o r e x o c r i n e t o t a l p r o t e i n , and f o r t h e enzymes amylase and l i p a s e . 44 Hormone Assays I n s u l i n Reagent s o u r c e s See T a b l e 5 i n t h e Appendix. Assay b u f f e r A phosphate b u f f e r (0.04 M, pH 7.5) was used t h r o u g h o u t and was p r e p a r e d by d i l u t i o n o f a t e n - f o l d c o n c e n t r a t e d s t o c k s o l u t i o n c o n t a i n i n g : N a H 2P0 4 0.4 M Na 2HP0 4 0.4 M o a d j u s t e d t o pH 7.5 and s t o r e d a t 4 C. D u r i n g d i l u t i o n o f t h e a s s a y b u f f e r s t o c k , hormone-free plasma (5%) was added t o p r e v e n t a d s o r p t i o n p e p t i d e l o s s e s . Hormone-free plasma A c t i v a t e d c h a r c o a l (5%) was added i n s u s p e n s i o n t o p o o l e d , o u t d a t e d human plasma. A f t e r 4 h, t h e c h a r c o a l was p e l l e t e d by c e n t r i f u g a t i o n and t h e now hormone-free plasma was s t o r e d o i n 10 ml a l i q u o t s a t -20 C. A n t i b o d y A n t i - i n s u l i n a n t i b o d y f o r t h e i n s u l i n RIA (GP01) was a g u i n e a p i g a n t i s e r u m t o i n s u l i n . T h i s was s t o r e d f r o z e n i n s o l u t i o n ( l : 2 x l 0 ~ 5 ) i n RIA phosphate b u f f e r and was u t i l i z e d a t a d i l u t i o n o f l : l x l 0 ~ 6 c a r r i e d o u t w i t h t h e RIA phosphate b u f f e r . 45 l z J I - I n s u l i n The c h l o r a m i n e - T method was u t i l i z e d f o r t h e r a d i o l a b e l l i n g p r o c e d u r e and most r e a g e n t s were made up i n sodium phosphate b u f f e r (0.2 M, pH 7.5). F i v e micrograms o f p o r c i n e i n s u l i n (Novo) were d i s s o l v e d i n HC1 (200 u l , 0.01 N) and br o u g h t t o a f i n a l c o n c e n t r a t i o n o f 5 ug/10 u l w i t h phosphate b u f f e r . Ten m i c r o l i t e r s o f t h e i n s u l i n s o l u t i o n (5 ug) was mixed w i t h 1 mCi o f 1 2 5 i and 10 ul o f sodium phosphate b u f f e r i n a s i l i c o n i z e d 12 x 75 mm g l a s s t u b e . A f t e r t h o r o u g h m i x i n g , c h l o r a m i n e - T (100 ug i n 25 u l o f phosphate b u f f e r ) was added, mixed, and t h e r e a c t i o n h a l t e d a f t e r 10 s e c w i t h a d d i t i o n o f sodium m e t a b i s u l f i t e (240 ug i n 100 u l b u f f e r ) . A f t e r a f u r t h e r 45 s e c , e x c e s s i o d i n e ( K I , 50 u l o f 1% s t o c k ) was added t o t h e r e a c t i o n m i x t u r e . The 1 2 5 I - i n s u l i n was p u r i f i e d by a d s o r p t i o n t o m i c r o f i n e s i l i c a (QUSO). The r e a c t i o n m i x t u r e was added t o QUSO (10 mg) t o g e t h e r w i t h phosphate b u f f e r (1.8 m l , 0.04 M) and a f t e r m i x i n g , QUSO was p e l l e t e d by c e n t r i f u g a t i o n a t 500c/. The QUSO was washed t w i c e w i t h d i s t i l l e d H 20 (3 ml) and r e c e n t r i f u g e d . Samples o f s u p e r n a t a n t from each wash were k e p t t o de t e r m i n e t h e degree o f 1 2 5 I - i n s u l i n i n c o r p o r a t i o n . A c i d i f i e d e t h a n o l , made up w i t h 100% e t h a n o l (150 m l ) , H 20 (50 m l ) , and HC1 (3 ml, I N ) , was used t o e l u t e t h e bound l a b e l . A c i d - e t h a n o l (5 ml) and H 20 (1.5 ml) were added t o res u s p e n d t h e QUSO and t h e QUSO was c e n t r i f u g e d f o r a f i n a l t i m e . The s u p e r n a t a n t c o n t a i n i n g t h e e l u t e d l a b e l was d e c a n t e d , sampled, and s t o r e d 46 s e c u r e l y a t 4 C i n a s h i e l d e d c o n t a i n e r . Samples of QUSO, each o f t h e wash s u p e r n a t a n t s , and a f i n a l e l u a n t sample were c o u n t e d t o e s t i m a t e i n c o r p o r a t i o n o f 1 2 5 i as 1 2 5 I - i n s u l i n . I n c o r p o r a t i o n o f 70% was common. I f adequate i n c o r p o r a t i o n was v e r i f i e d , s p e c i f i c a c t i v i t y was n o t measured. F o r some e x p e r i m e n t s such as e m u l s i o n a u t o r a d i o g r a p h s , a h i g h s p e c i f i c a c t i v i t y was e s s e n t i a l . The t e c h n i q u e f o r c a l c u l a t i o n o f s p e c i f i c a c t i v i t y assumed e q u i v a l e n t b i n d i n g o f n a t i v e p e p t i d e and t h e l a b e l l e d p e p t i d e t o t h e a n t i b o d y . On a s i n g l e p l o t , t h e r a t i o o f bound t o f r e e p e p t i d e ( o r d i n a t e ) was p l o t t e d a g a i n s t t h e mass o f t h e p e p t i d e ( a b s c i s s a ) . On t h e same a x e s , b o u n d / f r e e 1 2 5 I - p e p t i d e ( o r d i n a t e ) was p l o t t e d a g a i n s t t o t a l c o u n t s p e r min (cpm) (bound + f r e e = a b s c i s s a ) . I n t h e midrange o f B/F, t h e mass o f p e p t i d e c o r r e s p o n d i n g t o a c e r t a i n t o t a l cpm was c a l c u l a t e d f o r m u l t i p l e p o i n t s up and down t h e c u r v e and t h e n e x p r e s s e d as a f u n c t i o n o f t o t a l cpm ( a b s c i s s a ) . W i t h t h e known c o u n t i n g e f f i c i e n c y o f t h e gamma c o u n t e r , a s p e c i f i c a c t i v i t y (S.A.) c o u l d be c a l c u l a t e d a s : S.A. = 1 /slope X 1 /counting efficiency Sample p r e p a r a t i o n Samples were p l a c e d i n s i l i c o n i z e d 12 x 75 g l a s s t u b e s and o s t o r e d a t -20 C f o r l a t e r a s s a y , g e n e r a l l y c a r r i e d o u t w i t h i n a 2 week p e r i o d . E s t i m a t e s o f t h e a c t i v i t y l o s s o f i n s u l i n i n f r o z e n s o l u t i o n showed o n l y a s l i g h t f a l l o v e r p e r i o d s o f one 47 month. Samples were w i t h i n 15% o f i n i t i a l a c t i v i t y i f thawed, r e f r o z e n , and l a t e r r e a s s a y e d . S t a n d a r d s and C o n t r o l s Rat i n s u l i n (Novo, 21.4 - 27.5 i.u./mg) was o b t a i n e d i n 100 ug a l i q u o t s as a l y o p h i l i z e d powder i n a l b u m i n . A phosphate b u f f e r (pH = 7.5) made up w i t h : N a 2HP0 4 0.46 g NaH 2P0 4, H 20 0.105 g Albumin ( M i l e s ) 6.0 g Sodium m e r t h i o l a t e 0.024 g NaCI 60.6 g D i s t i l l e d H 20 100 ml was used d i s s o l v e and d i l u t e t h e i n s u l i n t o a f i n a l c o n c e n t r a t i o n o f 160 /iU/ml w h i c h was t h e n s t o r e d i n 2 ml o a l i q u o t s a t -20 C. D u r i n g t h e a s s a y , s t a n d a r d a l i q u o t s were thawed t h e n s e r i a l l y d i l u t e d i n a s s a y b u f f e r t o p r o v i d e s t a n d a r d i n s u l i n v a l u e s o f 0, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 /xU/ml, each c o n c e n t r a t i o n i n c l u d e d i n t r i p l i c a t e w i t h e v e r y c e n t r i f u g e r a c k o f samples. C h a r c o a l p r e p a r a t i o n A c t i v a t e d , d e x t r a n - c o a t e d c h a r c o a l was used t o s e p a r a t e a n t i b o d y - b o u n d from f r e e 1 2 5 I - i n s u l i n ( H e r b e r t 1965). T h i s was p r e p a r e d by d i s s o l v i n g d e x t r a n (0.5 g%, Pharmacia-Reagent grade) i n a s s a y phosphate b u f f e r (0.04 M) and a d d i n g a c t i v a t e d c h a r c o a l (5 g % ) . A f t e r a d d i t i o n , c h a r c o a l was k e p t i n s u s p e n s i o n by c o n t i n u o u s s t i r r i n g . 48 Technique o f as s a y The a s s a y was performed i n 12 x 75 mm g l a s s t u b e s , each tube c o n s i s t i n g o f s t a n d a r d o r sample p e r f u s a t e ( l O O u l ) , a n t i b o d y (100 u l ) , and 1 2 5 I - i n s u l i n (100 / i l c o n t a i n i n g 10,000 - 12,000 cpm) made up t o a t o t a l o f 900 u l . The 1 2 5 I - i n s u l i n was added t o t h e r e a c t i o n m i x t u r e 24 h a f t e r samples o r s t a n d a r d , b r i n g i n g t h e t o t a l volume t o 1 ml. S e p a r a t e a l i q u o t s o f 1 2 5 i -i n s u l i n were saved f o r c a l c u l a t i o n o f t h e bound/free r a t i o and a f t e r a f u r t h e r 24 h o f i n c u b a t i o n a t 4 C, unbound 1 2 5 i -i n s u l i n was s e p a r a t e d from bound by a d d i t i o n o f d e x t r a n c h a r c o a l (200 u l ) , i n c u b a t i o n f o r 10 min, and t h e n c e n t r i f u g a t i o n a t 2800 rpm f o r 30 min. The s u p e r n a t a n t was deca n t e d and t h e unbound 1 2 5 I - i n s u l i n t r a p p e d by t h e d e x t r a n c h a r c o a l was cou n t e d on a gamma c o u n t e r s e t f o r 1 2 5 I . A p p r o p r i a t e samples f o r n o n s p e c i f i c b i n d i n g (NSB, no added a n t i b o d y ) , s t a n d a r d s , and c o n t r o l s were i n c l u d e d . Data c a l c u l a t i o n N o n s p e c i f i c b i n d i n g was c a l c u l a t e d f o r each o f t h e s t a n d a r d s , c o n t r o l s , and samples, e x p r e s s e d a s : % NSB = ((total counts - NSB counts)/total counts) x 100% NSB v a l u e s were u s u a l l y i n t h e range o f 8-15%. The p e r c e n t o f 1 2 5 I - i n s u l i n bound f o r each s t a n d a r d was c a l c u l a t e d a s : % bound = (((total counts - mean standard counts)/total counts) x 100%) - % NSB 49 The s t a n d a r d c u r v e v a l u e s f o r % bound ( o r d i n a t e ) were p l o t t e d a g a i n s t i n s u l i n c o n c e n t r a t i o n ( a b s c i s s a ) on s e m i l o g paper and sample c o u n t s were t h e n e x t r a p o l a t e d t o d e t e r m i n e t h e sample i n s u l i n v a l u e s . A l l samples were measured i n d u p l i c a t e and t h e mean o f d u p l i c a t e c o u n t s was used t o c a l c u l a t e t h e % bound v a l u e a s : % bound = (((total counts - mean sample counts)/total counts) x 100%) - % NSB I f t h e % bound v a l u e s extended above t h e l i n e a r p o r t i o n o f t h e s t a n d a r d c u r v e , samples were a p p r o p r i a t e l y d i l u t e d and t h e as s a y r e p e a t e d . I n t h e l a t t e r p a r t o f t h i s work, a l o g i t c u r v e i n c o r p o r a t e d s t a n d a r d v a l u e s i n t o a r e g r e s s i o n e q u a t i o n t h a t a l l o w e d d i r e c t c a l c u l a t i o n o f i n s u l i n c o n c e n t r a t i o n s . V a l u e s from t h i s microcomputer program were p e r i o d i c a l l y checked a g a i n s t s t a n d a r d c u r v e s and found t o c o r r e s p o n d c l o s e l y . S e n s i t i v i t y and s p e c i f i c i t y I n t r a - and i n t e r - a s s a y v a r i a t i o n s were a s s e s s e d by i n c l u s i o n o f i n t e r n a l c o n t r o l s i n t h e i n s u l i n RIA. C o n t r o l s were p r e p a r e d by p o o l i n g p e r f u s a t e from s e v e r a l normal r a t s s t i m u l a t e d w i t h a r g i n i n e w h i c h was t h e n d i l u t e d , a l i q u o t e d , o and s t o r e d a t -20 C. I n each a s s a y , a c o n t r o l sample was r u n i n t r i p l i c a t e . The i n s u l i n a s s a y had a t h r e s h o l d s e n s i t i v i t y o f 2-3 jnU/ml and i n t r a - and i n t e r - a s s a y v a r i a t i o n o f 7% and 15%. There was no c r o s s - r e a c t i v i t y w i t h CCK, s e c r e t i n , V I P , o r s o m a t o s t a t i n . 50 S o m a t o s t a t i n Reagent s o u r c e s Reagent s o u r c e s a r e d e t a i l e d i n T a b l e 5 o f t h e Appendix. Assay b u f f e r Components o f t h e s o m a t o s t a t i n a s s a y b u f f e r i n c l u d e d : sodium b a r b i t a l 4.9 g NaCl 2.55 g NaCH3COO 0.32 g m e r t h i o l a t e 0.1 g H 20 700 ml and was b r o u g h t up t o 1000 ml w i t h d i s t i l l e d H 20, and a d j u s t e d o t o pH 7.4. The b u f f e r was s t o r e d a t 4 C and used g e n e r a l l y w i t h i n one week. F i n a l a s s a y b u f f e r c o n c e n t r a t i o n s were: sodium b a r b i t a l 23.8 mM NaCl 43.6 mM NaCH3COO 3.9 mM m e r t h i o l a t e 0.24mM A n t i b o d y A n t i b o d y f o r t h e s o m a t o s t a t i n a s s a y was d e r i v e d from a r a b b i t a n t i s e r u m , Go VI 3.2. A n t i b o d y f o r t h e a s s a y was s t o r e d as a o 1:100 ( s e r u m : d i s t i l l e d w a ter) d i l u t i o n a t -20 C i n 1 ml a l i q u o t s . I m m ediately p r i o r t o use, t h i s c o n c e n t r a t e was thawed and d i l u t e d 1:150 w i t h a s s a y b u f f e r , a c h i e v i n g a f i n a l c o n c e n t r a t i o n o f 1:6 x 1 0 4 i n t h e a s s a y r e a c t i o n m i x t u r e . C h a r c o a l s o l u t i o n The c h a r c o a l s u s p e n s i o n f o r t r a p p i n g o f unbound l a b e l was made up o f d e x t r a n T-70 (0.25 g%) d i s s o l v e d i n phosphate b u f f e r 51 (0.05 M), and a c t i v a t e d c h a r c o a l (1.25 g%) t o g e t h e r w i t h hormone-free plasma (100 u l % ) . The c h a r c o a l s u s p e n s i o n was s t i r r e d f o r a t l e a s t 1 h r p r i o r t o use i n t h e a s s a y . 1 2 5 I - S o m a t o s t a t i n As d e s c r i b e d above f o r i n s u l i n , t h e 1 2 5 I - S o m a t o s t a t i n was s y n t h e s i z e d by t h e c h l o r a m i n e - T method. T y r - 1 s o m a t o s t a t i n was d i s s o l v e d i n d i s t i l l e d H 20 (10 u l ) and added t o phosphate b u f f e r (10 u l , 0.5 M), 1 mCi o f Na 1 2 5 I ( i n 10 u l ) , and ch l o r a m i n e - T (10 u l o f 200 mg% s t o c k ) i n phosphate b u f f e r (0.05 M, pH 7.5). A f t e r m i x i n g f o r 30 s e c , sodium m e t a b i s u l f i t e (10 u l o f 500 mg% s t o c k ) was used t o s t o p t h e r e a c t i o n . Hormone-free plasma (1 ml) and QUSO (20 mg) were added t o t h e r e a c t i o n m i x t u r e and t h e QUSO was p e l l e t e d by c e n t r i f u g a t i o n a t 500g. The QUSO was washed t h r o u g h s u s p e n s i o n and c e n t r i f u g a t i o n w i t h H 20 t w i c e and a l i q u o t s o f s u p e r n a t a n t were p r e s e r v e d t o d e t e r m i n e t o t a l c o u n t s . The t h i r d wash w i t h a c e t i c a c i d / a c e t o n e (1:40 v:v d i l u t e d t o 50% w i t h H 20) e l u t e d t h e 1 2 5 I - s o m a t o s t a t i n w h i c h was t h e n d i l u t e d t o 500,000 cpm/10 u l i n a c e t i c a c i d (0.1 M) and BSA ( 0 . 5 % ) , and s e p a r a t e d i n t o 100 u l f r a c t i o n s f o r l y o p h i l i z a t i o n and s t o r a g e . On t h e day o f t h e a s s a y , t h e 1 2 5 I - s o m a t o s t a t i n was p u r i f i e d o v e r a c e l l u l o s e column. L y o p h i l i z e d a l i q u o t s were d i s s o l v e d i n ammonium a c e t a t e (0.002 M, pH 4.6) t h e n p l a c e d on a CM-52 c e l l u l o s e column p r e v i o u s l y e q u i l i b r a t e d w i t h ammonium a c e t a t e 52 (0.002 M) . A f t e r washing w i t h ammonium a c e t a t e (0.002 M) f o r 30 min (1 ml / m i n ) , t h e i o d i n a t e d p e p t i d e was e l u t e d w i t h ammonium a c e t a t e (0.2 M) onto an automated c o l l e c t i n g system and t h e t u b e s c o n t a i n i n g t h e peak and d e s c e n d i n g l i m b r a d i o a c t i v i t y were p o o l e d . The f i n a l 1 2 5 I - 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 was a d j u s t e d t o 3500 cpm/100 pi. S t a n d a r d s S t a n d a r d s f o r t h e s o m a t o s t a t i n a s s a y were p r e p a r e d by d i s s o l v i n g c y c l i c s o m a t o s t a t i n (1 mg) i n a c e t i c a c i d (5 m l , 0.1 M) c o n t a i n i n g BSA ( 0 . 0 5 % ) . F i f t y m i c r o l i t e r (10 /ig o f s o m a t o s t a t i n ) a l i q u o t s were t r a n s f e r r e d t o s i l i c o n i z e d 12 x 75 o mm g l a s s t u b e s and l y o p h i l i z e d p r i o r t o s t o r a g e a t -20 C. A t t h e t i m e o f s o m a t o s t a t i n a s s a y , t h e thawed s t a n d a r d was d i s s o l v e d i n a s s a y b u f f e r and s e r i a l l y d i l u t e d t o c o n c e n t r a t i o n s r a n g i n g from 3.9 pg/ml t o 250 pg/ml. T h i s d i l u t i o n range made up t h e s t a n d a r d range o f t h e s o m a t o s t a t i n a s s a y . Sample p r e p a r a t i o n and s t o r a g e Samples f o r s o m a t o s t a t i n a s s a y were o b t a i n e d from p e r f u s i o n o r a c i n i e x p e r i m e n t s . One ml a l i q u o t s o f t h e sample were added o t o t r a s y l o l (100 nl) and s t o r e d a t -20 C. E x t r a p e r f u s a t e w i t h added t r a s y l o l (1 m l / 10 ml p e r f u s a t e ) was a l s o s t o r e d f r o z e n f o r d i l u t i o n o f s t a n d a r d s d u r i n g t h e a s s a y . 53 A s s a y P r o c e d u r e s G l a s s 12 x 75 mm t u b e s were used f o r t h e a s s a y w i t h each p a i r o f r a c k s c o n t a i n i n g a s t a n d a r d c u r v e and s e t o f NSB v i a l s . The 400 ul t o t a l volume o f t h e a s s a y was made up o f s t a n d a r d o r sample (100 / i l ) , a n t i b o d y (100 u l ) , a s s a y b u f f e r (100 u l ) , and 1 2 5 I - s o m a t o s t a t i n (100 F ° r e s t i m a t e s o f t h e NSB, a n t i b o d y was r e p l a c e d by ass a y b u f f e r (100 M l ) . A f t e r m i x i n g , o v i a l s were s t o r e d a t 4 C f o r 72 h a f t e r w h i c h , s e p a r a t i o n o f bound from f r e e 1 2 5 I - s o m a t o s t a t i n was c a r r i e d o u t w i t h d e x t r a n - c o a t e d c h a r c o a l . A l i q u o t s o f c h a r c o a l - d e x t r a n s u s p e n s i o n (1 ml) were added t o each tube and a f t e r 30 min, t h e c h a r c o a l was p e l l e t e d by c e n t r i f u g a t i o n and t h e s u p e r n a t a n t d e c a n t e d . A f t e r d r y i n g o f t h e p e l l e t , r a d i o a c t i v e c o u n t s were measured on a gamma c o u n t e r w i t h a window s e t t i n g f o r 1 2 5 I . A l i q u o t s o f t h e i n i t i a l 1 2 5 I - s o m a t o s t a t i n were saved f o r measurement o f t o t a l c o u n t s , a v a l u e used i n t h e c a l c u l a t i o n o f sample s o m a t o s t a t i n c o n t e n t . T h i s c a l c u l a t i o n was i d e n t i c a l t o t h a t d e s c r i b e d f o r i n s u l i n ( v i d e s u p r a ) . S e n s i t i v i t y and s p e c i f i c i t y The s o m a t o s t a t i n radioimmunoassay had a s e n s i t i v i t y o f 2 pg/ml, and i n t r a - and i n t e r - a s s a y v a r i a t i o n s o f 5% and 11% r e s p e c t i v e l y . C o n t r o l s were o n l y i n t e r m i t t e n t l y used because d e c l i n e i n s o m a t o s t a t i n a c t i v i t y i n f r o z e n c o n t r o l s o l u t i o n s o v e r t i m e made them u n r e l i a b l e . Exocrine Assays Amylase S t a r c h - I o d i n e Reagents were p u r c h a s e d c o m m e r c i a l l y (Appendix, T a b l e 6 ) . Samples, u s u a l l y p a n c r e a t i c j u i c e , were c o l l e c t e d i n s i l i c o n i z e d 10 u l m i c r o p i p e t s and d e p o s i t e d i n t o s i l i c o n i z e d 12 x 75 mm t u b e s c o n t a i n i n g 0.5 t o 1.0 ml o f d i s t i l l e d H 20, d e p e n d i n g on t h e c h a r a c t e r o f t h e p r e p a r a t i o n and t h e e x p e c t e d o amylase c o n c e n t r a t i o n . Samples were s t o r e d a t -20 C u n t i l a s s a y , g e n e r a l l y w i t h i n 2 weeks. No a p p r e c i a b l e d e c l i n e i n a c t i v i t y was measured between c o n t r o l samples a s s a y e d i m m e d i a t e l y and t h o s e s t o r e d a t -20 C f o r two weeks. A t t h e t i m e o f a s s a y , samples were thawed and d i l u t e d w i t h s a l i n e by a f a c t o r o f 1:50 t o 1:100, and t h e n a s s a y e d i n d u p l i c a t e . T e s t and b l a n k 12 x 75 mm g l a s s v i a l s c o n t a i n i n g 0.5 ml o f o s t a r c h r e a g e n t were e q u i l i b r a t e d i n a water b a t h (37 C) t h e n 10 jul o f sample o r s a l i n e ( b l a n k ) was add^d t o each t u b e . V i a l s were mixed, i n c u b a t e d f o r 7.5 min, and i o d i n e r e a g e n t (0.5 ml) was t h e n added t o b o t h t h e t e s t and b l a n k f o l l o w e d by d i s t i l l e d H 20 (4 m l ) . An a b s o r b a n c e s p e c t r o p h o t o m e t e r (Beckman, F u l l e r t o n CA) s e t a t 660 nm was c a l i b r a t e d t o z e r o w i t h d i s t i l l e d H 20 and t h e absorbance (A) o f b l a n k and t e s t v i a l s was measured. Amylase a c t i v i t y was c a l c u l a t e d by: amylase (units) = [(A (blank) - A (test))/A (blank)] x 800 x % dilution I n t r a - and i n t e r - a s s a y v a r i a t i o n s f o r t h e s t a r c h - i o d i n e amylase a s s a y were 10% and 17%; b o t h were c r i t i c a l l y dependent 55 on t h e e x t e n t o f d i l u t i o n n e c e s s a r y . The advantages o f t h i s a s s a y were r a p i d i t y o f performance and t h e c a p a b i l i t y t o e x t e n d i t a c r o s s many d i f f e r e n t samples b u t a t t h e c o s t o f p r e c i s i o n . P r o c i o n y e l l o w - s t a r c h The p r o c i o n y e l l o w - s t a r c h a s s a y , due t o i t s g r e a t e r s e n s i t i v i t y and r e p r o d u c i b i l i t y , was used f o r a l l a c i n i p r e p a r a t i o n s . Reagent s o u r c e s a r e n o t e d i n t h e Appendix, T a b l e 6. P r o c i o n y e l l o w - s t a r c h was p r e p a r e d a c c o r d i n g t o t h e method o f Jung (1980). C o l d , d i s t i l l e d H 20 (1.8 1) was s t i r r e d and warmed w h i l e s t a r c h (200 g) was added, t h e n t h e s u s p e n s i o n was h e a t e d t o 50-55 C and p r o c i o n y e l l o w (2 g) i n o d i s t i l l e d H 20 (100 ml) a t 55 C was added t o t h e s t a r c h . W i t h o t h e t e m p e r a t u r e a t 55 C, t h e s u s p e n s i o n was s t i r r e d f o r 45 o min t h e n t h e t e m p e r a t u r e was r a p i d l y i n c r e a s e d t o 65 C f o r a n o t h e r 15 min. N a 2 C 0 3 (40 m l , 0.5 M) was t h e n added d r o p w i s e o w h i l e m a i n t a i n i n g t h e t e m p e r a t u r e a t 65 C f o r an a d d i t i o n a l 30 min. The s t a r c h s u s p e n s i o n was c o o l e d and a l l o w e d t o s e t t l e f o r 12 h. S u p e r n a t a n t was d i s c a r d e d and t h e s t a r c h was washed r e p e a t e d l y w i t h d i s t i l l e d H 20 by r e s u s p e n s i o n and c e n t r i f u g a t i o n u n t i l no f u r t h e r unbound dye c o l o r was e v i d e n t i n t h e s u p e r n a t a n t . Two methanol (100%) washes d e h y d r a t e d t h e powder p r i o r t o f i l t r a t i o n and t h e powder was a i r d r i e d f o r 12 h t h e n s t o r e d i n a d e s i c c a t o r . Components o f t h e a s s a y i n c l u d e d : 56 Assay Phosphate Buffer (pH 6.9) Na 2HP04 0.05 M NaCI 0.05 M Starch Reagent P r o c i o n Y e l l o w - S t a r c h 3% ( i n a s s a y b u f f e r ) Starch Reagent was m a i n t a i n e d i n s u s p e n s i o n and a l i q u o t e d (0.8 a ml) i n t o 13 x 100 mm g l a s s t u b e s i n a s h a k i n g water b a t h (37 C, 90 c y c / m i n ) i A t 30 second i n t e r v a l s , 20 ul o f sample ( o r b u f f e r f o r b l a n k s ) i n d u p l i c a t e were added t o a s s a y t u b e s , mixed, and r e p l a c e d i n t h e b a t h . A f t e r i n c u b a t i o n f o r 20 min, o HCI a t 4 C (1.6 m l , 0.1 N) was added t o quench t h e r e a c t i o n and t h e a s s a y v i a l s were k e p t on i c e u n t i l r e m a i n i n g p r o c i o n y e l l o w - s t a r c h was s e p a r a t e d by c e n t r i f u g a t i o n . The absorbance o f t h e s u p e r n a t a n t was measured on an absorbance s p e c t r o p h o t o m e t e r a t 4 20 nm a f t e r z e r o i n g w i t h a b l a n k . The p r o c i o n y e l l o w s t a r c h a s s a y r e l i a b l y measured amylase r e l e a s e a t 2-3% o f a c i n a r c e l l amylase c o n t e n t . I n t r a - and i n t e r - a s s a y v a r i a t i o n was measure i n s e l e c t e d e x p e r i m e n t s t o be about 1% and 5% r e s p e c t i v e l y . S e c r e t a g o g u e s (CCK, V I P , s e c r e t i n ) were w i t h o u t d i r e c t e f f e c t s on t h e amylase a s s a y i n c o n t r o l e x p e r i m e n t s . L i p a s e The l i p a s e a s s a y was c a r r i e d o u t u s i n g a m o d i f i c a t i o n o f t h e method f o r q u a n t i t a t i v e serum l i p a s e measurement d e v i s e d by T i e t z and F i e r e c k (1966). L i p a s e exposed t o a f a t s u b s t r a t e e m u l s i o n r e l e a s e d f a t t y a c i d s i n t o s o l u t i o n t h a t c o u l d be 57 q u a n t i t a t e d by back t i t r a t i o n . Components o f t h e ass a y i n c l u d e d : S u b s t r a t e o l i v e o i l e m u l s i o n 50 % T r i z m a b u f f e r t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e (pH 8.0) 0.2 M I n d i c a t o r t h y m o l p h t h a l e i n ( i n e t h a n o l ) 0.9 % NaOH 0.05 N The a s s a y r e a c t i o n m i x t u r e was made up w i t h d i s t i l l e d H 20 (250 / i l ) , s u b s t r a t e (300 nl) , and T r i z m a b u f f e r (100 ul) i n a 12 x 75 mm g l a s s t u b e . An a l i q u o t (10 ul) o f sample o r b u f f e r a p p r o p r i a t e l y d i l u t e d , was added, mixed, and i n c u b a t e d i n a o w a t e r b a t h (37 C, 60 cyc/min) i n d u p l i c a t e . B l a n k s c o n t a i n i n g b u f f e r r a t h e r t h a n samples were r u n b e f o r e and a f t e r a l l t e s t samples and b o t h were d i l u t e d 1:10 w i t h 0.9% s a l i n e p r i o r t o a s s a y . A f t e r 3 h, e t h a n o l (95%, 300 nl a t 4 C) was added t o s t o p t h e r e a c t i o n f o l l o w e d by i n d i c a t o r (20 / i l ) . The r e a c t i o n m i x t u r e was k e p t on i c e u n t i l back t i t r a t i o n w i t h NaOH (0.05 N) c o u l d be a c c o m p l i s h e d t o a s l i g h t b l u e - g r e e n c o l o r e n d p o i n t . U n i t s o f a c t i v i t y (U) were e x p r e s s e d as t h e volume o f NaOH (0.05 N) r e q u i r e d t o r e a c h t h e i n d i c a t o r e n d p o i n t , minus t h e b l a n k t i t r a t i o n volume. I n t r a - and i n t e r - a s s a y v a r i a t i o n s were 12% and 18% from s e l e c t e d e x p e r i m e n t s , t h e l a t t e r due t o d i l u t i o n s and i n c r e a s e d e r r o r w i t h a s m a l l sample volume. 58 T o t a l p r o t e i n T o t a l p r o t e i n was measure by t h e method o f Lowry ( 1 9 5 1 ) . Reagent s o u r c e s a r e l i s t e d i n T a b l e 6 o f t h e A p p e n d i x . Components o f t h e a s s a y i n c l u d e d : N a 2 C 0 3 ( i n d i s t i l l e d H 20) 2 % CuS0 4-5H 20 ( i n Na C i t r a t e ( 1 % ) ) 0.5 % F o l i n s Reagent 2 N NaOH 1 N Albumin 5 % The N a 2 C 0 3 s o l u t i o n was mixed w i t h t h e CuS0 4-5H 20 i n a r a t i o o f 49:1 (v:v) and was made up f r e s h f o r each a s s a y . The t o t a l p r o t e i n c o n t e n t o f samples was d e t e r m i n e d by c o m p a r i s o n t o an a l b u m i n s t a n d a r d p r e s e n t t h r o u g h s e r i a l d i l u t i o n s i n t r i p l i c a t e w h i c h p r o v i d e d a l i n e a r c u r v e r a n g i n g f r o m 0 ug t o 20 ug p e r r e a c t i o n t u b e . A f t e r t h a w i n g and m i x i n g , each sample (10 u l ) was added t o d i s t i l l e d H 20 t o make a f i n a l volume o f 200 u l w h i c h was t h e n measured i n d u p l i c a t e . To each v i a l , NaOH (200 u l , I N ) and sodium c a r b o n a t e / c o p p e r s u l f a t e - c i t r a t e s o l u t i o n (2 ml) were addecj. and m i x e d , f o l l o w e d 5 min l a t e r by F o l i n s r e a g e n t (200 u l , I N ) . A f t e r an assay r e a c t i o n i n t e r v a l o f 30 min a t room t e m p e r a t u r e , s t a n d a r d and sample ab s o r b a n c e s were r e a d on an absorbance s p e c t r o p h o t o m e t e r a t 750 nm. The p r o t e i n c o n c e n t r a t i o n i n each sample was c a l c u l a t e d from a f o r m u l a d e v e l o p e d by r e g r e s s i o n a n a l y s i s o f s t a n d a r d c u r v e v a l u e s . W i d e l y d i s s i m i l a r d u p l i c a t e samples were r a r e and were s u b m i t t e d f o r r e p e a t a s s a y . I n t r a - and i n t e r - a s s a y v a r i a t i o n s were s m a l l , 4 and 8% r e s p e c t i v e l y . 59 Glucose measurement G l u c o s e i n serum o r p e r f u s a t e was measured u s i n g a "Glucose A n a l y z e r 2" a p p a r a t u s and p r o p r i e t a r y o x i d i z i n g r e a g e n t (Beckman). I n t r a - and i n t e r - a s s a y v a r i a t i o n s were 5 and 8%. Osmolarity O s m o l a r i t y was measured on a Wescor Vapor P r e s s u r e Osmometer (mM/kg). T i s s u e S t a i n i n g and Immunocvtochemistrv S u p p l i e s f o r h i s t o l o g y and immunocytochemistry a r e l i s t e d i n T a b l e 7 o f t h e Appendix. P a n c r e a s s e c t i o n s f o r r o u t i n e h i s t o l o g i c e v a l u a t i o n were f i x e d i n b u f f e r e d f o r m a l i n (10%) and d e h y d r a t e d t h r o u g h an automated t i s s u e f i x a t o r ( T i s s u e Tek I I , M i l e s ) t o p a r a f f i n . A f t e r imbedding i n p a r a f f i n b l o c k s , 5 u s e c t i o n s f o r mounting on g l a s s s l i d e s were c u t on a microtome ( R e i c h e r t - J u n g , Cambridge I n s t r u m e n t s ) . S e c t i o n s were h e a t - f i x e d t o g l a s s s l i d e s t h e n p a s s e d t h r o u g h s o l u t i o n s o f x y l e n e (10 min) and EtOH (100%, 90%, and 70%, each f o r 5 min) t o d i s t i l l e d H 20. The s e c t i o n was s t a i n e d i n f i l t e r e d h e m a t o x y l i n (5 min) t h e n washed i n H 20 (5 m i n ) , a c i d - E t O H (0.1 N HCI i n 70% EtOH, 3 s e c ) , and H 20 (5 min) a g a i n . A f t e r d i p p i n g i n L i 2 C 0 3 t o enhance c o n t r a s t (12 s e c , s a t u r a t e d s o l u t i o n ) , s e c t i o n s were washed i n H 20 (10 min) a g a i n t h e n c o u n t e r s t a i n e d w i t h e o s i n (3 m i n ) . S t a i n e d s e c t i o n s were d e h y d r a t e d t h r o u g h EtOH (70%, 90%, and 100%) t o x y l e n e and a c o v e r s l i p was p l a c e d o v e r c l e a r cement. 60 P h o t o m i c r o g r a p h s were o b t a i n e d w i t h a camera a t t a c h e d t o t h e l i g h t p a t h o f a s t a n d a r d l i g h t m i c r o s c o p e . F o r immunocytochemical s t a i n i n g , s l i d e s were p r e p a r e d on a c r y o s t a t microtome ( T i s s u e Tek C r y o s t a t , M i l e s ) . F r o z e n t i s s u e imbedded i n a mounting medium was s e c t i o n e d a t 5 - 10 u s e c t i o n s and h e a t - f i x e d on g l a s s s l i d e s . S e c t i o n s t a k e n from p a r a f f i n s e c t i o n s were d e p a r a f f i n i z e d i n x y l e n e (5 min x 2 washes) and p e t r o l e u m e t h e r . S l i d e s were t h e n p l a c e d i n p h o s p h a t e - b u f f e r e d s a l i n e (PBS, 10 mM sodium phosphate i n 150 mM NaCl) w i t h a z i d e (1 mM) c o n t a i n i n g H 2 0 2 (1 ml o f 30% s o l u t i o n / 1 0 0 ml) f o r 30 min. The s l i d e s were t h e n washed w i t h PBS s e v e r a l t i m e s , d r i e d , and l a y e r e d w i t h t h e a p p r o p r i a t e f i r s t l a y e r a n t i b o d y , s p e c i f i c f o r t h e p e p t i d e b e i n g s t a i n e d . A n t i b o d i e s f o r i n s u l i n and s o m a t o s t a t i n were o f mouse o r i g i n . A f t e r 12 h i n a h u m i d i t y chamber t o p r e v e n t d r y i n g , t h e a n t i b o d y l a y e r was washed o f f w i t h PBS a z i d e . F o r f i r s t l a y e r a n t i b o d i e s d e r i v e d from mouse, r a b b i t anti-mouse IgG ( d i l u t e d 1:100) c o n j u g a t e d t o p e r o x i d a s e was d e p o s i t e d by d r o p p e r on s e c t i o n s and i n c u b a t e d f o r 45 min. A f t e r t h e p e r o x i d a s e t r e a t m e n t , s e c t i o n s were washed (x 3) w i t h P B S - a z i d e f o l l o w e d by t h e d i a m i n o b e n z i d i n e t e t r a h y d r o c h l o r i d e (DAB, 0.05%) r e a g e n t i n PBS (pH 7.3) t o g e t h e r w i t h H 2 0 2 (0.01%) f o r 5-10 min, u n t i l o p t i m a l s t a i n i n g had o c c u r r e d . The s l i d e s were t h e n c o u n t e r s t a i n e d w i t h h e m a t o x y l i n as p r e v i o u s l y d e s c r i b e d and d e h y d r a t e d t h r o u g h e t h a n o l ( 7 0 % , 80%, 100%) t o x y l e n e f o r mounting. 61 A u t o r a d i o g r a p h y ( I n s u l i n R e c e p t o r D i s t r i b u t i o n ^  1 2 5 I - i n s u l i n was p r e p a r e d t o a h i g h s p e c i f i c a c t i v i t y , c a l c u l a t e d t o be 71.3 u C i / u g by t h e t e c h n i q u e p r e v i o u s l y d e s c r i b e d . Based on p r i o r s t u d i e s by B e r g e r o n e t a l (1984) and Sakamoto and W i l l i a m s (1984) on i n s u l i n r e c e p t o r b i n d i n g from whole a n i m a l i n f u s i o n s o f 1 2 5 I - i n s u l i n , i s o l a t e d r a t p a n c r e a s e s (N = 4/group) were p e r f u s e d (2 ml/min) w i t h 50 X 1 0 6 cpm (22.5 uCi o r 0.3 ng) 1 2 5 I - i n s u l i n a f t e r a 10 min e q u i l i b r a t i o n p e r i o d . The t h r e e groups i n c l u d e d f o u r r a t s r e c e i v i n g 1 2 5 I - i n s u l i n a l o n e , 4 r a t s r e c e i v i n g 1 2 5 I - i n s u l i n w i t h p r i o r and c o n c o m i t a n t t r e a t m e n t w i t h g l u c o s e (18 mM) and a r g i n i n e (20 mM) t o s t i m u l a t e endogenous i n s u l i n r e l e a s e , and a t h i r d group r e c e i v i n g 1 2 5 I - i n s u l i n t o g e t h e r w i t h e x c e s s u n l a b e l l e d i n s u l i n (7.0 /ig/ml, 1 1 4 - f o l d g r e a t e r t h a n t h e molar dose o f 1 2 5 I - i n s u l i n a d m i n i s t e r e d ) . The s i d e a r m i n f u s i o n o f 1 2 5 I - i n s u l i n t o o k 3 min and was f o l l o w e d by c o n t i n u e d p e r f u s i o n w i t h normal (4.48 mM) o r h i g h (18 mM) g l u c o s e p e r f u s a t e o v e r 10 min t o wash o u t unbound 1 2 5 I - i n s u l i n . S u b s e q u e n t l y , t h e t i s s u e was f i x e d by p e r f u s i o n w i t h p a r a f o r m a l d e h y d e ( 4 % , 5 min) and p o s t - f i x e d i n t h e same s o l u t i o n f o r a f u r t h e r 30 min. F i x e d specimens were washed i n phosphate b u f f e r e d s a l i n e and d e h y d r a t e d by passage t h r o u g h EtOH t o x y l e n e , t h e n imbedded i n p a r a f f i n . A f t e r b e i n g c u t on t h e microtome, s e c t i o n s (5 u) were h e a t f i x e d t o s l i d e s . Kodak NTB3 e m u l s i o n was p r e p a r e d by m e l t i n g t h e s t o c k e m u l s i o n i n a darkroom w a t e r b a t h and d i l u t i n g 1:1 w i t h warmed, d i s t i l l e d H 20. A l i q u o t s o f t h e d i l u t e d e m u l s i o n were s t o r e d 62 i n l i g h t - t i g h t c o n t a i n e r s . A t t h e t i m e o f s l i d e p r e p a r a t i o n , d i l u t e d e m u l s i o n was m e l t e d and s l i d e s b e a r i n g s e c t i o n s were immersed i n e m u l s i o n t h e n d r i e d i n t h e darkroom. E m u l s i o n -o c o a t e d s l i d e s were s t o r e d i n l i g h t - t i g h t c o n t a i n e r s a t 4 C f o r 12 d p r i o r t o development bu t sample s l i d e s were d e v e l o p e d a t 4 and 8 days t o a s s e s s t h e degree o f r e s o l u t i o n o f t h e r a d i o a c t i v e t r a c k s i n t h e e m u l s i o n . A t t h e t i m e o f development, s l i d e s were warmed t o room t e m p e r a t u r e t h e n immersed i n Kodak Developer D-19 (1:1 d i l u t i o n ) f o r 4 min, washed, and f i x e d i n Kodak F i x a t i v e . P r o c e s s e d s l i d e s were washed a g a i n , s t a i n e d i n h e m a t o x y l i n (30-40 sec) t h r o u g h t h e e m u l s i o n , t h e n d e h y d r a t e d t h r o u g h EtOH (70, 90, and 100%) t o x y l e n e . C o v e r s l i p s were a p p l i e d o v e r cement. G r a i n c o u n t i n g was a c c o m p l i s h e d w i t h a microcomputer-based, image a n a l y s i s - d i g i t i z i n g system t h a t a l l o w e d d e s c r i p t i o n o f r e g i o n s f o r c o u n t i n g and i n c l u s i o n . The a r e a program o f a Model 3000 Image A n a l y z e r (Image Technology C o r p o r a t i o n , Deer P a r k , NY) was used t o a n a l y z e s e c t i o n s v i s u a l i z e d t h r o u g h a N i k o n D i a p h o t Reverse Phase M i c r o s c o p e ( N i k o n I n s t r u m e n t s , Garden C i t y , NY) Window s e t t i n g s o f H511 and V511 p i x e l s , w i t h s u i t a b l e m a g n i f i c a t i o n f o r 4 a c i n i p e r f i e l d (11.6 u f i e l d d i a m e t e r ) were u t i l i z e d . Counts were e x p r e s s e d as d e n s i t y u n i t s p e r m i c r o s c o p i c f i e l d . Comparisons were made between a c i n i r e g i o n s c l o s e t o i s l e t s and r e g i o n s more remote from t h e same i s l e t . The means o f absorbance v a l u e s f o r each a c i n i r e g i o n were compared between a n i m a l t r e a t m e n t groups 63 a f t e r d i s c o u n t i n g t h e e f f e c t s o f n o n s p e c i f i c b i n d i n g ( e x c e s s " c o l d " i n s u l i n g r o u p ) . M u l t i p l e s e c t i o n s and e m u l s i o n s were made from each o f t h e t r e a t e d a n i m a l s i n each group t o a l l o w measurement o f a l a r g e number o f c o u n t s w i t h i n each a n i m a l around many i s l e t s . R e a g e n t s / P e p t i d e s P e p t i d e s a r e l i s t e d i n T a b l e 8 o f t h e Appendix. S t a t i s t i c a l A n a l y s i s A l l v a l u e s i n t h e t e x t and f i g u r e s were e x p r e s s e d as t h e mean ± t h e s t a n d a r d e r r o r o f t h e mean (SEM). S i g n i f i c a n c e was c a l c u l a t e d by a n a l y s i s o f v a r i a n c e (ANOVA) f o r t i m e - s e r i e s and m u l t i p l e s e q u e n t i a l measurement d a t a ( f o r example, p e r f u s i o n s t u d i e s c a r r i e d o u t o v e r t i m e ) w h i l e t h e u n p a i r e d t - t e s t was used f o r com p a r i s o n o f i n d i v i d u a l measurements. M u l t i p l e c o m p a r i s o n s between s e v e r a l groups were c a r r i e d o u t w i t h ANOVA f o l l o w e d by Duncan's M u l t i p l e Range t e s t t o a s s i g n i n d i v i d u a l l y s i g n i f i c a n t d i f f e r e n c e s . S i g n i f i c a n c e was a s s e s s e d a t t h e p < 0.05 l e v e l u n l e s s o t h e r w i s e s t a t e d i n t h e t e x t . C a l c u l a t i o n s were c a r r i e d o u t u s i n g t h e c o m p i l e d BASIC f a m i l y o f programs, Northwest S t a t p a c ( Northwest A s s o c i a t e s , Bend, OR), and s i g n i f i c a n t d i f f e r e n c e s were g e n e r a l l y denoted by an a s t e r i s k (*) i n f i g u r e s and d i s c u s s e d i n t h e t e x t . 64 F i g u r e 1 Pancreatic Juice Volume Response to Increasing Doses of CCK-8 i n the Isolated Perfused Pancreas Pancreatic j u i c e volume (/il/min) was measured over time during perfusion of the i s o l a t e d pancreas preparation with increasing doses of CCK-8. CCK-8 was added v i a a sidearm to the heated, oxygenated perfusate to achieve f i n a l concentrations ranging from 0-1000 pM (see key). CCK-8 was administered during the i n t e r v a l indicated by the l a b e l l e d bar, a f t e r e q u i l i b r a t i o n and a basal c o l l e c t i o n period. Only one concentration of CCK was infused per r a t . The glucose concentration of the perfusate was 4.5 mM throughout. The N was 5 or greater per CCK dose. Values are given as the mean ± SEM; s i g n i f i c a n t d i f ferences are denoted by the asterisk (*) and discussed i n the text. 65 Figure 1 66 Figure z Pancreatic Juice Volume Response to Increasing Doses of Secretin i n the Isolated Perfused Pancreas Pancreatic ju i c e volume (al/min) was measured over time during perfusion of the i s o l a t e d pancreas preparation with increasing doses of s e c r e t i n , s e c r e t i n was added v i a a sidearm to the heated, oxygenated perfusate to achieve f i n a l concentrations ranging from 0-10 nM (see key). Secretin was administered during the i n t e r v a l indicated by the l a b e l l e d bar, a f t e r e q u i l i b r a t i o n and a basal c o l l e c t i o n period. Only one concentration of s e c r e t i n was infused per r a t . The glucose concentration of the perfusate was 4.5 mM throughout. The N was equal to 5 or greater per s e c r e t i n dose. Values are given as the mean ± SEM; s i g n i f i c a n t differences are denoted by the a s t e r i s k (*) and discussed i n the text. b7 0 20 40 60 Time (min) Figure 2 68 F i g u r e 3 Pancreatic Juice Volume Response to Increasing Doses of VIP i n the Isolated Perfused Pancreas Pancreatic j u i c e volume (ul/roin) was measured over time during perfusion of the i s o l a t e d pancreas preparation with increasing doses of VIP. VIP was added v i a a sidearm to the heated, oxygenated perfusate to achieve f i n a l concentrations ranging from 0-60 nM (see key). VIP was administered during the i n t e r v a l i n dicated by the l a b e l l e d bar, a f t e r e q u i l i b r a t i o n and a basal c o l l e c t i o n period. Only one concentration of VIP was infused per r a t . The glucose concentration of the perfusate was 4.5 mM throughout. The N was equal to 5 or greater per VIP dose. Values are given as the mean ± SEM; s i g n i f i c a n t d i f ferences are denoted by the a s t e r i s k (*) and discussed i n the text. [VIP] O 0 nM • 5 0 2 0 4 0 6 0 Time (min) Figure 3 70 Figure 4 Amylase Secretion Due to Increasing Doses of CCK-8 i n the Isolated Perfused Pancreas Amylase secretion i n pancreatic j u i c e (U x 10 _ 1/min) was measured over time during perfusion of the i s o l a t e d pancreas preparation with increasing doses of CCK-8. Conditions of the experiment are as described i n the caption f o r Figure 1 . 71 2 0 C C K c • r—t < X 15 10 5 -0 0 C C K - 8 Dose O 0 PM • 0.1 V 1 • 10 • 100 • 1000 20 40 Time (min) 60 Figure 4 72 Figure 5 Amylase Secretion Due to Increasing Doses of Secretin i n the Isolated Perfused Pancreas Amylase secretion i n pancreatic ju i c e (U x 10 _ 1/min) was measured over time during perfusion of the i s o l a t e d pancreas preparation with increasing doses of s e c r e t i n . Conditions of the experiment are as described i n the caption f o r Figure 2. 7 3 Figure 5 74 Figure 6 Amylase Secretion Due to Increasing Doses of VIP i n the Isolated Perfused Pancreas Amylase secretion i n pancreatic j u i c e (U x 10 _ 1/min) was measured over time during perfusion of the i s o l a t e d pancreas preparation with increasing doses of VIP. Conditions of the experiment are as described i n the caption f o r Figure 3. 75 Figure 6 76 Figure 7 T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n Response t o I n c r e a s i n g Doses o f CCK-8 i n t h e I s o l a t e d P e r f u s e d P a n c r e a s P a n c r e a t i c t o t a l p r o t e i n s e c r e t i o n (/ig X 10~ 2 /rain) i n p a n c r e a t i c j u i c e was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n w i t h i n c r e a s i n g doses o f CCK-8 . C o n d i t i o n s o f t h e e x p e r i m e n t a r e as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 1. 77 6 o w o 0 0 C C K CCK -8 Dose O 0 pM • 0 .1 V 1 • 1 0 • 1 0 0 • 1 0 0 0 20 40 Time (min) 60 Figure 7 78 F i g u r e g Total Protein Secretion i n Pancreatic Juice i n Response to Increasing Doses of Secretin i n the Isolated Perfused Pancreas Pancreatic t o t a l protein s e c retion (/ig x lO 'Vmin) i n pancreatic j u i c e was measured over time during perfusion of the i s o l a t e d pancreas preparation with increasing doses of s e c r e t i n . Conditions of the experiment are as described i n the caption f o r Figure 2. 7 9 0 20 40 Time (min) 60 80 Figure 9 Total Protein Secretion i n Pancreatic Juice i n Response to Increasing Doses of VIP i n the Isolated Perfused Pancreas Pancreatic t o t a l protein secretion (ug x 10~ 1/min) i n pancreatic j u i c e was measured over time during perfusion of the i s o l a t e d pancreas preparation with increasing doses of VIP. Conditions of the experiment are as described i n the caption f o r Figure 3. 82 F i g u r e I Q I n s u l i n Release into Perfusate (expressed per ml of perfusate) i n Response to Increasing Doses of CCK-8 i n the Isolated Perfused Pancreas The concentration of i n s u l i n released (/iU x lO 'Vial) into perfusate was measured over time during perfusion of the i s o l a t e d pancreas preparation with increasing doses of CCK-8. Conditions of the experiment are as described i n the caption f o r Figure 1. oo LO 84 Figure 11 I n s u l i n Release into Perfusate (expressed per min) i n Response to Increasing Doses of CCK-8 i n the Isolated Perfused Pancreas Ins u l i n release (/iU x 10" 1/min) into perfusate was measured over time during perfusion of the i s o l a t e d pancreas preparation with increasing doses of CCK-8. Conditions of the experiment are as described i n the caption f o r Figure 1. Comparison of Figures 4 and 5 show s i m i l a r patterns of response, i n d i c a t i n g that perfusion losses i n the experimental preparation were small and d i d not vary s i g n i f i c a n t l y over the course of the experiment. 10 C C K CCK-8 Dose O 0 pM — • 0.1 V 1 T 10 • 100 CD £ CO - r H CD 1 O 5 3 x 0 L 0 20 40 60 Time (min) F i g u r e 11 86 F i g u r e 1 2 I n s u l i n R e l e a s e i n t o P e r f u s a t e ( e x p r e s s e d p e r min) i n Response t o I n c r e a s i n g Doses o f S e c r e t i n i n t h e I s o l a t e d P e r f u s e d P a n c r e a s I n s u l i n r e l e a s e (uU x 1 0 - 1 / m i n ) i n t o p e r f u s a t e was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n w i t h i n c r e a s i n g doses o f s e c r e t i n . C o n d i t i o n s o f t h e e x p e r i m e n t a r e as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 2 . 87 Figure 12 88 Ficrure 13 I n s u l i n R e l e a s e i n t o P e r f u s a t e ( e x p r e s s e d p e r min) i n Response t o I n c r e a s i n g Doses o f V I P i n t h e I s o l a t e d P e r f u s e d P a n c r e a s I n s u l i n r e l e a s e (uU x 10" 2/min) i n t o p e r f u s a t e was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n w i t h i n c r e a s i n g doses o f V I P . C o n d i t i o n s o f t h e e x p e r i m e n t a r e as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 3. 89 0 L 20 40 60 Time (mm) F i g u r e 13 90 F i g u r e 14 S o m a t o s t a t i n R e l e a s e i n t o P e r f u s a t e ( e x p r e s s e d p e r ml o f p e r f u s a t e ) i n Response t o I n c r e a s i n g Doses o f CCK-8 i n t h e I s o l a t e d P e r f u s e d P a n c r e a s The c o n c e n t r a t i o n o f s o m a t o s t a t i n (pg x lO'Vffll) r e l e a s e d i n t o p e r f u s a t e was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n w i t h i n c r e a s i n g d o s e s o f CCK-8. C o n d i t i o n s o f t h e e x p e r i m e n t a r e as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 1 . 91 20 40 60 Time (min) F i g u r e 14 92 Figure 15 S o m a t o s t a t i n R e l e a s e i n t o P e r f u s a t e ( e x p r e s s e d p e r min) i n Response t o I n c r e a s i n g Doses o f CCK-8 i n t h e I s o l a t e d P e r f u s e d P a n c r e a s S o m a t o s t a t i n r e l e a s e (pg x 10" 2/min) i n t o p e r f u s a t e was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n w i t h i n c r e a s i n g doses o f CCK-8. C o n d i t i o n s o f t h e e x p e r i m e n t a r e as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 1. As n o t e d i n F i g u r e 5, c o m p a r i s o n o f F i g u r e s 6 and 7 show s i m i l a r p a t t e r n s o f r e s p o n s e , i n d i c a t i n g t h a t p e r f u s i o n l o s s e s i n t h e e x p e r i m e n t a l p r e p a r a t i o n were s m a l l and d i d n o t v a r y s i g n i f i c a n t l y o v e r t h e c o u r s e o f t h e e x p e r i m e n t . 93 CD m CO" CD % . — C 1 CO o -i-j 73 O X -»_> CO oo s o (71 2.5 -2.0 1.5 1.0 0.5 0.0 C C K C C K - 8 Dose O 0 pM • 0 .1 V 1 T 1 0 • 1 0 0 • 1 0 0 0 0 20 40 Time (min) 60 Figure 15 94 F i g u r e 16 S o m a t o s t a t i n R e l e a s e i n t o P e r f u s a t e ( e x p r e s s e d p e r min) i n Response t o I n c r e a s i n g Doses o f S e c r e t i n i n t h e I s o l a t e d P e r f u s e d P a n c r e a s S o m a t o s t a t i n r e l e a s e (pg x 1 0~ 2/min) i n t o p e r f u s a t e was, measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n w i t h i n c r e a s i n g doses o f s e c r e t i n . C o n d i t i o n s o f t h e e x p e r i m e n t a r e as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 2. 9 5 4 r S E C R E T I N ! [ S E C R E T I N ] O 0 nM • 0 5 V 1 T 5 • 10 CD m 73 O -(-) £0 O 00 C\2 X a 2 h 0 L 0 20 40 Time (min) 60 Figure 16 96 Figure 1 7 S o m a t o s t a t i n R e l e a s e i n t o P e r f u s a t e ( e x p r e s s e d p e r min) i n Response t o I n c r e a s i n g Doses o f VIP i n t h e I s o l a t e d P e r f u s e d P a n c r e a s S o m a t o s t a t i n r e l e a s e (pg x 10~ 2/min) i n t o p e r f u s a t e was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n w i t h i n c r e a s i n g doses o f V I P . C o n d i t i o n s o f t h e e x p e r i m e n t a r e as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 3. KM C H I—" 3 fD ? P O ro o o CO o 98 F i g u r e 18 P a n c r e a t i c J u i c e Volume Response t o I n c r e a s e d G l u c o s e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d Pancreas P a n c r e a t i c j u i c e volume (Ail/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d pancreas p r e p a r a t i o n under c o n s t a n t CCK s t i m u l a t i o n (10 pM, f i n a l c o n c e n t r a t i o n ) a d m i n i s t e r e d by s i d e a r m i n f u s i o n . A f t e r an e q u i l i b r a t i o n and b a s a l p e r i o d , t h e p e r f u s a t e c o n c e n t r a t i o n o f g l u c o s e was a l t e r e d ( 4 . 5 o r 1 7 . 9 mM, f i n a l c o n c e n t r a t i o n ) d u r i n g t h e " t e s t " p e r i o d by exchang ing t h e p e r f u s a t e ( see k e y ) . The N was e q u a l t o 5 o r g r e a t e r p e r g l u c o s e c o n d i t i o n . V a l u e s a r e g i v e n as t h e mean ± SEM; s i g n i f i c a n t d i f f e r e n c e s a r e d e n o t e d by the a s t e r i s k (*) and d i s c u s s e d i n t h e t e x t . 9 9 cd 9 ^ - a. CD s > Glucose (mM) 4.5 TEST 0 CCK 10 pM 0 4.5 [Glucose] for TEST O 4.5 mM • 17.9 20 40 Time (min) 60 F i g u r e 18 100 F i g u r e 19 E f f e c t o f I n c r e a s e d G l u c o s e on Amylase S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s Amylase (U x 10" 2/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under c o n s t a n t CCK s t i m u l a t i o n and d i f f e r e n t p e r f u s a t e g l u c o s e c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 18. 101 Glucose (mM) 4.5 1.0 -CD 1 0.5 x 3. 0.0 L TEST C C K 10 p M 4.5 [Glucose] for TEST O 4.5 mM • 179 0 20 40 Time (min) 60 Figure 19 102 Figure 20 E f f e c t o f I n c r e a s e d G l u c o s e on T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e CCK-S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s The t o t a l p r o t e i n i n p a n c r e a t i c j u i c e (uq x 10~ 2/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under c o n s t a n t CCK s t i m u l a t i o n and d i f f e r e n t p e r f u s a t e g l u c o s e c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 18. 103 Glucose (mM) 4.5 TEST 4.5 CCK 10 pM c .5 'CD S O cv u 1 1 cu o 1 CO o OfJ 3. [Glucose] for TEST O 4.5 mM • 179 0 L 0 20 40 60 Time (min) Figure 20 104 F i g u r e 21 E f f e c t o f I n c r e a s e d G l u c o s e on I n s u l i n R e l e a s e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s The i n s u l i n r e l e a s e i n t o p e r f u s a t e (jiU x 10"" 2/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under c o n s t a n t CCK s t i m u l a t i o n and d i f f e r e n t p e r f u s a t e g l u c o s e c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 18. 105 4.5 15 10 3 73 5 -0 L 0 Glucose (mM) T E S T CCK 1 0 pM 4.5 [Glucose] for TEST 20 40 60 T ime (min) F i g u r e 21 106 Figure ?2 E f f e c t o f I n c r e a s e d G l u c o s e on S o m a t o s t a t i n R e l e a s e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s The s o m a t o s t a t i n r e l e a s e i n t o p e r f u s a t e (pg X 10~ 2/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under c o n s t a n t CCK s t i m u l a t i o n and d i f f e r e n t p e r f u s a t e g l u c o s e c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 18. 107 1 G lucose (mM) : 4.5 I TEST 4.5 1.5 -CD 00 CO CD ~CD '5 l.o r £ 7 00 o cC t ! O (71 0.5 r 0.0 L C C K 10 p M [Glucose] for TEST O 4.5 mM • 17.9 0 20 40 60 Time (min) Figure 22 108 F i g u r e 23 E f f e c t of I n s u l i n on Pancreatic Juice Volume i n the CCK-Stimulated, Isolated Perfused Pancreas Pancreatic ju i c e volume (ul/min) was measured over time during perfusion of the i s o l a t e d pancreas preparation under constant CCK stimulation ( 1 0 pM, f i n a l concentration) administered by sidearm i n f u s i o n . A f t e r an e q u i l i b r a t i o n and basal period, i n s u l i n ( 0 or 2 0 0 uU/ml, f i n a l concentration) was added v i a sidearm inf u s i o n to the perfusate as indicated by the bar (see key). Perfusate glucose was maintained at 4 . 5 mM throughout. The N was equal to 5 or greater per i n s u l i n condition. Values are given as the mean ± SEM; s i g n i f i c a n t differences are denoted by the a s t e r i s k (*) and discussed i n the text. 109 CD CJ 3 o • r—* cC CD U , CJ C CO Cu 3. CCK 10 pM Insu l in | [ Insul in] ! O 0 /iV/ml 200 CD O > 0 L 0 2 0 4 0 Time (min ) 6 0 Figure 23 Figure 24 E f f e c t o f I n s u l i n on Amylase S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s Amylase (U x l 0 " 2 / m i n ) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under c o n s t a n t CCK s t i m u l a t i o n and two d i f f e r e n t i n s u l i n c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 23. I l l CCK 10 pM  Insu l in j [ Insul in] O 0 yuU/ml! • 200 j T * 0 20 40 60 Time (min) 112 Figure 25 E f f e c t o f I n s u l i n on T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s The t o t a l p r o t e i n i n p a n c r e a t i c j u i c e (/ig x 1 0 ~ 2 / r a i n ) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d -p a n c r e a s p r e p a r a t i o n under c o n s t a n t CCK s t i m u l a t i o n and two d i f f e r e n t i n s u l i n c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 23. 113 CCK 10 pM Insu l in r O W Cu O 3. [ Insul in] O 0 yuU/ml • 200 5 . * 0 L 0 j L 20 40 Time (min) 60 Figure 25 114 Figure 26 E f f e c t o f Added I n s u l i n on Measured I n s u l i n R e l e a s e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s I n s u l i n i n p e r f u s a t e (/iU x 10" 2/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under c o n s t a n t CCK s t i m u l a t i o n and two d i f f e r e n t i n s u l i n c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 23. These measurements c o n f i r m e d t h a t t h e i n t e n d e d dosage o f p e r f u s e d i n s u l i n was i n d e e d g i v e n . 115 10 -3 ' o D CCK 10 pM Insu l in [ Insul in] I O 0 / 2 U / m l I • 200 I 0 L 0 20 40 Time (min) 60 Figure 26 116 F i g u r e 27 E f f e c t o f I n s u l i n on S o m a t o s t a t i n R e l e a s e i n t h e C C K - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s The s o m a t o s t a t i n r e l e a s e i n t o p e r f u s a t e (pg x 10" 1/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under c o n s t a n t CCK s t i m u l a t i o n and two d i f f e r e n t i n s u l i n c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 23. 10 r I nsu l in s x a, 0 0 [ Insul in] O 0 yuU /ml • 200 20 40 Time (min) 60 F i g u r e 27 118 F i g u r e 28 P a n c r e a t i c J u i c e Volume Response t o I n c r e a s e d G l u c o s e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s P a n c r e a t i c j u i c e volume (/il/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n w i t h s e c r e t i n s t i m u l a t i o n ( 0 . 5 nM, f i n a l c o n c e n t r a t i o n ) a d m i n i s t e r e d by s i d e a r m i n f u s i o n d u r i n g t h e p e r i o d marked by t h e l a b e l l e d b a r . A f t e r an e q u i l i b r a t i o n and b a s a l p e r i o d , s e c r e t i n was s t a r t e d and 15 min l a t e r , t h e p e r f u s a t e c o n c e n t r a t i o n o f g l u c o s e was a l t e r e d ( 4 . 5 o r 17.9 mM, f i n a l c o n c e n t r a t i o n ) d u r i n g t h e " t e s t " p e r i o d by e x c h a n g i n g t h e p e r f u s a t e ( see k e y ) . The N was e q u a l t o 5 o r g r e a t e r p e r g l u c o s e c o n d i t i o n . V a l u e s a r e g i v e n as t h e mean ± SEM; s i g n i f i c a n t d i f f e r e n c e s a r e d e n o t e d by t h e a s t e r i s k (*) and d i s c u s s e d i n t h e t e x t . 119 Glucose (mM) 4~5 '[TEST i! 4~5 j SECRETIN 0.5 nM | 0 20 40 60 80 Time (min) Figure 28 L20 Figure 29 E f f e c t o f I n c r e a s e d G l u c o s e on Amylase S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s Amylase (U x 1 0 _ 1 / m i n ) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under s e c r e t i n s t i m u l a t i o n and two d i f f e r e n t " t e s t " p e r f u s a t e g l u c o s e c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 28. 121 Glucose (mM) ; 4.5 TEST 4.5 SECRETIN 0.5 nM ! 4 -c '5 CD C 00 \ ^ o >— I—I < X 2 h 0 [Glucose] for TEST O 4.5 mM • 17.9 0 20 40 60 80 Time (min) Figure 29 122 F i g u r e 30 E f f e c t o f I n c r e a s e d G l u c o s e on T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e S e c r e t i n -S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s The t o t a l p r o t e i n i n p a n c r e a t i c j u i c e (/*g x 10" 1/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under s e c r e t i n s t i m u l a t i o n and two d i f f e r e n t " t e s t " p e r f u s a t e g l u c o s e c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 28. 123 Glucose (mM) j 4.5 TEST 4.5 j SECRETIN 0.5 nM C o E— X 3. 4 E 3 2 -0 [Glucose] for TEST O 4.5 mM • 17.9 0 20 40 60 Time (min) 80 F i g u r e 30 F i g u r e 31 E f f e c t o f I n c r e a s e d G l u c o s e on I n s u l i n R e l e a s e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s The i n s u l i n r e l e a s e i n t o p e r f u s a t e (uU x l 0 ~ 2 / m i n ) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under s e c r e t i n s t i m u l a t i o n and under two d i f f e r e n t p e r f u s a t e g l u c o s e c o n c e n t r a t i o n s , i n i t i a l l y 4.5 mM t h e n 17.9 mM d u r i n g t h e " t e s t " p e r i o d , C o n d i t i o n s o f t h e e x p e r i m e n t were o t h e r w i s e as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 28. 125 Glucose (mM) ! 4.5 TEST 4.5 j Figure 31 F i g u r e 32 E f f e c t o f I n c r e a s e d G l u c o s e on S o m a t o s t a t i n R e l e a s e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s The s o m a t o s t a t i n r e l e a s e i n t o p e r f u s a t e (pg x 10~ 2/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under s e c r e t i n s t i m u l a t i o n and two d i f f e r e n t p e r f u s a t e g l u c o s e c o n c e n t r a t i o n s , 4.5 mM and t h e " t e s t " c o n d i t i o n o f 17.9 mM. C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 28. 127 10 Glucose (mM) 4.5 TEST 4.5 1 cu 73 CD o x tf s° c °-o 5 -0 SECRETIN 0.5 nM | O—O 0 20 40 60 Time (min) 80 Figure 32 128 Figure 33 E f f e c t o f I n s u l i n on P a n c r e a t i c J u i c e Volume i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s P a n c r e a t i c j u i c e volume ( u l / m i n ) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n . S e c r e t i n (0.5 nM, f i n a l c o n c e n t r a t i o n ) was a d m i n i s t e r e d by s i d e a r m i n f u s i o n d u r i n g t h e marked p e r i o d . A f t e r an e q u i l i b r a t i o n and b a s a l p e r i o d , i n s u l i n ( 0 o r 200 uU/ml, f i n a l c o n c e n t r a t i o n ) was added v i a s i d e a r m i n f u s i o n t o t h e p e r f u s a t e as i n d i c a t e d by t h e b a r (see k e y ) . P e r f u s a t e g l u c o s e was m a i n t a i n e d a t 4.5 mM t h r o u g h o u t . The N was e q u a l t o 5 o r g r e a t e r p e r i n s u l i n c o n d i t i o n . V a l u e s a r e g i v e n as t h e mean ± SEM; s i g n i f i c a n t d i f f e r e n c e s a r e d e n o t e d by t h e a s t e r i s k (*) and d i s c u s s e d i n t h e t e x t . 129 SECRETIN 0.5 nM CJ G 1 !-[ Insul in] r- 0 u U / n i l 200 o > 0 L 0 20 40 60 Time (min) 80 F i g u r e 33 F i g u r e 34 E f f e c t o f I n s u l i n on Amylase S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s Amylase (U x l O ^ / m i n ) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under s e c r e t i n s t i m u l a t i o n and two d i f f e r e n t i n s u l i n c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 33. 131 SECRETIN 0.5 nM [ insu l in ! ; : ' : { [ Insul in] [ J O 0 yuU/ml ' • 200 •> 1 1 l _ i ! i i_ 20 40 60 80 Time (min) F i g u r e 3 5 E f f e c t o f I n s u l i n on T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s The t o t a l p r o t e i n i n p a n c r e a t i c j u i c e (ug x l O - v m i n ) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under s e c r e t i n s t i m u l a t i o n and two d i f f e r e n t i n s u l i n c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 33. 133 6 QJ O '—. 13 -*-> o 14 x 2 CO) =1 0 L S E C R E T I N 0.5 nM Insu l in ; [ Insul in] 1 0 yUU . m i • 200 0 20 40 60 Time (min) 80 Figure 35 134 Figure 36 E f f e c t o f Added I n s u l i n on Measured I n s u l i n R e l e a s e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s I n s u l i n i n p e r f u s a t e (/JU X 1 0 ~ 2 / m i n ) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under s e c r e t i n s t i m u l a t i o n and two d i f f e r e n t i n s u l i n c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 3 3 . 135 10 SECRETIN 0.5 nM CD \ « o £ 3. 0 L 0 [ Insul in] 0 yuU/ml 200 20 40 60 Time (min) 80 Figure 36 136 Figure 37 E f f e c t o f I n s u l i n on S o m a t o s t a t i n R e l e a s e i n t h e S e c r e t i n - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s The s o m a t o s t a t i n r e l e a s e i n t o p e r f u s a t e (pg x 10" 2/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under s e c r e t i n s t i m u l a t i o n and two d i f f e r e n t i n s u l i n c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 33. 137 2 7} •-— 5 c o s * OD tf a, o 00 0 L ECRETIN 0.5 nM Insul in l [ Insul in] 0 fiU/ml > 200 4-/ 0 20 40 60 Time (min) 80 Figure 37 138 Figure 38 P a n c r e a t i c J u i c e Volume Response t o I n c r e a s e d G l u c o s e i n t h e V I P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s P a n c r e a t i c j u i c e volume ( u l / m i n ) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n w i t h VIP s t i m u l a t i o n (15 nM, f i n a l c o n c e n t r a t i o n ) a d m i n i s t e r e d by s i d e a r m i n f u s i o n d u r i n g t h e p e r i o d marked by t h e l a b e l l e d b a r . A f t e r an e q u i l i b r a t i o n and b a s a l p e r i o d , V I P was s t a r t e d and 15 min l a t e r , t h e p e r f u s a t e c o n c e n t r a t i o n o f g l u c o s e was a l t e r e d (4.5 o r 17.9 mM, f i n a l c o n c e n t r a t i o n ) d u r i n g t h e " t e s t 1 * p e r i o d by e x c h a n g i n g t h e p e r f u s a t e ( s e e k e y ) . The N was e q u a l t o 5 o r g r e a t e r p e r g l u c o s e c o n d i t i o n . V a l u e s a r e g i v e n as t h e mean ± SEM; s i g n i f i c a n t d i f f e r e n c e s a r e denoted by t h e a s t e r i s k (*) and d i s c u s s e d i n t h e t e x t . 139 Glucose (mM) 4.5 ! TEST f TE i i  | VIP 15 nM | 2 - ! [Glucose] I , i , i i I ;— 0 20 40 60 80 Time ( m i n ) Figure 38 F i g u r e 3 9 E f f e c t o f I n c r e a s e d G l u c o s e on Amylase S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e V I P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s Amylase (U x 10~ 2/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under V I P s t i m u l a t i o n and two d i f f e r e n t " t e s t " p e r f u s a t e g l u c o s e c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 38. 141 9 cd 01 1 L C r - l i C ^ I < X ZD 0 Glucose (mM) 4.5 TEST 4.5 VIP 15 nM [Glucose] for TEST O 4.5 mM • 17.9 0 20 40 60 Time (min) 80 F i g u r e 39 142 F i g u r e 40 E f f e c t o f I n c r e a s e d G l u c o s e on T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e V I P -S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s The t o t a l p r o t e i n i n p a n c r e a t i c j u i c e (ug x 10~ 2/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under V I P s t i m u l a t i o n and two d i f f e r e n t " t e s t " p e r f u s a t e g l u c o s e c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 38. 143 Glucose (mM) 4.5 TEST 4.5 1.5 c B 1.0 'o G O CJ C u O 03 x 0.5 OX) 3. 0.0 VIP 15 nM [Glucose] for TEST O 4.5 mM • 17.9 i / \ ! ! 1 0 20 40 60 Time (min) 80 Figure 40 F i g u r e 41 E f f e c t o f I n c r e a s e d G l u c o s e on I n s u l i n R e l e a s e i n t h e V I P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s The i n s u l i n r e l e a s e i n t o p e r f u s a t e (^U x 10~ 2/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under VIP s t i m u l a t i o n and two d i f f e r e n t p e r f u s a t e g l u c o s e c o n c e n t r a t i o n s , 4 .5 mM and t h e " t e s t " c o n d i t i o n o f 17.9 mM. C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 38. 145 <j • — V) £ tf CD \ — ro CD C£ O 8 6 4 0 Glucose (mM) 4.5 TEST 4.5 VIP 15 nM ! U \ Q ^ ' ^ y 0 20 40 60 Time (min) 80 F i g u r e 41 F i g u r e 42 E f f e c t o f I n c r e a s e d G l u c o s e on S o m a t o s t a t i n R e l e a s e i n t h e V I P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s The s o m a t o s t a t i n r e l e a s e i n t o p e r f u s a t e (pg x 10~ 2/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under VIP s t i m u l a t i o n and two d i f f e r e n t p e r f u s a t e g l u c o s e c o n c e n t r a t i o n s , 4.5 mM and t h e " t e s t " c o n d i t i o n o f 17.9 mM. C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 3tJ. 147 Glucose (mM) 4.5 TEST 4.5 4 CO 73 X OD o 00 0 L VIP 15 nM 0 20 40 60 Time (min) 80 Figure 42 148 F i g u r e 43 E f f e c t o f I n s u l i n on P a n c r e a t i c J u i c e Volume i n t h e V I P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s P a n c r e a t i c j u i c e volume ( u l / m i n ) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n . S e c r e t i n (0.5 nM, f i n a l c o n c e n t r a t i o n ) was a d m i n i s t e r e d by s i d e a r m i n f u s i o n d u r i n g t h e marked p e r i o d . A f t e r an e q u i l i b r a t i o n and b a s a l p e r i o d , i n s u l i n (0 o r 200 uU/ml, f i n a l c o n c e n t r a t i o n ) was added v i a s i d e a r m i n f u s i o n t o t h e p e r f u s a t e as i n d i c a t e d by t h e b a r (see k e y ) . P e r f u s a t e g l u c o s e was m a i n t a i n e d a t 4.5 mM t h r o u g h o u t . The N was e q u a l t o 5 o r g r e a t e r p e r i n s u l i n c o n d i t i o n . V a l u e s a r e g i v e n as t h e mean ± SEM; s i g n i f i c a n t d i f f e r e n c e s a r e d e n o t e d by t h e a s t e r i s k (*) and d i s c u s s e d i n t h e t e x t . 2 r VIP 15 nM Insulin [Insulin] O 0 / x U / m l • 200 6 i o o 20 40 60 Time (min) 80 F i g u r e 43 Figure 44 E f f e c t o f I n s u l i n on Amylase S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e V I P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s Amylase (U x 1 0 _ 1 / m i n ) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under VIP s t i m u l a t i o n and two d i f f e r e n t i n s u l i n c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 4 3 . 151 10 r CD -iH 7 c ^ < X D 0 L VIP 15 nM i Insu l in [ Insul in] O 0 fxl' m l • 200 0 20 40 60 Time (mm) 80 Figure 44 152 Figure 45 E f f e c t o f I n s u l i n on T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e V I P - S t i m u l a t e d , I s o l a t e d P e r f u s e d P a n c r e a s The t o t a l p r o t e i n i n p a n c r e a t i c j u i c e (ng x 10~ 1/min) was measured o v e r t i m e d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n under VIP s t i m u l a t i o n and two d i f f e r e n t i n s u l i n c o n c e n t r a t i o n s . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 43. 10 r o cu o cd x o QD 3. 0 0 VIP 15 n M I n s u l i n [ I n s u l i n ] O 0 / x U / m l • 200 20 40 60 Time (min) 80 Figure 45 154 Figure 46 E f f e c t of Added I n s u l i n on Measured Ins u l i n Release i n the VIP-Stimulated, Isolated Perfused Pancreas Insulin i n perfusate (uU x 10" 2/min) was measured over time during perfusion of the i s o l a t e d pancreas preparation under VIP stimulation and two d i f f e r e n t i n s u l i n concentrations. Conditions were as described i n the caption f o r Figure 43. 155 I VIP 15 nM I D R ZZZZZZT 1 ' Insulin; i ~ ~~i ' ' | [ Insul in] j O 0 /uU/ml j 0 20 40 60 80 Time (min) Figure 46 156 Figure 47 E f f e c t of I n s u l i n on Somatostatin Release i n the VIP-Stimulated, Isolated Perfused Pancreas The somatostatin release into perfusate (pg x 10~ 2/min) was measured over time during perfusion of the i s o l a t e d pancreas preparation under VIP stimulation and two d i f f e r e n t i n s u l i n concentrations. Conditions were as described i n the caption f o r Figure 4 3 . 157 VIP 15 nM 2 r CD 73 CO -CD 0 Cd 73 o CO c 1—1 c m x oo Insu l in I [ Insul in] j O 0 / zU/ml 200 I I / \ T T o - o - , -0 L 0 20 40 60 Time (mm) 80 F i g u r e 47 158 Figure 48. E f f e c t s o f A n o x i a on P a n c r e a t i c J u i c e Volume i n th e I s o l a t e d P e r f u s e d P a n c r e a s : L a c k o f E v i d e n c e f o r a P r o t e c t i v e E f f e c t f r om G l u c o s e o r I n s u l i n P r e t r e a t m e n t The e f f e c t o f p r e t r e a t m e n t w i t h i n c r e a s e d p e r f u s a t e g l u c o s e (17.9 mM, f i n a l c o n c e n t r a t i o n ) o r i n s u l i n (100 uU/ml, f i n a l c o n c e n t r a t i o n ) on p a n c r e a t i c j u i c e volume (/il/min) under c o n t i n u o u s CCK s t i m u l a t i o n (0.18 nM, f i n a l c o n c e n t r a t i o n ) was a s s e s s e d i n t h e i s o l a t e d p e r f u s e d p a n c r e a s . A f t e r an i n i t i a l e q u i l i b r a t i o n p e r i o d o f CCK i n f u s i o n , p e r f u s a t e g l u c o s e was i n c r e a s e d from a b a s e l i n e o f 4.5 mM, o r s i d e a r m i n s u l i n (made up i n p e r f u s a t e w i t h 4.5 mM g l u c o s e ) was s t a t e d f o r a 10 min " t e s t " p e r i o d (see k e y ) . A f t e r t h e t e s t p e r i o d , a n o x i a ( p e r f u s i o n w i t h p e r f u s a t e e q u i l i b r a t e d w i t h N 2 -95% C0 2-5%) was i n d u c e d f o r 20 m i n u t e s t h e n normal o x y g e n a t i o n o f t h e p e r f u s a t e was r e - e s t a b l i s h e d . N = 5 o r more i n e a c h t r e a t m e n t g r o u p . R e s u l t s a r e e x p r e s s e d as means ± SEM; s i g n i f i c a n t d i f f e r e n c e s a r e i n d i c a t e d by t h e a s t e r i s k (*) and d i s c u s s e d i n t h e t e x t . 159 Glucose 4.5 mM ; Test Glucose 4.5 mM 9 ~ o P c \ cC — •CD > 3 9 -0 L CCK 0.18 nM Anoxia Test Anoxia Glucose 4.5 mM Glucose 17.9 mM Insulin 100 /nil/ml 0 20 40 Time (min) 60 Figure 48 160 F i g u r e 49 E f f e c t s o f A n o x i a on Amylase S e c r e t i o n i n t h e I s o l a t e d P e r f u s e d P a n c r e a s Amylase s e c r e t i o n i n t o t h e p a n c r e a t i c j u i c e (U x 10" 1 / r a i n ) was measured under c o n d i t i o n s o f c o n s t a n t CCK s t i m u l a t i o n and a n o x i a . The e f f e c t s o f p r e t r e a t m e n t w i t h i n c r e a s e d g l u c o s e o r i n s u l i n were a s s e s s e d i n t h e i s o l a t e d p e r f u s e d p a n c r e a s under c o n d i t i o n s d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 48. 161 G l u c o s e 4 o m M Test• G l u c o s e 4.5 m M 10 r CCK 0.18 n M Anoxia cu 'fl — CQ ; C r •-4 < 0 -Test A n o x i a i C G l u c o s e 4.5 m M • \7 G l u c o s e 17.9 m M • I n s u l i n 100 yub'/ml -0 20 40 60 Time (mm) Figure 4 9 162 Figure so E f f e c t s o f A n o x i a on T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e i n t h e I s o l a t e d P e r f u s e d P a n c r e a s T o t a l p r o t e i n s e c r e t i o n i n t o t h e p a n c r e a t i c j u i c e (ug x lCVmin) was measured under c o n d i t i o n s o f c o n s t a n t CCK s t i m u l a t i o n and a n o x i a . The e f f e c t s o f p r e t r e a t m e n t w i t h i n c r e a s e d g l u c o s e o r i n s u l i n were a s s e s s e d i n t h e i s o l a t e d p e r f u s e d p a n c r e a s under c o n d i t i o n s d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 48. 163 Glucose 4.5 mM j Test i Glucose 4.5 mM i I CCK 0.18 nM 10 Anoxia O _ D r 0 Test Anoxia O Glucose 4.5 mM I I V Glucose 17 9 mM Insulin 1 0 0 uU/ml 0 20 40 60 T ime ( m i n ) Figure 50 164 Figure 51 E f f e c t s of Anoxia on I n s u l i n Release into Perfusate i n the Isolated Perfused Pancreas Insulin release i n t o the perfusate (jiU x 10~ 2/min) was measured under conditions of constant CCK stimulation and anoxia. The e f f e c t s of pretreatment with increased glucose or i n s u l i n were assessed i n the i s o l a t e d perfused pancreas under conditions described i n the caption f o r Figure 48. 165 Glucose 4 .5 mM ITestj Glucose 4 .5 mM CCK 0.18 nM j Anoxia ! 5 r j Test Anoxia i 7 j O Glucose 4.5 mM ,'i ! V Glucose 17.9 mM 0 20 40 60 Time ( m i n ) ;ure 5 1 166 Figure 52 E f f e c t s o f A n o x i a on S o m a t o s t a t i n R e l e a s e i n t o P e r f u s a t e i n t h e I s o l a t e d P e r f u s e d P a n c r e a s S o m a t o s t a t i n r e l e a s e i n t o t h e p e r f u s a t e (pg x 10~ 2/min) was measured under c o n d i t i o n s o f c o n s t a n t CCK s t i m u l a t i o n and a n o x i a . The e f f e c t s o f p r e t r e a t m e n t w i t h i n c r e a s e d g l u c o s e o r i n s u l i n were a s s e s s e d i n t h e i s o l a t e d p e r f u s e d p a n c r e a s under c o n d i t i o n s d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 48. 167 Glucose 4.5 mM jTesti Glucose 4.5 mM CCK 0.18 nM r Anoxia j CD 73 CC 3 9 i_ 73 O •71 CD £ 1 0 !-Test Anoxia Glucose 4.5 mM i i v ' Glucose 17.9 mM ' Insulin 1 0 0 / i U / m l 0 20 40 60 Time (min) Figure 52 ~1 F i g u r e s 53 and 54 Amylase R e l e a s e from A c i n i i n Response t o I n c r e a s i n g Doses o f CCK-8 D i s p e r s e d p a n c r e a t i c a c i n i were p r e p a r e d as d e s c r i b e d i n t h e "Methods" s e c t i o n from normoglycemic r a t s and suspended i n a HEPES i n c u b a t i o n b u f f e r ( [ g l u c o s e ] = 11.1 mM, [ C a + 2 ] = 1.69 mM). I n c r e a s i n g doses o f CCK-8 (0 - 1000 pM, f i n a l c o n c e n t r a t i o n ) were added and a c i n i were i n c u b a t e d f o r 20 min. F o l l o w i n g i n c u b a t i o n , amylase i n t h e i n c u b a t i o n medium was a s s a y e d by t h e p r o c i o n y e l l o w - s t a r c h method and n e t amylase s e c r e t i o n due t o t h e p r e s e n c e o f t h e s e c r e t a g o g u e was c a l c u l a t e d . Each d a t a p o i n t r e p r e s e n t s t h e r e s u l t s o f t h r e e t o f o u r e x p e r i m e n t s c o n d u c t e d i n t r i p l i c a t e , and t h e r e s u l t s a r e p r e s e n t e d as t h e mean % of t o t a l c e l l amylase content released ± SEM ( F i g u r e 53) and t h e mean t o t a l amount of amylase released as a function of c e l l mass (uU x 10~4/ug c e l l protein) ± SEM ( F i g u r e 54) v e r s u s dosage o f CCK (Log-Lp s c a l e a f t e r t h e 0 pM CCK p o i n t ) . Reasons f o r e x p r e s s i n g amylase s e c r e t i o n i n two forms a r e d i s c u s s e d i n t h e t e x t . When n o t v i s i b l e , t h e SEM f o r an i n d i v i d u a l p o i n t was below t h e r e s o l u t i o n o f t h e o r d i n a t e . 169 Figure 53 I ' V —< < X ID =1 0 r-. 1 , x unstimulated / Z. ! , i_ \ 'A 0 1 2 3 [ CCK-8 ] ( Log 1 Q (Dose in pM)) F i g u r e 54 171 F i g u r e s 55 and 56 Amylase R e l e a s e from A c i n i i n Response t o I n c r e a s i n g Doses o f S e c r e t i n Under i n c u b a t i o n and a s s a y c o n d i t i o n s i d e n t i c a l t o t h o s e d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e s 53 and 54, t h e amylase s e c r e t o r y r e s p o n s e o f d i s p e r s e d p a n c r e a t i c a c i n i t o i n c r e a s i n g doses o f s e c r e t i n (0 - 100 nM) was measured. F o u r e x p e r i m e n t s were c a r r i e d o u t , e a ch i n t r i p l i c a t e , and t h e r e s u l t s a r e p r e s e n t e d as t h e mean % of t o t a l c e l l amylase content release ± SEM ( F i g u r e 55) and t h e mean t o t a l amylase released as a function of c e l l mass (uU x 10~4/ng c e l l protein) ± SEM ( F i g u r e 56) v e r s u s dosage o f s e c r e t i n . 30 r p 1 2 3 4 5 [Secre t in ] (Log (Dose i n pM)) F i g u r e 55 173 Figure 56 174 F i g u r e s 57 a,nd, 58 Amylase R e l e a s e from A c i n i i n Response t o I n c r e a s i n g Doses o f VIP The amylase s e c r e t o r y r e s p o n s e o f d i s p e r s e d p a n c r e a t i c a c i n i t o i n c r e a s i n g doses o f V I P (0 - 100 nM) was a s s e s s e d . C o n d i t i o n s were as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e s 53 and 54. Three e x p e r i m e n t s were c a r r i e d o u t , each i n t r i p l i c a t e , and t h e r e s u l t s a r e p r e s e n t e d as t h e mean % of t o t a l c e l l amylase content release ± SEM ( F i g u r e 57) and t h e mean t o t a l amylase released as a function of c e l l mass (uU x 10~4/ng c e l l protein) ± SEM ( F i g u r e 58) v e r s u s dosage o f V I P . 30 -0 -10 L T 0 unstimulated 1 2 3 4 5 [VIP] (Log 1 Q (Dose i n pM)) F i g u r e 57 w T. < A . , - Y unstimulated 0 -7 /— 1 2 4 [VIP] ( L o g 1 0 ( D o s e i n pM)) Figure 58 1 7 7 F i g u r e s 59 and 60 Amylase Release from A c i n i i n Response t o I n c r e a s i n g Doses of M e t h a c h o l i n e The amylase s e c r e t o r y response o f d i s p e r s e d p a n c r e a t i c a c i n i t o i n c r e a s i n g doses o f methacholine (0 - 100 nM) was a s s e s s e d . C o n d i t i o n s were as d e s c r i b e d i n the c a p t i o n f o r F i g u r e s 53 and 54. Three experiments were c a r r i e d out, each i n t r i p l i c a t e , and t h e r e s u l t s are pre s e n t e d as the mean % of total cell amylase content release ± SEM ( F i g u r e 59) and t h e mean total amylase released as a function of cell mass (nU x 10~4/ng cell protein) ± SEM ( F i g u r e 60) v e r s u s dosage o f methacholine. 71 6\ 30 --r 20 j-178 0 L unstimulated 0 1 2 3 4 5 [ iMethachol ine] ( Log 1 Q (Dose in nM)) F i g u r e 59 0 1 2 3 4 5 [Methacho l ine ] (Log (Dose in nM)) Figure 60 180 Figures 61 and 62 S i g n i f i c a n c e o f I s l e t Fragments i n t h e D i s p e r s e d P a n c r e a t i c A c i n i P r e p a r a t i o n : The Effect of Increasing Glucose Concentration on I n s u l i n and Amylase Release into the Incubation Medium I n s u l i n r e l e a s e (uU/mg a c i n a r c e l l p r o t e i n , F i g u r e 61) and amylase s e c r e t i o n (uU x 10" 3/>g c e l l p r o t e i n , F i g u r e 62) were measured i n t h e i n c u b a t i o n medium o f a c i n i f r o m n o r m o g l y c e m i c r a t s . A c i n i p u r i f i e d i n t h e normal f a s h i o n were v a r i o u s l y exposed t o a c o n s t a n t s e c r e t o r y s t i m u l u s , e i t h e r s e c r e t i n (10 nM) o r CCK-8 (100 pM) and g l u c o s e i n t h e medium was v a r i e d between 0 and 20 mM. R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM o f s i x e x p e r i m e n t s r u n i n t r i p l i c a t e . 181 1 0 0 - , S e c r e to r y S t i m u l u s ; C Se c r e t i n 10 nM : • C C K - 8 100 pM i i ' . . - • / _^_\ • ; , -r y  0 2 4 6 10 20 30 [Glucose] (mM) 182 10 -Secretory S t imu lu s C S ec r e t i n 10 nM • C C K - 8 100 pM o i l O r 1 1 X 0 0 2 4 6 10 20 [Glucose] (mM) 30 Figure 62 183 Figure 63 S i g n i f i c a n c e o f I s l e t Fragments i n t h e D i s p e r s e d P a n c r e a t i c A c i n i P r e p a r a t i o n : The Effect of Increasing CCK-8 Doses on Insulin Release into the Medium I n s u l i n r e l e a s e (/uU/mg a c i n a r c e l l p r o t e i n ) was measured i n t h e i n c u b a t i o n medium o f a c i n i from f a s t e d , n ormoglycemic r a t s . A c i n i were p r e p a r e d as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e s 61 and 62 and i n c u b a t e d w i t h i n c r e a s i n g dosages o f CCK-8 (0-1000 pM) w h i l e g l u c o s e was m a i n t a i n e d a t 11.2 mM. R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM o f t h r e e e x p e r i m e n t s r u n i n t r i p l i c a t e . 184 100 y . 80 h 60 h-20 I Glucose = 11.2 mM unstimulated 0 L. 0 1 2 3 4 [CCK -8] (Log1 0(Dose in pM)) Figure 63 185 F i g u r e 64 S i g n i f i c a n c e o f I s l e t Fragments i n t h e D i s p e r s e d P a n c r e a t i c A c i n i P r e p a r a t i o n : The Effect of GIP and/or Glucose on I n s u l i n Release into the Medium I n s u l i n r e l e a s e (jiU/mg a c i n a r c e l l p r o t e i n ) was measured i n t h e i n c u b a t i o n medium o f a c i n i from normoglycemic r a t s . A c i n i were p r e p a r e d as d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e s 61 and 62 and i n c u b a t e d under t h r e e d i f f e r e n t s e c r e t o r y c o n d i t i o n s : a b a s a l c o n d i t i o n , s e c r e t i n (10 nM), and CCK-8 (100 pM). D i f f e r e n t c o m b i n a t i o n s o f GIP and g l u c o s e i n t h e medium were a s s e s s e d f o r an e f f e c t on i n s u l i n r e l e a s e ( s e e f i g u r e k e y ) . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r t h r e e e x p e r i m e n t s r u n i n t r i p l i c a t e . C H (D O N 4 > f/J (D O •1 CD rf O 1 I — CD p CO 0> f/J fD o 09 O o O O I CO Insulin Release from l s l e l Fragments (/j.U/nig cell protein) o o fO o o o OD Cu 187 Figures 65 and 66 E f f e c t o f I n s u l i n P r e s e n t O n l y D u r i n g t h e I n c u b a t i o n P e r i o d on C C K - S t i m u l a t e d Amylase S e c r e t i o n Amylase r e l e a s e e x p r e s s e d as % c e l l content ( F i g u r e 65) and uU x 10~3/ixg c e l l protein ( F i g u r e 66) was measured i n C C K - s t i m u l a t e d a c i n i p r e p a r e d f r o m normoglycemic r a t s . The amylase r e s p o n s e t o i n c r e a s i n g doses o f CCK-8 (0-1000 pM) under two d i f f e r e n t i n c u b a t i o n c o n d i t i o n s o f i n s u l i n ( s e e f i g u r e key) was s t u d i e d . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r t h r e e e x p e r i m e n t s r u n i n t r i p l i c a t e . 188 | I nsu l in T r e a t m e n t 30 - I Incubation (2000 uU .ml) ; 0 1 2 3 [ C C K - 8 ] (Log (Dose in pM)) Figure 65 30 -- 20 r I I nsu l in T r e a t m e n t I Incubation (2000 yul'/ml) 10 r X U n s t i m u l a t e d 0 L 0 2 3 [CCK-8] (Log (Dose in pM)) Figure 66 190 F i g u r e s 67 and 68 E f f e c t o f I n s u l i n P r e s e n t O n l y D u r i n g t h e P R E i n c u b a t i o n P e r i o d on C C K - S t i m u l a t e d Amylase S e c r e t i o n Amylase r e l e a s e e x p r e s s e d as % c e l l content ( F i g u r e 67) and uU x 10~3/ng c e l l protein ( F i g u r e 68) was measured i n C C K - s t i m u l a t e d a c i n i p r e p a r e d f r o m normoglycemic r a t s . The amylase r e s p o n s e t o i n c r e a s i n g doses o f CCK-8 (0-1000 pM) under two d i f f e r e n t p r e i n c u b a t i o n c o n d i t i o n s o f i n s u l i n ( s e e f i g u r e k ey) was s t u d i e d . P r e i n c u b a t i o n w i t h i n s u l i n was m a i n t a i n e d f o r 3^ h w i t h one exchange o f medium t h a t c o n t a i n e d f r e s h i n s u l i n . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r t h r e e e x p e r i m e n t s r u n i n d u p l i c a t e . 191 20 I n su l in T r e a t m e n t Preincubation (20,000 /uL", ml) ' — -7) CD cL* — J CD „ • i o CD •7} — < 0 L U n s t i m u l a t e d 0 1 3 [CCK] (Log 1 Q (Dose in pM)) Figure 67 192 . _ •fl 1 o -J] „ w „ .1- c o I— 1 H < X 20 0 -I n su l i n T r e a t m e n t . Preincubation j (20,000 fxU/ml) U n s t i m u l a t e d 0 1 2 3 [CCK] ( Log 1 Q (Dose in pM)) Figure 68 193 F i g u r e s 69 and 70 E f f e c t o f I n s u l i n P r e s e n t D u r i n g B o t h t h e P R E i n c u b a t i o n and I n c u b a t i o n P e r i o d s on CCK S t i m u l a t e d Amylase S e c r e t i o n Amylase r e l e a s e e x p r e s s e d as * c e l l content ( F i g u r e 69) and ixU x 10~3/ng c e l l protein ( F i g u r e 70) was measured i n C C K - s t i m u l a t e d a c i n i p r e p a r e d from n o r m o g l y c e m i c r a t s . The amylase r e s p o n s e a t t h r e e dose l e v e l s o f CCK-8 ( 0 , 1, 100 pM) under f o u r d i f f e r e n t c o n d i t i o n s o f i n s u l i n t r e a t m e n t ( s e e f i g u r e k e y ) was s t u d i e d . The d u r a t i o n o f p r e i n c u b a t i o n w i t h i n s u l i n was 40 min i n t h e s e s t u d i e s . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r t h r e e e x p e r i m e n t s r u n i n t r i p l i c a t e . 'i Basal i. • • CCK (1 pM) \ -7 CCK (100 pM) T I A B C D I n s u l i n T r e a t m e n t G r o u p Group I n s u l i n ( 2 0 Q 0 A^U/ml) Incubation Preincubation A B C D 30 - 195 Basal • CCK (1 pM) v CCK (100 p M ) •i. on r$ — ~ U Ti \ E ~ < X 10 I--7- - V 7 0 A B C D I n s u l i n T r e a t m e n t G r o u p Group A B C D Insulin (2000 /zU/ml) Incubation Preincubation Figure 70 1 9 6 F i g u r e s 71 and 72 E f f e c t o f I n s u l i n P r e s e n t O n l y D u r i n g t h e P R E i n c u b a t i o n P e r i o d on S e c r e t i n - S t i m u l a t e d Amylase S e c r e t i o n Amylase r e l e a s e e x p r e s s e d as % cell content ( F i g u r e 71) and uU x 10~3/ng cell protein ( F i g u r e 72) was measured i n s e c r e t i n - s t i m u l a t e d a c i n i p r e p a r e d from normoglycemic r a t s . The amylase r e s p o n s e t o i n c r e a s i n g doses o f s e c r e t i n (0-100 nM) under two d i f f e r e n t p r e i n c u b a t i o n c o n d i t i o n s o f i n s u l i n ( s e e f i g u r e key) was s t u d i e d . P r e i n c u b a t i o n w i t h i n s u l i n was m a i n t a i n e d f o r 2\ h w i t h one exchange o f medium t h a t c o n t a i n e d f r e s h i n s u l i n . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r t h r e e e x p e r i m e n t s r u n i n d u p l i c a t e . 197 Insu l in T r ea tmen t P r e i n c u b a t i o n (20.000 m l ) r- — 10 CD _T/ CD CC CD D \ U n s t i m u l a t e d 0 L 0 1 2 [ Secret in ] (Log 1 Q (Dose in nM)) Figure 71 198 I nsu l in T r e a t m e n t Preincubation (20 .000 / x U / m l ) w cu ti _ cu — X -3. 10 -U n s t i m u l a t e d 0 Z. L 0 1 2 [Secretin] (Log1Q(Dose in nM)) Figure 72 199 F i g u r e s 73 and 74 E f f e c t o f I n s u l i n P r e s e n t D u r i n g B o t h t h e P R E i n c u b a t i o n and I n c u b a t i o n P e r i o d s on S e c r e t i n -S t i m u l a t e d Amylase S e c r e t i o n Amylase r e l e a s e e x p r e s s e d as % c e l l content ( F i g u r e 73) and all x 10~3/\ig c e l l protein ( F i g u r e 74) was measured i n s e c r e t i n - s t i m u l a t e d a c i n i p r e p a r e d from n ormoglycemic r a t s . The amylase r e s p o n s e a t f i v e dose l e v e l s o f s e c r e t i n (0 - 400 nM) under f o u r d i f f e r e n t c o n d i t i o n s o f i n s u l i n t r e a t m e n t ( s e e f i g u r e key) was s t u d i e d . The d u r a t i o n o f p r e i n c u b a t i o n w i t h i n s u l i n was 40 min i n t h e s e s t u d i e s . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r t h r e e e x p e r i m e n t s r u n i n d u p l i c a t e . 200 I nsu l in T r e a t m e n t Incubation Preincubation (4000 /LtU/ml) (2000 /xU ml) 7n a; 00 CO O O r. \ w CJ 15 r S = 10 D 0 U n s t i m u l a t e d T i 0 1 2 3 [Secretin] (Log Q(Dose in nM)) Figure 73 201 10 Insu l in T r e a t m e n t Incubation Preincubation (4000 /xU/ ml) (2000 /xU/ml) y 5 r o x i-U n s t i m u l a t e d 0 V Z L 0 1 2 3 [Secret in ] ( Log 1 0 (Dose i n nM)) Figure 74 F i g u r e s 75 and 76 E f f e c t o f I n s u l i n P r e s e n t D u r i n g B o t h t h e P R E i n c u b a t i o n and I n c u b a t i o n P e r i o d s on S e c r e t i n -and t h e C o m b i n a t i o n o f S e c r e t i n - and CCK-S t i m u l a t e d Amylase S e c r e t i o n Amylase r e l e a s e e x p r e s s e d as % c e l l content ( F i g u r e 75) and uU x 10~3/ug c e l l protein ( F i g u r e 76) was measured i n s e c r e t i n - and combined s e c r e t i n - and C C K - s t i m u l a t e d a c i n i p r e p a r e d from normoglycemic r a t s . The amylase r e s p o n s e t o t h e t h r e e s e c r e t o r y c o n d i t i o n s under f o u r d i f f e r e n t c o n d i t i o n s o f i n s u l i n t r e a t m e n t (see f i g u r e key) was s t u d i e d . The d u r a t i o n o f p r e i n c u b a t i o n w i t h i n s u l i n was 40 min i n t h e s e s t u d i e s . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r t h r e e e x p e r i m e n t s r u n i n d u p l i c a t e . 203 15 -Basa l S e c r e t i n (10 nM) S e c r e t i n (10 nM) + CCK ( 10 pM) * W — 4 I i i w 10 r V i / CO O O K O 0 \ 0 L A B C D I n s u l i n T r e a t m e n t G r o u p Group Insu l in (2000 /uU/ml) Incubation Preincubation Figure 75 r o CO 3. 15 -_ 10 r O r 0 L C Basal • Secretin (10 nM) v Secretin (10 nM) + CCK ( 10 pM) A B C D I n s u l i n T r e a t m e n t G r o u p Group Insulin (2000 yuU/ml) Incubation Preincubation A B - -C — + D 4- 4-Figure 76 E f f e c t o f I n s u l i n P r e s e n t D u r i n g P R E i n c u b a t i o n and I n c u b a t i o n P e r i o d s on M e t h a c h o l i n e - S t i m u l a t e d Amylase S e c r e t i o n Amylase r e l e a s e e x p r e s s e d as % c e l l content ( F i g u r e 77) and uU x 10~3/ng c e l l protein ( F i g u r e 78) was measured i n m e t h a c h o l i n e - s t i m u l a t e d a c i n i p r e p a r e d from n o r m o g l y c e m i c r a t s . The amylase r e s p o n s e t o i n c r e a s i n g d oses o f m e t h a c h o l i n e ( 0 , 10 nM, 1 /iM) under f o u r d i f f e r e n t c o n d i t i o n s o f i n s u l i n t r e a t m e n t ( s e e f i g u r e key) was s t u d i e d . P r e i n c u b a t i o n w i t h i n s u l i n was o v e r a 40 min p e r i o d i n t h e s e s t u d i e s . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r t h r e e e x p e r i m e n t s r u n i n t r i p l i c a t e . 206 2 0 -Basa l Me thacho l i ne (10 nM) Methacho l i ne ( 1 /u\l) Cv m i CJ CJ CJ r V )—1 *~' CJ o Ui zz •CJ >, CJ < Kp 15 - ? 10 5 0 A B C D I n s u l i n T r e a t m e n t G r o u p Group I nsu l in (2000 /xU/ml) Incubation Preincubation A _ — B + -C - + D + -r Figure 77 207 3 20 -IT* r . O CO o < X ZD 3 0 -O Basa l • Me thacho l i ne (10 nM) ~ Me thacho l i ne ( 1 /^M) A B C D I n s u l i n T r e a t m e n t G r o u p Group Insu l in (2000 /xU/ml) Incubation Preincubation A. — B +• -C - + D + + Figure 78 208 F i g u r e s 79 and 80 E f f e c t o f Zymogen D e p l e t i o n and I n s u l i n Dosage on P o t e n t i a t i o n o f B a s a l and S t i m u l a t e d Amylase S e c r e t i o n Amylase r e l e a s e e x p r e s s e d as % c e l l content ( F i g u r e 79) and uU x 10"3/ug c e l l protein ( F i g u r e 80) was measured under b a s a l and two s e c r e t o r y c o n d i t i o n s ( s e e f i g u r e key) i n a c i n i p r e p a r e d from normoglycemic r a t s . A l l a c i n i were p r e i n c u b a t e d f o r a t o t a l o f 3s* h and d u r i n g t h e f i r s t 2 h, a l l a c i n i were exposed t o CCK (100 pM) t o d e p l e t e c e l l u l a r zymogen s t o r e s . The e f f e c t s o f i n s u l i n on a c i n i d e p l e t e d o f zymogen by a c h r o n i c s t i m u l u s were a s s e s s e d by i n s u l i n p r e i n c u b a t i o n i n groups c and D. The s e c r e t o r y r e s p o n s e t o h i g h e r doses and a l o n g e r d u r a t i o n o f i n s u l i n e x p o s u r e were a s s e s s e d o v e r a l l by t h e f o u r d i f f e r e n t c o n d i t i o n s o f t r e a t m e n t (A-D, see f i g u r e k e y ) . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r t h r e e e x p e r i m e n t s r u n i n t r i p l i c a t e . j C Basa l j • S e c r e t i n 20 nM V CCK 100 pM 7 77 — 0 O 0 _1 I 1_ A B C D Insulin Treatment A No insulin B Insulin (2000 / *U/ml) incubation C Insulin (2000 /LtU/ml) preincubation D Insulin (20,000 /zU/ml) preincubation V O 210 i S e c re to r y S ta tus i ~ I O Basa l | • Secretin 20 nM | v CCK 100 pM S3 CU cu CC o jO £ < ? 1 0 r cu a "a o —; 3. 5 T V I # O v 0 o 3. 0 A B C D Insulin Treatment A No insulin B Insulin (2000 /zU/ml) incubation C Insulin (2000 ,uU/ml) preincubation D Insulin (20,000 u\J/ml) preincubation Figure 80 211 F i g u r e s 81 and 82 E f f e c t o f S h o r t - t e r m D i a b e t e s and I n s u l i n Dosage on P o t e n t i a t i o n o f B a s a l and S t i m u l a t e d Amylase S e c r e t i o n Amylase r e l e a s e e x p r e s s e d as * cell content ( F i g u r e 81) and x 10~3/ug cell protein ( F i g u r e 82) was measured under b a s a l and two s e c r e t o r y c o n d i t i o n s ( s ee f i g u r e key) i n a c i n i p r e p a r e d from h y p e r g l y c e m i c r a t s . R a t s were r e n d e r e d d i a b e t i c w i t h STZ (60 mg/kg i . p . ) 3-4 d p r i o r t o h a r v e s t i n g o f a c i n i . A l l a c i n i were p r e i n c u b a t e d f o r 50 min. The s e c r e t o r y r e s p o n s e t o h i g h e r d o s e s and a l o n g e r d u r a t i o n o f i n s u l i n e x p o s u r e was a s s e s s e d o v e r a l l by t h e s i x d i f f e r e n t c o n d i t i o n s o f t r e a t m e n t (A-F, see f i g u r e k e y ) . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r f o u r e x p e r i m e n t s r u n i n d u p l i c a t e . 30 :-C 73 J*< | - c 9Q _ — O ' 73 c 5 10 r 7 o v i i V Sec re to r y S ta tus O Basa l • Sec re t i n 20 nM v CCK 50 pM O o o 0 L B C D E Insulin Treatment A No insulin B Insulin (200 yuU/ml) incubation C Insulin (2000 ^ U / m l ) incubation D Insulin (2000 /aU/ml) preincubation E Insulin incubation (200 /LiU/ml) and preincubation (2000 /zU/ml) F Insulin (2000 AtU/ml) incubation and preincubation Figure 81 213 C 'cu c cu w (0 cu cu ac 10 3 5 r T i i l i f T v 1 9 Sec r e to r y S ta tus O Basa l • S e c r e t i n 20 nM v CCK 50 pM M i l 3 0 A B C D E F Insulin Treatment A No insulin B Insulin (200 /zU/ml) incubation C Insulin (2000 /LiU/ml) incubation D Insulin (2000 /i.U/ml) preincubation E Insulin incubation (200 ynU/ml) and preincubation (2000 /uU/ml) F Insulin (2000 ^ U / m l ) incubation and preincubation F i g u r e 82 214 F i g u r e 83 Body Weight Changes i n Response t o C h r o n i c a l l y A l t e r e d C i r c u l a t i n g I n s u l i n C o n c e n t r a t i o n s A n i m a l body w e i g h t , e x p r e s s e d as a p e r c e n t o f i n i t i a l w e i g h t i s d i s p l a y e d a g a i n s t t i m e i n d a y s . I n i t i a l body w e i g h t s f o r most g r o u p s was 280-300 g. The f o u r t r e a t m e n t g r o u p s a r e i n d i c a t e d i n t h e f i g u r e key and d e t a i l s o f t r e a t m e n t a r e d i s c u s s e d i n t h e Methods s e c t i o n . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM w i t h 8-16 a n i m a l s / g r o u p . S i g n i f i c a n t d i f f e r e n c e s between t r e a t m e n t g r o u p s a r e i n d i c a t e d by t h e a s t e r i s k (*) a t t h e p<0.05 l e v e l . 215 | A n i m a l T r e a t m e n t Groups ! | O C o n t r o l | • C h l o r p r o p a m i d e - T r e a t e d ; I v Diabetic ! ; • I n s u l i n - T r e a t e d j 120 - r ' T 0 5 10 T i m e (days) Figure 83 216 F i g u r e s 84 and 85 F a s t e d Plasma G l u c o s e and I n s u l i n i n Response t o C h r o n i c a l l y A l t e r e d C i r c u l a t i n g I n s u l i n C o n c e n t r a t i o n s Plasma g l u c o s e (mM, F i g u r e 84) and i n s u l i n (uU/ml, F i g u r e 85) were measured a t t h r e e t i m e p o i n t s b e f o r e and a f t e r i n i t i a t i o n o f t r e a t m e n t s d e s i g n e d t o a l t e r c i r c u l a t i n g i n s u l i n c o n c e n t r a t i o n s ( s e e f i g u r e k e y ) . The l a s t t i m e p o i n t i n d i c a t e s t h e day o f s a c r i f i c e f o r p r e p a r a t i o n o f a c i n i . R e s u l t s i n 5-8 a n i m a l s / g r o u p a r e e x p r e s s e d as t h e mean ± t h e SEM. O n l y some a n i m a l s i n each s t u d y g r o u p had g l u c o s e and i n s u l i n samples t a k e n though a l l members o f each g r o u p were f a s t e d i n a s i m i l a r f a s h i o n . 217 A n i m a l T r e a t m e n t Groups C o n t r o l • C h l o r p r o p a m i d e - T r e a t e d v Diabetic • I n s u l i n - T r e a t e d 30 -c 71 20 cu -—-O <-U C O — 10 0 -0 5 10 T i m e (days) Figure 84 Animal Treatment Groups O Control # Chlorpropamide-Treated V Diabetic T Insulin-Treated 0 5 10 T ime (days) Figure 85 219 Figures 8g and 37 E f f e c t o f I n s u l i n i n vitro on B a s a l and S t i m u l a t e d Amylase S e c r e t i o n i n C o n t r o l , N o r m o i n s u l i n e m i c R a t s Amylase r e l e a s e e x p r e s s e d as % cell content ( F i g u r e 86) and uU x 10~3/ng cell protein ( F i g u r e 87) was measured under b a s a l and two s e c r e t o r y c o n d i t i o n s ( s ee f i g u r e key) i n a c i n i p r e p a r e d f rom CONTROL r a t s . A l l a c i n i were p r e i n c u b a t e d f o r a t o t a l o f 3*i h. D u r i n g t h e f i r s t 2 h, a l l a c i n i were exposed t o CCK (10 pM). The e f f e c t s o f i n s u l i n p r e i n c u b a t i o n on a c i n i s e c r e t i o n were a s s e s s e d by i n s u l i n p r e i n c u b a t i o n i n gro u p s C and D. P o t e n t i a l c u m u l a t i v e e f f e c t s o f i n s u l i n e x p o s u r e d u r i n g t h e p r e i n c u b a t i o n p e r i o d , d u r i n g i n c u b a t i o n , and d u r i n g b o t h p r e i n c u b a t i o n and i n c u b a t i o n were a s s e s s e d by t h e f o u r d i f f e r e n t c o n d i t i o n s o f t r e a t m e n t (A-D, see f i g u r e k e y ) . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r t h r e e e x p e r i m e n t s r u n i n t r i p l i c a t e . 220 C o n t r o l A n i m a l s CD V) Cu CU cj c CJ o o CJ CJ >> tf £ o < ^ &s 0 20 r 15 -10 r O r 0 L O o Secretory Status O Basal • Secretin 20 nM v CCK 100 pM v i o o A B C D Insulin Treatment A No insulin B Insulin (2000 /uU/ml) incubation C Insulin (2000 uU/ml) preincubation D Insulin (2000 ^ u/ml) incubation and preincubation Figure 86 221 Cont ro l An imals a oo CU •fl CO CD CU CU w \ CO ^ ' o c — < X D c 15 -o j £ ! - 10 h 0 L o Sec r e to r v S ta tus • • 1 | O Basa l j • S e c r e t i n 2 0 nM j v CCK 1 0 0 pM | T i # o i o o A B C D Insulin Treatment A No insulin B Insulin (2000 /xU/ml) incubation C Insulin (2000 j*U/ml) preincubation D Insulin (2000 /iU/ml) incubation and preincubation Figure 87 222 F i g u r e s 88 and 89 E f f e c t o f I n s u l i n in v i t r o on B a s a l and S t i m u l a t e d Amylase S e c r e t i o n i n S T Z - D i a b e t i c R a t s Amylase r e l e a s e e x p r e s s e d as * c e l l content ( F i g u r e 88) and u£7 x 10~2/ng c e l l protein ( F i g u r e 89) was measured under b a s a l and two s e c r e t o r y c o n d i t i o n s ( s e e f i g u r e key) i n a c i n i p r e p a r e d from d i a b e t i c r a t s . E x c e p t f o r t h e d i f f e r e n c e i n a n i m a l c o n d i t i o n , a c i n i s t i m u l a t i o n and in v i t r o i n s u l i n t r e a t m e n t were i d e n t i c a l t o t h o s e d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e s 86 and 87. R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r f o u r e x p e r i m e n t s r u n i n t r i p l i c a t e . 223 ^ ' T Z - D i a b e t i c A n i m a l s C/3 CD cn o CD CD 'JI J2 >. S < CD O 15 15 --± 10 L ID 0 v o Secretory Status O Basal • Secretin 20 nM v CCK 100 pM v v O O A B C D Insulin Treatment A No insulin 6 Insulin (2000 / /U/ml ) incubation C Insulin (2000 AiU/ml) preincubation D Insulin (2000 /j,U/ml) incubation and preincubation Figure 88 224 ^ T Z - D i a b e t i c A n i m a l s , 3 0 a o m cC CD CD CJ CD 1 cn o E X < 3 20 r ^ 1 5 r D 0 V # 0 Secretory Status O Basal • Secretin 20 nM 7 CCK 100 pM i v 0 # 0 A B C D Insulin Treatment A No insulin B Insulin (2000 /aU/ml) incubation C Insulin (2000 /uU/ml) preincubation D Insulin (2000 /uU/ml) incubation and preincubation Figure 89 225 F i g u r e s 90 and 91 E f f e c t o f I n s u l i n in v i t r o on B a s a l and S t i m u l a t e d Amylase S e c r e t i o n i n H y p e r i n s u l i n e m i c , C h l o r p r o p a m i d e - t r e a t e d R a t s Amylase r e l e a s e e x p r e s s e d as % c e l l content ( F i g u r e 90) and uU x 10~ 3/ixg c e l l protein ( F i g u r e 91) was measured under b a s a l and two s e c r e t o r y c o n d i t i o n s ( s e e f i g u r e key) i n a c i n i p r e p a r e d f r o m c h l o r p r o p a m i d e - t r e a t e d r a t s . E x c e p t f o r t h e d i f f e r e n c e i n a n i m a l c o n d i t i o n , a c i n i s t i m u l a t i o n and in v i t r o i n s u l i n t r e a t m e n t were i d e n t i c a l t o t h o s e d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e s 86 and 87. R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r f o u r e x p e r i m e n t s r u n i n t r i p l i c a t e . 226 C h l o r p r o p a m i d e - t r e a t e d A n i m a l s — J r -CD CD CO CO CD O CD o 02 1 1 CD CD 00 CJ JO . o < 20 ,-15 k 9 10 0 o o Sec re to r y S ta tus O Basa l • S e c r e t i n 20 nM v CCK 100 pM T v 1 T i v I o o A B C D Insulin Treatment A No insulin B Insulin (2000 uU/ml) incubation C Insulin (2000 uU/ml) preincubation D Insulin (2000 /uU/ml) incubation and preincubation F i g u r e 90 227 C h l o r p r o p a m i d e - T r e a t e d A n i m a l s c 15 r CD -<-> O CD Vi CC CD <D os CD . w crj C J < x - 10 i-CD O OO o -0 L T i V O Secretory Status O Basal • Secretin 20 nM v CCK 100 pM T V X T 1 V I O O O A B C D Insulin Treatment A No insulin B Insulin (2000 /xU/ml) incubation C Insulin (2000 yuU/ml) preincubation D Insulin (2000 /xU/ml) incubation and preincubation Figure 91 F i g u r e s 92 and 93 E f f e c t o f I n s u l i n in v i t r o on B a s a l and S t i m u l a t e d Amylase S e c r e t i o n i n H y p e r i n s u l i n e m i c , NPH I n s u l i n - t r e a t e d R a t s Amylase r e l e a s e e x p r e s s e d as % c e l l content ( F i g u r e 92) and \iU x 1Q~ 3/\xq c e l l protein ( F i g u r e 93) was measured under b a s a l and two s e c r e t o r y c o n d i t i o n s (see f i g u r e key) i n a c i n i p r e p a r e d from NPH i n s u l i n - t r e a t e d r a t s . E x c e p t f o r t h e d i f f e r e n c e i n a n i m a l c o n d i t i o n , a c i n i s t i m u l a t i o n and in v i t r o i n s u l i n t r e a t m e n t were i d e n t i c a l t o t h o s e d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e s 86 and 87. R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r f o u r e x p e r i m e n t s r u n i n t r i p l i c a t e . 229 I n s u l i n - T r e a t e d A n i m a l s CD CD 'Ji "c o % o — CD CD V) O JS _ CC S _ J 0 < -1—> 20 ^ 15 h = 10 0 Secretory Status j O Basal I i • Secretin 20 nM j v CCK 100 pM i v 1 A B C D Insulin Treatment A No insulin B Insulin (2000 /zU/ml) incubation C Insulin (2000 /x\J/ml) preincubation D Insulin (2000 /xU/ml) incubation and preincubation Figure 92 230 I n s u l i n - T r e a t e d A n i m a l s c 10 o oo CO — 0) CD on CD CD 00 \ jO « >> o 6 -< X D 3. r o i v 1 ? Secretory Status I O Basal | • Secretin 20 nM j v CCK 100 pM I V 1 T V A B C D Insulin Treatment A No insulin B Insulin (2000 /zU/ml) incubation C Insulin (2000 /uU/ml) preincubation D Insulin (2000 /iU/ml) incubation and preincubation Figure 93 231 F i g u r e 94 Body Weight Changes i n Response t o S T Z - D i a b e t e s W i t h and W i t h o u t I n s u l i n Treatment A n i m a l body w e i g h t , e x p r e s s e d as a p e r c e n t o f i n i t i a l w e i g h t i s d i s p l a y e d a g a i n s t t i m e i n day s . I n i t i a l body w e i g h t s f o r most groups was 260-300 g. The t h r e e t r e a t m e n t g r o u p s were c o n t r o l s , d i a b e t i c r a t s , and i n s u l i n - t r e a t e d d i a b e t i c r a t s . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM w i t h 8-10 a n i m a l s / g r o u p . S i g n i f i c a n t d i f f e r e n c e s between t r e a t m e n t g r o u p s a r e i n d i c a t e d by t h e a s t e r i s k (*) a t t h e p<0.05 l e v e l . A n i m a l T r e a t m e n t G r o u p s O C o n t r o l j • U n t r e a t e d D i a b e t i c i v I n s u l i n - T r e a t e d D i a b e t i c 140 120 O K 3 100 • 1 6 6 9 6 T ^ V 1 0 0 10 T i m e (days) 20 Figure 94 233 F i g u r e 95 E f f e c t o f t h e D u r a t i o n o f U n t r e a t e d D i a b e t e s on T o t a l , C C K - S t i m u l a t e d Amylase R e l e a s e Amylase s e c r e t i o n s t i m u l a t e d by i n c r e a s i n g d o s e s o f CCK (0-1000 pM) was e x p r e s s e d as iiU x 10"3/y,g c e l l protein. A c i n a r s e c r e t i o n f r om a n i m a l s r e n d e r e d d i a b e t i c f o r 7 and 15 days r e s p e c t i v e l y were compared t o c o n t r o l s . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r f o u r e x p e r i m e n t s r u n i n d u p l i c a t e . 234 CD 00 Cu CD c 'CD o ^ 1 CD CD CD < ^ 00 ^ ^ I >^ o < X ZD 15 -Animal Treatment Groups O Control • Diabetic (Day 7 after STZ) V Diabetic (Day 15 after STZ) — 10 ^ Unstimulated y 0 v i / 0 1 2 3 [CCK] (Log1 0(Dose in pM)) Figure 95 235 F i g u r e 96 E f f e c t o f t h e D u r a t i o n o f U n t r e a t e d D i a b e t e s on T o t a l , S e c r e t i n - S t i m u l a t e d Amylase R e l e a s e Amylase s e c r e t i o n s t i m u l a t e d by i n c r e a s i n g d o s e s o f s e c r e t i n ( 0-400 nM) was e x p r e s s e d as nU x 10~3/ng c e l l protein. A c i n a r s e c r e t i o n f r om a n i m a l s r e n d e r e d d i a b e t i c f o r 7 and 15 days r e s p e c t i v e l y were compared t o c o n t r o l s . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r f o u r e x p e r i m e n t s r u n i n d u p l i c a t e . 236 Animal Treatment Groups 0 Control ' 7///^~ ' ' 1 1  0 1 2 3 [Secretin] (Log1Q(Dose in nM)) Figure 96 237 F i g u r e 97 E f f e c t o f t h e D u r a t i o n o f U n t r e a t e d D i a b e t e s on S e c r e t i n - and t h e C o m b i n a t i o n o f CCK- and S e c r e t i n - S t i m u l a t e d Amylase R e l e a s e Amylase s e c r e t i o n s t i m u l a t e d by f o u r d i f f e r e n t t r e a t m e n t c o n d i t i o n s (A-D) was e x p r e s s e d as uU x 10" 3/ng c e l l protein. A c i n a r s e c r e t i o n f rom a n i m a l s r e n d e r e d d i a b e t i c f o r 7 and 15 days r e s p e c t i v e l y were compared t o c o n t r o l s . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r f o u r e x p e r i m e n t s r u n i n d u p l i c a t e . Where n o t v i s i b l e , t h e SEM was below t h e r e s o l u t i o n o f t h e p l o t . 238 CD 73 CO CU CD OS CD 73 CO CD O 03 O OX) 3. CO < O X D 3. 20 r '~ 15 -10 0 Animal Treatment Groups j O Control j • Diabetic (Day 7 after STZ) i V Diabetic (Day 15 after STZ) i A B C D Secretory Stimulus Secretory Stimulus A Basal B Secretin (10 nM) C Secretin (10 nM) + CCK (10 pM) D Secretin (10 nM) + CCK (100 pM) Figure 97 F i g u r e 98 E f f e c t o f I n s u l i n Treatment on T o t a l , CCK-S t i m u l a t e d Amylase R e l e a s e from D i a b e t i c A n i m a l A c i n i Amylase s e c r e t i o n s t i m u l a t e d by i n c r e a s i n g d o s e s o f CCK (0-1000 pM) was e x p r e s s e d as uU x 10~3/ug c e l l protein. A n i m a l s were r e n d e r e d d i a b e t i c f o r 7 days t h e n t r e a t e d w i t h i n s u l i n t o o a c h i e v e normoglycemia. A c i n i were p r e p a r e d from i n s u l i n - t r e a t e d , d i a b e t i c a n i m a l s t h a t were s a c r i f i c e d on days 1, 2, 3, 5, and 8 o f i n s u l i n t r e a t m e n t . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r two r e p r e s e n t a t i v e e x p e r i m e n t s r u n i n t r i p l i c a t e . 240 cu 73 S3 c cu o L. cu o a. cu . w ~^ ' o 3. Unstimulated 6 r r 4 0 Animal Treatment Groups O Diabetic (Day 7 after STZ) # Diabetic (Day 1 of insulin treatment) V Diabetic (Day 2 of insulin treatment) • Diabetic (Day 3 of insulin treatment) • Diabetic (Day 5 of insulin treatment) • Diabetic (Day 6 of insulin treatment) - V 0 1 2 3 [CCK] (Log1 0(Dose in pM)) Figure 98 241 F i g u r e 99 E f f e c t o f I n s u l i n Treatment on T o t a l , S e c r e t i n -S t i m u l a t e d Amylase R e l e a s e from D i a b e t i c A n i m a l A c i n i Amylase s e c r e t i o n s t i m u l a t e d by i n c r e a s i n g doses o f s e c r e t i n (0-400 nM) was e x p r e s s e d as pU x 10~3/ng c e l l protein. C o n d i t i o n s were i d e n t i c a l t h o s e d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 98. R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r two r e p r e s e n t a t i v e e x p e r i m e n t s r u n i n t r i p l i c a t e . Animal Treatment Croups 0 Diabetic (Day 7 after STZ) # Diabetic (Day 1 of insulin treatment) V Diabetic (Day 2 of insulin treatment) • Diabetic (Day 3 of insulin treatment) • Diabetic (Day 5 of insulin L - / / _ J 1 1 1 0 1 2 3 [Secretin] (Log1Q(Dose in nM)) Figure 99 F i g u r e 100 E f f e c t o f I n s u l i n T reatment on S e c r e t i n - and t h e C o m b i n a t i o n o f CCK- and S e c r e t i n - S t i m u l a t e d Amylase R e l e a s e From D i a b e t i c A n i m a l A c i n i Amylase s e c r e t i o n s t i m u l a t e d by f o u r d i f f e r e n t t r e a t m e n t c o n d i t i o n s (A-D) was e x p r e s s e d a s \OJ x 10~ 3/ng c e l l protein. C o n d i t i o n s were i d e n t i c a l t h o s e d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 98. R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r two r e p r e s e n t a t i v e e x p e r i m e n t s r u n i n t r i p l i c a t e . i Animal Treatment Groups ! , i O Diabetic (Day 7 after STZ) j # Diabetic (Day I of insulin j treatment) j V Diabetic (Day 2 of insulin ! treatment) I • Diabetic (Day 3 of insulin j treatment) [ • Diabetic (Day 5 of insulin i treatment) j • Diabetic (Day 8 of insulin ; treatment) : A B C D Secretory Stimulus Secretory Stimulus A Basal B Secretin (10 nM) C Secretin (10 nM) + CCK (10 pM) D Secretin (10 nM) + CCK (100 pM) 245 F i g u r e I Q I I n f l u e n c e o f An ima l Age on t h e Plasma G l u c o s e Response t o a G l u c o s e C h a l l e n g e : GTT The g l u c o s e c o n c e n t r a t i o n i n p lasma (mM) o f f a s t e d , h e a l t h y r a t s was measured a t i n t e r v a l s a f t e r a 1 g/kg i . p . g l u c o s e c h a l l e n g e . The e f f e c t s o f a g l u c o s e c h a l l e n g e were compared i n w e a n l i n g v s o l d e r r a t s as a measure o f t h e i n s u l i n r e s p o n s i v e n e s s o f t h e t i s s u e s i n e a c h g r o u p . N = 5 p e r g r o u p . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM and s i g n i f i c a n t d i f f e r e n c e s a r e i n d i c a t e d by t h e a s t e r i s k ( * ) . 246 e 00 cC C CD 00 o o a 3 Glucose 1 g/kg i.p. Groups  O Weanl ing ra ts (56 ± 1 g body wt.) • Older (4-5 mos) rats (432 ± 12 g body wt.) 20 r 10 0 0 20 40 60 80 Time (min) 100 Figure 101 247 Figure 1Q2 I n f l u e n c e o f A n i m a l Age on t h e Plasma I n s u l i n Response t o a G l u c o s e C h a l l e n g e : GTT The i n s u l i n c o n c e n t r a t i o n i n p lasma (uU x 10~ 2 /ml) o f f a s t e d , h e a l t h y r a t s was measured a t i n t e r v a l s a f t e r a 1 g/kg i . p . g l u c o s e c h a l l e n g e . C o n d i t i o n s were d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e 101 . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM and a g a i n , s i g n i f i c a n t d i f f e r e n c e s a r e i n d i c a t e d by t h e a s t e r i s k ( * ) . 248 Glucose 1 g /kg i.p. i , Groups O Weanling rats i . i , i i i i 1 , 1 0 20 40 60 80 100 Time (min) Figure 102 249 F i g u r e s 103 and 104 E f f e c t o f I n s u l i n on C C K - S t i m u l a t e d Amylase S e c r e t i o n from A c i n i D e r i v e d f r o m W e a n l i n g R a t s Amylase r e l e a s e e x p r e s s e d as % c e l l content ( F i g u r e 103) and \)&J x 10~3/ng c e l l protein ( F i g u r e 104) was measured i n C C K - s t i m u l a t e d a c i n i p r e p a r e d f r o m w e a n l i n g r a t s . The amylase r e s p o n s e a t t h r e e dose l e v e l s o f CCK-8 ( 0 , 1, 100 pM) under t h r e e d i f f e r e n t c o n d i t i o n s o f i n s u l i n t r e a t m e n t ( s e e f i g u r e k ey) was s t u d i e d . P r e i n c u b a t i o n w i t h i n s u l i n was c a r r i e d o u t f o r 3% h i n t h e s e s t u d i e s . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r f o u r e x p e r i m e n t s r u n i n d u p l i c a t e . 250 20 o r Insulin Treatment Incubation Preincubation (2000 /LtU/ml) (20,000 /xU/ml) 0J ^ CO >-cu CU ~& C Pi 0) c3 O O 10 cu o Unstimulated 0 L 7 <A 0 [CCK-8] (Log1Q(Dose in pM)) F i g u r e 103 251 Insulin Treatment j Incubation Preincubation (2000 MU/ml) (20.000 uU.'ml) o 10 - V [CCK-8] (Log (Dose in pM)) Figure 104 252 F i g u r e s 105 and 106 E f f e c t o f I n s u l i n on S e c r e t i n - S t i m u l a t e d Amylase S e c r e t i o n from A c i n i D e r i v e d f r o m W e a n l i n g R a t s Amylase r e l e a s e e x p r e s s e d as % c e l l content ( F i g u r e 105) and uU x 10~3/ng c e l l protein ( F i g u r e 106) was measured i n s e c r e t i n - s t i m u l a t e d a c i n i p r e p a r e d from w e a n l i n g r a t s . The amylase r e s p o n s e a t two dosage l e v e l s o f s e c r e t i n (0 and 20 nM) under t h r e e d i f f e r e n t c o n d i t i o n s o f i n s u l i n t r e a t m e n t ( s e e f i g u r e k ey) was s t u d i e d . P r e i n c u b a t i o n w i t h a maximal dose o f i n s u l i n was c a r r i e d o u t f o r 3*s h t o a m p l i f y any i n s u l i n e f f e c t i n t h e s e s t u d i e s . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r f o u r e x p e r i m e n t s r u n i n d u p l i c a t e . 253 Figure 105 254 Figure 106 255 F i g u r e s 107 and 108 E f f e c t o f I n s u l i n on S e c r e t i n - and V I P - S t i m u l a t e d Amylase S e c r e t i o n from A c i n i D e r i v e d f rom W e a n l i n g R a t s Amylase r e l e a s e e x p r e s s e d as % c e l l content ( F i g u r e 107) and uU x 10~3/ng c e l l protein ( F i g u r e 108) was measured under t h r e e d i f f e r e n t s e c r e t o r y c o n d i t i o n s (see f i g u r e key) i n a c i n i p r e p a r e d f rom w e a n l i n g r a t s . The amylase r e s p o n s e under f o u r d i f f e r e n t c o n d i t i o n s o f i n s u l i n t r e a t m e n t (see f i g u r e key) was s t u d i e d . As d e s c r i b e d i n t h e c a p t i o n f o r F i g u r e s 86 and 87, p o t e n t i a l c u m u l a t i v e e f f e c t s o f i n s u l i n e x p o s u r e d u r i n g t h e p r e i n c u b a t i o n p e r i o d , d u r i n g i n c u b a t i o n , and d u r i n g b o t h p r e i n c u b a t i o n and i n c u b a t i o n were a s s e s s e d by t h e f o u r d i f f e r e n t c o n d i t i o n s o f t r e a t m e n t (A-D, s e e f i g u r e k e y ) . P r e i n c u b a t i o n w i t h i n s u l i n was c a r r i e d o u t f o r 40 min i n t h e s e s t u d i e s . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r t h r e e e x p e r i m e n t s r u n i n t r i p l i c a t e . 256 CJ 00 cC CJ IJ cj 00 cC CJ c O o CJ o >> cC I ° 30 r 20 -10 0 v o v O Se c r e to r y S ta tus O Basa l • S e c r e t i n 1 0 nM v VIP 1 0 nM O o A B C D I n s u l i n T r e a t m e n t A No insulin B Insulin (2000 /xU/ml) incubation C Insulin (2000 ^ U / m l ) preincubation D Insulin (2000 uV/ml) incubation and preincubation F i g u r e 107 257 C 'cu _J O cu — w cu ti CJ cu _ cu as 00 cd ti o an 15 -10 r o o Secretory Status j O Basal | • Secretin 10 nM j v VIP 10 nM v I 3. 0 A B C D Insulin Treatment A No insulin B Insulin (2000 / x U / m l ) incubation C Insulin (2000 / l U / m l ) preincubation D Insulin (2000 M n/ml) incubation and preincubation Figure 1Q8 258 F i g u r e s 109 and 110 I n s u l i n E f f e c t s on S e c r e t i n - S t i m u l a t e d Amylase R e l e a s e : Effect of the Calcium Concentration of the Medium During Incubation Amylase r e l e a s e e x p r e s s e d as % c e l l content ( F i g u r e 109) and nU x 10~3/(Mg c e l l protein ( F i g u r e 110) was measured u n d e r s i x d i f f e r e n t c o n d i t i o n s o f added s e c r e t i n and i n s u l i n ( s e e f i g u r e key) i n a c i n i p r e p a r e d f r om h e a l t h y a d u l t r a t s . A l l s i x t r e a t m e n t c o n d i t i o n s were s t u d i e d a t t h r e e c a l c i u m c o n c e n t r a t i o n s : 0, 1.69, and 4.5 mM. Note t h e b r o k e n and expanded o r d i n a t e s c a l e . S t a n d a r d e r r o r b a r s were o m i t t e d f o r c l a r i t y b u t were i n t h e ra n g e o f 5-10% o f t h e measured v a l u e . R e s u l t s a r e e x p r e s s e d as t h e mean f o r t h r e e e x p e r i m e n t s c a r r i e d o u t i n d u p l i c a t e . 259 CD CO c CD c o CD O Di ^ CD 'a; W O cO 1>% lo E o 10 8 6 / 0 -0 2 4 6 [Ca] (mM) Trea tment Groups [Secretin] Insulin Insulin (nM) preincubation incubation (2000 /zU/ml) (4000 /xU/ml) o 0 - -• 0 - + • 10 - -V 10 + -• 10 - + • 10 + + Figure 109 260 0 2 4 [Ca] (mM) 6 T r e a t m e n t G r o u p s [Secretin] Insulin Insulin (nM) preincubation incubation (2000 jxU/ml) (4000 juU/ml) o 0 • 0 -V 10 + -• 10 - -• 10 - + • 10 + 4-Figure 110 F i g u r e s 111 and 112 I n s u l i n E f f e c t s on S t i m u l a t e d Amylase R e l e a s e : Effect of Colchicine Treatment During Acini Preincubation Amylase r e l e a s e e x p r e s s e d as % cell content ( F i g u r e 111) and nU x 10~3/ng cell protein ( F i g u r e 112) was measured under t h r e e d i f f e r e n t s e c r e t o r y c o n d i t i o n s ( s e e f i g u r e key) i n a c i n i p r e p a r e d from h e a l t h y , a d u l t r a t s . C o l c h i c i n e p r e i n c u b a t i o n was c a r r i e d o u t w i t h and w i t h o u t i n s u l i n i n c u b a t i o n i n f o u r d i f f e r e n t t r e a t m e n t c o m b i n a t i o n s (A-D , s e e f i g u r e k e y ) . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r t h r e e e x p e r i m e n t s c a r r i e d o u t i n d u p l i c a t e . 262 C CD C o CD O CD I D tf _ ^ tf 30 r 20 r 10 -0 T V O o Secretory Status O Basal • Secretin 20 nM v CCK 50 pM O O B C D Treatment Group A No insulin B Insulin (1000 ^U /ml) incubation C Colchicine (100 A * M ) preincubation D Colchicine and Insulin Figure 111 263 o a Ul cO —i cu cu 'v CJ GO 3. Ul \ ^ 03 CO 1 1 O E i < X 10 -5 -0 T v 1 Sec r e to r y S ta tus O Basa l • S e c r e t i n 20 nM v CCK 50 pM T 1 T I T V 1 A B C D Treatment Group A No i n s u l i n B I n s u l i n (1000 jxU/ml) i n c u b a t i o n C C o l c h i c i n e (100 (J.IA) p r e i n c u b a t i o n D C o l c h i c i n e a n d I n s u l i n Figure 112 264 F i g u r e s 113 and 114 I n s u l i n E f f e c t s on S t i m u l a t e d Amylase R e l e a s e : Effect of Cycloheximide Treatment During Acini Incubation Amylase r e l e a s e e x p r e s s e d as % c e l l content ( F i g u r e 111) and uU x 10~3/ng c e l l protein ( F i g u r e 112) was measured under t h r e e d i f f e r e n t s e c r e t o r y c o n d i t i o n s ( s e e f i g u r e key) i n a c i n i p r e p a r e d from h e a l t h y , a d u l t r a t s . C y c l o h e x i m i d e t r e a t m e n t d u r i n g i n c u b a t i o n was c a r r i e d o u t i n f o u r d i f f e r e n t t r e a t m e n t c o m b i n a t i o n s w i t h i n s u l i n (A-D, see f i g u r e k e y ) . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r t h r e e e x p e r i m e n t s c a r r i e d o u t i n d u p l i c a t e . 265 CD C/3 CO CD c CD c _ o CD CJ 02 — CD "to w cj tf —, *>% tf E o < ^ 2^ 30 r 20 10 0 o O Secretory Status O Basal • Secretin 20 nM v CCK 50 pM T V x O T V 1 O B C D T r e a t m e n t G r o u p A No insulin B Insulin (1000 yuU/ml) incubation C Cycloheximide (1 Mg/ml) D Insulin and cycloheximide Figure 113 266 c '53 o CD 73 CO _QJ CD O oo CD :t 73 JO C3 1 1 o s 1—1 < X D 3 10 r 5 -0 T v 1 T v 1 # Secretory Status O Basal • Secretin 20 nM v CCK 50 pM B T v 1 T v 1 D Treatment Group A No insulin B Insulin (1000 /xU/ml) incubation C Cycloheximide (1 /xg/ml) D Insulin and cycloheximide Figure 114 F i g u r e s 115 and 116 D i f f e r e n t i a l R e s p o n s i v e n e s s o f The V e n t r a l and D o r s a l P a n c r e a s t o C C K - S t i m u l a t e d Amylase S e c r e t i o n Amylase r e l e a s e e x p r e s s e d as % c e l l content ( F i g u r e 115) and uZJ x 10~3/ug c e l l protein ( F i g u r e 116) was measured i n C C K - s t i m u l a t e d a c i n i p r e p a r e d from e i t h e r t h e v e n t r a l ( d u o d e n a l ) o r d o r s a l ( g a s t r o s p l e n i c ) p a n c r e a s l o b e o f h e a l t h y , a d u l t r a t s . The amylase r e s p o n s e a t f o u r dose l e v e l s o f CCK-8 ( 0 , 1, 100, and 300 pM) under two d i f f e r e n t c o n d i t i o n s o f i n s u l i n t r e a t m e n t ( s e e f i g u r e key) was s t u d i e d . R e s u l t s a r e e x p r e s s e d as t h e mean ± SEM f o r t h r e e e x p e r i m e n t s r u n i n t r i p l i c a t e . 268 20 r Ventral Pancreas cu 00 CO CD "cu 02 CD 00 CO CD £ < 10 CD 0 £ O CD ^ 2 0 r Unstimulated 0 Dorsal Pancreas 10 Insulin Treatment Incubation (2000 AtU/ml) o 0 Unstimulated 0 1 2 3 [CCK] (Log1 Q(Dose in pM)) Figure 115 269 'CD CO o as CD — "CD o CD VI CO 1 E O < X 3 20 -15 -10 -5 0 Ventral Pancreas 15 5 0 Unstimulated 0 Dorsal Pancreas Insulin Treatment Incubation (2000 AtU/ml) o Unstimulated /-^ l — 0 1 2 3 [CCK] (Log1Q(Dose in pM)) Figure 116 M i c r o s c o p i c Appearance o f a Normal Rat P a n c r e a t i c I s l e t S t a i n e d w i t h H e m a t o x y l i n and E o s i n o r Immunostained f o r I n s u l i n (600x) Normal r a t p a n c r e a s was h a r v e s t e d and p r e p a r e d as d e s c r i b e d i n t h e Methods s e c t i o n . Cut s e c t i o n s were s t a i n e d w i t h h e m a t o x y l i n and e o s i n (H & E ) ( F i g u r e 117) o r immunostained f o r i n s u l i n by t h e a n t i b o d y - l i n k e d p e r o x i d a s e method ( F i g u r e 118). R e p r e s e n t a t i v e p h o t o m i c r o g r a p h s showing p a n c r e a t i c i s l e t s were chosen from among many s t a i n e d s e c t i o n s d e r i v e d from 5 normal p a n c r e a s e s . 271 273 F i g u r e s 119 and 120 M i c r o s c o p i c Appearance o f an S T Z - D i a b e t i c R a t P a n c r e a t i c I s l e t (a) d i s p l a y e d b e s i d e Normal I s l e t s ( b ) , b o t h S t a i n e d w i t h e i t h e r H e m a t o x y l i n and E o s i n o r Immunostained f o r I n s u l i n (600x) S T Z - d i a b e t i c r a t p a n c r e a s e s were h a r v e s t e d seven days a f t e r i n i t i a t i o n o f d i a b e t e s and p r e p a r e d as d e s c r i b e d i n t h e Methods s e c t i o n . Cut s e c t i o n s were s t a i n e d w i t h H & E ( F i g u r e 119) o r immunostained f o r i n s u l i n by t h e a n t i b o d y - l i n k e d p e r o x i d a s e method ( F i g u r e 1 2 0 ) . R e p r e s e n t a t i v e p h o t o m i c r o g r a p h s showing i s l e t s and s u r r o u n d i n g a c i n a r t i s s u e were chosen from among many s e c t i o n s so p r e p a r e d from 4 d i a b e t i c a n i m a l s and a r e d i s p l a y e d , f o r purposes o f c o m p a r i s o n , b e s i d e s i m i l a r l y t r e a t e d normal r a t p a n c r e a s s e c t i o n s . 275 F i g u r e s 121 and 122 M i c r o s c o p i c Appearance o f an I n s u l i n - T r e a t e d , D i a b e t i c R a t P a n c r e a t i c I s l e t (a) d i s p l a y e d b e s i d e a Normal I s l e t ( b ) , S t a i n e d w i t h e i t h e r H e m a t o x y l i n and E o s i n o r Immunostained f o r I n s u l i n (600x) I n s u l i n - t r e a t e d , d i a b e t i c r a t p a n c r e a s e s were h a r v e s t e d and p r e p a r e d as d e s c r i b e d i n t h e Methods s e c t i o n . Cut s e c t i o n s were s t a i n e d w i t h H & E ( F i g u r e 121) o r immunostained f o r i n s u l i n by t h e a n t i b o d y - l i n k e d p e r o x i d a s e method ( F i g u r e 1 2 2 ) . R e p r e s e n t a t i v e p h o t o m i c r o g r a p h s showing i s l e t s and s u r r o u n d i n g a c i n a r t i s s u e were chosen from among many s e c t i o n s so p r e p a r e d from 5 i n s u l i n - t r e a t e d , d i a b e t i c a n i m a l s and a r e d i s p l a y e d i n F i g u r e 121, f o r pur p o s e s o f c o m p a r i s o n , b e s i d e s i m i l a r l y t r e a t e d normal r a t p a n c r e a s s e c t i o n s . 277 2 7 8 f . 6 . i l l 279 F i g u r e s 1 2 3 a n d 1 2 4 M i c r o s c o p i c A p p e a r a n c e o f a N o n d i a b e t i c , N P H I n s u l i n - T r e a t e d R a t P a n c r e a t i c I s l e t ( a ) d i s p l a y e d b e s i d e a N o r m a l I s l e t ( b ) , b o t h S t a i n e d w i t h e i t h e r H e m a t o x y l i n a n d E o s i n o r I m m u n o s t a i n e d f o r I n s u l i n ( 6 0 0 x ) P a n c r e a s e s f r o m 1 0 d N P H i n s u l i n - t r e a t e d r a t s w e r e h a r v e s t e d a n d a n d p r e p a r e d a s d e s c r i b e d i n t h e M e t h o d s s e c t i o n . C u t s e c t i o n s w e r e s t a i n e d w i t h H & E ( F i g u r e 1 2 3 ) o r i m m u n o s t a i n e d f o r i n s u l i n b y t h e a n t i b o d y -l i n k e d p e r o x i d a s e m e t h o d ( F i g u r e 1 2 4 ) . R e p r e s e n t a t i v e p h o t o m i c r o g r a p h s s h o w i n g i s l e t s a n d s u r r o u n d i n g a c i n a r t i s s u e w e r e c h o s e n f r o m a m o n g m a n y s e c t i o n s s o p r e p a r e d f r o m 5 i n s u l i n - t r e a t e d , n o n d i a b e t i c a n i m a l s a n d a r e d i s p l a y e d , f o r p u r p o s e s o f c o m p a r i s o n , b e s i d e s i m i l a r l y t r e a t e d n o r m a l r a t p a n c r e a s s e c t i o n s . 280 282 F i g u r e s 125 and 126 M i c r o s c o p i c Appearance o f a C h l o r p r o p a m i d e -T r e a t e d Rat P a n c r e a t i c I s l e t (a) d i s p l a y e d b e s i d e a Normal I s l e t ( b ) , b o t h S t a i n e d w i t h e i t h e r H e m a t o x y l i n and E o s i n o r Immunostained f o r I n s u l i n (600x) P a n c r e a s e s from 10 d c h l o r p r o p a m i d e - t r e a t e d r a t s were h a r v e s t e d and p r e p a r e d as d e s c r i b e d i n t h e Methods s e c t i o n . Cut s e c t i o n s were s t a i n e d w i t h H & E ( F i g u r e 125) o r immunostained f o r i n s u l i n by t h e a n t i b o d y -l i n k e d p e r o x i d a s e method ( F i g u r e 1 2 6 ). R e p r e s e n t a t i v e p h o t o m i c r o g r a p h s showing i s l e t s and s u r r o u n d i n g a c i n a r t i s s u e were chosen from among many s e c t i o n s so p r e p a r e d from 5 c h l o r p r o p a m i d e - t r e a t e d a n i m a l s and a r e d i s p l a y e d , f o r pur p o s e s o f c o m p a r i s o n , b e s i d e s i m i l a r l y t r e a t e d , normal r a t p a n c r e a s s e c t i o n s . 283 Fie llff 2 8 4 F i g u r e 127 The E f f e c t o f I n c r e a s e d Endogenous I n s u l i n R e l e a s e and D i s t a n c e from t h e I s l e t on B i n d i n g of 1 2 5 I - I n s u l i n t o A c i n i : R e s u l t s o f an A u t o r a d i o g r a p h i c Study A f t e r 1 2 ^ I - I n s u l i n I n f u s i o n o f t h e I s o l a t e d P e r f u s e d P a n c r e a s 1 2 5 I - I n s u l i n was p e r f u s e d t h r o u g h t h e i s o l a t e d p e r f u s e d pancreas under t h r e e c o n d i t i o n s : normal g l u c o s e c o n c e n t r a t i o n (4.5 mM), g l u c o s e 18 mM + a r g i n i n e 20 mM, and g l u c o s e 4.5 mM + " c o l d " i n s u l i n (114x e x c e s s ) w i t h f o u r a n i m a l s per group. The p a n c r e a s e s were t h e n p r o c e s s e d as d e s c r i b e d i n t h e Methods. D e n s i t y o f r a d i o a c t i v e c o u n t s i n a c i n a r t i s s u e was d e t e r m i n e d by a m i c r o s c o p i c a r e a mapping program and measured i n 4 r e g i o n s d i f f e r e n t i a t e d by r a d i a l d i s t a n c e from i s l e t s , e x p r e s s e d i n a c i n i d i a m e t e r s : 0-4, 5-8, 9-12, and 13-15 a c i n i d i a m e t e r s from t h e i s l e t . N o n s p e c i f i c 1 2 5 i -i n s u l i n b i n d i n g , d e t e r m i n e d by t h e group p e r f u s e d w i t h e x c e s s " c o l d " i n s u l i n , was d i s c o u n t e d from t h e o t h e r two groups and t h e r e s u l t s d i s p l a y e d a g a i n s t r a d i a l d i s t a n c e from t h e i s l e t . R e s u l t s a r e e x p r e s s e d as t h e mean absorbance p e r f i e l d ± SEM and s i g n i f i c a n c e (p<0.05) i s i n d i c a t e d by t h e a s t e r i s k (*). O Glucose 4.5 mM # Glucose 18 mM + Arginine 20 mM 0 - 4 5 - 8 9 - 12 13 - 15 Radial Distance f r o m Islet (in § of acini) 287 R E S U L T S S t u d i e s i n t h e I s o l a t e d P e r f u s e d P a n c r e a s Exocrine Response to Secretagogues T h e e x o c r i n e r e s p o n s i v e n e s s o f t h e i s o l a t e d p e r f u s e d p a n c r e a s w a s s t u d i e d u s i n g t h r e e s e c r e t a g o g u e s : C C K - 8 , s e c r e t i n , a n d V I P . T h e p u r p o s e o f t h e s e s t u d i e s w a s t o v e r i f y t h a t t h e i s o l a t i o n p r o c e d u r e a n d p e r f u s i o n t e c h n i q u e p r e s e r v e d f u n c t i o n i n t h e e x o c r i n e p a n c r e a s a n d d i d n o t i m p a i r t h e e x o c r i n e p a n c r e a t i c r e s p o n s e t o s e c r e t a g o g u e s , t h u s v a l i d a t i n g t h e m o d e l s y s t e m . P a n c r e a s J u i c e V o l u m e R e s p o n s e T h e f l o w r a t e o f p a n c r e a t i c j u i c e s t i m u l a t e d b y i n c r e a s i n g d o s e s o f C C K - 8 , s e c r e t i n , a n d V I P d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s i s s h o w n i n F i g u r e s 1 - 3 . T h e f l o w r a t e o f j u i c e w a s s t a b l e a t a b a s e l i n e o f 0 . 1 t o 0 . 2 j i l / m i n i n u n s t i m u l a t e d a n i m a l s . C h a n g e s i n t h e f l o w r a t e i n r e s p o n s e t o t h e v a r i o u s s e c r e t a g o g u e s w e r e r a p i d , o c c u r r i n g w i t h i n m i n u t e s o f i n f u s i o n a n d p e r s i s t i n g f o r u p t o 2 0 m i n u t e s a f t e r s e c r e t a g o g u e i n f u s i o n w a s s t o p p e d , p a r t i c u l a r l y a f t e r h i g h e r s e c r e t a g o g u e c o n c e n t r a t i o n s . A t l o w e r s e c r e t a g o g u e c o n c e n t r a t i o n s , t h e f l o w r a t e r e t u r n e d t o b a s e l i n e b e f o r e t e r m i n a t i o n o f t h e e x p e r i m e n t . On a m o l a r b a s i s , C C K - 8 i n c r e a s e d t h e f l o w r a t e s i g n i f i c a n t l y m o r e t h a n s e c r e t i n o r V I P w i t h i n t h e d o s e r a n g e s e m p l o y e d . M a x i m u m s t i m u l a t o r y d o s e s f o r V I P a n d C C K w e r e n o t a c h i e v e d i n t h e s e s t u d i e s . C C K a n d V I P b o t h s t i m u l a t e d p a n c r e a t i c j u i c e f l o w i n a d o s e -288 r e l a t e d f a s h i o n w h i l e s e c r e t i n appeared t o r e a c h a maximal s t i m u l a t o r y dose a t l nM. Amylase S e c r e t i o n i n P a n c r e a t i c J u i c e S e c r e t i o n o f p a n c r e a t i c amylase s t i m u l a t e d by i n c r e a s i n g doses o f CCK-8, s e c r e t i n , and VIP d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s i s shown i n F i g u r e s 4-6. B a s a l amylase s e c r e t i o n v a r i e d from 10 t o 50 U/min. On a m o l a r b a s i s , CCK was f i v e - f o l d more p o t e n t a s t i m u l u s f o r amylase s e c r e t i o n t h a n s e c r e t i n o r V I P . I n c r e a s i n g doses o f CCK r e s u l t e d i n i n c r e a s i n g amylase s e c r e t i o n b u t t h i s was o n l y c l e a r l y d i s t i n g u i s h a b l e from t h e b a s e l i n e above 10 pM. The cause o f v a r i a b i l i t y i n t h e b a s e l i n e (0 pM) amylase s e c r e t i o n was n o t c l e a r . Amylase was s e c r e t e d i n r e s p o n s e t o s e c r e t i n b u t , as seen w i t h t h e s e c r e t i n e f f e c t on t h e f l o w r a t e o f p a n c r e a t i c j u i c e , no c l e a r d o s e - r e l a t e d i n c r e a s e i n s e c r e t i o n was e v i d e n t . R a t h e r , a l l c o n c e n t r a t i o n s o f s e c r e t i n above 0.5 nM appeared t o s t i m u l a t e amylase s e c r e t i o n i n a s i m i l a r f a s h i o n . One e x p l a n a t i o n f o r t h e l a c k o f a s e c r e t i n dose r e s p o n s e e f f e c t on amylase s e c r e t i o n might be t h a t t h e amylase d e t e c t e d was a conseguence o f washout by i n c r e a s e d j u i c e f l o w . But t h e s u s t a i n e d amylase r e s p o n s e a t a l l doses s u g g e s t s t h a t i n c r e a s e d c e l l u l a r s e c r e t i o n o f amylase must a l s o have o c c u r r e d . VIP a l s o s t i m u l a t e d amylase r e l e a s e b u t o n l y a t doses above 30 nM, i n d i c a t i n g d e c r e a s e d p o t e n c y r e l a t i v e t o s e c r e t i n . A f t e r t h e s t i m u l u s p e r i o d o f each s e c r e t a g o g u e , amylase 289 s e c r e t i o n r e t u r n e d t o t h e b a s e l i n e p r i o r t o c o m p l e t i o n o f t h e e x p e r i m e n t , though w i t h a s i g n i f i c a n t d e l a y (15 min) a t h i g h e r doses o f CCK. T h i s i n d i c a t e d t h a t t h e amylase r e l e a s e was n o t an a r t i f a c t o f c e l l u l a r d e g e n e r a t i o n w i t h i n t h e p a n c r e a s b u t r a t h e r , a t r u e s e c r e t o r y r e s p o n s e . T o t a l P r o t e i n S e c r e t i o n i n P a n c r e a t i c J u i c e S e c r e t i o n o f e x o c r i n e t o t a l p r o t e i n s t i m u l a t e d by i n c r e a s i n g doses o f CCK-8, s e c r e t i n , and VIP d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s i s shown i n F i g u r e s 7-9. B a s a l t o t a l p r o t e i n s e c r e t i o n v a r i e d from 10 t o 50 /zg/min. As p r e v i o u s l y n o t e d , t h e i n c r e m e n t a l dosage e f f e c t s o f t h e t h r e e s e c r e t a g o g u e s on p a n c r e a t i c t o t a l p r o t e i n s e c r e t i o n were most c l e a r l y e v i d e n t f o r CCK and V I P . On a m o l a r b a s i s , CCK was about t e n - f o l d more p o t e n t t h a n VIP o r s e c r e t i n a t s t i m u l a t i n g p r o t e i n s e c r e t i o n . S i n c e amylase s e c r e t i o n was o n l y f i v e - f o l d i n c r e a s e d by CCK o v e r t h e o t h e r s e c r e t a g o g u e s , t h i s f i n d i n g s u g g e s t s t h a t CCK may s t i m u l a t e p r e f e r e n t i a l / s e c r e t i o n o f e x o c r i n e p r o t e i n s o t h e r t h a n amylase. The s t i m u l u s t h r e s h o l d f o r CCK was about 10 pM and h i g h e r doses s t i m u l a t e d b o t h g r e a t e r p r o t e i n s e c r e t i o n and had a l o n g e r d u r a t i o n o f a c t i o n . As was f o u n d w i t h amylase s e c r e t i o n , i n c r e a s i n g doses o f s e c r e t i n d i d n o t produce i n c r e m e n t a l p r o t e i n s e c r e t i o n , r a t h e r a l l doses o f s e c r e t i n appeared t o s t i m u l a t e s e c r e t i o n o f p r o t e i n a t a s i m i l a r r a t e . I n c r e a s i n g d oses o f VIP r e s u l t e d i n i n c r e m e n t a l s e c r e t i o n o f p r o t e i n a t a t h r e s h o l d dose o f 5 nM. S e c r e t i o n o f e x o c r i n e p r o t e i n 290 r e t u r n e d t o b a s e l i n e f o r each o f t h e s e c r e t a g o g u e doses t e s t e d b u t o n l y a f t e r a v a r i a b l e d e l a y . The 1000 pM CCK s t i m u l u s was t h e s o l e e x c e p t i o n t o t h i s o b s e r v a t i o n and p r o t e i n s e c r e t i o n was s t i l l above b a s e l i n e 20 min a f t e r t h e CCK i n f u s i o n was s t o p p e d , a t t h e t e r m i n a t i o n o f t h e e x p e r i m e n t . Endocrine Response to Exocrine Secretagogues I n s u l i n R e l e a s e i n P e r f u s a t e I n s u l i n r e l e a s e i n p e r f u s a t e , i n r e s p o n s e t o i n c r e a s i n g doses o f CCK-8, s e c r e t i n , and VIP d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s , i s shown i n F i g u r e s 10-13. I n s u l i n r e l e a s e i n r e s p o n s e t o i n c r e a s i n g doses o f CCK was e x p r e s s e d as t h e c o n c e n t r a t i o n o f i n s u l i n i n p e r f u s a t e (uU x 1 0 _ 1 / m l , F i g u r e 10) and as t h e t o t a l amount o f i n s u l i n r e l e a s e d o v e r t i m e (/xU x 1 0 _ 1 / m i n , F i g u r e 1 1 ) . S i n c e t e c h n i c a l f a c t o r s l e a d i n g t o v a r i a b i l i t y i n p e r f u s a t e f l o w r a t e o r l e a k a g e from t h e p r e p a r a t i o n might i n t r o d u c e a r t i f a c t u a l changes i n hormone measurements e x p r e s s e d p e r u n i t t i m e , b o t h c o n c e n t r a t i o n and t o t a l hormone r e l e a s e were e v a l u a t e d . I f t h e p e r f u s a t e l e a k a g e had i n c r e a s e d o v e r t h e c o u r s e o f t h e e x p e r i m e n t , i n s u l i n c o n c e n t r a t i o n s might have d e c l i n e d i n p e r f u s a t e as i n f u s i o n f l o w was a d j u s t e d t o m a i n t a i n a c o n s t a n t o u t f l o w r a t e . However, no d i f f e r e n c e s i n t h e p a t t e r n o f b a s e l i n e measurement b e f o r e o r d u r i n g C C K - s t i m u l a t i o n were e v i d e n t between t h e two f i g u r e s , s u g g e s t i n g t h a t e x p r e s s i o n o f i n s u l i n r e l e a s e o v e r t i m e was a c c u r a t e . The p o s s i b i l i t y o f 291 s uch an a r t i f a c t o f measurement was a l s o a s s e s s e d f o r s o m a t o s t a t i n as d i s c u s s e d i n t h e n e x t s e c t i o n . V a r i a b i l i t y i n b a s e l i n e measurements o f i n s u l i n r e l e a s e was e v i d e n t f o r a l l t h r e e s e c r e t a g o g u e s s t u d i e d . S i g n i f i c a n t (*) d i f f e r e n c e s were d e t e r m i n e d by comparing i n s u l i n r e l e a s e b e f o r e and d u r i n g s e c r e t a g o g u e s t i m u l a t i o n f o r t h e same a n i m a l s r a t h e r t h a n s t a t i s t i c a l l y comparing t e s t groups t o c o n t r o l s . Only w i t h VIP was t h e r e any s u g g e s t i o n o f an i n s u l i n r e s p o n s e d u r i n g s e c r e t a g o g u e i n f u s i o n and t h i s was o n l y s i g n i f i c a n t f o r t h e 5 and 30 nM doses due t o b a s a l v a r i a b i l i t y . S o m a t o s t a t i n R e l e a s e i n P e r f u s a t e S o m a t o s t a t i n r e l e a s e i n p e r f u s a t e , i n r e s p o n s e t o i n c r e a s i n g doses o f CCK-8, s e c r e t i n , and VIP d u r i n g p e r f u s i o n o f t h e i s o l a t e d p a n c r e a s , i s shown i n F i g u r e s 14-17. As d i s c u s s e d p r e v i o u s l y f o r i n s u l i n , s o m a t o s t a t i n r e l e a s e d u r i n g CCK s t i m u l a t i o n was e x p r e s s e d as t h e c o n c e n t r a t i o n o f hormone i n p e r f u s a t e (pg x 1 0 _ 1 / m l , F i g u r e 14) and as t o t a l s o m a t o s t a t i n r e l e a s e o v e r t i m e (pg x 10~ 2/min, F i g u r e 15) t o d e t e c t s y s t e m a t i c e r r o r s due t o p e r f u s a t e l o s s from t h e system. There were no d i f f e r e n c e s between t h e p a t t e r n s o f r e s p o n s e i n F i g u r e s 14 and 15. CCK s t i m u l a t e d s o m a t o s t a t i n r e l e a s e b u t o n l y a t t h e h i g h e s t dosage used, 1000 pM. V a r i a b i l i t y o f t h e b a s e l i n e s o m a t o s t a t i n r e l e a s e f o r s e c r e t i n and VIP groups was e v i d e n t b u t s o m a t o s t a t i n was n o t s t i m u l a t e d a t any dosage o f e i t h e r s e c r e t a g o g u e . 292 Effects of Glucose and Exogenous I n s u l i n on Stimulated Exocrine Secretion S e p a r a t e e x p e r i m e n t s were d e s i g n e d t o a s s e s s 1) t h e e f f e c t s o f endogenous i n s u l i n r e l e a s e , s t i m u l a t e d by i n c r e a s e d p e r f u s a t e g l u c o s e c o n c e n t r a t i o n , and 2) t h e e f f e c t s o f exogenous i n s u l i n on e x o c r i n e p a n c r e a t i c f u n c t i o n under c o n d i t i o n s o f c o n t i n u o u s e x o c r i n e s t i m u l a t i o n by CCK, s e c r e t i n , o r VIP. C C K - S t i m u l a t e d E x o c r i n e S e c r e t i o n The e x o c r i n e e f f e c t s o f endogenous i n s u l i n r e l e a s e d by a g l u c o s e c h a l l e n g e i n t h e C C K - s t i m u l a t e d , p e r f u s e d p a n c r e a s were examined i n s t u d i e s shown i n F i g u r e s 18-22. C o n t i n u o u s CCK s t i m u l a t i o n was p r e s e n t a t a dose o f 10 pM. I n c r e a s i n g t h e p e r f u s a t e g l u c o s e f o u r - f o l d from 4.5 t o 17.9 mM d u r i n g t h e t e s t p e r i o d i n d i c a t e d by t h e key b a r f a i l e d t o a l t e r t h e p a n c r e a t i c j u i c e f l o w r a t e ( F i g u r e 1 8 ) , o r amylase s e c r e t i o n ( F i g u r e 1 9 ) , b u t d i d l e a d t o a s i g n i f i c a n t i n c r e a s e i n e x o c r i n e p r o t e i n s e c r e t i o n as compared t o b o t h t h e c o n t r o l group and b a s e l i n e s e c r e t i o n (p<0.05, F i g u r e 2 0 ) . The c h a r a c t e r i s t i c b i m o d a l s e c r e t o r y p a t t e r n o f i n s u l i n r e l e a s e s t i m u l a t e d by i n t r a v e n o u s g l u c o s e was e v i d e n t i n F i g u r e 21. I n s u l i n r e l e a s e r e a c h e d a peak o f 1000 uU/min a t t h e end o f t h e t e s t p e r i o d i n d i c a t i n g a s i g n i f i c a n t endogenous i n s u l i n r e s p o n s e t o t h e g l u c o s e c h a l l e n g e employed. S o m a t o s t a t i n was a l s o r e l e a s e d i n t o p e r f u s a t e i n t h e t e s t group ( F i g u r e 22) b u t t h e r i s e i n s o m a t o s t a t i n was p r o p o r t i o n a t e l y s m a l l e r , o n l y a f r a c t i o n o f t h e i n s u l i n r e s p o n s e . Under t h e chosen c o n d i t i o n s o f CCK s t i m u l a t i o n i n t h e i s o l a t e d p e r f u s e d p a n c r e a s , endogenous i n s u l i n p o t e n t i a t e d some a s p e c t s o f e x o c r i n e f u n c t i o n b u t o v e r a l l t h e e f f e c t was m i n i m a l . Under i d e n t i c a l C C K - s t i m u l a t i o n c o n d i t i o n s as d e s c r i b e d f o r t h e p r e v i o u s g l u c o s e s t u d i e s , t h e e f f e c t s o f exogenous i n s u l i n on e x o c r i n e and e n d o c r i n e f u n c t i o n i n t h e p e r f u s e d p a n c r e a s were s t u d i e d and a r e shown i n F i g u r e s 23-27. I n s u l i n was added v i a a s i d e a r m t o p e r f u s a t e d u r i n g t h e t e s t p e r i o d (200 juU/ml = 1.3 nM i n s u l i n f i n a l c o n c e n t r a t i o n , see f i g u r e key) and had no s i g n i f i c a n t e f f e c t on p a n c r e a t i c j u i c e f l o w r a t e ( F i g u r e 23) b u t d i d s i g n i f i c a n t l y p o t e n t i a t e amylase r e l e a s e (p<0.05, F i g u r e 2 4 ) . Perhaps due t o t h e marked v a r i a b i l i t y i n b a s e l i n e v a l u e s , no i n s u l i n e f f e c t was e v i d e n t on t h e C C K - s t i m u l a t e d t o t a l p r o t e i n s e c r e t i o n ( F i g u r e 2 5 ) . The magnitude o f t h e i n s u l i n i n f u s i o n was c o n f i r m e d by m e a suring p e r f u s a t e i n s u l i n c o n c e n t r a t i o n s ( F i g u r e 2 6 ) . A t an average p e r f u s i o n f l o w r a t e o f 3 ml/min, 600 /LtU/min o f i n s u l i n s h o u l d t h e o r e t i c a l l y be i n f u s e d d u r i n g t h e t e s t p e r i o d and i n d e e d , about 600 /iU/min were r e c o v e r e d . These d a t a i n d i c a t e d t h a t m i n i m a l a d s o r p t i o n o r o t h e r l o s s e s o f i n s u l i n had o c c u r r e d d u r i n g passage t h r o u g h t h e s i d e a r m assembly and p a n c r e a s p r e p a r a t i o n . F i n a l l y , exogenous i n s u l i n had no e f f e c t s on s o m a t o s t a t i n r e l e a s e under c o n d i t i o n s o f c o n t i n u o u s CCK s t i m u l a t i o n ( F i g u r e 2 7 ) . 294 S e c r e t i n - S t i m u l a t e d E x o c r i n e S e c r e t i o n The e f f e c t s o f endogenous i n s u l i n r e l e a s e d by a g l u c o s e c h a l l e n g e d u r i n g s e c r e t i n - s t i m u l a t e d e x o c r i n e f u n c t i o n were a s s e s s e d i n s t u d i e s shown i n F i g u r e s 28-32. S e c r e t i n was i n f u s e d as i n d i c a t e d by t h e f i g u r e b a r a t a dose o f 0.5 nM and p e r f u s a t e g l u c o s e was i n c r e a s e d f o u r - f o l d f r om 4.5 t o 17.9 mM d u r i n g t h e t e s t p e r i o d . The i n c r e a s e i n p e r f u s a t e g l u c o s e c o n c e n t r a t i o n s i g n i f i c a n t l y p o t e n t i a t e d p a n c r e a t i c j u i c e f l o w (p<0.05, F i g u r e 28) and amylase s e c r e t i o n (p<0.05, F i g u r e 2 9 ) , b u t d i d n o t s i g n i f i c a n t l y a l t e r t o t a l p r o t e i n s e c r e t i o n ( F i g u r e 3 0 ) . I t i s i m p o r t a n t t o n o t e t h a t t h e magnitude o f b a s e l i n e amylase and t o t a l p r o t e i n s e c r e t i o n i n t h e s e s e c r e t i n s t u d i e s ( F i g u r e s 29, 30, 34, and 35) was p r o p o r t i o n a t e l y s m a l l e r t h a n i n t h e p r e v i o u s CCK s t u d y ( F i g u r e s 19, 20, 24, and 25) and t h i s , r a t h e r t h a n t h e s e c r e t a g o g u e i t s e l f , may be a r e a s o n f o r d i f f e r e n c e s i n t h e degree o f i n s u l i n p o t e n t i a t i o n . The magnitude o f t h e i n s u l i n i r e s p o n s e t o g l u c o s e was e v i d e n t i n F i g u r ^ 31 and, perhaps due t o t h e i n s u l i n o t r o p i c e f f e c t s o f CCK, was o n l y o n e - t h i r d t h e s i z e o f t h e i n s u l i n r e s p o n s e i n t h e p r i o r CCK s t u d y ( F i g u r e s 31 vs 2 1 ) . The i n c r e m e n t i n s o m a t o s t a t i n o v e r b a s e l i n e d u r i n g g l u c o s e i n f u s i o n was, however, g r e a t e r t h a n t h a t f o u n d i n t h e CCK s t u d y ( F i g u r e 3 2 ) . I n c o n t r a s t t o t h e s m a l l p o t e n t i a t i o n o f j u i c e f l o w and amylase d u r i n g g l u c o s e c h a l l e n g e , exogenous i n s u l i n m a r k e d l y p o t e n t i a t e d s e c r e t i n - s t i m u l a t e d e x o c r i n e s e c r e t i o n (0.5 nM, F i g u r e s 32-37). I n s u l i n c a u s e d a s i g n i f i c a n t i n c r e a s e i n t h e 295 j u i c e f l o w r a t e o v e r c o n t r o l (p<0.05, F i g u r e 3 3 ) , an e f f e c t t h a t was s i m i l a r l y e v i d e n t f o r amylase s e c r e t i o n (p<0.05, F i g u r e 34) and e x o c r i n e t o t a l p r o t e i n s e c r e t i o n (p<0.05, F i g u r e 3 5 ) . The dose o f p e r f u s e d i n s u l i n was c o n f i r m e d and a p p r o x i m a t e d t h a t seen i n t h e p r i o r CCK s t u d y (600-700 /iU/min, F i g u r e 3 6 ) . Exogenous i n s u l i n had no e f f e c t on s o m a t o s t a t i n r e l e a s e ( F i g u r e 3 7 ) . V I P - S t i m u l a t e d E x o c r i n e S e c r e t i o n The e f f e c t s o f a g l u c o s e c h a l l e n g e on V I P - s t i m u l a t e d e x o c r i n e f u n c t i o n were a l s o a s s e s s e d i n s t u d i e s shown i n F i g u r e s 38-42. VIP (15 nM) was i n f u s e d as i n d i c a t e d by t h e f i g u r e b a r and p e r f u s a t e g l u c o s e was i n c r e a s e d f o u r - f o l d from 4.5 t o 17.9 mM d u r i n g t h e t e s t p e r i o d . The p a n c r e a t i c j u i c e f l o w r a t e ( F i g u r e 38) i n c r e a s e d n o n s i g n i f i c a n t l y b u t amylase s e c r e t i o n , s u f f e r i n g from a v a r i a b l e b a s e l i n e , n e v e r t h e l e s s showed p o t e n t i a t i o n by t h e g l u c o s e i n f u s i o n (p<0.05, F i g u r e 3 9 ) . T o t a l p r o t e i n s e c r e t i o n was s i g n i f i c a n t l y i n c r e a s e d o v e r c o n t r o l by t h e g l u c o s e c h a l l e n g e (p<0.05, F i g u r e 4 0 ) . The i n s u l i n r e s p o n s e t o g l u c o s e was s i m i l a r t o t h a t seen f o r s e c r e t i n b u t was l e s s t h a n h a l f t h e magnitude o f t h e r e s p o n s e seen under c o n d i t i o n s o f CCK s t i m u l a t i o n ( F i g u r e 41 vs F i g u r e 2 1 ) . As was found i n t h e p r i o r s e c r e t i n s t u d y , g l u c o s e s t i m u l a t e d marked s o m a t o s t a t i n r e l e a s e d u r i n g t h e t e s t p e r i o d ( F i g u r e 4 2 ) . E v i d e n c e f o r i n c r e a s e d s o m a t o s t a t i n r e l e a s e i n r e s p o n s e t o g l u c o s e w i t h b o t h s e c r e t i n - and V I P - , b u t n o t C C K - s t i m u l a t e d e x o c r i n e s e c r e t i o n , s u g g e s t s t h a t e i t h e r b o t h 296 s e c r e t i n and VIP p o t e n t i a t e d t h e e f f e c t s o f g l u c o s e on s o m a t o s t a t i n r e l e a s e , o r CCK has an i n h i b i t o r y i n f l u e n c e on s o m a t o s t a t i n r e l e a s e . W h i l e t h e pa r a m e t e r s o f t h i s s t u d y do no t a l l o w t h i s i s s u e t o be r e s o l v e d , t h e p r e s e n c e o f such i n t e r a c t i o n s between e x o c r i n e s e c r e t a g o g u e s and i s l e t hormone s e c r e t i o n i s f u r t h e r e v i d e n c e f o r i n t e r d e p e n d e n t e n d o c r i n e and e x o c r i n e c o n t r o l mechanisms. The e f f e c t s o f exogenous i n s u l i n on V I P - s t i m u l a t e d e x o c r i n e s e c r e t i o n i n t h e p e r f u s e d p a n c r e a s a r e d i s p l a y e d i n F i g u r e s 43-47. I n c o n t r a s t t o t h e p r i o r s e c r e t i n s t u d y , exogenous i n s u l i n f a i l e d t o p o t e n t i a t e t h e s e c r e t o r y a c t i o n s o f VIP. I n s u l i n appeared t o have l i t t l e e f f e c t on V I P - s t i m u l a t e d p a n c r e a t i c j u i c e f l o w r a t e ( F i g u r e 43) o r e x o c r i n e t o t a l p r o t e i n s e c r e t i o n ( F i g u r e 4 5 ) , and i n d e e d , may have i n h i b i t e d amylase s e c r e t i o n (p<0.05, F i g u r e 4 4 ) . The s t a n d a r d e r r o r f o r t h e s e s t u d i e s i s such t h a t s i g n i f i c a n t d i f f e r e n c e s o n l y e x i s t e d f o r amylase s e c r e t i o n . C a u t i o n must however be e x e r c i s e d i n i n t e r p r e t i n g t h e s e d a t a s i n c e i n F i g u r e s 43-5, i t i s e v i d e n t t h a t t h e b a s e l i n e between t e s t and c o n t r o l groups d i f f e r e d even p r i o r t o a d d i n g i n s u l i n , i n d i c a t i n g non-random v a r i a t i o n i n t h e VIP r e s p o n s e between t h e two groups. Assay o f t h e p e r f u s a t e f o r i n s u l i n showed t h e e x p e c t e d c o n c e n t r a t i o n s o f i n s u l i n i n t h e group r e c e i v i n g t h e i n f u s i o n ( F i g u r e 46) and s o m a t o s t a t i n r e l e a s e d i d not change w i t h i n s u l i n i n f u s i o n ( F i g u r e 47) s u g g e s t i n g t h a t s o m a t o s t a t i n r e l e a s e i n r e s p o n s e t o g l u c o s e i s n o t a se c o n d a r y e f f e c t o f 297 i n c r e a s e d i n s u l i n r e l e a s e . Reasons f o r t h e d i f f e r e n c e between s e c r e t o r y e f f e c t s o f endogenous, g l u c o s e - s t i m u l a t e d i n s u l i n r e l e a s e and exogenous i n s u l i n i n t h e s e s t u d i e s remain u n c l e a r b u t may r e p r e s e n t t h e e f f e c t o f o t h e r hormonal f a c t o r s s uch as s o m a t o s t a t i n a l s o r e l e a s e d by g l u c o s e , o r a d i r e c t g l u c o s e e f f e c t on t h e e x o c r i n e p a n c r e a s . What i s c l e a r i s t h a t t h e p o t e n t i a t i n g e f f e c t s o f i n s u l i n on e x o c r i n e s e c r e t i o n a r e n o t u n i v e r s a l , b u t r a t h e r v a r y s i g n i f i c a n t l y by s e c r e t a g o g u e . Effects of Anoxia on CCK-Stimulated Exocrine Secretion P a r a m e t e r s o f t h e e x o c r i n e i n t e g r i t y o f t h e i s o l a t e d , p e r f u s e d p a n c r e a s were f u r t h e r i n v e s t i g a t e d by e x p e r i m e n t s d e s i g n e d t o a s s e s s t h e r o l e o f p e r f u s a t e o x y g e n a t i o n i n e x o c r i n e f u n c t i o n o f t h e i s o l a t e d p a n c r e a s . An a n o x i c p e r i o d was i n t r o d u c e d d u r i n g p e r f u s i o n o f t h e C C K - s t i m u l a t e d , i s o l a t e d p a n c r e a s w i t h exchange o f normal oxygenated p e r f u s a t e f o r p e r f u s a t e e q u i l i b r a t e d w i t h N 2 95%-C0 2 5%. R e s u l t s from t h e s e s t u d i e s a r e summarized i n F i g u r e s 48-52. P r i o r t o t h e a n o x i c p e r i o d , c o n d i t i o n s o f i n c r e a s e d p e r f u s a t e g l u c o s e (17.9 mM, see f i g u r e key b a r ) o r i n c r e a s e d i n s u l i n (100 /iU/ml) were i n t r o d u c e d t o d e t e r m i n e i f e i t h e r o f t h e s e t r e a t m e n t s might a m e l i o r a t e t h e e f f e c t s o f a n o x i c s t r e s s . D e s p i t e wide d i f f e r e n c e s i n t h e b a s e l i n e f l o w r a t e o f p a n c r e a t i c j u i c e , when each group was compared t o i t s e l f d u r i n g t h e b a s e l i n e p e r i o d , a n o x i a markedly reduced t h e f l o w r a t e w i t h an e f f e c t t h a t l a s t e d t h e d u r a t i o n o f t h e 298 e x p e r i m e n t ( F i g u r e 4 8 ) . P r e t r e a t m e n t w i t h g l u c o s e o r i n s u l i n d i d n o t a l t e r t h i s r e s p o n s e . The amylase d a t a ( F i g u r e 49) a r e somewhat h a r d e r t o i n t e r p r e t due t o v a r i a b i l i t y o f t h e b a s e l i n e C C K - s t i m u l a t e d amylase s e c r e t i o n . However, i n a t l e a s t t h e i n s u l i n - t r e a t e d group, a n o x i a markedly d e c r e a s e d amylase s e c r e t i o n w i t h o u t any a p p a r e n t e f f e c t from t h e added i n s u l i n . T o t a l e x o c r i n e p r o t e i n r e s p o n s e s i n F i g u r e 50 had a more c o n s i s t e n t b a s e l i n e t h a n amylase b u t were a l l markedly d e c r e a s e d d u r i n g and a f t e r t h e a n o x i c p e r i o d w i t h no a p p a r e n t e f f e c t from t h e g l u c o s e o r i n s u l i n p r e t r e a t m e n t . I n s u l i n r e l e a s e measured i n p e r f u s a t e from t h e p r e p a r a t i o n a c h i e v e d t h e e x p e c t e d l e v e l s due t o g l u c o s e o r i n s u l i n i n f u s i o n ( F i g u r e 5 1 ) . Of n o t e , t h e a n o x i c p e r i o d d i d n o t a p p a r e n t l y r e s u l t i n B - c e l l damage s i n c e no i n s u l i n l e a k a g e (as from d i s r u p t e d B - c e l l s ) was d e t e c t e d d u r i n g o r a f t e r t h e a n o x i c p e r i o d . I n c o n t r a s t , s o m a t o s t a t i n r e l e a s e i n t h e a n o x i c group w i t h o u t p r e t r e a t m e n t ( f i l l e d c i r c l e ) was s i g n i f i c a n t l y i n c r e a s e d ( F i g u r e 5 2 ) , an e f f e c t t h a t was a p p a r e n t l y s u p p r e s s e d by g l u c o s e o r i n s u l i n p r e t r e a t m e n t . P o s s i b l e e x p l a n a t i o n s f o r i n c r e a s e d s o m a t o s t a t i n r e l e a s e w i t h a n o x i a i n c l u d e d i s i n h i b i t i o n o r a c t u a l s t i m u l a t i o n o f s o m a t o s t a t i n r e l e a s e , o r p o s s i b l y D - c e l l i n j u r y w i t h hormone l e a k a g e from c e l l s though t h e a b o l i t i o n o f t h e r e s p o n s e by i n s u l i n and g l u c o s e p r e t r e a t m e n t s u g g e s t s t h a t t h e mechanism i s o t h e r t h a n s i m p l y an i n j u r y r e s p o n s e . 299 S t u d i e s i n P u r i f i e d . D i s p e r s e d P a n c r e a t i c A c i n i S t u d i e s i n p a n c r e a t i c a c i n i were c a r r i e d o u t t o v a l i d a t e t h e e x o c r i n e r e s p o n s i v e n e s s o f t h e system and t o i n v e s t i g a t e i n s u l i n e f f e c t s on e x o c r i n e p a n c r e a t i c f u n c t i o n a t t h e c e l l u l a r l e v e l . As d i s c u s s e d i n t h e Methods s e c t i o n , an i m p o r t a n t purpose o f t h e s e s t u d i e s was t o d e t e c t changes i n t h e t o t a l c e l l enzyme c o n t e n t as w e l l as t h e f r a c t i o n o f t h a t c o n t e n t r e l e a s e d by a s e c r e t o r y s t i m u l u s under t h e d i f f e r e n t e x p e r i m e n t a l i n s u l i n - t r e a t m e n t c o n d i t i o n s employed. Thus i n many o f t h e a c i n i s t u d i e s , f i g u r e s e x p r e s s i n g b o t h t h e % o f t h e c e l l amylase c o n t e n t r e l e a s e d and t h e amount o f amylase i n u n i t s p e r mass o f c e l l p r o t e i n a r e p r o v i d e d . I t s h o u l d a l s o be n o t e d t h a t a l t h o u g h l i p a s e r e l e a s e was a measurable f a c t o r i n a number o f a c i n i s t u d i e s ( t h e l i p a s e a s s a y i s d e s c r i b e d i n t h e Methods s e c t i o n ) , no l i p a s e r e s u l t s a r e p r o v i d e d because c o n t a m i n a t e d r e a g e n t s made r e s u l t s o f t h o s e s t u d i e s i n a c c u r a t e . When p o s s i b l e , t h e compromised s t u d i e s were s u b s e q u e n t l y r e p e a t e d b u t w i t h o u t p e r f o r m i n g t h e t i m e -consuming l i p a s e a s s a y . Exocrine Response to Secretagogues D i s p e r s e d p a n c r e a t i c a c i n i responded t o a l l s e c r e t a g o g u e s t e s t e d i n an i n c r e m e n t a l , d o s e - r e l a t e d f a s h i o n w h i c h was e x p r e s s e d a g a i n s t a l o g 1 0 dose s c a l e o f t h e s e c r e t a g o g u e w i t h a s e p a r a t e , u n s t i m u l a t e d b a s a l (0 dose) p o i n t i n each f i g u r e . B a s a l s e c r e t i o n f o r a l l s e c r e t a g o g u e s o v e r t h e 40 min o f i n c u b a t i o n was about 5% o f t h e t o t a l c e l l amylase c o n t e n t o r 500 uM/ug c e l l p r o t e i n . S e c r e t i o n o f amylase due t o CCK 300 ( F i g u r e s 53 and 54) was d e t e c t a b l e a t 10 pM and maximal a t 100 pM, h i g h e r doses b e i n g p a r t i a l l y i n h i b i t o r y t o t h e amylase r e l e a s e . S e c r e t i o n i n r e s p o n s e t o s e c r e t i n ( F i g u r e s 55 and 56) was d e t e c t a b l e a t 100 pM and d i d n o t a c h i e v e a maximum i n t h e dose range t e s t e d though t h e maximum s e c r e t i n -s t i m u l a t e d amylase s e c r e t i o n measured exceeded t h a t o f CCK. S i m i l a r l y , V I P - s t i m u l a t e d s e c r e t i o n ( F i g u r e s 57 and 58) d i d not a c h i e v e a maximum i n t h e dose range t e s t e d . Amylase s e c r e t i o n due t o b o t h s e c r e t i n and VIP r o s e s h a r p l y between t h e 1 0 4 and 1 0 5 pM doses as i f a t h r e s h o l d dose was exceeded. S e c r e t i o n due t o m e t h a c h o l i n e ( F i g u r e s 59 and 60) was d e t e c t a b l e between 1 0 1 and 1 0 2 nM and, i n a p a t t e r n s i m i l a r t o CCK, r e a c h e d a maximal r e s p o n s e a t 1 0 3 nM w i t h a s u b t l e b u t d e c l i n i n g s e c r e t i o n a t h i g h e r doses. On a molar b a s i s and l i k e l y a t dose ranges t h a t might be c o n s i d e r e d p h y s i o l o g i c a l , CCK was t h e most p o t e n t s e c r e t a g o g u e t e s t e d by a f a c t o r o f 1.8 w i t h r e s p e c t t o s e c r e t i n o r V I P , and was s e v e r a l o r d e r s o f magnitude more p o t e n t t h a n m e t h a c h o l i n e . S i g n i f i c a n c e of I s l e t Fragments in the Dispersed Pancreatic Acini Preparation I n s p e c t i o n o f p u r i f i e d a c i n i w i t h t r y p a n b l u e commonly demo n s t r a t e d i s l e t s and i s l e t f r a g m e n t s . The purpose o f t h e s e s t u d i e s was 1) t o d e t e r m i n e t h e c o n c e n t r a t i o n s o f i n s u l i n and s o m a t o s t a t i n i n a c i n i medium r e l e a s e d by t h e s e i s l e t f r a g m e n t s , and 2) t o d e t e r m i n e whether v i a b l e , f u n c t i o n i n g e n d o c r i n e t i s s u e was p r e s e n t and c a p a b l e o f s e c r e t i n g i s l e t hormones i n t o t h e a c i n a r i n c u b a t i o n medium, 301 p o s s i b l y i n f l u e n c i n g e x o c r i n e f u n c t i o n . T h i s l a t t e r o b j e c t i v e was p a r t i c u l a r l y i m p o r t a n t i n t h e c o n t e x t o f o t h e r a c i n a r s t u d i e s t o be d i s c u s s e d because i s l e t hormones p r e s e n t i n t h e a c i n i p r e p a r a t i o n might i n t r o d u c e a c o n f o u n d i n g e r r o r , o b s c u r i n g t h e e x o c r i n e r e s p o n s e t o exogenous hormone and l e a d i n g t o e r r o n e o u s l y n e g a t i v e r e s u l t s . A c i n i were p r e p a r e d i n t h e u s u a l f a s h i o n and samples o f i n c u b a t i o n medium f o r i n s u l i n o r s o m a t o s t a t i n radioimmunoassay were d i l u t e d w i t h s a l i n e o r a p r o t o n i n (350 KlU/ml) t h e n b o i l e d f o r 2-3 min t o i n a c t i v a t e enzymes p r i o r o t o s t o r a g e a t -20 C. B o i l e d samples had no measurable amylase a c t i v i t y and w h i l e no immunoreactive s o m a t o s t a t i n c o u l d be d e m o n s t r a t e d , i n s u l i n i m m u n o r e a c t i v i t y was p r e s e n t a t between 60 and 160 /iU/mg a c i n a r c e l l p r o t e i n . I n one e x p e r i m e n t (n = 18 s a m p l e s ) , i n s u l i n was r e c o v e r e d a t 158 ± 11 /iU/mg c e l l p r o t e i n . i i C o n t r o l s t u d i e s t o d e t e r m i n e t h e f a t e o f r a t i n s u l i n added' t o i n c u b a t i o n medium showed t h a t v i r t u a l l y c o m p l e t e r e c o v e r y o f a l l added i n s u l i n was p o s s i b l e i f t h e i n c u b a t i o n medium was n o t b o i l e d . W i t h b o i l i n g however, o n l y 30% o f i n s u l i n added t o t h e i n c u b a t i o n medium c o u l d be r e c o v e r e d (n = 6 ) . When a c i n i were p r e s e n t i n t h e medium and i n s u l i n was added, immediate r e c o v e r y o f t h e i n s u l i n from t h e medium a f t e r b o i l i n g was i n c r e a s e d t o 48% (n = 12) a t s e v e r a l dose r a n g e s . C o n t r o l v i a l s ( a c i n i s u s p e n s i o n b u t no added i n s u l i n ) showed t h a t t h i s e f f e c t was n o t due t o endogenous i n s u l i n . A f t e r 302 i n c u b a t i o n f o r 40 m i n u t e s , r e c o v e r y o f i n s u l i n added t o a c i n i was s t i l l i n t h e range of 35 t o 45% (n = 1 2 ) . A l t h o u g h subsequent i n s u l i n measurements from t h e a c i n i medium were n o t c o r r e c t e d f o r t h e e f f e c t s o f b o i l i n g on r e c o v e r y o f i n s u l i n , t h e s e d a t a i m p l y t h a t b o i l i n g r e d u c e d t h e e f f i c i e n c y o f t h e i n s u l i n a s s a y by 50% and t h a t t r u e i n s u l i n v a l u e s i n t h e i n c u b a t i o n medium a s s a y e d i n t h i s f a s h i o n might be as much as d o u b l e t h e i r measured v a l u e . The f a t e o f i s l e t f r agments d u r i n g a c i n i p r e p a r a t i o n was a s s e s s e d by m e asuring t h e p r o p o r t i o n o f t o t a l p a n c r e a t i c i n s u l i n ( c o n s i d e r e d t o be p r o p o r t i o n a l t o B - c e l l mass) r e m a i n i n g a t each s t a g e o f a c i n i p r e p a r a t i o n as d e s c r i b e d i n t h e Methods s e c t i o n . Two s e p a r a t e r e c o v e r y e x p e r i m e n t s were c o n d u c t e d , t h e f i r s t showing 34 ± 12% (n = 5) and t h e second showing o n l y 10 ± 1.4% (n = 5) o f t h e t o t a l p a n c r e a t i c i n s u l i n i n i t i a l l y p r e s e n t t o be c a r r i e d t h r o u g h t o t h e f i n a l washed a c i n i p r e p a r a t i o n . The g r e a t e s t p r o p o r t i o n o f t h e i s l e t t i s s u e (60-90%) was s e p a r a t e d i n t h e c o u r s e of p u r i f y i n g a c i n i t h r o u g h 4% BSA d e n s i t y c e n t r i f u g a t i o n . These d a t a s u g g e s t t h a t up t o one t h i r d o f t h e i s l e t t i s s u e may p e r s i s t i n t h e a c i n i p r e p a r a t i o n and t h a t , from p r e p a r a t i o n t o p r e p a r a t i o n , wide d i f f e r e n c e s may e x i s t . To i n v e s t i g a t e whether i s l e t f r agments i n t h e a c i n i p r e p a r a t i o n r e t a i n f u n c t i o n , i n s u l i n r e l e a s e d i n t o t h e i n c u b a t i o n medium i n r e s p o n s e t o i n c r e a s i n g g l u c o s e c o n c e n t r a t i o n s ( 0 - 2 0 mM) was measured ( F i g u r e 6 1 ) . No 303 s i g n i f i c a n t e f f e c t on i n s u l i n c o n c e n t r a t i o n i n t h e i n c u b a t i o n medium was d e m o n s t r a t e d . The e f f e c t s o f g l u c o s e on e x o c r i n e f u n c t i o n were a l s o i n v e s t i g a t e d d u r i n g t h e same e x p e r i m e n t and changes i n g l u c o s e f a i l e d t o a l t e r amylase s e c r e t e d i n r e s p o n s e t o s e c r e t i n o r CCK ( F i g u r e 6 2 ) . T h i s r e s u l t i m p l i e d t h a t g l u c o s e e f f e c t s i n t h e p e r f u s e d p a n c r e a s n o t e d e a r l i e r were u n l i k e l y t o be a p r i m a r y r e s p o n s e t o g l u c o s e , r a t h e r t h e y were more l i k e l y t o be s e c o n d a r y e f f e c t s o f g l u c o s e -i n d u c e d i n s u l i n r e l e a s e . E m p l o y i n g t h e i n s u l o i n o t r o p i c e f f e c t o f CCK n o t e d e a r l i e r i n t h e p e r f u s i o n s t u d i e s , i n s u l i n c o n c e n t r a t i o n was measured under c o n d i t i o n s o f i n c r e a s i n g CCK s t i m u l a t i o n (0 - 1 0 3 pM) and tho u g h a t r e n d t o w a r d i n c r e a s e d i n s u l i n was n o t e d a t h i g h e r c o n c e n t r a t i o n s , no s i g n i f i c a n t e f f e c t was d e t e c t e d ( F i g u r e 6 2 ) . F i n a l l y , t h e e f f e c t s o f GIP and g l u c o s e on i n s u l i n r e l e a s e f r o m i s l e t f r a g m e n t s i n t h e a c i n i medium were a s s e s s e d under s e v e r a l e x o c r i n e s e c r e t o r y c o n d i t i o n s ( F i g u r e 6 4 ) . A g a i n , no s i g n i f i c a n t i n s u l i n o t r o p i c e f f e c t s were n o t e d . These d a t a i n d i c a t e t h a t i s l e t f r a g m e n t s a r e v a r i a b l y c a r r i e d t h r o u g h t h e p u r i f i c a t i o n p r o c e d u r e f o r p a n c r e a t i c a c i n i b u t a r e n o n f u n c t i o n a l . I n s u l i n ( b u t n o t s o m a t o s t a t i n ) i s p r e s e n t i n t h e medium, may come from f r a g m e n t e d o r i n j u r e d c e l l s , and may be o f s u f f i c i e n t c o n c e n t r a t i o n t o i n f l u e n c e e x o c r i n e f u n c t i o n and o b s c u r e r e s p o n s e s t o a d d i t i o n o f more i n s u l i n . The e x c e l l e n t r e c o v e r y o f added i n s u l i n even a f t e r a p r o l o n g e d p e r i o d o f i n c u b a t i o n s u g g e s t s t h a t added i n s u l i n i s 304 n o t r a p i d l y degraded when added t o an a c i n i s u s p e n s i o n and, i f i m m u n o r e a c t i v i t y equates t o b i o a c t i v i t y , s h o u l d remain b i o l o g i c a l l y a c t i v e f o r p r o l o n g e d p e r i o d s o f t i m e . Effect of I n s u l i n on CCK-Stimulated Acinar Secretion The in v i t r o e f f e c t s o f r a t i n s u l i n on C C K - s t i m u l a t e d a c i n a r s e c r e t i o n were a s s e s s e d f o r a range o f CCK doses and under a v a r i e t y o f i n s u l i n t r e a t m e n t c o n d i t i o n s . A t a l l dose l e v e l s o f CCK s t i m u l a t i o n , i n s u l i n added d u r i n g t h e i n c u b a t i o n p e r i o d had no e f f e c t on amylase r e l e a s e ( F i g u r e s 65 and 6 6 ) . When added a t h i g h dose (20,000 uU/ml) f o r a p r o l o n g e d p e r i o d o f p r e i n c u b a t i o n ( i n c u b a t i o n and p r e i n c u b a t i o n p e r i o d s d i s c u s s e d i n t h e Methods s e c t i o n ) , i n s u l i n s i g n i f i c a n t l y i n h i b i t e d t h e peak C C K - s t i m u l a t e d amylase r e s p o n s e as measured by b o t h % c e l l amylase r e l e a s e ( F i g u r e 67) and juU p e r /ig o f c e l l p r o t e i n ( F i g u r e 68) w i t h o u t s h i f t i n g t h e c u r v e s . I n h i b i t i o n was t h e r e f o r e o f a n o n c o m p e t i t i v e n a t u r e . However when i n s u l i n was added d u r i n g b o t h i n c u b a t i o n and p r e i n c u b a t i o n p e r i o d s b u t a t a l o w e r c o n c e n t r a t i o n (2000 MU/ml), no a d d i t i v e e f f e c t s o r s i g n i f i c a n t i n f l u e n c e on amylase r e l e a s e was p r e s e n t ( F i g u r e s 69 and 7 0 ) . Effect of I n s u l i n on Secretin-Stimulated Acinar Secretion S i m i l a r s t u d i e s t o t h o s e j u s t r e v i e w e d f o r CCK were a l s o c a r r i e d o u t f o r s e c r e t i n . P r o l o n g e d , h i g h dose i n s u l i n p r e i n c u b a t i o n w i t h i n s u l i n (20,000 /xU/ml, F i g u r e s 71 and 72) and a c o m b i n a t i o n o f i n s u l i n i n c u b a t i o n and p r e i n c u b a t i o n a t l o w e r c o n c e n t r a t i o n s ( F i g u r e s 73 and 74) o v e r a range o f 305 s e c r e t i n d o s e s d i d n o t a l t e r a m y l a s e r e l e a s e s i g n i f i c a n t l y . I n d e e d , t h e o n l y s i g n i f i c a n t e f f e c t o f i n s u l i n o n s e c r e t i n -s t i m u l a t e d s e c r e t i o n d e t e c t e d i n t h e s e s t u d i e s w a s f o u n d w i t h i n s u l i n t r e a t m e n t d u r i n g b o t h p r e i n c u b a t i o n a n d i n c u b a t i o n p e r i o d s i n t h e s t u d y d e p i c t e d i n F i g u r e 7 6 . T h e m o r e p h y s i o l o g i c a l a c i n a r s t i m u l u s o f b o t h C C K a n d s e c r e t i n ( F i g u r e s 7 5 a n d 7 6 ) w a s n o t i n f l u e n c e d b y a n y i n s u l i n t r e a t m e n t . T h u s , in v i t r o i n s u l i n e f f e c t s o n s e c r e t i n -s t i m u l a t e d p a n c r e a t i c a c i n a r s e c r e t i o n w a s d e t e c t a b l e b u t v a r i a b l e , a n d s m a l l . Effect of I n s u l i n on Methacholine-Stimulated Acinar Secretion In v i t r o i n s u l i n e f f e c t s o n m e t h a c h o l i n e - s t i m u l a t e d p a n c r e a t i c a c i n a r s e c r e t i o n w e r e s t u d i e d a t s e v e r a l d o s e l e v e l s o f m e t h a c h o l i n e a n d u n d e r t h e p r e v i o u s l y d i s c u s s e d c o n d i t i o n s o f e i t h e r i n s u l i n i n c u b a t i o n , p r e i n c u b a t i o n , o r t h e c o m b i n a t i o n o f i n c u b a t i o n a n d p r e i n c u b a t i o n ( F i g u r e s 7 7 a n d 7 8 ) . N o s i g n i f i c a n t i n s u l i n e f f e c t w a s s e e n . Effect of I n s u l i n Dosage on Basal and Stimulated Secretion G i v e n t h e l a c k o f in v i t r o i n s u l i n e f f e c t s o n s t i m u l a t e d s e c r e t i o n f r o m n o r m a l a c i n i , a n a t t e m p t w a s m a d e t o a m p l i f y i n s u l i n e f f e c t s b y a l t e r a t i o n o f t h e a c i n i . T w o e x p e r i m e n t a l a p p r o a c h e s w e r e u t i l i z e d , 1 ) z y m o g e n - d e p l e t e d a c i n i ( F i g u r e s 7 9 a n d 8 0 ) , a n d 2 ) a c i n i h a r v e s t e d f r o m n e w l y d i a b e t i c a n i m a l s w h e r e c i r c u l a t i n g i n s u l i n c o n c e n t r a t i o n s h a d d e c r e a s e d t h e r e b y p o t e n t i a l l y s e n s i t i z i n g a c i n i t o i n s u l i n 306 ( F i g u r e s 8 1 a n d 8 2 ) . A s d i s c u s s e d i n t h e I n t r o d u c t i o n s e c t i o n , i n s u l i n h a s b e e n s h o w n t o p r o m o t e p r o t e i n s y n t h e s i s a s o n e o f i t s m a n y a c t i o n s . I n F i g u r e s 7 9 a n d 8 0 , a c i n i w e r e c h r o n i c a l l y s t i m u l a t e d w i t h C C K ( 1 0 0 p M f o r 2 h ) d u r i n g t h e p r e i n c u b a t i o n p e r i o d t o d e p l e t e z y m o g e n a n d t h e r e b y p o t e n t i a l l y i n d u c e i n c r e a s e d c e l l u l a r p r o t e i n s y n t h e s i s . T h e c o n v e r s e o f t h i s e x p e r i m e n t a l a p p r o a c h , a s s e s s i n g i n s u l i n e f f e c t s u n d e r c o n d i t i o n s o f p r o t e i n s y n t h e s i s i n h i b i t i o n ( w i t h c y c l o h e x i m i d e ) , w a s a l s o c a r r i e d o u t a n d i s d i s c u s s e d l a t e r . U n d e r s e v e r a l c o n d i t i o n s o f i n s u l i n t r e a t m e n t , t h e o n l y s i g n i f i c a n t e f f e c t o f i n s u l i n w a s p o t e n t i a t i o n o f C C K -s t i m u l a t e d s e c r e t i o n w h e n i n s u l i n w a s a d d e d d u r i n g t h e i n c u b a t i o n p e r i o d . S i n c e t h i s r e s p o n s e w a s s i g n i f i c a n t f o r b o t h t h e % c e l l a m y l a s e a n d t o t a l a m y l a s e r e l e a s e d , s e c r e t i o n o f n e w l y s y n t h e t i z e d a m y l a s e a l o n e c o u l d n o t a c c o u n t f o r t h i s e f f e c t . I n t h e s e c o n d i n s u l i n s t u d y o n a c i n i h a r v e s t e d f r o m d i a b e t i c a n i m a l s , s i x s e p a r a t e c o n d i t i o n s o f i n s u l i n t r e a t m e n t o n b a s a l a n d s t i m u l a t e d a m y l a s e s e c r e t i o n h a d n o s i g n i f i c a n t p o t e n t i a t i n g e f f e c t ( F i g u r e s 8 1 a n d 8 2 ) . S i n c e s h o r t - t e r m d i a b e t e s c o u l d b e e x p e c t e d t o r e d u c e t h e c o n c e n t r a t i o n o f e n d o g e n o u s i n s u l i n c o n t a m i n a t i n g t h e a c i n a r p r e p a r a t i o n , o n e i m p l i c a t i o n o f t h i s s t u d y w a s t h a t e n d o g e n o u s i n s u l i n ( f r o m c o n t a m i n a t i n g i s l e t f r a g m e n t s ) d i d n o t h a v e a s u b t l e , c o n f o u n d i n g i n f l u e n c e a s h a d b e e n p r e v i o u s l y h y p o t h e s i z e d . D i a b e t e s o f s h o r t d u r a t i o n a p p a r e n t l y d i d n o t i n c r e a s e a c i n a r 307 r e s p o n s i v e n e s s t o i n s u l i n , a t l e a s t a s m e a s u r a b l e b y a c u t e e f f e c t s o f i n s u l i n d u r i n g s t i m u l a t e d a c i n a r s e c r e t i o n . Effect of Altered C i r c u l a t i n g I n s u l i n Concentrations in the Rat on Subsequent Acinar Responsiveness to I n s u l i n in  v i t r o T h e p o s s i b i l i t y t h a t a l t e r a t i o n o f c i r c u l a t i n g i n s u l i n c o n c e n t r a t i o n i n t h e a n i m a l m i g h t i n f l u e n c e t h e i n s u l i n -r e s p o n s i v e n e s s o f s u b s e q u e n t l y i s o l a t e d a c i n i w a s f u r t h e r e x p l o r e d i n a s e r i e s o f s t u d i e s u t i l i z i n g n o r m a l , c o n t r o l a n i m a l s , S T Z - d i a b e t i c a n i m a l s , a n d a n i m a l s t r e a t e d w i t h e i t h e r N P H - i n s u l i n o r t h e d r u g c h l o r p r o p a m i d e t o i n c r e a s e c i r c u l a t i n g i n s u l i n c o n c e n t r a t i o n s . T r e a t m e n t r e g i m e n s a r e d i s c u s s e d i n t h e M e t h o d s s e c t i o n . T r e a t m e n t s w e r e i n i t i a t e d i n a s t a g g e r e d f a s h i o n t o p e r m i t s e q u e n t i a l p r e p a r a t i o n a n d t e s t i n g o f p a n c r e a t i c a c i n i f r o m m e m b e r s o f e a c h t r e a t m e n t g r o u p . F i g u r e 8 3 i n d i c a t e s t h e w e i g h t c h a n g e s w i t h t r e a t m e n t a n d s h o w s t h a t t h e h y p e r p h a g i c , i n s u l i n - t r e a t e d r a t s g a i n e d s t r i k i n g a m o u n t s o f w e i g h t o v e r a s h o r t p e r i o d w h i l e t h e d i a b e t i c a n i m a l s l o s t w e i g h t s i g n i f i c a n t l y . T h e t r e a t m e n t s h a d t h e i r i n t e n d e d e f f e c t i n t h a t p l a s m a g l u c o s e w a s s i g n i f i c a n t l y i n c r e a s e d i n d i a b e t i c a n i m a l s a n d d e c r e a s e d i n t h e c h l o r p r o p a m i d e a n d i n s u l i n - t r e a t e d g r o u p s c o m p a r e d t o c o n t r o l a n i m a l s ( F i g u r e 8 4 ) d u e t o i n c r e a s e d c i r c u l a t i n g i n s u l i n c o n c e n t r a t i o n s i n t h e i n s u l i n a n d a n d c h l o r p r o p a m i d e g r o u p s , a n d d e c r e a s e d c i r c u l a t i n g i n s u l i n i n t h e d i a b e t i c g r o u p ( F i g u r e 8 5 ) . A l t e r a t i o n o f c i r c u l a t i n g i n s u l i n 308 c o n c e n t r a t i o n s a l s o a f f e c t e d p a n c r e a s w e i g h t as d emonstrated i n T a b l e 1. T a b l e 1 P a n c r e a s Weights from A n i m a l s T r e a t e d t o A l t e r C i r c u l a t i n g I n s u l i n C o n c e n t r a t i o n s Treatment Group Weight (% body wt^ C o n t r o l 0.35 ± 0.02 S T Z - D i a b e t i c 0.32 ± 0.02 C h l o r p r o p a m i d e 0.32 ± 0.02 N P H - I n s u l i n 0.36 ± 0.04 D i a b e t i c and c h l o r p r o p a m i d e - t r e a t e d a n i m a l s had a m a r g i n a l l y s m a l l e r g l a n d i n p r o p o r t i o n t o body w e i g h t w h i l e t h e i n s u l i n -t r e a t e d a n i m a l s d e v e l o p e d p a n c r e a t i c e nlargment. I n t h i s s t u d y , i n s u l i n i n c u b a t i o n ( I n s u l i n Treatment Group B) p o t e n t i a t e d C C K - s t i m u l a t e d amylase r e l e a s e f o r b o t h t h e c o n t r o l ( F i g u r e s 86 and 87) and d i a b e t i c ( F i g u r e s 88 and 89) groups w h i l e t h e l o n g e r d u r a t i o n o f i n s u l i n e xposure d u r i n g combined p r e i n c u b a t i o n and i n c u b a t i o n p e r i o d s ( I n s u l i n Treatment Group D) p r e v e n t e d t h i s p o t e n t i a t i n g e f f e c t . A l t h o u g h d i a b e t i c a n i m a l s s e c r e t e d t h e same p r o p o r t i o n o f c e l l u l a r amylase i n r e s p o n s e t o t h e b a s a l and s t i m u l a t e d s e c r e t o r y c o n d i t i o n s , t h e t o t a l amount o f amylase s e c r e t e d was an o r d e r o f magnitude l e s s t h a n c o n t r o l a n i m a l s under a l l c o n d i t i o n s ( F i g u r e s 89 vs 8 7 ) , s u g g e s t i n g t h a t i n s u l i n has a p o w e r f u l i n f l u e n c e on amylase c o n t e n t i n t h e normal p a n c r e a s . The i n c r e a s e d c i r c u l a t i n g i n s u l i n c o n c e n t r a t i o n s o f t h e 309 c h l o r p r o p a m i d e - ( F i g u r e s 90 and 91) and i n s u l i n - t r e a t m e n t groups ( F i g u r e s 92 and 93) a p p a r e n t l y r e d u c e d t h e i n s u l i n r e s p o n s i v e n e s s o f t h e p a n c r e a s so t h a t i n s u l i n t r e a t m e n t o f a c i n i from t h e s e two groups was w i t h o u t e f f e c t . F u r t h e r , perhaps as a consequence o f t h e h y p e r p h a g i c s t a t e , a c i n i from a n i m a l s t r e a t e d w i t h NPH i n s u l i n had a marked a t t e n u a t i o n o f C C K - s t i m u l a t e d amylase r e l e a s e . A l t e r a t i o n o f c i r c u l a t i n g i n s u l i n c o n c e n t r a t i o n i n t h i s s e r i e s o f e x p e r i m e n t s appeared t o change t h e s e n s i t i v i t y o f a c i n i t o b o t h i n s u l i n and o t h e r s e c r e t a g o g u e s . The d i a b e t i c s t a t e m a r k e d l y r e d u c e d c e l l u l a r amylase c o n t e n t and t h e r e f o r e , t h e amount o f amylase a v a i l a b l e f o r s e c r e t i o n w h i l e h y p e r i n s u l i n i s m , perhaps v i a i n d i r e c t mechanisms, mar k e d l y a t t e n u a t e d t h e p o t e n c y o f CCK. Effect of Streptozotocin-Diabetes and Subsequent Exogenous I n s u l i n Replacement on the Acinar Response to I n s u l i n in  v i t r o Based upon t h e p r o f o u n d e f f e c t o f d i a b e t e s on amylase c o n t e n t o b s e r v e d i n t h e p r i o r s t u d y , a n o t h e r s e r i e s o f i n v e s t i g a t i o n s were d e s i g n e d t o measure t h e e f f e c t o f u n t r e a t e d and i n s u l i n -t r e a t e d d i a b e t e s on amylase c o n t e n t , c e r t a i n l y t h e most p r o f o u n d though c h r o n i c e f f e c t o f i n s u l i n on t h e e x o c r i n e p a n c r e a s d e t e c t e d t h u s f a r . D i a b e t e s was i n d u c e d w i t h s t r e p t o z o t o c i n and seven days l a t e r , NPH i n s u l i n t r e a t m e n t s were s t a r t e d a t a dosage chosen t o a c h i e v e normal plasma g l u c o s e c o n c e n t r a t i o n s . A n i m a l body w e i g h t ( e x p r e s s e d as a % o f i n i t i a l wt, F i g u r e 94) i n t h e d i a b e t i c groups i n i t i a l l y 310 d e c l i n e d b u t t h e i n s u l i n - t r e a t e d group e v e n t u a l l y g a i n e d w e i g h t . F a s t i n g plasma g l u c o s e i n t h e c o n t r o l group was 8.1 ± 0.4 mM compared t o 40 ± 2.3 mM i n t h e u n t r e a t e d d i a b e t i c g r o u p , and 5.4 ± 0.9 mM i n t h e i n s u l i n - t r e a t e d , d i a b e t i c group. S i m i l a r l y , f a s t i n g plasma i n s u l i n f o r t h e c o n t r o l a n i m a l s averaged 76.9 ± 2 /iU/ml w h i l e d i a b e t i c a n i m a l s had a low plasma i n s u l i n o f 15.4 ± 1.7 juU/ml w h i c h m a r k e d l y i n c r e a s e d t o 440 ± 80 /iU/ml a f t e r i n s u l i n t r e a t m e n t . A t t h e t i m e o f a c i n i p r e p a r a t i o n , t h e mass o f t h e p a n c r e a s d e c r e a s e d w i t h d i a b e t e s b u t t h e n i n c r e a s e d back t o normal upon subsequent i n s u l i n t r e a t m e n t , as shown i n T a b l e 2. P a n c r e a s Weights from C o n t r o l , D i a b e t i c , and I n s u l i n - t r e a t e d D i a b e t i c A n i m a l s a t t h e Time o f A c i n i P r e p a r a t i o n T a b l e 2 Treatment Group Weight f% body wt) C o n t r o l 0.40 ± 0.01 S T Z - D i a b e t i c 0.33 ± 0.02 I n s u l i n - T r e a t e d D i a b e t i c (Day 1) 0.35 ± 0.02 I n s u l i n - T r e a t e d D i a b e t i c (Day 3) 0.33 ± 0.02 I n s u l i n - T r e a t e d D i a b e t i c (Day 5) 0.41 ± 0.05 I n s u l i n - T r e a t e d D i a b e t i c (Day 8) 0.46 ± 0.03 Amylase s e c r e t i o n was measured i n r e s p o n s e t o i n c r e a s i n g doses o f CCK ( F i g u r e 9 5 ) , s e c r e t i n ( F i g u r e 9 6 ) , o r b o t h ( F i g u r e 97) i n a c i n i o b t a i n e d from c o n t r o l , 7-day, and 15-day 311 d i a b e t i c a n i m a l s . W h i l e t h e magnitude o f t h e amylase r e s p o n s e was markedly d e c r e a s e d i n b o t h d i a b e t i c groups as compared t o c o n t r o l s , t h e CCK and s e c r e t i n doses s t i m u l a t i n g a maximal r e s p o n s e were t h e same f o r a l l g r o u p s . The amount of amylase p r e s e n t f o r s e c r e t i o n was reduced b u t t h e s e c r e t o r y mechanism appeared t o be p r e s e r v e d . W i t h i n i t i a t i o n o f i n s u l i n t r e a t m e n t a t day 7 o f d i a b e t e s , t h e i n c r e m e n t i n amylase c o n t e n t and s l o w improvement i n t o t a l amylase s e c r e t o r y r e s p o n s e t o s e v e r a l s e c r e t a g o g u e s ( F i g u r e s 98, 99, and 100) i n d i c a t e d t h a t s e v e r a l days were r e q u i r e d f o r f u l l e x p r e s s i o n o f t h e i n s u l i n e f f e c t . By day 8, i n s u l i n t r e a t m e n t had a c h i e v e d about 50% o f c o n t r o l amylase c o n t e n t , as d e t e r m i n e d by com p a r i s o n o f amylase r e l e a s e from c o n t r o l and i n s u l i n - t r e a t e d d i a b e t i c a n i m a l s . Whether i n s u l i n t r e a t m e n t c o u l d e v e n t u a l l y r e s t o r e t h e d i a b e t i c p a n c r e a s t o normal amylase c o n c e n t r a t i o n s was n o t e v i d e n t from t h i s s t u d y . Effect of I n s u l i n on Acini from Weanling Rats The plasma and i n s u l i n r e s p o n s e s t o an i n t r a p e r i t o n e a l g l u c o s e c h a l l e n g e were s t u d i e d i n w e a n l i n g and o l d e r r a t s t o d e t e r m i n e t h e c o m p a r a t i v e i n s u l i n s e n s i t i v i t y o f w e a n l i n g a n i m a l s . F a s t i n g a d u l t a n i m a l s had a s i g n i f i c a n t l y h i g h e r and more p r o l o n g e d plasma g l u c o s e r e s p o n s e t h a n w e a n l i n g s r e c e i v i n g t h e same dose o f g l u c o s e ( F i g u r e 101). When plasma i n s u l i n v a l u e s from t h e same e x p e r i m e n t were compared i n F i g u r e 102, t h e a d u l t a n i m a l s a l s o had a much h i g h e r and 312 p r o l o n g e d plasma i n s u l i n r e s p o n s e compared t o w e a n l i n g s . These d a t a i n d i c a t e d t h a t w e a n l i n g a n i m a l s appeared t o m e t a b o l i z e g l u c o s e more q u i c k l y and a t l o w e r i n s u l i n c o n c e n t r a t i o n s t h a n a d u l t a n i m a l s , s u g g e s t i n g an i n c r e a s e d i n s u l i n s e n s i t i v i t y i n t h e w e a n l i n g a n i m a l s . F o r t h i s r e a s o n and by way o f c o n t r a s t t o many o f t h e p e r v i o u s a c i n i s t u d i e s c a r r i e d o u t u s i n g o l d e r , a d u l t a n i m a l s , t h e e f f e c t o f i n s u l i n on b a s a l and s t i m u l a t e d e x o c r i n e s e c r e t i o n i n w e a n l i n g r a t s was d e t e r m i n e d . However, in v i t r o i n s u l i n f a i l e d t o i n f l u e n c e CCK- ( F i g u r e s 103 and 104), s e c r e t i n - ( F i g u r e s 105 and 1 0 6), o r V I P - s t i m u l a t e d s e c r e t i o n ( F i g u r e s 107 and 108) i n a c i n i d e r i v e d from w e a n l i n g r a t s . The i n c r e a s e d s e n s i t i v i t y t o i n s u l i n d e t e c t e d i n t h e w e a n l i n g GTT d i d n o t appear t o e x t e n d t o t h e s e c r e t o r y r e s p o n s e o f t h e e x o c r i n e p a n c r e a s . Effect of Calcium Concentration in Incubation Medium on the Acinar Response to I n s u l i n S i n c e i n s u l i n i s t h o u g h t t o a c t , a t l e a s t i n p a r t , t h r o u g h c a l c i u m second messenger systems, t h e e f f e c t o f changes i n c a l c i u m c o n c e n t r a t i o n o f t h e i n c u b a t i o n medium were s t u d i e d i n s e c r e t i n - s t i m u l a t e d , i s o l a t e d a c i n i and a r e shown i n F i g u r e s 109 and 110. S e c r e t i n was chosen as t h e s e c r e t a g o g u e s i n c e i t does n o t r e l y on c a l c i u m as a second messenger. A l t h o u g h i n c r e a s i n g c a l c i u m i n t h e medium i n c r e a s e d amylase r e l e a s e m a r g i n a l l y , t h e r e was no s i g n i f i c a n t e f f e c t from t h e d i f f e r e n t i n s u l i n i n c u b a t i o n and p r e i n c u b a t i o n t r e a t m e n t c o n d i t i o n s . T h i s s u g g e s t e d t h a t t h e absence o f in v i t r o 313 i n s u l i n e f f e c t s s e e n i n m a n y a c i n i e x p e r i m e n t s w a s n o t a c o n s e q u e n c e o f i n s u f f i c i e n t c a l c i u m i n t h e m e d i u m . Effects of Microtubule ( c o l c h i c i n e ) and Protein-Synthesis (cycloheximide) I n h i b i t o r s on the Acinar Response to I n s u l i n A c e l l u l a r m i c r o t u b u l e i n h i b i t o r , c o l c h i c i n e w a s a d d e d t o t h e m e d i u m d u r i n g p r e i n c u b a t i o n w i t h a n d w i t h o u t i n s u l i n t o d e t e r m i n e i f p r e i n c u b a t i o n o f a c i n i w i t h i n s u l i n i n d u c e d i n t r a c e l l u l a r t r a n s p o r t o f p r o t e i n t h a t m i g h t b e p r e v e n t e d b y c o l c h i c i n e t h u s a l t e r i n g s e c r e t o r y r e s p o n s i v e n e s s o f t h e a c i n i ( F i g u r e s 1 1 1 a n d 1 1 2 ) . F o r s i m i l a r r e a s o n s , a p r o t e i n s y n t h e s i s i n h i b i t o r , c y c l o h e x i m i d e , w a s a d d e d t o a c i n i d u r i n g t h e i n c u b a t i o n s t e p t o d e t e r m i n e i f a n y p o r t i o n o f t h e a m y l a s e r e l e a s e d i n t h e p r e s e n c e o f a d d e d i n s u l i n r e p r e s e n t e d n e w l y s y n t h e s i z e d e n z y m e ( F i g u r e s 1 1 3 a n d 1 1 4 ) . B o t h s t u d i e s w e r e c o n d u c t e d u n d e r t h e s a m e t h r e e c o n d i t i o n s o f b a s a l a n d s t i m u l a t e d s e c r e t i o n ( s e c r e t i n 2 0 n M , C C K 5 0 p M ) a n d f a i l e d i t o s h o w a n y s i g n i f i c a n t e f f e c t d u e t o i n s u l i n t r e a t m e n t . I t w o u l d a p p e a r t h a t w i t h i n t h e t i m e c o u r s e o f t h e s e a c u t e s t u d i e s o n p a n c r e a t i c a c i n i ( 1 - 4 h ) , e n z y m e s e c r e t i o n i s d e p e n d e n t o n a v a i l a b l e c e l l u l a r e n z y m e s t o r e s , p r e s e n t i n c l o s e p r o x i m i t y t o t h e a p i c a l m e m b r a n e a n d n o t d e p e n d e n t o n i n s u l i n . Effect of I n s u l i n on Stimulated Secretion of Acini derived from Dorsal and Ventral Pancreas D o r s a l a n d v e n t r a l p a n c r e a s c o n t a i n i s l e t s o f d i f f e r e n t c o m p o s i t i o n t h a t m a y d i f f e r e n t i a l l y p o t e n t i a t e e x o c r i n e 314 s e c r e t i o n . A c i n i were p r e p a r e d from d o r s a l and v e n t r a l p a n c r e a s specimens r e s p e c t i v e l y and s u b j e c t e d t o CCK-s t i m u l a t i o n (0 - 500 pM) under two c o n d i t i o n s o f i n s u l i n i n c u b a t i o n t o d e t e r m i n e i f , i n d e e d , s t i m u l a t e d a c i n i r e s p o n d d i f f e r e n t l y d e p e nding on o r i g i n and i f one p a r t o f t h e p a n c r e a s might be more i n s u l i n r e s p o n s i v e t h a n t h e o t h e r . Maximal s t i m u l a t i o n o f a c i n i from b o t h d o r s a l and v e n t r a l p a n c r e a s o c c u r r e d a t a CCK dose o f 100 pM b u t t h e d o r s a l p a n c r e a s s e c r e t e d a p r o p o r t i o n a t e l y s m a l l e r p e r c e n t a g e of c e l l amylase c o n t e n t t h a n t h e v e n t r a l p a n c r e a s . The t o t a l amylase s e c r e t e d ( p e r /ig c e l l p r o t e i n ) f o r b o t h d o r s a l and v e n t r a l p a n c r e a s was i d e n t i c a l and t h e r e was no a p p r e c i a b l e r e s p o n s e t o i n s u l i n ( F i g u r e s l l 5 and 116). These d i f f e r e n c e s i n s t i m u l a t e d s e c r e t i o n between v e n t r a l and d o r s a l a c i n i s u g g e s t t h a t f u n c t i o n a l d i f f e r e n c e s e x i s t between t h e two e m b r y o l o g i c p a r t s o f t h e p a n c r e a s and may be a consequence o f i n s u l o a c i n a r e f f e c t s f rom t h e d i f f e r e n t i s l e t p o p u l a t i o n s p r e s e n t . H i s t o l o g i c and Immunocvtochemical S t u d i e s Normal Adult Rat Pancreas The h i s t o l o g i c appearance o f a normal r a t p a n c r e a t i c i s l e t s t a i n e d w i t h h e m a t o x y l i n and e o s i n i s shown i n F i g u r e 117 and a s i m i l a r s e c t i o n s t a i n e d i m m u n o c y t o c h e m i c a l l y f o r i n s u l i n i s shown i n F i g u r e 118. I s l e t t i s s u e , marked by a c l e a r c y t o p l a s m and d i f f e r e n t a r c h i t e c t u r e , was c l e a r l y d i s t i n g u i s h e d from t h e s u r r o u n d i n g a c i n a r e x o c r i n e t i s s u e . 315 I s l e t c e l l s were o r i e n t e d i n c o r d s i n an i r r e g u l a r p a t t e r n t h a t was emphasized by t h e p e r o x i d a s e - i n s u l i n s t a i n . I n t h i s normal i s l e t , t h e m a j o r i t y o f t h e i s l e t b u l k was made up o f B - c e l l s and t h e i n t i m a t e a p p r o x i m a t i o n of i s l e t t o s u r r o u n d i n g a c i n a r t i s s u e was e v i d e n t . F i g u r e 118 i n d i c a t e d f a i r l y symmetric s t a i n i n g f o r i n s u l i n t h r o u g h t h e c o r e of t h e i s l e t , t h e c l e f t s between s t a i n e d B - c e l l s e m p h a s i z i n g t h e c e l l u l a r o r g a n i z a t i o n o f t h e i s l e t c e l l s and l i k e l y r e p r e s e n t i n g e lements o f t h e i n t r a i s l e t " g l o m e r u l a r " c a p i l l a r y system. I n b o t h f i g u r e s , t h e c e l l u l a r o r g a n i z a t i o n o f e x o c r i n e t i s s u e i n t o a c i n i was a l s o e v i d e n t . B a s a l l y o r i e n t e d n u c l e i a t t h e p e r i p h e r y o f each a c i n u s e n c i r c l e d an a p i c a l c y t o p l a s m r i c h l y endowed w i t h e o s i n - s t a i n e d , zymogen m a t e r i a l . Even on a s i m p l e , q u a l i t a t i v e l e v e l , b o t h a c i n a r c e l l s i z e and t h e p r o p o r t i o n o f e o s i n - s t a i n e d c y t o p l a s m i c m a t e r i a l appeared more promine n t i n a c i n i n e a r t h e i s l e t as compared t o a c i n i a t a d i s t a n c e from t h e i s l e t . T h i s h i s t o l o g i c e v i d e n c e f o r a p e r i - i s l e t t r o p h i c e f f e c t has been p r e v i o u s l y n o t e d by o t h e r s and was d i s c u s s e d i n t h e I n t r o d u c t i o n as t h e " h a l o e f f e c t " . A c i n i r e s p o n s i b l e f o r t h e " h a l o e f f e c t " seemed t o be c l u s t e r e d w i t h i n 3-4 a c i n i d i a m e t e r s o f t h e i s l e t though i t must be r e c a l l e d t h a t o t h e r i s l e t s , t h r e e - d i m e n s i o n a l l y o r i e n t e d s u p e r f i c i a l o r deep t o t h e p l a n e o f s e c t i o n , remain an u n q u a n t i f i e d s o u r c e of i n f l u e n c e . 316 Streptozotocin-Diabetic Rat Pancreas I s l e t s from r a t s t r e a t e d w i t h t h e i s l e t - c e l l t o x i n , s t r e p t o z o t o c i n , a r e shown b e s i d e normal i s l e t s i n H & E s t a i n e d s e c t i o n s i n F i g u r e 119 and p e r o x i d a s e - i n s u l i n s t a i n e d s e c t i o n s i n F i g u r e 120. The n u c l e i o f c e l l s i n t h e H & E s t a i n e d d i a b e t i c i s l e t appeared c l u s t e r e d more c l o s e l y t o g e t h e r and t h e o r g a n i z a t i o n i n t o c o r d s was l e s s c l e a r . A l t h o u g h s t i l l p e r c e p t i b l e , t h e prominence o f e o s i n - s t a i n e d c y t o p l a s m i n p e r i - i s l e t a c i n i was l e s s i n many b u t n o t a l l o f th e S T Z - t r e a t e d p a n c r e a t i c s e c t i o n s . V a r i a b i l i t y i n i s l e t damage and t h e temporary e f f e c t s o f i n c r e a s e d i n s u l i n r e l e a s e from damaged c e l l s i m m e d i a t e l y a f t e r STZ exposure may be r e s p o n s i b l e f o r t h i s i n c o n s i s t e n c y . However t h e s e a l t e r a t i o n s i n c y t o p l a s m i c prominence were t h e o n l y h i s t o l o g i c e v i d e n c e f o r a change i n t h e e x o c r i n e p a n c r e a s as a consequence o f d i a b e t e s t h r o u g h t h e many d i a b e t i c p a n c r e a s s e c t i o n s s t u d i e d . The r e l a t i v e degree o f B - c e l l d e s t r u c t i o n was emphasized by t h e p e r o x i d a s e - i n s u l i n s t a i n . I n most i s l e t s , o n l y s c a t t e r e d c e l l s , most on t h e p e r i p h e r y o f t h e B-c e l l c o r e , s t i l l c o n t a i n e d i n s u l i n . Insulin-Treated, Diabetic Rat Pancreas I s l e t s from d i a b e t i c r a t s t r e a t e d w i t h i n s u l i n a r e shown b e s i d e normal i s l e t s i n H & E s t a i n e d s e c t i o n s i n F i g u r e 121 and p e r o x i d a s e - i n s u l i n s t a i n e d s e c t i o n s i n F i g u r e 122. Crowding and d i s o r g a n i z a t i o n o f t h e i s l e t c e l l n u c l e i t o g e t h e r w i t h a s u g g e s t i o n o f l e s s prominent e x o c r i n e p e r i -i s l e t c y t o p l a s m were e v i d e n t i n F i g u r e 121, unchanged from 317 F i g u r e 119. p e r o x i d a s e - i n s u l i n s t a i n e d s e c t i o n s o f t h e i n s u l i n - t r e a t e d a n i m a l showed t h a t i n s u l i n t r e a t m e n t a f t e r i n i t i a t i o n o f d i a b e t e s d i d n o t change t h e e f f e c t s o f t h e i s l e t t o x i n on t h e B c e l l s ; i n d e e d , perhaps even fewer i n s u l i n - c o n t a i n i n g c e l l s remained i n t h e d i s r u p t e d i s l e t c o r e a f t e r t h e i n s u l i n t r e a t m e n t . Insulin-Treated, Nondiabetic Rat Pancreas By way o f c o n t r a s t t o i n s u l i n - t r e a t e d d i a b e t i c a n i m a l s , i n s u l i n t r e a t m e n t i n normal a n i m a l s i n d u c e d s e v e r a l changes i n i s l e t h i s t o l o g i c appearance. An H & E s t a i n e d s e c t i o n from a t r e a t e d a n i m a l i s d i s p l a y e d b e s i d e a normal r a t s e c t i o n i n F i g u r e 123 and t h e p e r o x i d a s e - i n s u l i n s t a i n e d s e c t i o n i s d i s p l a y e d i n F i g u r e 124. N u c l e i w i t h i n t h e i s l e t o f t h e i n s u l i n - t r e a t e d a n i m a l were crowded compared t o t h e normal i s l e t b u t t h e c o r d a r c h i t e c t u r e was r e t a i n e d . A c i n i up t o t h e p e r i p h e r y o f t h e i s l e t had r e d u c e d c y t o p l a s m and t h e r e was no p e r c e p t i b l e change i n prominence o f t h e a c i n a r c y t o p l a s m w i t h r a d i a l d i s t a n c e from t h e i s l e t . The p e r o x i d a s e - i n s u l i n s t a i n showed t h a t w h i l e t h e r e was s t i l l i n s u l i n p r e s e n t t h r o u g h o u t c e l l s o f t h e i s l e t c o r e , t h e d e n s i t y o f s t a i n i n g was m a r kedly d e c r e a s e d , s u g g e s t i n g a r e d u c e d i n t r a c e l l u l a r c o n c e n t r a t i o n o f i n s u l i n . The h i s t o l o g i c and immunostain f i n d i n g s i n t h i s s t u d y s u g g e s t e d t h a t exogenous i n s u l i n , perhaps v i a normal feedback mechanisms, r e d u c e d i s l e t i n s u l i n s y n t h e s i s ( d e c r e a s e d i n s u l i n c o n t e n t i n d i c a t e d by t h e d e c r e a s e d s t a i n i n g ) and 318 r e l e a s e . The changes i n p e r i - i s l e t a c i n a r appearance s t r e n g t h e n t h e e v i d e n c e t h a t t h e " h a l o e f f e c t " i s due t o i n s u l i n r a t h e r t h a n a n o t h e r o f t h e i s l e t hormones. Chlorpropamide-Treated Rat Pancreas C h l o r p r o p a m i d e - t r e a t e d p a n c r e a t i c i s l e t s a r e d i s p l a y e d w i t h an H & E s t a i n i n F i g u r e 125, and t h e p e r o x i d a s e - i n s u l i n immunostain i n F i g u r e 126, b o t h b e s i d e normal c o n t r o l s e c t i o n s s i m i l a r l y s t a i n e d . C h l o r p r o p a m i d e had s i m i l a r e f f e c t s t o e x o g e n o u s l y a d m i n i s t e r e d i n s u l i n ; c e l l a r c h i t e c t u r e was unchanged b u t t h e B - c e l l s were d e p l e t e d o f i n s u l i n as i n d i c a t e d by t h e a t t e n u a t e d d e n s i t y o f t h e i n s u l i n immunostained s e c t i o n . No c o n s i s t e n t change i n t h e appearance o f a c i n i c o u l d be a t t r i b u t e d t o c h l o r p r o p a m i d e t r e a t m e n t . A u t o r a d i o g r a p h i c S t u d i e s A l t h o u g h c a p i l l a r y b r anches f o r m i n g an anatomic c o n n e c t i o n between t h e i s l e t and t h e s u r r o u n d i n g a c i n a r t i s s u e have been d e s c r i b e d as have p e r i - i s l e t h a l o s , no d i r e c t e v i d e n c e has been p r o v i d e d i n t h e l i t e r a t u r e t h a t i n s u l i n p r e f e r e n t i a l l y r e a c h e s p e r i - i s l e t a c i n i a t h i g h c o n c e n t r a t i o n d u r i n g c o n d i t i o n s o f i s l e t i n s u l i n r e l e a s e . An a u t o r a d i o g r a p h i c e x p e r i m e n t u t i l i z i n g p a n c r e a s e s p e r f u s e d w i t h 1 2 5 I - i n s u l i n was d e s i g n e d t o e x p l o r e t h i s q u e s t i o n . I s o l a t e d p e r f u s e d p a n c r e a s e s were a l l p e r f u s e d w i t h 1 2 5 I - i n s u l i n and one group was s t i m u l a t e d t o s e c r e t e i n s u l i n b o t h p r i o r t o and d u r i n g t h e 1 2 5 I - i n s u l i n , t o d i s p l a c e t h e r a d i o a c t i v e l y l a b e l l e d 319 hormone from a c i n a r i n s u l i n r e c e p t o r s . The r e s u l t s o f t h i s e x p e r i m e n t a f t e r d i s c o u n t i n g n o n s p e c i f i c b i n d i n g a r e d i s p l a y e d i n F i g u r e 127. A l t h o u g h c o n d u c t e d as a p i l o t s t u d y w i t h o n l y f o u r a n i m a l s p e r group, a s i g n i f i c a n t d i f f e r e n c e i n t h e b i n d i n g o f 1 2 5 i -i n s u l i n was e v i d e n t . Under c o n d i t i o n s o f endogenous i n s u l i n s t i m u l a t i o n , 1 2 5 I - i n s u l i n b i n d i n g was s i g n i f i c a n t l y d e c r e a s e d but o n l y i n a c i n i h i s t o l o g i c a l l y l o c a t e d w i t h i n 4 a c i n i d i a m e t e r s o f t h e i s l e t as compared t o t h e c o n t r o l , n o n s t i m u l a t e d group. Beyond 4 a c i n i from t h e i s l e t , no d i f f e r e n c e i n 1 2 5 I - i n s u l i n b i n d i n g was e v i d e n t between gr o u p s . These d a t a s u g g e s t t h a t i n s u l i n b i n d s t o s p e c i f i c a c i n i r e c e p t o r s on a c i n a r c e l l s , and t h a t endogenous i n s u l i n may be p r e s e n t i n p a r t i c u l a r l y h i g h c o n c e n t r a t i o n w i t h i n 4 a c i n i d i a m e t e r s o f t h e i s l e t , f o r m i n g a d e c l i n i n g g r a d i e n t o f i n s u l i n c o n c e n t r a t i o n w i t h i n c r e a s e d r a d i a l d i s t a n c e from t h e i s l e t . 320 DISCUSSION The a c t i o n s o f i n s u l i n i n t h e mammal a r e c r i t i c a l f o r m e t a b o l i c f u n c t i o n and growth i n many t i s s u e s . G l u c o s e t r a n s p o r t and m e t a b o l i s m , and t h e s y n t h e s i s o f g l y c o g e n , p r o t e i n , and f a t a r e p r i n c i p l e f u n c t i o n s i n many c e l l s w h i l e s t i m u l a t i o n o f DNA s y n t h e s i s , c e l l d i v i s i o n , d i f f e r e n t i a t i o n , and growth a r e prominent i n s u l i n e f f e c t s i n o t h e r s (Denton 1986). Kahn (1985) has p o i n t e d o u t t h a t f o r most t i s s u e s , m e t a b o l i c e f f e c t s o f i n s u l i n o c c u r r a p i d l y a f t e r exposure o f c e l l s t o t h e hormone, u s u a l l y a t low c o n c e n t r a t i o n , w h i l e t h e g r o w t h - p r o m o t i n g a c t i o n s r e q u i r e h o u r s o r days t o become e v i d e n t and g e n e r a l l y r e q u i r e much h i g h e r c o n c e n t r a t i o n s o f t h e hormone. W i t h i n t h e p a n c r e a s , i n s u l i n i n h i g h c o n c e n t r a t i o n i s s e c r e t e d i n t o i s l e t c a p i l l a r i e s and c a r r i e d , a t l e a s t i n p a r t , t o t h e e x o c r i n e t i s s u e where a c i n i a r e exposed t o h i g h b u t m i n u t e - t o - m i n u t e v a r y i n g c o n c e n t r a t i o n s o f i n s u l i n . The c o n d i t i o n s under w h i c h t h e e x o c r i n e p a n c r e a s i s exposed t o i n s u l i n a r e t h e r e f o r e d i f f e r e n t from a l l o t h e r i n s u l i n - r e s p o n s i v e o r g a n s . R e a l i z i n g t h i s u n i q u e r e l a t i o n s h i p o f e x o c r i n e t o e n d o c r i n e p a n c r e a s , t h e i n t e n t o f t h i s work was t o e x p l o r e t h e r e l a t i v e s i g n i f i c a n c e o f l o n g -term t r o p h i c and more a c u t e , m e t a b o l i c e f f e c t s o f i n s u l i n on normal e x o c r i n e p a n c r e a t i c f u n c t i o n . I n s u l i n c l e a r l y has i m p o r t a n t l o n g - t e r m e f f e c t s on e x o c r i n e p a n c r e a t i c f u n c t i o n and i n t e g r i t y . The p h y s i o l o g i c a l s i g n i f i c a n c e o f t h e s e e f f e c t s has been s u g g e s t e d by C o r r i n g 321 (1987) who showed t h a t n u t r i e n t - s p e c i f i c a l t e r a t i o n i n p a n c r e a t i c enzyme l e v e l s o c c u r r e d w i t h i n days of a d i e t change b u t c o u l d n o t be c o r r e l a t e d t o changes i n c i r c u l a t i n g l e v e l s o f CCK, s e c r e t i n , PP, o r s o m a t o s t a t i n , s t r o n g l y i m p l y i n g a p h y s i o l o g i c a l r o l e f o r i n s u l i n . S t u d i e s i n d i a b e t i c a n i m a l s by S o l i n g e t a l (1971) and o t h e r s ( K o r c 1981a) have c o n f i r m e d t h a t i n s u l i n r e g u l a t e s amylase s y n t h e s i s a t t h e l e v e l o f t r a n s c r i p t i o n , i n c r e a s i n g amylase s y n t h e s i s w i t h i n 2 hours o f i n s u l i n t r e a t m e n t i n d i a b e t i c r a t s and i n f l u e n c i n g mRNA t r a n s c r i p t i o n f o r o t h e r enzymes as w e l l (StSckmann 1980). However, S o l i n g e t a l (1971) c o n c l u d e d from p a r a l l e l in v i t r o s t u d i e s t h a t i n s u l i n has no a c u t e r e g u l a t o r y e f f e c t s on e x o c r i n e s e c r e t i o n . T h i s c o n c l u s i o n has been c h a l l e n g e d by s e v e r a l more r e c e n t in vivo s t u d i e s . C o u t u r e e t a l (1972) showed t h a t i n s u l i n t r e a t m e n t i n t h e i n t a c t , a n e s t h e t i z e d r a t c a u sed an immediate i n c r e a s e i n p a n c r e a t i c amylase s e c r e t i o n and t o t a l a c i n a r p r o t e i n s y n t h e s i s , b u t s t u d i e s by t h e same i n v e s t i g a t o r s f a i l e d t o show any d i r e c t , in v i t r o e f f e c t s o f i n s u l i n on t h e p a n c r e a s . They c o n c l u d e d t h a t i n s u l i n must a c t i n d i r e c t l y , perhaps t h r o u g h v a g a l a f f e r e n t s . Other s t u d i e s c o n d u c t e d i n a s i m i l a r f a s h i o n i n t h e in vivo a n e s t h e t i z e d r a t i n d i c a t e d t h a t i n s u l i n i n j e c t e d s e l e c t i v e l y i n t o t h e c e l i a c a r t e r y -t h u s r e a c h i n g t h e p a n c r e a s a t r e l a t i v e l y h i g h c o n c e n t r a t i o n -s t i m u l a t e d e x o c r i n e s e c r e t i o n , b u t o n l y by p o t e n t i a t i n g t h e 322 a c t i o n s o f o t h e r s e c r e t a g o g u e s such as CCK (Tseng 1984). I n one o f t h e more r e c e n t r e p o r t s r e g a r d i n g i n s u l i n e f f e c t s on e x o c r i n e s e c r e t i o n , Lee e t a l (1990) found t h a t r a b b i t a n t i -i n s u l i n a n t i s e r u m a c u t e l y i n h i b i t e d meal, s e c r e t i n , and CCK-s t i m u l a t e d p a n c r e a t i c s e c r e t i o n i n t h e c o n s c i o u s r a t , from w h i c h t h e a u t h o r s c o n c l u d e d t h a t i n s u l i n has an i m p o r t a n t p e r m i s s i v e r o l e i n a l l a s p e c t s o f n o r m a l , s t i m u l a t e d s e c r e t i o n . Most s t u d i e s from t h e i n t a c t c o n s c i o u s a n i m a l t o p u r i f i e d a c i n a r c e l l s i n d i c a t e t h a t i n s u l i n by i t s e l f i s n o t a c t i n g s i m p l y as a s e c r e t a g o g u e . One p o s s i b l e e x p l a n a t i o n f o r t h e i n s u l i n e f f e c t i s a r a p i d s t i m u l a t i o n o f zymogen p r o t e i n s y n t h e s i s , perhaps by i n c r e a s e d i n t r a c e l l u l a r t r a n s p o r t o f s u b s t r a t e . But t h e c o n d i t i o n s under w h i c h i n s u l i n i n f l u e n c e s amino a c i d t r a n s p o r t a l s o remain c o n t r o v e r s i a l . Some groups have r e p o r t e d i n c r e a s e d n e u t r a l amino a c i d t r a n s p o r t a t t h e b a s o l a t e r a l membrane a f t e r i n s u l i n t r e a t m e n t (Mann 1985) w h i l e o t h e r s have found no e f f e c t ( B i e g e r 1979). Thus a l t h o u g h i n s u l i n seems t o e x e r t a c u t e e f f e c t s on e x o c r i n e s e c r e t i o n i n t h e in vivo p a n c r e a s , whether t h i s i s a d i r e c t (as i m p l i e d by t h e i n s u l o a c i n a r h y p o t h e s i s ) o r i n d i r e c t e f f e c t on t h e a c i n a r c e l l and by what c e l l u l a r mechanism s e c r e t i o n i s p o t e n t i a t e d remains c o n t r o v e r s i a l . One purpose o f t h i s s t u d y was t o d e v e l o p and c h a r a c t e r i z e in v i t r o model systems t h a t might be used t o f u r t h e r i n v e s t i g a t e t h e d i r e c t , s h o r t - t e r m r e g u l a t o r y f u n c t i o n s o f i n s u l i n on s e c r e t i o n o f 323 t h e e x o c r i n e p a n c r e a s . S e v e r a l r e v i e w s o f t h e p h y s i o l o g i c a l s i g n i f i c a n c e o f t h e i n s u l o a c i n a r a x i s have been p u b l i s h e d ( S c h a p i r o 1981, Henderson 1981, and W i l l i a m s 1985). Model Systems Two in v i t r o model systems f o r t h e s t u d y o f e x o c r i n e p a n c r e a t i c s e c r e t i o n were d e v e l o p e d and c h a r a c t e r i z e d f o r e x o c r i n e r e s p o n s i v e n e s s i n t h e r a t , t h e i s o l a t e d p e r f u s e d r a t p a n c r e a s and a p r e p a r a t i o n o f d i s p e r s e d p a n c r e a t i c a c i n i . These two models p r o v i d e d t h e means f o r a s s e s s i n g d i r e c t e f f e c t s o f i s l e t hormones on e x o c r i n e f u n c t i o n b u t each had some i n t r i n s i c l i m i t a t i o n s . Use o f t h e i s o l a t e d p e r f u s e d p a n c r e a s system a l l o w e d c o m p l e t e c o n t r o l o f g l u c o s e , hormone, and s e c r e t a g o g u e c o n c e n t r a t i o n s r e a c h i n g t h e p a n c r e a s so t h a t t h e s e p a r a m e t e r s were n o t t h e r e l a t i v e l y unknown, d y n a m i c a l l y v a r y i n g q u a n t i t i e s p r e s e n t i n t h e p r e v i o u s l y d i s c u s s e d in vivo s t u d i e s . A l t h o u g h s e v e r e d from c e n t r a l autonomic c o n t r o l , i n t r i n s i c r e f l e x ( e n t e r o p a n c r e a t i i c ) pathways o f t h e autonomic nervous system ( H o i s t 1980), t h e r e t a i n e d i s l e t s o f Langerhans, p a n c r e a t i c d u c t s c a p a b l e o f m o d i f y i n g a c i n a r s e c r e t i o n , and i n j u r y due t o t h e i s o l a t i o n p r o c e d u r e - were a l l p o t e n t i a l i n f l u e n c e s on t h e r e s p o n s e o f t h e " i s o l a t e d " p a n c r e a s . " D i r e c t " e f f e c t s o f s e c r e t a g o g u e s , i n s u l i n , o r o t h e r hormones when s t u d i e d t h r o u g h use o f an i s o l a t e d p e r f u s e d p a n c r e a s model had t o be i n t e r p r e t e d i n l i g h t o f t h e s e o t h e r i n f l u e n c e s . 324 I n c o n t r a s t , t h e d i s p e r s e d a c i n i p r e p a r a t i o n a l s o a l l o w e d c o m plete c o n t r o l o f s t i m u l a t o r y p arameters but a t t h e c o s t o f a more s t r i n g e n t i s o l a t i o n p r o c e d u r e . A c i n i r e t a i n e d c e l l u l a r o r i e n t a t i o n a f t e r t h e p r o c e d u r e and were more r e s p o n s i v e t h a n t h e even more s t r i n g e n t l y t r e a t e d , i s o l a t e d a c i n a r c e l l s ( W i l l i a m s 1978). Other t h a n changes i n c e l l u l a r r e s p o n s i v e n e s s from t h e d i s p e r s i o n p r o c e s s , t h e o n l y o t h e r p o t e n t i a l s o u r c e of s y s t e m a t i c i n t e r f e r e n c e a f f e c t i n g a c i n i was t h e p r e s e n c e o f i s l e t f r agments t h a t remained d e s p i t e t h e p u r i f i c a t i o n p r o c e d u r e . A s u b t l e i n f l u e n c e from t h e s e f r a g m e n t s was an o n g o i n g c o n c e r n and prompted s p e c i f i c i n v e s t i g a t i o n o f t h e i r e f f e c t s . N e v e r t h e l e s s , t h e p r e p a r a t i o n o f d i s p e r s e d a c i n i p r o v i d e d t h e most a c c u r a t e measure of a c i n a r c e l l r e s p o n s e s t o t h e v a r i o u s t r e a t m e n t s employed. Exocrine and endocrine function in the isolated, perfused pancreas I n t h i s s t u d y , t h e i s o l a t e d p e r f u s e d p a n c r e a s responded t o e x o c r i n e s e c r e t a g o g u e s by s e c r e t i n g i n c r e a s e d p a n c r e a t i c j u i c e , amylase, and p r o t e i n . CCK was, on a m o l a r b a s i s , t h e most p o w e r f u l s e c r e t o r y s t i m u l u s f o r a l l t h r e e measured parameters and w h i l e b o t h CCK and VIP showed s e c r e t o r y r e s p o n s e s t h a t were dose-dependent, s e c r e t i n d i d n o t i n t h e dose range employed. S t a b l e b a s e l i n e c o n t r o l groups f o r each s e c r e t a g o g u e , and t h e r e t u r n t o b a s e l i n e a f t e r t h e s t i m u l a t i o n p e r i o d , c o n f i r m e d t h e i n t e g r i t y and u t i l i t y o f t h e model as s u g g e s t e d by r e p o r t s from o t h e r i n v e s t i g a t o r s . 3 2 5 Kanno and S a i t o (1980) w o r k i n g w i t h t h e i s o l a t e d k i t t e n p a n c r e a s showed t h a t CCK i n d u c e d a dose-dependent enzyme s e c r e t i o n b u t l i t t l e enzyme was s e c r e t e d i n r e s p o n s e t o VIP o r s e c r e t i n . The l a t t e r two s e c r e t a g o g u e s , i n f u s e d a t a c o n c e n t r a t i o n a p p r o x i m a t i n g t h a t found i n t h e p o r t a l v e i n , d i d however cause a dose-dependent i n c r e a s e i n s e c r e t i o n o f b i c a r b o n a t e and w a t e r . I s o l a t e d p a n c r e a t i c p e r f u s i o n w i t h VIP o r s e c r e t i n i n t h e p i g a l s o r e s u l t e d i n dose-dependent e x o c r i n e s e c r e t i o n b u t t h e s e e f f e c t s were l a r g e l y volume and b i c a r b o n a t e s i n c e p r o t e i n c o n c e n t r a t i o n i n t h e e f f l u e n t a c t u a l l y d e c r e a s e d a t h i g h e r s e c r e t a g o g u e doses ( L i n d k a e r J e n s e n 1978a and b ) . The r e p o r t most comparable t o t h e p r e s e n t s t u d y has come from O t s u k i e t a l (1981, 1986) who c a r r i e d o u t a s e r i e s o f i s o l a t e d p a n c r e a s p e r f u s i o n s i n t h e r a t . As i n t h e p r e s e n t s t u d y , O t s u k i r e p o r t e d b o t h i n c r e a s e d f l o w and enzyme s e c r e t i o n i n r e s p o n s e t o s e c r e t i n i n t h e r a t . I n c o n t r a s t t o t h e p r e s e n t s t u d y however, dose-dependent s e c r e t i n s e c r e t i o n was e v i d e n t w i t h a maximal r e s p o n s e a t 1 /xg/ml (327 nM) , s u g g e s t i n g t h a t t h e s e c r e t o r y ranges t e s t e d i n t h e p r e s e n t s t u d y may have s i m p l y been i n a d e q u a t e t o show a d o s e - r e s p o n s e . The e f f e c t s o f CCK-8 on e x o c r i n e f u n c t i o n i n t h e p e r f u s e d r a t pan c r e a s were a l s o s t u d i e d by O t s u k i (1986) w i t h f i n d i n g s t h a t c o n f i r m t h e dose-dependency and e f f e c t i v e s e c r e t o r y range r e p o r t e d h e r e . E n d o c r i n e hormone r e l e a s e from t h e p e r f u s e d r a t p a n c r e a s i n re s p o n s e t o e x o c r i n e s e c r e t a g o g u e s was a l s o a s s e s s e d i n t h e 3 2 6 p r e s e n t s t u d y . I n s u l i n r e l e a s e was m o n i t o r e d i n p e r f u s a t e t o q u a n t i f y endogenous o r e x o g e n o u s l y d e l i v e r e d i n s u l i n a v a i l a b l e t o i n f l u e n c e s e c r e t i o n , v e r i f y i n g t h e p r e s e n c e o f i n s u l i n under t h e v a r i o u s i n f u s i o n o r s t i m u l a t o r y c o n d i t i o n s u t i l i z e d . S o m a t o s t a t i n was a l s o a s s a y e d i n p e r f u s a t e because o f i t s s i m i l a r s t i m u l u s - r e l e a s e p a t t e r n w i t h i n s u l i n , and because o f i n c r e a s i n g e v i d e n c e t h a t s o m a t o s t a t i n may i n f l u e n c e b o t h e n d o c r i n e ( i n s u l i n r e l e a s e (Marco 1983)) and e x o c r i n e f u n c t i o n l o c a l l y . R e c e p t o r s f o r s o m a t o s t a t i n have been found on a c i n a r c e l l s ( E s t e v e 1984) and t h e hormone has been shown t o s u p p r e s s b o t h e x o c r i n e s e c r e t i o n (Boden 1975) and t h e n u t r i e n t r e l e a s e o f s e c r e t a g o g u e s ( s e c r e t i n and CCK, S c h l e g e l ( 1 9 7 7 ) ) , s u g g e s t i n g t h a t s o m a t o s t a t i n might a l s o be an i m p o r t a n t component o f t h e i n s u l o a c i n a r a x i s . I n a c o m p a r i s o n o f a s s a y methods, t h e p a t t e r n o f i s l e t hormone r e l e a s e f o r i n s u l i n and s o m a t o s t a t i n was r e l i a b l y e x p r e s s e d as t h e t o t a l hormone o u t p u t o v e r t i m e . VIP s t i m u l a t e d i n s u l i n r e l e a s e w h i l e CCK i n d u c e d s o m a t o s t a t i n r e l e a s e i n t o p e r f u s a t e , b u t t h e l a t t e r o n l y a t h i g h e r dose r a n g e s . S e c r e t i n had no e f f e c t on e i t h e r i n s u l i n o r s o m a t o s t a t i n r e l e a s e . The g l u c o s e dependence o f t h e s e e f f e c t s has been emphasized by o t h e r s ( L i n d k a e r J e n s e n 1978, O t s u k i 1981) b u t was n o t a s s e s s e d i n t h e c u r r e n t i n v e s t i g a t i o n . The low g l u c o s e c o n c e n t r a t i o n (4.5 mM) used t h r o u g h o u t t o m i n i m i z e endogenous i n s u l i n e f f e c t s l i k e l y a c c o u n t s f o r b o t h t h e s m a l l e f f e c t o f VIP on i n s u l i n r e l e a s e , 327 and t h e absence o f an e x p e c t e d CCK-induced i n s u l i n s e c r e t i o n ( O t s u k i 1986). Indeed, i n p e r f u s i o n e x p e r i m e n t s where g l u c o s e was used t o s t i m u l a t e endogenous i n s u l i n , t h e magnitude of t h e i n s u l i n r e s p o n s e d u r i n g CCK s t i m u l a t i o n ( F i g u r e s 18-23) was much g r e a t e r t h a n t h a t seen d u r i n g VIP ( F i g u r e s 38-42) o r s e c r e t i n ( F i g u r e s 28-32) s t i m u l a t i o n , v e r i f y i n g t h e i n c r e t i n a c t i o n s o f CCK i n t h i s p r e p a r a t i o n (Sakamoto 1982). The i n t e g r i t y o f e x o c r i n e and e n d o c r i n e components i n t h e p e r f u s e d p a n c r e a s model was f u r t h e r t e s t e d i n e x p e r i m e n t s u t i l i z i n g an a n o x i c s t r e s s . A p e r i o d o f a n o x i a ( N 2 - C 0 2 e q u i l i b r a t e d p e r f u s a t e ) m a r k e d l y d e p r e s s e d p a n c r e a t i c j u i c e volume, p r o t e i n , and amylase s e c r e t i o n w i t h an e f f e c t t h a t was p e r s i s t e n t t h r o u g h t o t h e end o f t h e e x p e r i m e n t . Other w o r k e r s have a l s o n o t e d t h e dependence of e x o c r i n e f u n c t i o n on oxygen ( H o i s t 1980), showing t h a t a c i n a r c e l l s e c r e t i o n ( H a r p e r 1986) and c e l l u l a r h y p e r p o l a r i z a i i o n due t o CCK a r e c o m p l e t e l y dependent on o x i d a t i v e m e t a b o l i s m (Kanno 1976a). As p r e v i o u s l y n o t e d , B - c e l l b a s a l s e c r e t i o n was m i n i m a l and d i d n o t i n c r e a s e w i t h a n o x i a . N a r i m i y a e t a l (1982) s t u d i e d i n s u l i n s e c r e t i o n d u r i n g h y p o x i a i n t h e p e r f u s e d r a t p a n c r e a s and showed t h a t i t d e p l e t e d h i g h energy s u b s t r a t e s (ATP) and r e d u c e d i n s u l i n r e l e a s e , a r e s p o n s e n o t d e t e c t e d i n t h i s s t u d y perhaps due t o t h e low b a s a l s e c r e t i o n r a t e . N e i t h e r h i g h g l u c o s e n o r i n s u l i n i n t h e p r e s e n t s t u d y a l t e r e d t h e e f f e c t s o f a n o x i a . 328 I n c o n t r a s t t o t h e l a c k o f an i n s u l i n r e s p o n s e , s o m a t o s t a t i n r e l e a s e was m a r k e d l y i n c r e a s e d by t h e a n o x i c s t r e s s i n a r e s p o n s e t h a t c o u l d have been due t o e i t h e r d i s i n h i b i t i o n of s o m a t o s t a t i n r e l e a s e , a c t u a l s t i m u l a t i o n o f s e c r e t i o n , o r c e l l u l a r damage w i t h l e a k a g e from D c e l l s . L a u t t e t a l (1982) r e p o r t e d s i m i l a r f i n d i n g s o f d e p r e s s e d i n s u l i n and g l u c a g o n s e c r e t i o n b u t markedly i n c r e a s e s o m a t o s t a t i n r e l e a s e d u r i n g h e m o r r h a g i c shock i n c a t s . They h y p o t h e s i z e d t h a t s o m a t o s t a t i n may have been a c t i n g i n a p a r a c r i n e manner t o i n h i b i t A- and B - c e l l f u n c t i o n , s u g g e s t i n g t h a t t h e s o m a t o s t a t i n r e s p o n s e was r e g u l a t e d r a t h e r t h a n due t o D c e l l i n j u r y . C e r t a i n l y , t h e e f f e c t s o f g l u c o s e and i n s u l i n i n s u p p r e s s i n g s o m a t o s t a t i n r e l e a s e d u r i n g a n o x i a would s u g g e s t t h a t s o m a t o s t a t i n i s r e l e a s e d as p a r t o f a r e g u l a t e d r e s p o n s e . F u r t h e r , s o m a t o s t a t i n might have had i n h i b i t o r y a c t i o n s w i t h i n t h e i s l e t o r c o n t r i b u t e d t o t h e s u p p r e s s i o n o f e x o c r i n e s e c r e t i o n o b s e r v e d . These s t u d i e s o f a n o x i c s t r e s s s e r v e d t o emphasize t h e f r a g i l e i n t e g r i t y o f t h e i s o l a t e d p a n c r e a s p r e p a r a t i o n and i t s dependence on adequate p e r f u s i o n s u b s t r a t e s . F u r t h e r s t u d y o f t h e c a u s e s and e f f e c t s o f h y p o x i c s o m a t o s t a t i n r e l e a s e a l t h o u g h n o t c a r r i e d out i n t h i s s t u d y might be o f c l i n i c a l i m p o r t a n c e p a r t i c u l a r l y i n an e r a o f p a n c r e a t i c t r a n s p l a n t a t i o n w i t h p r o l o n g e d h y p o x i c p e r i o d s (Limmer 1990). 329 Pancreatic acini preparation P a n c r e a t i c a c i n i w e r e p r e p a r e d a s d e s c r i b e d v a r i o u s l y b y W i l l i a m s ( 1 9 7 8 ) a n d L i d d l e ( 1 9 8 4 ) , a n d d e m o n s t r a t e d r e s p o n s i v e n e s s t o e x o c r i n e s e c r e t a g o g u e s c o m p a r a b l e t o t h a t d e s c r i b e d i n t h e c i t e d s t u d i e s . C a l c i u m - m e d i a t e d s e c r e t a g o g u e s ( v i d e s u p r a ) : C C K , c a e r u l e i n , a n d m e t h a c h o l i n e a l l s h o w e d t h e c h a r a c t e r i s t i c s u p r a m a x i m a l i n h i b i t i o n o f a m y l a s e r e l e a s e t h a t h a s b e e n d e s c r i b e d i n d i s p e r s e d a c i n i b y o t h e r s . I n c o n t r a s t , n o m a x i m a l s t i m u l a t o r y d o s e f o r s e c r e t i n o r V I P w a s a c h i e v e d . P o s s i b l y b e c a u s e o f t h e p h e n o m e n o n o f s u p r a m a x i m a l i n h i b i t i o n , b o t h s e c r e t i n a n d V I P a p p e a r e d t o s t i m u l a t e m o r e a m y l a s e r e l e a s e a t h i g h d o s e r a n g e s t h a n a n y c o n c e n t r a t i o n o f t h e c a l c i u m - m e d i a t e d s e c r e t a g o g u e s , a f i n d i n g i n s h a r p c o n t r a s t t o t h e m o r e m o d e r a t e e f f i c a c y o f t h e s a m e t w o s e c r e t a g o g u e s i n t h e i s o l a t e d p e r f u s e d p a n c r e a s . I n d e e d , t h e e f f e c t o f t h e s e s e c r e t a g o g u e s m a y b e s p e c i e s - s p e c i f i c s i n c e i s o l a t e d a c i n i f r o m t h e m o u s e h a v e b e e n r e p o r t e d t o s h o w n e g l i g i b l e s e c r e t o r y r e s p o n s e s t o V I P a n d s e c r e t i n a t d o s e s u p t o 1 0 0 -1 0 0 0 n M ( B u r n h a m 1 9 8 3 ) . A n a l t e r n a t i v e e x p l a n a t i o n f o r t h i s f i n d i n g i s s u g g e s t e d b y r e p o r t s t h a t s e c r e t i n a c t s s y n e r g i s t i c a l l y w i t h C C K , p o t e n t i a t i n g s e c r e t i o n b e y o n d a n y s i m p l e a d d i t i v e e f f e c t ( L e e 1 9 7 9 ) . T h e m a g n i t u d e o f t h e s e c r e t i n a n d V I P r e s p o n s e s f o u n d i n t h i s s t u d y m a y b e d u e t o p o s t r e c e p t o r m o d u l a t i o n o f c A M P - m e d i a t e d ( O l i n g e r 1 9 7 9 ) s e c r e t a g o g u e e f f e c t s b y i n c r e a s e d i n t r a c e l l u l a r c a l c i u m ( C o l l e n 1 9 8 2 ) . T h e f a c t t h a t a c i n i r e t a i n e d s u r f a c e i n s u l i n 330 r e c e p t o r s , even a f t e r s t r i n g e n t p u r i f i c a t i o n ( W i l l i a m s 1981) and were r e s p o n s i v e t o e x o c r i n e s e c r e t a g o g u e s a t doses c o n s i d e r e d t o be p h y s i o l o g i c a l i n d i c a t e d t h a t d i s p e r s e d a c i n i m i ght p r o v i d e a v e r s a t i l e model t o i n v e s t i g a t e s e c r e t a g o g u e dependency o f i n s u l i n p o t e n t i a t i o n . As mentioned e a r l i e r , t h e p r e s e n c e o f i s l e t f r a g m e n t s w i t h i n s u s p e n s i o n s o f " p u r i f i e d " a c i n i was o f c o n c e r n as a s o u r c e o f i s l e t hormones t h a t might i n t e r f e r e w i t h s t u d i e s on i s l e t hormone e f f e c t s . A l t h o u g h i n s u l i n was p r e s e n t i n t h e medium o f t h e p u r i f i e d , d i s p e r s e d a c i n i , o n l y about 10 t o 30% o f t h e t o t a l p a n c r e a t i c i n s u l i n was p r e s e n t a t t h e end o f t h e p u r i f i c a t i o n p r o c e d u r e . No s o m a t o s t a t i n was measurable and e f f o r t s a t s t i m u l a t i n g i n s u l i n s e c r e t i o n from i s l e t f ragments by g l u c o s e o r i n c r e t i n s (GIP and CCK) were u n s u c c e s s f u l . The s i g n i f i c a n c e o f i s l e t f r agments i n t h e d i s p e r s e d a c i n i p r e p a r a t i o n has been s t u d i e d by o t h e r s u s i n g a d i f f e r e n t , a c i d - a l c o h o l t e c h n i q u e o f e x t r a c t i o n . T h e i r f i n d i n g s were s i m i l a r t o t h e p r e s e n t s t u d y , showing an 8 - f o l d e n r i c h m e n t o f a c i n i by p u r i f i c a t i o n t h r o u g h d e n s i t y c e n t r i f u g a t i o n (Mossner 1984). The same group a l s o found t h a t removing g l u c o s e from t h e medium o r t r e a t m e n t w i t h x y l a z i n e t o i n h i b i t B - c e l l i n s u l i n r e l e a s e was w i t h o u t e f f e c t on i n s u l i n l e v e l s , i m p l y i n g t h a t l e a k a g e r a t h e r t h a n s e c r e t i o n was r e s p o n s i b l e f o r t h e f r e e i n s u l i n p r e s e n t i n t h e medium. I n t h e same r e p o r t , i n s u l i n r e c e p t o r b i n d i n g s t u d i e s i n normal and d i a b e t i c a c i n i showed t h a t i n s u l i n might d o w n r e g u l a t e i n s u l i n 331 r e c e p t o r d e n s i t y , an i m p o r t a n t c o n s i d e r a t i o n i n t h e h i g h ambient i n s u l i n environment p r o v i d e d by t h e i n s u l o a c i n a r a x i s and a n o t h e r r e a s o n t h a t exogenous i n s u l i n m i g h t be i n e f f e c t i v e i n p o t e n t i a t i n g s e c r e t i o n from s u s p e n s i o n s o f d i s p e r s e d a c i n i . T h i s c o n c e r n , and t h e marked d e c r e a s e i n c o n t a m i n a t i n g i n s u l i n found i n a c i n i p r e p a r e d from d i a b e t i c a n i m a l s , p r o v i d e d a r a t i o n a l e f o r s t u d y i n g d i a b e t i c a c i n i i s o l a t e d e a r l y a f t e r t h e o n s e t o f d i a b e t e s , b e f o r e m a s s i v e amylase d e p l e t i o n had o c c u r r e d . A l t h o u g h n e u t r a l t h i o l p r o t e a s e s (an i n s u l i n d e g r a d i n g enzyme p r e s e n t on a c i n i ( G o l d f i n e 1984)) were a l s o o f c o n c e r n i n t h e d i s p e r s e d a c i n i p r e p a r a t i o n , t h e r e was e x c e l l e n t p e r s i s t e n c e and r e c o v e r y o f exogenous im m u n o r e a c t i v e i n s u l i n i n b o t h t h e p r e s e n t s t u d y and a n o t h e r r e p o r t (Sankaran 1981) even a f t e r l o n g i n c u b a t i o n . I n s u l i n added t o p r e p a r a t i o n s o f a c i n i was t h e r e f o r e p r e s e n t f o r p r o l o n g e d p e r i o d s and a v a i l a b l e t o a c t on t h e a c i n i . j D i s t r i b u t i o n o f endogenous i n s u l i n t o a c i n a r t i s s u e ;  a u t o r a d i o g r a p h i c s t u d y F o r i n s u l i n t o e x e r t an e f f e c t on t h e e x o c r i n e p a n c r e a s , i t must r e a c h t h e a c i n a r c e l l . C u r r e n t e v i d e n c e s u g g e s t s t h a t i n s u l i n - and l i k e l y t h o s e components of t h e e x o c r i n e p a n c r e a s r e s p o n s i v e t o i n s u l i n - a r e n o t d i s t r i b u t e d u n i f o r m l y t h r o u g h o u t t h e g l a n d . Henderson (1979) and o t h e r s (Bonner Weir 1982) have d e s c r i b e d a network o f p e r i - i s l e t c a p i l l a r i e s t h a t v a r i a b l y , d epending on t h e s i z e o f t h e i s l e t , form p o r t a l c o n n e c t i o n s t o t h e s u r r o u n d i n g a c i n a r t i s s u e . A c i n a r 3 3 2 c e l l s i n p r o x i m i t y t o i s l e t s have a g r e a t e r n u c l e a r s i z e and n u c l e o l a r volume (Hellman 1962), i n c o r p o r a t e more [ 3 H ] l e u c i n e t h a n s u r r o u n d i n g a c i n i (Kramer 1968), and c o n t a i n a d i s t i n c t i v e p a t t e r n o f h y d r o l a s e enzymes ( M a l a i s s e - L a g a e 1975) t h a t has been a t t r i b u t e d t o i s l e t hormonal i n f l u e n c e s . T h i s p a t t e r n o f t o p o g r a p h i c a l p a r t i t i o n f o r h y d r o l a s e a c t i v i t y has a l s o been c o n f i r m e d i m m u n o h i s t o c h e m i c a l l y (Bendayan 1987). D e s p i t e t h e s e f i n d i n g s , no d i r e c t e v i d e n c e t h a t i n s u l i n r e a c h e s t h e p e r i - i s l e t e x o c r i n e p a n c r e a s has been p r o v i d e d i n t h e l i t e r a t u r e . To a d d r e s s t h i s q u e s t i o n , an a u t o r a d i o g r a p h i c s t u d y i n t h e p e r f u s e d r a t p a n c r e a s was c a r r i e d o u t . As shown i n F i g u r e 127, s t i m u l a t i o n o f endogenous i n s u l i n r e l e a s e by g l u c o s e and a r g i n i n e appeared t o s i g n i f i c a n t l y d i s p l a c e 1 2 5 I - i n s u l i n from a c i n a r c e l l s w i t h i n a 4 a c i n i p e r i m e t e r o f t h e i s l e t . A l t h o u g h o n l y c a r r i e d o u t i n a s m a l l number o f a n i m a l s , t h i s s t u d y seemed t o c o r r e l a t e w i t h t h e " h a l o " v i s i b l e i n F i g u r e s 117 and 118 and r e p o r t e d by M a l a i s s e - L a g a e ( 1 9 7 6 ) , s u g g e s t i n g t h a t endogenous i n s u l i n r e a c h e s t h e e x o c r i n e p a n c r e a s b u t i n a n o n u n i f o r m d i s t r i b u t i o n . P r o x i m i t y o f t h e e x o c r i n e t o e n d o c r i n e p o r t i o n s o f t h e p a n c r e a s , i n a d d i t i o n t o i s l e t s i z e and s e c r e t i o n , a r e p r o b a b l y i m p o r t a n t p a r a m e t e r s o f t h e i n s u l o a c i n a r a x i s . 333 A c t i o n s o f i n s u l i n A c u t e e f f e c t s of i n s u l i n I n t h e p r e s e n t s t u d y , a c u t e e f f e c t s o f i n s u l i n were a s s e s s e d i n b o t h t h e i s o l a t e d , p e r f u s e d p a n c r e a s and i n a c i n i under a v a r i e t y o f e x o c r i n e s e c r e t o r y s t i m u l i . Endogenous, g l u c o s e -s t i m u l a t e d i n s u l i n and exogenous r a t i n s u l i n p o t e n t i a t e d CCK-s t i m u l a t e d s e c r e t i o n b u t had a much more pr o m i n e n t e f f e c t d u r i n g s e c r e t i n - s t i m u l a t e d s e c r e t i o n . D e s p i t e t h e ap p a r e n t p o t e n t i a t i o n o f V I P - s t i m u l a t e d s e c r e t i o n by endogenous, g l u c o s e - s t i m u l a t e d s e c r e t i o n i n t h e p e r f u s e d r a t model, exogenous i n s u l i n f a i l e d t o p o t e n t i a t e and i n d e e d , may even have had an i n h i b i t o r y i n f l u e n c e on V I P - s t i m u l a t e d s e c r e t i o n . D i f f e r e n c e s between t h e c o n t r o l and t e s t group V I P r e s p o n s e s o f t h e s e groups make e x a c t i n t e r p r e t a t i o n d i f f i c u l t , b u t c l e a r l y t h e r e were s i g n i f i c a n t d i f f e r e n c e s i n i n s u l i n p o t e n t i a t i o n d u r i n g s e c r e t i n - and V I P - s t i m u l a t e d s e c r e t i o n . Exogenous i n s u l i n doses t e s t e d were based on p r i o r s t u d i e s of b a s a l and s t i m u l a t e d i n s u l i n c o n c e n t r a t i o n s i n t h e p o r t a l v e i n o f mammals (Kanazawa 1968). I t seemed u n l i k e l y t h a t any of t h e endogenous i n s u l i n e f f e c t s c o u l d be a s c r i b e d t o g l u c o s e a l o n e , s i n c e g l u c o s e f a i l e d t o i n f l u e n c e a c i n a r f u n c t i o n i n t h e p r e v i o u s l y d i s c u s s e d s t u d y o f i s l e t fragment e f f e c t s from p r e p a r a t i o n s o f d i s p e r s e d a c i n i . The i s o l a t e d p e r f u s e d p a n c r e a s has been used by o t h e r i n v e s t i g a t o r s t o examine e f f e c t s o f i n s u l i n on e x o c r i n e f u n c t i o n . S a i t o e t a l (1980) showed t h a t s t i m u l a t i n g endogenous i n s u l i n o r i n f u s i n g i n s u l i n i n t h e C C K - s t i m u l a t e d , 334 p e r f u s e d r a t p a n c r e a s p o t e n t i a t e d e x o c r i n e s e c r e t i o n . Of i n t e r e s t , t h e y c o n t r o l l e d f o r d i r e c t g l u c o s e e f f e c t s by showing t h a t o t h e r s u g a r s , x y l o s e and g a l a c t o s e , f a i l e d t o s t i m u l a t e i n s u l i n r e l e a s e o r p o t e n t i a t e enzyme s e c r e t i o n . T r i m b l e e t a l (1985) found t h a t t r e a t m e n t s t o r e duce endogenous i n s u l i n w i t h e i t h e r a n t i i n s u l i n a n t i s e r u m o r u s i n g i n s u l i n - t r e a t e d d i a b e t i c r a t s ( t o n o r m a l i z e e x o c r i n e c o n t e n t ) , r e s u l t e d i n r e d u c e d c a e r u l e i n - and V I P - s t i m u l a t e d enzyme r e l e a s e . The f a c t t h a t i n t h i s s t u d y i n s u l i n seemed t o c o n t r i b u t e t o s e c r e t i o n s t i m u l a t e d by b o t h c a l c i u m and a t l e a s t one cAMP-mediated s e c r e t a g o g u e i m p l i e d t h a t i t ' s p o t e n t i a t i n g a c t i o n was e i t h e r i n d i r e c t , o r a d i r e c t e f f e c t b u t v i a a second messenger o t h e r t h a n c a l c i u m o r cAMP. However a n o t h e r s t u d y a l s o by T r i m b l e ' s group i n t h e i s o l a t e d p e r f u s e d p a n c r e a s o f normal r a t s showed t h a t endogenous and exogenous i n s u l i n c o u l d a l s o i n h i b i t b o t h b a s a l and c a e r u l e i n - s t i m u l a t e d e x o c r i n e s e c r e t i o n i n a g l u c o s e -dependent manner (Bruzzone 1984), though t h e r e s u l t s were not c o m p l e t e l y c o n s i s t e n t w i t h i n t h a t s t u d y . C o n t r a d i c t o r y r e s u l t s as t o t h e e f f e c t o f i n s u l i n on e x o c r i n e s e c r e t i o n have a l s o been r e p o r t e d i n o t h e r whole organ p a n c r e a s p r e p a r a t i o n s ( D a n i e l s s o n 1974). Perhaps t h e o n l y c o n c l u s i o n s t h a t can be drawn from t h e s e d a t a a r e t h a t i n s u l i n a f f e c t s s e c r e t i o n from t h e i n t a c t p a n c r e a s but i n an i n c o n s i s t e n t , p o s s i b l y , i n d i r e c t manner, and t h a t s p e c i f i c p a rameters d e t e r m i n i n g whether i n s u l i n w i l l s t i m u l a t e , i n h i b i t , o r have no e f f e c t on s e c r e t i o n remain u n c l e a r . Data from t h e p r e s e n t 335 s t u d y a l s o showed t h a t i n t h e p e r f u s e d p a n c r e a s , i n s u l i n p o t e n t i a t e d s t i m u l a t e d s e c r e t i o n i n a v a r i a b l e manner. For CCK and s e c r e t i n as p r i n c i p l e s e c r e t a g o g u e s o f t h e mammalian p a n c r e a s , a s m a l l b u t r e p r o d u c i b l e p o t e n t i a t i o n o f s t i m u l a t e d s e c r e t i o n was e v i d e n t . I n t e r e s t i n g l y , t h e s e c r e t o r y e f f e c t s o f V I P - a hormone w i t h a l e s s c l e a r l y d e f i n e d r o l e i n a c i n a r s e c r e t i o n - were n o t p o t e n t i a t e d by i n s u l i n , r e i n f o r c i n g t h e p o s s i b i l i t y o f an i n d i r e c t i n s u l i n e f f e c t . W i t h t h e s e f i n d i n g s , s t u d i e s were t h e r e f o r e c a r r i e d o ut i n d i s p e r s e d a c i n i t o d e t e r m i n e whether and under what c o n d i t i o n s i n s u l i n -p o t e n t i a t e d s e c r e t i o n might be a d i r e c t e f f e c t o f i n s u l i n on t h e a c i n a r c e l l . A c u t e t r e a t m e n t o f a c i n i w i t h i n s u l i n d u r i n g CCK s t i m u l a t i o n f a i l e d t o a l t e r amylase s e c r e t i o n i n one s e t o f e x p e r i m e n t s ( F i g u r e s 65 and 66) b u t d i d p o t e n t i a t e s e c r e t i o n i n a n o t h e r s t u d y where a n i m a l s were s u b j e c t e d t o t r e a t m e n t s a l t e r i n g c i r c u l a t i n g i n s u l i n c o n c e n t r a t i o n s ( F i g u r e 8 6 ) . When t h e d u r a t i o n o f exposure was p r o l o n g e d , i n s u l i n seemed t o i n h i b i t C C K - s t i m u l a t e d s e c r e t i o n from a c i n i b u t t h e magnitude o f i n h i b i t i o n v a r i e d from 20% ( F i g u r e 67) t o an i n s i g n i f i c a n t l e v e l o f 5% ( F i g u r e 6 9 ) . T h i s f i n d i n g was i n marked c o n t r a s t t o t h e p r i o r s t u d i e s by W i l l i a m s (1981) where i n s u l i n i n c r e a s e d C C K - s t i m u l a t e d amylase r e l e a s e , b u t c o r r e s p o n d e d t o t h e p e r f u s e d p a n c r e a s e f f e c t s o f i n h i b i t i o n r e p o r t e d by Bruzzone (1984). S e c r e t i n - and m e t h a c h o l i n e - s t i m u l a t e d s e c r e t i o n a t a l l doses were u n a f f e c t e d by i n s u l i n 336 p r e i n c u b a t i o n o r c o n c o m i t a n t i n s u l i n t r e a t m e n t d u r i n g t h e s e c r e t a g o g u e e x p o s u r e . Indeed, e x c e p t f o r t h e i n c o n s i s t e n t f i n d i n g s d u r i n g a c u t e i n s u l i n t r e a t m e n t o f C C K - s t i m u l a t e d s e c r e t i o n , t h e o n l y o t h e r s e c r e t a g o g u e dose o r c o m b i n a t i o n i n f l u e n c e d by i n s u l i n t r e a t m e n t i n normal r a t a c i n i was t h e c o m b i n a t i o n o f s e c r e t i n and CCK, an e f f e c t t h a t b a r e l y a c h i e v e d s i g n i f i c a n c e ( F i g u r e 7 6 ) . These d a t a seemed t o i n d i c a t e t h a t i n s u l i n e f f e c t s on e x o c r i n e f u n c t i o n were dependent on t h e s e c r e t a g o g u e employed and on t h e d u r a t i o n o f i n s u l i n e x p o s u r e , b u t t h a t because o f v a r i a b i l i t y i n t h e r e s p o n s e under s i m i l a r c o n d i t i o n s i n d i f f e r e n t e x p e r i m e n t s , f a c t o r s o t h e r t h a n t h o s e c o n t r o l l e d i n t h e s e e x p e r i m e n t s were i m p o r t a n t . The l a r g e s t r e p o r t e d e x p e r i e n c e from a s i n g l e group e x p l o r i n g d i r e c t e f f e c t s o f i n s u l i n on t h e a c i n a r c e l l o f t h e p a n c r e a s has come from W i l l i a m s and G o l d f i n e (1986). E a r l y i n t h e i r i n v e s t i g a t i o n s , t h e y r e p o r t e d t h a t i n s u l i n r e c e p t o r s on t h e i s o l a t e d mouse a c i n a r c e l l e x i s t e d i n two a f f i n i t y s t a t e s w i t h h a l f - m a x i m a l b i n d i n g a f t e r 2 min, and maximal b i n d i n g t h a t o c c u r r e d a f t e r 30 min ( K o r c 1978). Once bound, i n s u l i n was c a p a b l e o f a c u t e l y s t i m u l a t i n g b o t h g l u c o s e u p t a k e ( W i l l i a m s 1982) and p r o t e i n s y n t h e s i s (Mossner 1985) i n a c i n a r c e l l s . I n t h e s e s t u d i e s , maximal e f f e c t s o f i n s u l i n o c c u r r e d a t a c o n c e n t r a t i o n o f 100 nM (15,000 /iU/ml) t h a t m ight be p h y s i o l o g i c a l l y r e l e v a n t i n t h e u n i q u e m i c r o e n v i r o n m e n t o f t h e i n s u l o a c i n a r a x i s b u t m i g h t a l s o 337 i n d i c a t e some measure o f a c i n a r compromise. A l t h o u g h i n s u l i n had no d i r e c t e f f e c t on amylase s e c r e t i o n i n t h e i r e x p e r i m e n t s , W i l l i a m s and h i s c o w o r k e r s d i d d e m o n s t r a t e t h a t i n s u l i n p r e t r e a t m e n t o f a c i n i a t h i g h c o n c e n t r a t i o n (22,000 uU/ml) f o r 2 h s i g n i f i c a n t l y p o t e n t i a t e d C C K - s t i m u l a t e d a c i n a r s e c r e t i o n and c o n c o m i t a n t l y , s t i m u l a t e d i n t r a c e l l u l a r p r o t e i n p h o s p h o r y l a t i o n w i t h o u t m o b i l i z i n g C a + 2 ( W i l l i a m s 1981). I n a d d i t i o n t o m e t a b o l i c c e l l u l a r e f f e c t s , t h e y a l s o r e p o r t e d t h a t i n s u l i n seemed t o i n f l u e n c e r e c e p t o r f u n c t i o n f o r s e v e r a l d i f f e r e n t d i r e c t hormonal a g o n i s t s o f a c i n a r c e l l s e c r e t i o n . D i a b e t e s i n c r e a s e d CCK r e c e p t o r number ( O t s u k i 1983) b u t r e d u c e d CCK e f f i c a c y t h r o u g h a r e v e r s i b l e p o s t r e c e p t o r d e f e c t ( O t s u k i 1984) w h i l e i n s u l i n was shown t o d o w n r e g u l a t e t h e number o f c e l l s u r f a c e i n s u l i n r e c e p t o r s w i t h o u t a l t e r i n g t h e I C 5 0 / t h e r e c e p t o r b i n d i n g a f f i n i t y f o r i n s u l i n (Mossner 1984). A number o f o t h e r workers have a l s o i n v e s t i g a t e d t h e a c u t e e f f e c t s o f i n s u l i n on a c i n a r c e l l f u n c t i o n b u t w i t h v a r i a b l e r e s u l t s . I n s u l i n , CCK, and c a r b a c h o l each i n d u c e d a s i m i l a r p a t t e r n o f zymogen p r o t e i n s y n t h e s i s a f t e r 60 m i n u t e s o f i n c u b a t i o n w i t h normal r a t a c i n i , an e f f e c t t h a t seemed t o be p o s t t r a n s c r i p t i o n a l i n n a t u r e i n a s t u d y r e p o r t e d by L a h a i e ( 1 9 8 6 ) . S i n g h (1983) and T r i m b l e e t a l (1985) b o t h f o u n d t h a t a l t h o u g h i n s u l i n appeared t o a l t e r c e l l m e t a b o l i c f u n c t i o n , i n t h e former s t u d y i n c r e a s i n g cAMP l e v e l s , t h e r e was no e f f e c t on b a s a l o r s t i m u l a t e d amylase s e c r e t i o n . Y e t 338 Kanno and S a i t o (1976) found t h a t i n s u l i n b o t h i n d u c e d c e l l h y p e r p o l a r i z a t i o n and p o t e n t i a t e d C C K - s t i m u l a t e d amylase r e l e a s e f rom a c i n i , b u t o n l y a t c e r t a i n low c o n c e n t r a t i o n s o f CCK. Thus, w h i l e i n s u l i n e f f e c t s on r e c e p t o r b i n d i n g , m o d u l a t i o n o f p o s t r e c e p t o r a c t i o n s , and s u b s t r a t e a v a i l a b i l i t y have been c o n s i s t e n t l y d e m o n s t r a t e d i n t h e a c i n a r c e l l (summarized by W i l l i a m s ( 1 9 8 5 ) ) , no good e x p l a n a t i o n f o r t h e p r e v i o u s l y d i s c u s s e d , a c u t e in vivo and i s o l a t e d p e r f u s e d p a n c r e a s in v i t r o e f f e c t s o f i n s u l i n on enzyme s e c r e t i o n has been p r o v i d e d , n o r does t h e r e appear t o be a con s e n s u s as t o whether i n s u l i n has any r o l e i n p h y s i o l o g i c a l l y m o d u l a t i n g a c i n a r s e c r e t i o n f rom minute t o mi n u t e . The r e s u l t s o f t h e p r e s e n t s t u d y were s i m i l a r l y c o n t r a d i c t o r y , showing an i n s u l i n r e s p o n s e o n l y under s e l e c t e d c o n d i t i o n s i n some groups o f r a t s . Of i m p o r t a n c e however, was t h e f i n d i n g t h a t t h i s v a r i a b i l i t y e x i s t e d between groups o f a n i m a l s w i t h i n one laboratory. Thus some part o f t h e v a r i a b l e r e s p o n s e t o i n s u l i n i s i n t r i n s i c t o t h e r a t model and s h o u l d n o t s i m p l y be a s c r i b e d t o d i f f e r e n c e s i n t e c h n i q u e between t h e v a r i o u s l a b o r a t o r i e s t h a t have s t u d i e d t h e i n s u l o a c i n a r a x i s . A s y n t h e s i s o f d a t a from t h i s and t h e o t h e r s t u d i e s c i t e d s u g g e s t s t h a t a c u t e t r e a t m e n t w i t h i n s u l i n does i n f l u e n c e s e c r e t i o n b u t o n l y when s t i m u l a t e d by some t y p e s o f s e c r e t a g o g u e s i n a r e s p o n s e t h a t l i k e l y depends on m u l t i p l e p a r a m e t e r s . I n s u l i n dose, t h e d u r a t i o n o f i n s u l i n t r e a t m e n t , 339 t h e t y p e and dose o f t h e p r i m a r y s e c r e t a g o g u e , m e t a b o l i c p r o c e s s e s o f p r o t e i n s y n t h e s i s and i n t r a c e l l u l a r s u b s t r a t e t r a n s p o r t , and t h e i n f l u e n c e o f p r o l o n g e d a l t e r a t i o n s i n t h e c i r c u l a t i n g i n s u l i n e nvironment ( i n s u l i n e x c e s s and d e f i c i e n c y s t a t e s ) were among t h e p a r a m e t e r s o f t h e i n s u l o a c i n a r a x i s s u b s e q u e n t l y i n v e s t i g a t e d . Factors modulating i n s u l i n responsiveness of the exocrine pancreas E f f e c t o f a l t e r a t i o n s i n t h e r a t e o f p r o t e i n s y n t h e s i s I n s u l i n s t i m u l a t e s p r o t e i n s y n t h e s i s by s e v e r a l mechanisms i n c l u d i n g p r o m o t i o n of mRNA t r a n s c r i p t i o n , r i b o s o m a l number and e f f i c i e n c y , and t h e p r o v i s i o n o f e n e rgy and s t r u c t u r a l (amino a c i d ) s u b s t r a t e s ( J e f f e r s o n 1980). I n t h e p a n c r e a s , in vivo s t u d i e s have shown t h a t i n s u l i n i s an i m p o r t a n t s t i m u l u s f o r s y n t h e s i s o f s e v e r a l p a n c r e a t i c s e c r e t o r y p r o t e i n s ( C o u t u r e 1972, A d l e r 1975). I n s u l i n was f o u n d t o s t i m u l a t e [ 3 H ] l e u c i n e i n c o r p o r a t i o n i n t o j a c i n a r p r o t e i n i n s ubsequent in v i t r o mouse a c i n i s t u d i e s b u t had no e f f e c t on amino a c i d t r a n s p o r t ( K o r c 1981b). A l t h o u g h i n s u l i n appeared t o s t i m u l a t e a c i n a r s e c r e t o r y p r o t e i n s y n t h e s i s , a c i n i f o r t h e s e s t u d i e s came from d i a b e t i c a n i m a l s and f u r t h e r , t h e t i m e dependency o f t h e i n s u l i n e f f e c t was n o t t e s t e d . F o r t h e p u r p o s e o f t h e p r e s e n t s t u d y , i t was h y p o t h e s i z e d t h a t i n s u l i n e f f e c t s on zymogen p r o t e i n s y n t h e s i s m ight be a mechanism f o r a c u t e m o d u l a t i o n o f s t i m u l a t e d p a n c r e a t i c s e c r e t i o n . To p l a c e t h i s i n c o n t e x t , i n s u l i n i s r e s p o n s i b l e 3 4 0 f o r o n l y 30% o f t h e t o t a l c e l l p r o t e i n s y n t h e s i s o f t h e p a n c r e a s ( A d l e r 1975) and a c i n a r p r o t e i n s y n t h e s i s l i k e l y o c c u r s i n r e s p o n s e t o c o o r d i n a t e d a c t i o n s o f a number of t r o p h i c f a c t o r s . Indeed, CCK as a p r i n c i p l e enzyme s e c r e t a g o g u e has a l s o been shown t o d i r e c t l y s t i m u l a t e p r o t e i n s y n t h e s i s i n b o t h normal and d i a b e t i c a c i n i ( K o r c 1 9 81c), a c c e l e r a t i n g i n t r a c e l l u l a r t r a n s p o r t , p a c k a g i n g , and : g r a n u l e d i s c h a r g e a f t e r a s t i m u l a t i o n p e r i o d o f o n l y 3 h ( L i i t c k e 1987). W i t h t h e s e background d a t a , a s t u d y was d e s i g n e d t o t e s t whether i n s u l i n might a c u t e l y i n f l u e n c e s e c r e t i o n from a c i n i t r e a t e d by p r o l o n g e d CCK s t i m u l a t i o n , d e p l e t e d o f zymogen and a c t i v e l y s y n t h e s i z i n g p r o t e i n . As shown i n F i g u r e s 79 and 80, b a s a l and s e c r e t i n - s t i m u l a t e d s e c r e t i o n from zymogen-depleted a c i n i d i d not show any change i n s e c r e t o r y r e s p o n s e t o added i n s u l i n . Only a c u t e i n s u l i n t r e a t m e n t of C C K - s t i m u l a t e d a c i n i , a t a dose o f 2000 /iU/ml (13.3 nM), showed s i g n i f i c a n t p o t e n t i a t i o n o f s e c r e t i o n though t h e r e was a l s o a t r e n d t o s i g n i f i c a n t p o t e n t i a t i o n i n t h e C C K - s t i m u l a t e d group p r e i n c u b a t e d w i t h t h e same dose of i n s u l i n (2000 /iU/ml). I n c o n t r a s t t o t h e r e p o r t by O t s u k i e t a l ( 1 9 83), a h i g h e r dose of i n s u l i n (20,000 /xU/ml = 133 nM) i n t h e p r e s e n t s t u d y d i d n o t i n d u c e g r e a t e r amylase r e l e a s e b u t r a t h e r seemed t o i n h i b i t t h e e f f e c t s seen a t t h e l o w e r dose. Companion e x p e r i m e n t s u t i l i z i n g c y c l o h e x i m i d e , a p r o t e i n s y n t h e s i s i n h i b i t o r (Vasquez 1974) ( F i g u r e s 113 and 114) and c o l c h i c i n e , a m i c r o t u b u l e i n h i b i t o r (Benz 1972) ( F i g u r e s 111 and 112) b o t h f a i l e d t o a l t e r s t i m u l a t e d 341 s e c r e t i o n from normal a c i n i , r e g a r d l e s s of t h e p r e s e n c e of i n s u l i n . From t h e s e d a t a , i t appeared t h a t s t i m u l a t e d s e c r e t i o n from normal a c i n i was not dependent on r a p i d p r o t e i n s y n t h e s i s o r m i c r o t u b u l e t r a n s p o r t mechanisms, and, as n o t e d e a r l i e r , was r e l a t i v e l y i n s e n s i t i v e t o i n s u l i n . However, i n t h e a c t i v e l y s e c r e t i n g , p r o t e i n - s y n t h e s i z i n g p a n c r e a s , i n s u l i n appeared c a p a b l e o f p o t e n t i a t i n g s e c r e t i o n b u t o n l y a t c e r t a i n doses. The p a r a d o x i c a l r e v e r s a l o f p o t e n t i a t i o n found i n t h e 20,000 /iU/ml t r e a t m e n t group may have r e p r e s e n t e d occupancy o f a l o w e r a f f i n i t y , i n h i b i t o r y p o p u l a t i o n o f i n s u l i n r e c e p t o r s by t h e h i g h i n s u l i n dose, p a r a l l e l i n g t h e b i p h a s i c s e c r e t o r y r e s p o n s e seen i n CCK-t r e a t e d a c i n i ( ( W i l l i a m s 1981) and c o n f i r m e d i n CCK-dose r e s p o n s e d a t a from t h i s s t u d y ( F i g u r e s 53 and 5 4 ) ) . Indeed, b i n d i n g s t u d i e s have i d e n t i f i e d two p o p u l a t i o n s o f i n s u l i n r e c e p t o r s ( h i g h a f f i n i t y - K d 1.6 nM, and low a f f i n i t y - K d 84 nM) t h a t might be t h e b a s i s f o r h i g h dose, i n h i b i t o r y i n s u l i n e f f e c t s . The p h y s i o l o g i c a l consequences of i n s u l i n b i n d i n g t o low a f f i n i t y r e c e p t o r s has n o t been p r e v i o u s l y r e p o r t e d ( W i l l i a m s 1981). F o l l o w - u p s t u d i e s d u p l i c a t i n g t h e c o n d i t i o n s found i n F i g u r e s 79 and 80 b u t w i t h t h e a d d i t i o n o f c y c l o h e x i m i d e i n some groups d u r i n g t h e p r e i n c u b a t i o n phase would be of i n t e r e s t t o d e t e r m i n e c o n c l u s i v e l y whether p r o t e i n s y n t h e s i s a c c o u n t s f o r p a r t o f t h e i n s u l i n e f f e c t s i n t h e d e p l e t e d zymogen model. 342 E f f e c t o f C a + 2 c o n c e n t r a t i o n i n t h e medium A l t h o u g h t h e p r i n c i p l e a c t i o n s o f i n s u l i n do n o t appear t o r e s u l t s o l e l y from i n t r a c e l l u l a r c a l c i u m m o b i l i z a t i o n (Denton 1986), c l e a r l y c a l c i u m remains an i m p o r t a n t p e r m i s s i v e f a c t o r i n enzyme r e l e a s e ( W i l l i a m s 1975, S c h u l z 1981). I t has been shown t o modulate t h e a c t i o n s o f b o t h CCK (Dormer 1981, Gardner 1979) and cAMP-mediated s e c r e t a g o g u e s on enzyme r e l e a s e ( C o l l e n 1982). G i v e n t h e c r i t i c a l r o l e o f c a l c i u m i n enzyme s e c r e t i o n and i n p o t e n t i a t i n g i n t e r a c t i o n s between s e c r e t a g o g u e s , t h e p o s s i b i l i t y t h a t i n s u l i n might have a s i m i l a r c a l c i u m dependency f o r i t s e f f e c t s on enzyme s e c r e t i o n was s t u d i e d i n e x p e r i m e n t s d e p i c t e d i n F i g u r e s 109 and 110 u t i l i z i n g s e c r e t i n as t h e s e c r e t a g o g u e . A l t h o u g h no s i g n i f i c a n t d i f f e r e n c e s between groups were n o t e d , one group seemed t o show a t r e n d t oward a c a l c i u m - d e p e n d e n t i n s u l i n p o t e n t i a t i o n , t h a t group p r e i n c u b a t e d w i t h i n s u l i n and s t i m u l a t e d by s e c r e t i n (20 nM, open t r i a n g l e s ) . However t h e magnitude o f t h e r e s p o n s e was s m a l l . Under t h e e x p e r i m e n t a l c o n d i t i o n s o u t l i n e d , i t d i d n o t appear t h a t t h e p o t e n t i a t i n g e f f e c t s o f i n s u l i n were p r i m a r i l y due t o a l t e r a t i o n o f c e l l u l a r c a l c i u m f l u x o r c o n t e n t o r t h a t i n s u f f i c i e n t c a l c i u m i n t h e a c i n i p r e p a r a t i o n medium a c c o u n t e d f o r t h e i n c o n s i s t e n t magnitude o f i n s u l i n p o t e n t i a t i o n . E f f e c t o f s h o r t - t e r m d i a b e t e s A l t h o u g h t h e i s o l a t i o n p r o c e d u r e f o r a c i n i r e d u c e d t h e i s l e t p o p u l a t i o n i n t h e f i n a l s u s p e n s i o n o f a c i n i , i s l e t fragments and i n s u l i n were s t i l l p r e s e n t as n o t e d p r e v i o u s l y . To 343 i n v e s t i g a t e whether i n s u l i n from t h e s e fragments o b s c u r e d a r e s p o n s e due t o exogenous i n s u l i n , a c i n i were p r e p a r e d from a n i m a l s r e n d e r e d d i a b e t i c f o r a s h o r t p e r i o d (3 d a y s ) . T h i s t r e a t m e n t r e d u c e d i n t e r f e r e n c e from endogenous i n s u l i n w i t h o u t s i g n i f i c a n t l y r e d u c i n g t h e amylase c o n t e n t o f t h e a c i n i . However, when s t u d i e d under c o n d i t i o n s o f s e c r e t i n and CCK s t i m u l a t i o n , i n s u l i n i n c u b a t i o n o r p r e i n c u b a t i o n a t s e v e r a l doses had no s i g n i f i c a n t e f f e c t on amylase s e c r e t i o n , though n e i t h e r s e c r e t a g o g u e was a t maximal dose ( F i g u r e s 81 and 8 2 ) . A l t h o u g h not c o n c l u s i v e , t h i s s t u d y s u g g e s t e d t h a t i n c o n s i s t e n t i n s u l i n p o t e n t i a t i o n was u n l i k e l y t o be due t o i n t e r f e r e n c e from endogenous i n s u l i n e f f e c t s . Use o f d i a b e t i c a n i m a l s a t a l a t e r t i m e a f t e r t h e d i a b e t o g e n i c s t i m u l u s (which i n d u c e s a l a r g e r e l e a s e o f i n s u l i n t r a n s i e n t l y ) and t r e a t e d w i t h i n s u l i n t o m a i n t a i n enzyme c o n t e n t , t o g e t h e r w i t h a w i d e r range o f b o t h s e c r e t a g o g u e and i n s u l i n doses might be h e l p f u l i n c o n f i r m i n g t h e s e c o n c l u s i o n s . E f f e c t o f a g e - r e l a t e d . i n s u l i n s e n s i t i v i t y I n c o n j u n c t i o n w i t h t h e use o f d i a b e t i c and c h l o r p r o p r a m i d e -t r e a t e d a n i m a l s b o t h o f which have i n c r e a s e d s e n s i t i v i t y t o g l u c o r e g u l a t o r y e f f e c t s o f i n s u l i n , s t u d i e s were c a r r i e d o ut i n young, w e a n l i n g r a t s t o d e t e r m i n e t h e i r i n s u l i n s e n s i t i v i t y and t h e age dependency, i f any, o f i n s u l i n e f f e c t s on s e c r e t i o n . W h i l e t h e s t u d i e s i n d i c a t e d i n F i g u r e s 101 and 102 showed t h a t w e a n l i n g a n i m a l s a r e more i n s u l i n -344 s e n s i t i v e w i t h r e s p e c t t o g l u c o s e m e t a b o l i s m t h a n o l d e r r a t s ( l e s s i n s u l i n c o n t r o l l i n g c i r c u l a t i n g g l u c o s e l e v e l s more t i g h t l y ) , t h i s d i d n o t appear t o t r a n s l a t e i n t o i n c r e a s e d a c i n a r s e n s i t i v i t y t o i n s u l i n when t e s t e d w i t h CCK-, s e c r e t i n - , o r V I P - s t i m u l a t e d s e c r e t i o n ( F i g u r e s 103 t o 108). The a g e - r e l a t e d r e s p o n s i v e n e s s o f t h e e x o c r i n e p a n c r e a s t o i n s u l i n has n o t been e x p l o r e d e l s e w h e r e i n t h e l i t e r a t u r e but i t appears u n l i k e l y from d a t a i n t h e p r e s e n t s t u d y t h a t age i s an i m p o r t a n t d e t e r m i n a n t . I n summary, a l t h o u g h s e c r e t i o n o f t h e e x o c r i n e p a n c r e a s i s a c u t e l y r e s p o n s i v e t o i n s u l i n in vivo and i n t h e in vitro, p e r f u s e d p a n c r e a s , t h e r e s p o n s e o f a c i n i t o i n s u l i n was s m a l l e r i n magnitude and l e s s c o n s i s t e n t . E f f o r t s a t m o d i f y i n g t h e parameters o f a c i n a r s e c r e t i o n by p r o m o t i n g o r i n h i b i t i n g p r o t e i n s y n t h e s i s , i n h i b i t i n g m i c r o t u b u l e f u n c t i o n , a l t e r i n g c a l c i u m l e v e l s , o r u t i l i z i n g younger, more i n s u l i n r e s p o n s i v e a n i m a l s - l a r g e l y f a i l e d t o a m p l i f y an i n s u l i n e f f e c t on s t i m u l a t e d s e c r e t i o n . T h i s l a c k o f r e s p o n s i v e n e s s d i d n o t appear t o be due t o i n t e r f e r e n c e from i s l e t f r a g m e n t s p r e s e n t i n t h e a c i n i p r e p a r a t i o n ; r a t h e r two p o s s i b l e e x p l a n a t i o n s remain. E i t h e r some p o r t i o n o f t h e i n s u l i n p o t e n t i a t i n g e f f e c t seen i n t h e i n t a c t p a n c r e a s was i n d i r e c t , p o s s i b l y v i a r e l e a s e o f o t h e r hormones o r a c t i v a t i o n o f t h e e n t e r o p a n c r e a t i c nervous system, o r t h e r e s p o n s i v e n e s s o f t h e a c i n i u t i l i z e d was compromised by t h e d i s p e r s i o n t e c h n i q u e i n ways n o t t e s t e d by t h e p r e c e d i n g 345 sequence of e x p e r i m e n t s . An example of such an u n q u a n t i f i e d e f f e c t on a c i n i might be v a r i a b l e damage o r l o s s o f r e c e p t o r s f o r i n s u l i n s i n c e e f f i c a c y o f b i n d i n g was not s y s t e m a t i c a l l y measured i n t h e p r e s e n t s t u d i e s . R e s o l u t i o n o f t h i s c o n t r o v e r s y w i l l l i k e l y a w a i t more d e t a i l e d s t u d i e s i n t h e p e r f u s e d system t o e l u c i d a t e p o s s i b l e i n d i r e c t m e d i a t o r s of i n s u l i n a c t i o n , and s t u d i e s i n t h e a c i n a r c e l l t o r e v e a l how, by a s t i l l u n d e f i n e d mechanism, i n s u l i n might promote i n c r e a s e d s e c r e t i o n a c u t e l y . E f f e c t s o f a l t e r e d c i r c u l a t i n g i n s u l i n c o n c e n t r a t i o n s on  i n s u l i n r e s p o n s i v e n e s s A l t h o u g h t h e a c u t e r e s p o n s i v e n e s s o f i s o l a t e d a c i n i t o i n s u l i n was s m a l l and i n c o n s i s t e n t , many p r i o r s t u d i e s had shown t h a t i n s u l i n p l a y s a c r i t i c a l r o l e i n m a i n t a i n i n g t h e i n t e g r i t y o f t h e e x o c r i n e p a n c r e a s . W i t h i n t h e normal p a n c r e a s , r e g i o n a l d i f f e r e n c e s e x i s t about each i s l e t as i s d i s c u s s e d below i n t h e a u t o r a d i o g r a p h i c s t u d y . But d i f f e r e n c e s i n i s l e t hormone s e c r e t i o n a l s o e x i s t between t h e two e m b r y o l o g i c p o r t i o n s o f t h e p a n c r e a s . T r i m b l e e t a l (1981) showed t h a t d o r s a l i s l e t s s e c r e t e d more i n s u l i n and g l u c a g o n i n r e s p o n s e t o a g l u c o s e s t i m u l u s t h a n d i d v e n t r a l i s l e t s w h i l e s o m a t o s t a t i n r e l e a s e from b o t h was s i m i l a r . Subsequent s t u d i e s from t h e same group showed t h a t b o t h t h e in vivo and in v i t r o p e r f u s e d p a n c r e a s s e c r e t e d p r o p o r t i o n a t e l y more amylase from t h e d o r s a l l o b e t h a n t h e v e n t r a l l o b e o f t h e p a n c r e a s ( B r u z z o n e 1986) though t h e y a l s o r e p o r t e d t h a t a c i n i from each l o b e had t h e same s e n s i t i v i t y 346 t o s e c r e t a g o g u e s t i m u l a t i o n . Q u a n t i f i c a t i o n o f enzyme c o n t e n t by a n o t h e r group found i n c r e a s e d amylase i n t h e d o r s a l ( s p l e n i c ) l o b e ( M a l a i s s e - L a g a e 1983). G i v e n t h e s e d a t a s u g g e s t i n g d i f f e r e n t i a l l y g r e a t e r i n s u l i n and amylase s e c r e t i o n o v e r t i m e from t h e d o r s a l p a n c r e a s , i t was h y p o t h e s i z e d t h a t a c u t e i n s u l i n e f f e c t s on s e c r e t i o n n o t e d i n t h e i n t a c t g l a n d might be due t o p r e f e r e n t i a l s e c r e t i o n from one p a r t o f t h e p a n c r e a s o r t h e o t h e r and t h a t s u c h r e s p o n s i v e n e s s might be s u b s e q u e n t l y o b s c u r e d by m i x i n g o f a c i n i from a l l p a r t s o f t h e p a n c r e a s d u r i n g p r e p a r a t i o n o f d i s p e r s e d a c i n i . To t e s t t h i s h y p o t h e s i s , a c i n i d e r i v e d s e p a r a t e l y from e i t h e r t h e v e n t r a l o r d o r s a l l o b e s o f t h e p a n c r e a s were s t u d i e d f o r s e c r e t a g o g u e (CCK) and i n s u l i n s e n s i t i v i t y . These r e s u l t s a r e d e p i c t e d i n F i g u r e s 115 and 116 and showed t h a t a l t h o u g h d o r s a l a c i n i s e c r e t e d t h e same amount o f amylase as v e n t r a l a c i n i ( F i g u r e 116, uU/ug c e l l p r o t e i n ) , t h e maximum p e r c e n t a g e of c e l l u l a r amylase s e c r e t e d was l e s s i n a c i n i from t h e d o r s a l p a n c r e a s ( F i g u r e 115) c o n f i r m i n g t h e p r e v i o u s r e p o r t o f a h i g h e r t o t a l amylase c o n t e n t i n t h e d o r s a l p a n c r e a s . U n f o r t u n a t e l y , i n s u l i n t r e a t m e n t produced no d i f f e r e n c e i n a c i n a r s e c r e t i o n from e i t h e r p a r t o f t h e p a n c r e a s . D i f f e r e n c e s between d o r s a l and v e n t r a l p a n c r e a t i c e x o c r i n e s e c r e t i o n r e p r e s e n t a n a t u r a l e x p e r i m e n t w i t h a p r o p o r t i o n a t e l y i n c r e a s e d d o r s a l g l a n d s e c r e t o r y c a p a c i t y t h a t may be due t o t h e u n i q u e , l o n g - t e r m i n f l u e n c e o f 347 r e g i o n a l i s l e t s . But a t t h e a c i n a r l e v e l , t h e l a c k o f a d i f f e r e n t i a l i n s u l i n r e s p o n s e made i t u n l i k e l y t h a t p r e f e r e n t i a l s e c r e t i o n from one l o b e o r t h e o t h e r was r e s p o n s i b l e f o r a c u t e i n s u l i n e f f e c t s on s e c r e t i o n i n t h e i n t a c t p a n c r e a s . To f u r t h e r e x p l o r e t h e e f f e c t s o f c h r o n i c a l l y a l t e r e d c i r c u l a t i n g i n s u l i n c o n c e n t r a t i o n s in vivo on subsequent in v i t r o a c i n a r r e s p o n s i v e n e s s t o i n s u l i n , a n i m a l s were t r e a t e d t o i n c r e a s e o r d e c r e a s e i n a c h r o n i c f a s h i o n c i r c u l a t i n g i n s u l i n c o n c e n t r a t i o n s i n e x p e r i m e n t s o u t l i n e d i n F i g u r e s 83 t o 93. H i s t o l o g i c and immunocytochemical consequences o f t h e s e t r e a t m e n t s were d e s c r i b e d i n F i g u r e s 117 t o 126. As p r e v i o u s l y n o t e d , exogenous i n s u l i n o r c h l o r p r o p a m i d e t r e a t m e n t i n c r e a s e d plasma i n s u l i n , r e d u c e d plasma g l u c o s e , and r e n d e r e d t h e a c i n i from t h e s e groups of a n i m a l s i n s e n s i t i v e t o i n s u l i n when compared t o c o n t r o l s . The c h l o r p r o p a m i d e e f f e c t was somewhat s u r p r i s i n g s i n c e t h i s d r u g i s known t o i n c r e a s e p e r i p h e r a l s e n s i t i v i t y ( G r e e n f i e l d 1982) f o r i n s u l i n e f f e c t s on g l u c o s e . The l a c k o f a r e s p o n s e t o i n s u l i n by e i t h e r group s u g g e s t e d t h a t s e n s i t i v i t y t o i n s u l i n e f f e c t s was i n d e e d m o d i f i e d by c h a n g i n g t h e ambient i n s u l i n c o n c e n t r a t i o n . A n o t h e r i m p o r t a n t consequence of a l t e r a t i o n s i n c i r c u l a t i n g i n s u l i n l e v e l s was a marked a t t e n u a t i o n i n C C K - s t i m u l a t e d amylase s e c r e t i o n i n t h e i n s u l i n - t r e a t e d group, an e f f e c t t h a t c o u l d n o t be e x p l a i n e d by t h e c u r r e n t d a t a b u t may have been due t o c h r o n i c s t i m u l a t i o n from t h e 348 h y p e r p h a g i c s t a t e o f t h e a n i m a l s , o r perhaps due t o t h e p r e v i o u s l y d e s c r i b e d , d o w n r e g u l a t o r y i n f l u e n c e o f i n s u l i n on a c i n a r CCK r e c e p t o r s ( O t s u k i 1983). I n c o n t r a s t t o t h e e f f e c t s o f i n c r e a s e d c i r c u l a t i n g i n s u l i n , s e c r e t i o n from a c i n i o f c o n t r o l and d i a b e t i c a n i m a l s was p o t e n t i a t e d by i n s u l i n b u t o n l y when t h e i n s u l i n was added d u r i n g s e c r e t a g o g u e s t i m u l a t i o n . P r e i n c u b a t i o n w i t h i n s u l i n a b o l i s h e d t h i s e f f e c t . A l t h o u g h o t h e r w o r k e r s have j u s t i f i e d use o f d i a b e t i c a c i n i because of i n c r e a s e d i n s u l i n s e n s i t i v i t y o v e r normal a n i m a l s (Mossner 1984), t h e c o n t r o l s i n t h i s e x p e r i m e n t had a g r e a t e r p r o p o r t i o n a l i n s u l i n r e s p o n s i v e n e s s t h a n t h e d i a b e t i c a n i m a l s . Based on t h e r e s u l t s o f t h i s s t u d y , i t seemed r e a s o n a b l e t o propose t h a t t h e e x o c r i n e p a n c r e a t i c r e s p o n s e s t o i n s u l i n and perhaps a l s o CCK were, a t l e a s t under t h e c o n d i t i o n s o f t h i s e x p e r i m e n t , i n v e r s e l y dependent on c i r c u l a t i n g i n s u l i n l e v e l s t h a t p r e v a i l w i t h i n t h e h o s t . Comparing t h e r e s u l t s o f t h i s e x p e r i m e n t w i t h o t h e r s p r e v i o u s l y d i s c u s s e d emphasized t h e i n c o n s t a n t n a t u r e o f t h e i n s u l i n e f f e c t and s u g g e s t e d ( a f t e r t h e s e s t u d i e s were completed) t h a t d i f f e r e n c e s between groups might be due t o a v a r i a b l e t h r e s h o l d f o r i n s u l i n r e s p o n s i v e n e s s t h a t , from a n i m a l group t o group, might have i n t e r m i t t e n t l y exceeded t h e 1000 - 2000 juU/ml i n s u l i n dose commonly employed (Kanazawa 1968). F o l l o w - u p s t u d i e s t o s y s t e m a t i c a l l y t e s t a c u t e e f f e c t s o f h i g h e r i n s u l i n c o n c e n t r a t i o n s would be u s e f u l i n a n s w e r i n g t h i s q u e s t i o n . 3 4 9 E f f e c t s o f i n s u l i n - t r e a t e d and u n t r e a t e d d i a b e t e s A l t h o u g h t h e p h y s i o l o g i c s i g n i f i c a n c e o f a c u t e i n s u l i n e f f e c t s on p a n c r e a t i c s e c r e t i o n remained u n c l e a r d e s p i t e t h e i n v e s t i g a t i o n s o u t l i n e d , ample e v i d e n c e e x i s t s t h a t i n s u l i n has an i m p o r t a n t r o l e i n m a i n t a i n i n g e x o c r i n e p a n c r e a t i c i n t e g r i t y ( L a n k i s c h 1982). E x p e r i m e n t a l d i a b e t e s has been shown t o reduce enzyme and b i c a r b o n a t e s e c r e t i o n i n s e v e r a l s p e c i e s ( B a l k 1975, Grossman 1946) and l e a d t o a l t e r a t i o n s i n c o n t e n t and d i s t r i b u t i o n o f o t h e r g u t hormones t h a t modulate e x o c r i n e f u n c t i o n ( B a l l m a n n 1985). D i a b e t e s i n c r e a s e d t r y p s i n o g e n and d e c r e a s e d amylase s e c r e t i o n i n t h e r a t ( S o f r a n k o v a 1983) w h i l e s t u d i e s i n t h e p e r f u s e d p a n c r e a s found b o t h r e d u c e d enzyme c o n t e n t and s e c r e t a g o g u e (CCK) r e s p o n s i v e n e s s (Okabayashi 1988). These e f f e c t s were a l s o s t u d i e d a t a c e l l u l a r l e v e l where enzyme c o n t e n t and s e c r e t i o n , as w e l l as s e n s i t i v i t y t o CCK were a l s o a l t e r e d i n d i a b e t i c a c i n i ( O t s u k i 1982). S i m i l a r e f f e c t s on e x o c r i n e f u n c t i o n have been d e s c r i b e d i n t h e s p o n t a n e o u s l y d i a b e t i c Zucker r a t ( T r i m b l e 1987). I n t h e p r e s e n t s t u d y , t h e t i m e -c o u r s e o f changes i n s e c r e t o r y c a p a b i l i t y a f t e r t h e o n s e t and subsequent i n s u l i n - t r e a t m e n t o f d i a b e t i c a n i m a l s was s t u d i e d . CCK and s e c r e t i n - s t i m u l a t e d s e c r e t i o n i n u n t r e a t e d d i a b e t i c a n i m a l s were compared because of t h e p r e v i o u s l y n o t e d , s e l e c t i v e e f f e c t s o f d i a b e t e s on CCK r e s p o n s i v e n e s s ( F i g u r e s 95 and 9 7 ) . A marked d e c l i n e i n d o s e - r e s p o n s i v e amylase r e l e a s e by day 7 o f d i a b e t e s was e v i d e n t , an e f f e c t l i k e l y due t o d e p l e t i o n o f enzyme r a t h e r t h a n reduced s e c r e t a g o g u e 350 s e n s i t i v i t y s i n c e b o t h s e c r e t i n and CCK were e q u a l l y a f f e c t e d . The c o m b i n a t i o n ( F i g u r e 97) o f s e c r e t a g o g u e s was no more e f f e c t i v e a s t i m u l u s . Once i n s u l i n t r e a t m e n t s were i n s t i t u t e d , t h e r e appeared t o be a b i p h a s i c i n c r e a s e i n amylase s e c r e t i o n o v e r t i m e . A s l o w i n c r e a s e i n peak amylase s e c r e t i o n o c c u r r e d o v e r t h e f i r s t 5 days t h e n a b r u p t l y i n c r e a s e d t o 50% o f c o n t r o l a t day 8 ( F i g u r e 9 8 ) . T h i s was a l s o e v i d e n t f o r t h e s e c r e t i n - and c o m b i n a t i o n o f s e c r e t i n and C C K - s t i m u l a t e d groups ( F i g u r e 99 and 100) and s u g g e s t e d t h a t enzyme c o n t e n t changes n o t a change i n r e s p o n s i v e n e s s t o any s p e c i f i c s e c r e t a g o g u e a c c o u n t e d f o r i n c r e a s e i n amylase s e c r e t i o n a f t e r i n s u l i n t r e a t m e n t . A p a r a l l e l s t u d y has been r e p o r t e d by Ko r c e t a l (1981) where amylase a c t i v i t y and amylase mRNA l e v e l s were compared i n i n s u l i n - t r e a t e d d i a b e t i c r a t s and demon s t r a t e d t h a t amylase mRNA l e v e l s r o s e s i g n i f i c a n t l y f a s t e r t h a n t h e c o r r e s p o n d i n g amylase v a l u e s . I n c o n t r a s t t o t h e p r e s e n t d a t a , K o r c found t h a t amylase l e v e l s n o r m a l i z e d a f t e r o n l y 7 days o f i n s u l i n t r e a t m e n t however t h i s e f f e c t may be due t o d i f f e r e n c e s i n t h e i n s u l i n t r e a t m e n t regimen. Both s t u d i e s i n d i c a t e d t h a t i n t h e d i a b e t i c a n i m a l i n s u l i n has a p r o f o u n d though l a t e n t e f f e c t on amylase b i o s y n t h e s i s v i a mechanisms t h a t a r e u n l i k e l y t o c o n t r i b u t e t o any a c u t e , s h o r t - t e r m e f f e c t of i n s u l i n i n t h e normal i n t a c t p a n c r e a s . On t h e b a s i s o f s t u d i e s c a r r i e d o u t i n t h e c o u r s e o f t h i s t h e s i s , i n s u l i n was found t o be a t r o p h i c s t i m u l u s f o r t h e 351 p a n c r e a s . I n s u l i n had a c r i t i c a l r o l e i n t h e development and maintenance o f zymogen r e s e r v e s a v a i l a b l e f o r r e l e a s e by a p p r o p r i a t e s e c r e t o r y s t i m u l i . Even t h e e f f i c a c y o f c e l l -s u r f a c e a c t i o n s f o r some s e c r e t a g o g u e s such as CCK were modulated by i n s u l i n . But i n t h e a c u t e r e g u l a t i o n o f enzyme s e c r e t i o n , d a t a from t h i s work a l s o showed t h a t i n s u l i n had an e v a n e s c e n t r o l e t h a t appeared t o be dependent on t h e s e c r e t o r y s t a t u s o f t h e p a n c r e a s , t h e dosage and d u r a t i o n of i n s u l i n e x p o s u r e , t h e p r o x i m i t y t o i s l e t s , and o t h e r , s t i l l u n d e s c r i b e d f a c t o r s . 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I n : The e x o c r i n e p a n c r e a s . e d i t e d by Go, V.L.W., Gardner, J.D., B r o o k s , F.P., L e b e n t h a l , E., DiMagno, E.P. and S c h e e l e , G.A. New York: Raven P r e s s , 1986, p. 347-360. APPENDIX T a b l e 1 Reagents for Isolated Pancreas Perfusion Reacrent Source dextran Sigma bovine serum albumin Sigma KCl Fisher CaCl2 Fisher MgS04-7H20 Fisher KH2P04 Fisher NaHC03 Fisher NaCl Fisher D50 I.V. Glucose Travenol porcine heparin LyoKed pentobarbital Abbott insulin (rat) Novo Grade Reagent grad RIA grade Reagent grad 369 T a b l e 2 Dissection Instruments s c i s s o r s , Hetzenbaum and i r i s forceps, blunt and sharp clamps- bulldog C r i l e (x3) Halsted (x3) Kelly tubing, polyethylene blunt needles (x2) (PE-50, 90, 120) T a b l e 3 Isolated Acini Equipment pipets, disposable (5 ml) pipets, disposable (10 ml) centrifuge tubes, Eppendorf (1.5 ml) centrifuge tubes, polycarbonate (12 ml) centrifuge tubes, polyethylene (12 ml) Gilson Pipetman (adjustable) 2 ul 200 jil 1000 jjl Pipetman t i p s 2 - 200 jil 1000 jil Nytex c l o t h pipet (10 ml) ation Equipment Source Western i> II it II Handel M n II n B & S.H.Thompson Quebec Canlab Reagent Source e s s e n t i a l amino acid concentrate (no L-glutamine) Gibco L-glutamine Gibco D-glucose (anhydrous) Sigma tr y p s i n i n h i b i t o r (soy bean) Sigma HEPES c r y s t a l l i n e free acid Sigma bovine serum albumin Sigma collagenase Cooper Bioied Study Reagents Source cyclohexamide Sigma co l c h i c i n e Sigma C a t . No. 53300-443 53300-545 20170-545 21009-284 21008-598 GF23600 GF23601 GF23602 GF23802 GF23812 150 mesh P4473-10 C a t . No. 320-1135 G3126 G5000 T9003 H3375 CLSTA T a b l e 4 Animal Treatment Reagents for A l t e r a t i o n of C i r c u l a t i I n s u l i n Levels Reagent Source Grade streptozotocin Calbiochem Grade A insulin (NPH) Novo chlorpropamide Squibb (JSP TABLE 5 Radioimmunossay Reagents I n s u l i n R e a g e n t NaH2P04 Na2HP04 KI HC1 NaCl ethanol chloramine-T sodium metabisulfite microfine s i l i c a (QUSO) sodium merthiolate activated charcoal BSA Na 1 2 5 I dextran albumin (outdated) human plasma antiinsulin antibody (GP01) S o u r c e Fisher Fisher Fisher Fisher Fisher Fisher Sigma Sigma Sigma Sigma Sigma Sigma Amersham Pharmacia Miles Regional Blood Bank Regulatory Peptides Group G r a d e Reagent grade RIA grade Reagent grade RIA Grade S o m a t o s t a t i n R e a g e n t sodium barbital NaCl NaCH3CO0 ammonium acetate acetic acid merthiolate sodium metabisulfite BSA trasylol dextran T-70 Na 1 2 5 I CH-52 cellulose cyclic somatostatin S o u r c e Fisher Fisher Fisher Fisher Fisher Sigma Sigma Sigma Sigma Pharmacia Amersham Pharmacia Kabi G r a d e Reagent grade RIA Grade Injectable RIA grade T a b l e 6 Perfusion Amylase Assay Harleco Division EH Industries Starch Reagent(pH 7.0) 64191A Iodine Reagent (0.01 N) 64MB Acini Amylase Assay Reagent procion yellow dye (HX-86) cornstarch Na2C03-H20 methanol NaP04 NaCI Source Grade Polysciences Kingsford Sigma Fisher Reagent grade Fisher Fisher Lipase Assay Reagent Sigma Diagnostics Kit NaOH Total Protein Reagent Na 2C0 3-H 20 CuS04-5H20 NaOH sodium citrate Folins Reagent BSA Source Grade Cat f 800 Fisher Reagent Grade Source Grade Fisher Fisher Fisher Fisher Sigma Sigma Reagent Grade RIA grade 374 T a b l e 7 Reagents for Histology/Immunocytochemistry H i s t o l o g y Reagent p a r a f f i n hematoxylin eosin L i 2 C 0 3 Na 2HP0 4 HaH 2P0 4 EtOH xylene NaN 3 Source Canlab Sigma Sigma Fisher Fisher Fisher Fisher Fisher Fisher Sigma P e r o x i d a s e - A n t i p e r o x i d a s e Reagents mouse a n t i - i n s u l i n Ab peroxidase linked rabbit anti-mouse Ab diaminobenzidine tetrahydrochloride (DAB) reagent Sigma A u t o r a d i o g r a p h y Reagent " l i q u i d " emulsion developer f i x a t i v e Source Kodak Kodak Kodak C a t e g o r y NTB3 D-19 Equipment Area Program Image Analyser Reverse Phase Microscope Source I .T .c i II Nikon C a t e g o r y 3000 Diaphot I.T.C.- Image Technology Corporation T a b l e 8 Regulatory Peptides Reagent CCK-8 VIP Secretin Insulin (Rat) Methacholine Source Peninsula GIH (Karolinska) GIH (Karolinska) and Peninsula Novo Sigma 

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