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On the role of the enteroinsular axis in obesity Chan, Catherine Barbara 1985

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ON THE ROLE OF THE ENTEROINSULAR AXIS IN OBESITY By CATHERINE BARBARA CHAN B . S c , The U n i v e r s i t y o f B r i t i s h C o l u m b i a ^ 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES D e p a r t m e n t 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 O c t o b e r 1985 © C a t h e r i n e B a r b a r a Chan, 1985 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 1956 Main Mall Vancouver, Canada V6T 1Y3 Date /8£fJ>A*JrfA. 2Q_ / f f e DE-6(3/81) ABSTRACT The e n t e r o i n s u l a r a x i s r e f e r s to the e f f e c t s of n u t r i e n t , hormonal and nervous inputs from the gut on p a n c r e a t i c endocrine s e c r e t i o n . The o b j e c t i v e of these s t u d i e s was to examine the r o l e of the e n t e r o i n s u l a r a x i s i n the development and maintenance of o b e s i t y , using the Zucker f a t t y r a t as an animal model. Onset of g l u c o s e - s t i m u l a t e d h y p e r i n s u l i n e m i a at 21 days i n f a / f a r a t s was preceded by the appearance of enlarged p a n c r e a t i c i s l e t s at 7 days of age. -The m e t a b o l i c events t r i g g e r i n g i n s u l i n h y p e r s e c r e t i o n c o u l d not be r e p l i c a t e d i n v i t r o and d i d not appear to i n v o l v e GIP (glucose-dependent i n s u l i n o t r o p i c p o l y p e p t i d e ; g a s t r i c i n h i b i t o r y polypeptide) or c h o l i n e r g i c a g o n i s t s . F a s t i n g h y p e r i n s u l i n e m i a was measurable by 5 weeks of age, developing concomitantly with a l o s s of the glucose t h r e s h o l d f o r the i n s u l i n o t r o p i c a c t i v i t y of GIP. A c a u s a l r e l a t i o n s h i p was p o s t u l a t e d . T h i s c o n d i t i o n p e r s i s t e d i n a d u l t r a t s and was hypothesized to c o n t r i b u t e to the maintenance of f a s t i n g h y p e r i n s u l i n e m i a i n obese r a t s . In a second study, a d u l t r a t s were t r e a t e d with j e j u n o i l e a l bypass (JIB) surgery and the e f f e c t s on the e n t e r o i n s u l a r a x i s were examined. H y p e r i n s u l i n e m i a of obese r a t s was reduced by JIB, but not to l e v e l s of l e a n c o n t r o l s . However, t h i s d i d not appear to be an e f f e c t of a l t e r a t i o n s i n the n u t r i e n t or known hormonal components of the e n t e r o i n s u l a r a x i s . JIB d i d not a l t e r i n v i t r o p a n c r e a t i c s e n s i t i v i t y t o glucose or GIP, nor was the glucose t h r e s h o l d f o r the i n s u l i n o t r o p i c a c t i o n o f GIP r e s t o r e d by t h i s treatment. The reduced h y p e r i n s u l i n e m i a observed i n obese JIB r a t s c o u l d not be accounted f o r by weight l o s s , as i n s u l i n l e v e l s of obese r a t s s u b j e c t e d to prolonged f a s t i n g were normal both i n v i v o and i n v i t r o . Changes i n endocrine c e l l p o p u l a t i o n o f the continuous and bypassed sma l l bowel were observed and may c o n t r i b u t e t o a l t e r a t i o n s i n a p p e t i t e , weight l o s s and hormone s e c r e t i o n observed i n obese r a t s . In p a r t i c u l a r , a strong c o r r e l a t i o n between enteroglucagon-c e l l number and plasma g l u c a g o n - l i k e immunoreactivity was noted. JIB s t u d i e s a l s o demonstrated t h a t d a i l y p e r f u s i o n of the bypassed loop o f JIB r a t s with a n u t r i e n t s o l u t i o n c o u l d s t i m u l a t e the r e l e a s e o f GIP and p o s s i b l y other hormones, r e s u l t i n g i n a r e d u c t i o n of p a n c r e a t i c s e n s i t i v i t y to GIP. The involvement o f n e u r a l aspects o f the e n t e r o i n s u l a r a x i s i n the h y p e r i n s u l i n e m i a i n Zucker r a t s was a l s o i n v e s t i g a t e d . No evidence was shown to support a hypothesis of h y p e r a c t i v i t y of the vagus nerves. The pancreata of obese Zucker r a t s responded t o the c h o l i n e r g i c a n t a g o n i s t s , hexamethonium and a t r o p i n e , i n a manner i d e n t i c a l t o that of the pancreata of l e a n animals. A second hormonal system i n f l u e n c e d by vaga l i n n e r v a t i o n i s g a s t r i n s e c r e t i o n from the stomach. A d u l t obese Zucker r a t s were shown to be h y p e r g a s t r i n e m i c , but i n v i t r o g a s t r i n s e c r e t i o n responded normally to a t r o p i n e a d m i n i s t r a t i o n both b a s a l l y and when st i m u l a t e d by v a g a l e x c i t a t i o n . A n t r a l h y p e r p l a s i a of G-c e l l s was noted and may cause hypergastrinemia i n obese r a t s . D - c e l l p o p u l a t i o n and so m a t o s t a t i n s e c r e t i o n appeared normal. The p h y s i o l o g i c s i g n i f i c a n c e of hypergastrinemia i n o b e s i t y remains unknown. From these s t u d i e s of the hormonal, n u t r i e n t and nervous components of the e n t e r o i n s u l a r a x i s , i t was not p o s s i b l e to deduce the cause of the onset of h y p e r i n s u l i n e m i a i n obese Zucker r a t s . However, n u t r i e n t s and the hormone GIP may make important c o n t r i b u t i o n s t o the maintenance of f a s t i n g h y p e r i n s u l i n e m i a of these animals due to the notable absence of a minimum glucose c o n c e n t r a t i o n r e q u i r e d f o r GIP to s t i m u l a t e i n s u l i n r e l e a s e . U n l i k e obese VMH-lesioned r a t s , the c h o l i n e r g i c nervous system d i d not appear to be important i n the development or maintenance of h y p e r i n s u l i n e m i a i n Zucker f a t t y r a t s . A second o b j e c t i v e o f these s t u d i e s was to i n v e s t i g a t e the p o s s i b l e presence i n plasma of i n c r e t i n s other than GIP. Plasma c o l l e c t e d from r a t s was e x t r a c t e d using immunoadsorbent chromatography and r e s u l t a n t f r a c t i o n s were assayed f o r i n c r e t i n a c t i v i t y employing the p e r f u s e d r a t pancreas b i o a s s a y . The data p r o v i d e d evidence t h a t plasma of l e a n r a t s c o n t a i n e d more i n c r e t i n s than plasma of obese r a t s , but no data was shown f o r an i n c r e a s e i n i n c r e t i n i v a c t i v i t y with aging i n normal Wistar r a t s . Improved methodology i s r e q u i r e d f o r c l a r i f i c a t i o n of these s t u d i e s . v TABLE OP CONTENTS PAGE ABSTRACT i i LIST OF TABLES x v i i i LIST OF FIGURES xx ACKNOWLEDGEMENTS xxv GENERAL INTRODUCTION 1 GENERAL METHODOLOGY 24 I. PEPTIDE QUANTIFICATION 24 A. Radioimmunoassay 24 1. C a l c u l a t i o n s 24 2. I n s u l i n 24 a. I o d i n a t i o n 24 b. Assay b u f f e r 26 c. Antiserum 26 d. Standards 27 e. C o n t r o l s 27 f . P r o t o c o l 28 g. S e p a r a t i o n 28 3. Glucagon 29 a. Io d i n a t e d glucagon 29 b. Assay b u f f e r 29 c. Antibody 29 d. Standards 30 v i e. P r o t o c o l 30 f. S e p a r a t i o n 30 4. G a s t r i n 31 a. I o d i n a t i o n 31 b. Assay b u f f e r 32 c. Antiserum 32 d. Standards 33 e. C o n t r o l s 34 f . P r o t o c o l 34 g. S e p a r a t i o n 34 5. Glucose-dependent i n s u l i n o t r o p i c p o l y p e p t i d e 34 a. I o d i n a t i o n 35 b. Assay b u f f e r 36 c. Antiserum 36 d. Standards 36 e. C o n t r o l s 37 f . P r o t o c o l 37 g. S e p a r a t i o n 37 6. Somatostatin 38 a. I o d i n a t i o n 38 b. Assay b u f f e r 39 c. Antiserum 39 d. Standards 40 e. Samples 40 f . P r o t o c o l 40 g. S e p a r a t i o n 40 v i i I I . ANIMAL STUDIES 41 A. I s o l a t e d Perfused Organ P r e p a r a t i o n s 41 1. S o l u t i o n s and Reagents 41 a. Krebs-Ringer b i c a r b o n a t e b u f f e r 41 b. Sidearm i n f u s i o n s 42 2. Procedure 42 a. Apparatus 42 b. E q u i l i b r a t i o n 46 c. S u r g i c a l p r e p a r a t i o n 46 i . A n e s t h e s i a 46 i i . Pancreas 46 i i i . Stomach 47 B. Glucose Tolerance T e s t s 48 1. O r a l 48 C. I d e n t i f i c a t i o n o f Obese Zucker Pups 49 1. C o l o n i c Temperature Measurement 49 2. Pancreas H i s t o l o g y 49 D. Zucker Rat Colony 50 1. Breeding 50 2. Housing 50 3. N u t r i t i o n 51 4. I d e n t i f i c a t i o n 51 I I I . IMMUNOCYTOCHEMISTRY 51 A. R a t i o n a l e 51 B. P e r o x i d a s e - a n t i p e r o x i d a s e (PAP) Immunostaining 52 1. F i x a t i o n o f T i s s u e 52 v i i i 2. Dehydration 52 3. Embedding 52 4. C u t t i n g s e c t i o n s 53 5. D e p a r a f f i n i z a t i o n 53 6. Immunostaining Procedures 53 a. F i r s t l a y e r antibody 53 b. B l o c k i n g 53 c. Second l a y e r antibody 54 d. T h i r d l a y e r antibody 54 e. Peroxidase r e a c t i o n 57 7. Q u a n t i f i c a t i o n 57 a. Pancreas 57 b. Antrum 58 c. I n t e s t i n e 58 IV. STATISTICAL INTERPRETATION 59 APPENDIX TO METHODS—CHEMICAL SOURCES 60 APPENDIX TO METHODS—LIST OF ABBREVIATIONS 62 CHAPTER ONE: THE ROLE OF GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE IN THE DEVELOPMENT AND MAINTENANCE OF HYPERINSULINEMIA IN THE OBESE ZUCKER RAT 63 I. INTRODUCTION 63 I I . METHODS 66 A. Glucose Tolerance T e s t s 66 1. Intravenous 66 2. I n t r a p e r i t o n e a l 67 I I I . RESULTS 68 i x Development of Hyperinsulemia: The Role of GIP 68 1. I d e n t i f i c a t i o n of S u c k l i n g and Weanling Rats 68 a. Weight g a i n 68 b. C o l o n i c temperature 68 c. P a n c r e a t i c morphology 68 2. H y p e r i n s u l i n e m i a i n S u c k l i n g and Weanling f a / f a Rats 78 a. F a s t i n g plasma i n s u l i n 78 b. G l u c o s e - s t i m u l a t e d plasma i n s u l i n 78 c. F a s t i n g plasma GIP 78 d. The e f f e c t of GIP on 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 from the i n v i t r o pancreas..78 e. The e f f e c t of GIP on i n s u l i n r e l e a s e under b a s a l glucose c o n d i t i o n s i n the p e r f u s e d pancreas 79 3. A d u l t r a t s 86 a. P a n c r e a t i c morphology 86 b. Q u a n t i f i c a t i o n of GIP c e l l number 86 c. O r a l glucose t o l e r a n c e i n young (8-12-wk-old) a d u l t r a t s 86 d. O r a l glucose t o l e r a n c e i n o l d e r (6-8-month-old) a d u l t r a t s 87 e. Intravenous glucose t o l e r a n c e 88 f . The e f f e c t of a GIP g r a d i e n t on the i n s u l i n response to glucose of the p e r f u s e d pancreas o f lea n and obese r a t s 104 g(. The e f f e c t o f GIP on the i n s u l i n response of the p e r f u s e d pancreas of lean and obese r a t s t o a glucose g r a d i e n t 104 h. The e f f e c t o f GIP on the i n s u l i n response of the p e r f u s e d pancreas of le a n and obese r a t s t o b a s a l glucose 105 i . The e f f e c t o f a GIP g r a d i e n t on the i n s u l i n response to b a s a l glucose of the p e r f u s e d pancreas of l e a n and obese r a t s 105 IV. DISCUSSION 115 CHAPTER TWO: THE ROLE OF THE CHOLINERGIC NERVOUS SYSTEM IN THE DEVELOPMENT AND MAINTENANCE OF HYPERINSULINEMIA IN OBESE ZUCKER RATS 128 I. INTRODUCTION 128 I I . METHODS 131 I I I . RESULTS 131 A. S u c k l i n g Rats 131 1. The E f f e c t of A c e t y l c h o l i n e on I n s u l i n S e c r e t i o n of S u c k l i n g Rats under Hyperglycemic C o n d i t i o n s 131 B. A d u l t r a t s 134 1. I n s u l i n S e c r e t i o n from the I s o l a t e d Perfused Pancreas 134 x i a. The e f f e c t o f a t r o p i n e on 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 of l e a n and obese r a t s 134 b. The e f f e c t of hexamethonium on 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 of l e a n and obese r a t s 134 c. The e f f e c t of a t r o p i n e on 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 L - a r g i n i n e 135 d. The e f f e c t of a t r o p i n e on i n s u l i n r e l e a s e of le a n and obese r a t s s t i m u l a t e d by glucose p l u s GIP 135 e. The e f f e c t of hexamethonium on i n s u l i n r e l e a s e of l e a n and obese r a t s s t i m u l a t e d by gl u c o s e p l u s GIP 136 2. G a s t r i n S e c r e t i o n i n Lean and Obese Rats: C h o l i n e r g i c I n f l u e n c e 147 a. F a s t i n g plasma g a s t r i n 147 b. In v i t r o g a s t r i n r e l e a s e from the i s o l a t e d stomach of l e a n and obese Zucker r a t s 149 i . B a s a l g a s t r i n r e l e a s e 149 i i . The e f f e c t of a t r o p i n e on b a s a l g a s t r i n r e l e a s e of l e a n and obese Zucker r a t s . . . 1 4 9 i i i . The e f f e c t o f a t r o p i n e on v a g a l l y -s t i m u l a t e d g a s t r i n r e l e a s e of l e a n and obese Zucker r a t s 149 c. B a s a l s o m a t o s t a t i n r e l e a s e from the x i i i s o l a t e d stomach of l e a n and obese Zucker r a t s 157 d. A n t r a l c e l l morphology 160 IV. DISCUSSION 166 CHAPTER THREE: THE EFFECT ON THE ENTEROINSULAR AXIS OF JEJUNOILEAL BYPASS SURGERY IN ZUCKER RATS 174 I. INTRODUCTION 174 I I . METHODS 177 A. J e j u n o i l e a l Bypass Surgery 177 1. "Conventional" S u r g i c a l Technique 177 2. A Revised S u r g i c a l Approach to J e j u n o i l e a l Bypass 177 B. Glucose Tolerance T e s t s 181 1. I n t r a j e j u n a l 181 I I I . RESULTS 182 A. The E f f e c t of J e j u n o i l e a l Bypass on the E n t e r o i n s u l a r A x i s o f Lean versus Obese Rats....182 1. A Comparison of Two Methods of J e j u n o i l e a l Bypass Surgery 182 a. O r a l glucose t o l e r a n c e 183 b. In v i t r o 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 glucose and a g r a d i e n t of GIP 183 2. The E f f e c t of JIB i n Lean versus Obese Zucker r a t s 188 a. Weight change 188 b. Weekly plasma measurements of x i i i g l u c o s e , i n s u l i n and GIP 188 c. O r a l glucose t o l e r a n c e 189 d. Plasma glucagon 191 e. In v i t r o i n s u l i n r e l e a s e i n response to glucose and a GIP g r a d i e n t 200 f. The i n v i t r o i n s u l i n response to a glucose g r a d i e n t p l u s GIP 200 g. A d a p t a t i o n of the gut f o l l o w i n g JIB i n l e a n and obese r a t s 210 i . E p i t h e l i a l h e i g h t 210 i i . Endocrine c e l l q u a n t i f i c a t i o n 210 The E f f e c t of JIB on the E n t e r o i n s u l a r A x i s i n Animals not F a s t e d Overnight One Night Per Week 221 a. Weight change 221 b. O r a l glucose t o l e r a n c e 221 c. In v i t r o i n s u l i n response to glucose p l u s GIP 222 The E f f e c t on the E n t e r i n s u l a r A x i s of P e r f u s i n g the Bypassed Loop of Lean and Obese JIB Rats with N u t r i e n t s 231 a. Weight change.... 231 b. Weekly plasma glucose measurement 232 c. Weekly i n t r a j e j u n a l g lucose s t i m u l a t i o n . . . 232 d. O r a l glucose t o l e r a n c e 233 e. In v i t r o i n s u l i n s e c r e t i o n i n response to glucose p l u s a GIP g r a d i e n t 234 x i v 5. The e f f e c t on the e n t e r o i n s u l a r a x i s of weight l o s s by s t a r v a t i o n o f l e a n and and obese Zucker r a t s 247 a. Weight change 247 b. O r a l glucose t o l e r a n c e 247 e. In v i t r o i n s u l i n s e c r e t i o n i n response to glucose p l u s a GIP g r a d i e n t 248 IV. DISCUSSION 259 CHAPTER FOUR: INCRETINS IN PLASMA—CHARACTERIZATION BY IMMUNOREACTIVITY AND BIOACTIVITY 284 I. INTRODUCTION 284 I I . METHODS 286 A. Iramunoadsorbent A f f i n i t y Chromatography 286 1. R a t i o n a l e 286 2. I n s u l i n 286 a. Antibody 286 b. P r e p a r a t i o n o f column 287 i . G e l matrix 287 i i . C o upling r e a c t i o n 288 c. E l u t i o n b u f f e r s 288 d. D e t e c t i o n of e l u t e d m a t e r i a l 289 3. Rat Plasma P r e p a r a t i o n 289 4. Bioassay 290 I I I . RESULTS 291 A. P r e p a r a t i o n of the I n s u l i n Immunoadsorbent Column 291 xv 1. C h a r a c t e r i z a t i o n of I n s u l i n Antibody 291 2. C a p a c i t y of the I n s u l i n Immuno-adsorbent Column 291 C. Measurement of I n c r e t i n A c t i v i t y i n Rat Plasma E x t r a c t s . . . 300 1. C o n t r o l s 300 a. The e f f e c t of i n c r e a s i n g p r o t e i n on p e r f u s a t e o s m o l a r i t y and i n s u l i n output from the i n v i t r o pancreas 300 b. The e f f e c t of T r i s HCl b u f f e r on i n s u l i n output from the i n v i t r o pancreas 301 2. The E f f e c t of Age on the I n c r e t i n A c t i v i t y of Wistar Rat Plasma 304 a. O r a l g lucose t o l e r a n c e 304 b. In v i t r o i n s u l i n response of o l d and young r a t s to a g lucose g r a d i e n t p l u s GIP 304 c. I n c r e t i n a c t i v i t y of plasma from o l d and young r a t s 305 i . Standard curve f o r known p o r c i n e GIP c o n c e n t r a t i o n s 305 i i . Rat plasma e x t r a c t s 306 4. I n c r e t i n A c t i v i t y of Plasma E x t r a c t s from Lean and Obese Zucker Rats 315 a. I n c r e t i n a c t i v i t y of plasma e x t r a c t s 315 IV. DISCUSSION 320 x v i SUMMARY AND CONCLUSIONS REFERENCES CITED LIST OF TABLES NUMBER TITLE PAGE I. Antibody sources ( f i r s t l a y e r ) 55 I I . Antibody sources (second l a y e r ) 56 I I I . Body weight of l e a n and obese Zucker r a t s 70 IV. I d e n t i f i c a t i o n of f a / f a r a t s by c o l o n i c temperature at 20 days 71 V. Plasma i n s u l i n c o n c e n t r a t i o n s of l e a n and obese Zucker r a t s 80 VI. Plasma GIP c o n c e n t r a t i o n s of l e a n and obese Zucker r a t s 81 VI I . I s l e t s i z e and composition i n 8-12 week o l d Zucker r a t s 93 V I I I . F a s t i n g plasma g a s t r i n c o n c e n t r a t i o n s i n lea n and obese Zucker r a t s 148 IX. Endocrine c e l l q u a n t i f i c a t i o n of the antrum i n l e a n and obese Zucker r a t s 161 X. Body weight changes i n l e a n and obese Zucker r a t s f o l l o w i n g j e j u n o i l e a l bypass surgery 192 XI. G l u c a g o n - l i k e immunoreactivity (GLI) of l e a n and obese r a t s f i v e weeks f o l l o w i n g j e j u n o i l e a l bypass surgery 199 XII. E p i t h e l i a l h e i g h t i n l e a n and obese r a t s t r e a t e d with j e j u n o i l e a l bypass surgery 212 X I I I . Q u a n t i f i c a t i o n of endocrine c e l l s i n the x v i i i jejunum of le a n and obese JIB r a t s 219 XIV. ( Q u a n t i f i c a t i o n of endocrine c e l l s i n the ileum o f l e a n and obese JIB r a t s 220 XV. A summary of the e f f e c t s on the e n t e r o i n s u l a r a x i s o f JIB, JIB-NS, JIB-L and f a s t i n g to 80% of the o r i g i n a l body weight compared t o c o n t r o l s i n l e a n and obese Zucker r a t s 269 XVI. I n s u l i n b i n d i n g to an i n s u l i n immunoadsorbent column 299 XVII. The e f f e c t of p e r f u s a t e o s m o l a r i t y on i n s u l i n output from the i s o l a t e d , p e r f u s e d r a t pancreas 302 XVIII. The e f f e c t of T r i s - H C l on i n s u l i n output from the i s o l a t e d , p e r f u s e d r a t pancreas 303 XIX. A comparison of f a s t e d young (2 month) and o l d (6 month) Wistar r a t s 307 XX. Determination of the i n c r e t i n a c t i v i t y of r a t plasma e x t r a c t s 314 XXI. Plasma e x t r a c t s from l e a n and obese Zucker r a t s : B i o a s s a y a b l e i n c r e t i n vs radioimmunoassayable GIP 317 x i x LIST OF FIGURES NUMBER TITLE PAGE 1. Apparatus f o r the p e r f u s i o n o f the i s o l a t e d r a t pancreas 44 2. P a n c r e a t i c i s l e t s of 7-day-old Zucker pups s t a i n e d f o r i n s u l i n 72 3. P a n c r e a t i c i s l e t s of 21-day-old Zucker pups s t a i n e d f o r i n s u l i n 74 4. P a n c r e a t i c i s l e t area i n 7-day-old l e a n and obese Zucker pups 76 5. The response of the i s o l a t e d pancreas of Zucker pups to glucose p l u s GIP 82 6. The response of the i s o l a t e d pancreas of Zucker pups to b a s a l glucose p l u s GIP 84 7. P a n c r e a t i c i s l e t s o f a d u l t Zucker r a t s 90 8. GIP-containing c e l l s i n Zucker r a t s 94 9. The plasma g l u c o s e , i n s u l i n and GIP responses to o r a l glucose i n young a d u l t Zucker r a t s 96 10. The plasma g l u c o s e , i n s u l i n and GIP responses to o r a l glucose i n o l d a d u l t Zucker r a t s 98 11. The plasma glucose and i n s u l i n responses to intravenous glucose i n young a d u l t Zucker rats....100 12. The i n t e g r a t e d g l u c o s e , i n s u l i n and GIP responses to o r a l and intravenous glucose i n a d u l t Zucker r a t s 102 13. The i n s u l i n response of the i s o l a t e d pancreas of Zucker r a t s to glucose i n the absence or presence of a GIP g r a d i e n t 107 14. The i n s u l i n response of the i s o l a t e d pancreas o f Zucker r a t s to a glucose g r a d i e n t alone or i n the presence of GIP 109 15. The i n s u l i n response of the i s o l a t e d pancreas of Zucker r a t s to xx b a s a l glucose i n the presence of GIP 111 16. The i n s u l i n response of the i s o l a t e d pancreas of Zucker r a t s to b a s a l glucose i n the presence of a GIP g r a d i e n t . . . 113 17. The response of the i s o l a t e d pancreas of Zucker pups to glucose p l u s a c e t y l c h o l i n e 132 18. The i n s u l i n response of the i s o l a t e d pancreas of Zucker r a t s to glucose i n the presence of a t r o p i n e 137 19. The i n s u l i n response of the i s o l a t e d pancreas of Zucker r a t s to glucose i n the presence of hexamethonium 139 20. The i n s u l i n response of the i s o l a t e d pancreas of Zucker r a t s t o a r g i n i n e i n the presence of a t r o p i n e 141 21. The i n s u l i n response of the i s o l a t e d pancreas of Zucker r a t s to glucose p l u s GIP i n the presence of a t r o p i n e 143 22. The i n s u l i n response of the i s o l a t e d pancreas of Zucker r a t s to glucose p l u s GIP i n the presence of hexamethonium 145 23. G a s t r i n s e c r e t i o n from the i s o l a t e d stomach of Zucker r a t s under b a s a l c o n d i t i o n s 151 24. The e f f e c t of a t r o p i n e on b a s a l g a s t r i n s e c r e t i o n from the i s o l a t e d stomach of Zucker r a t s 153 25. The e f f e c t of a t r o p i n e on g a s t r i n s e c r e t i o n e l i c i t e d by s t i m u l a t i o n of the vag a l trunks from the i s o l a t e d stomach of Zucker r a t s 155 26. Somatostatin r e l e a s e from the i s o l a t e d stomach of Zucker r a t s under b a s a l c o n d i t i o n s 158 27. A n t r a l endocrine c e l l s c o n t a i n i n g g a s t r i n i n Zucker r a t s 162 28. A n t r a l endocrine c e l l s c o n t a i n i n g s o m a t o s t a t i n i n Zucker r a t s 164 29. Two methods of j e j u n o i l e a l bypass surgery 179 x x i 30. The gl u c o s e , i n s u l i n and GIP responses to o r a l glucose i n Wistar r a t s t r e a t e d with two types o f JIB surgery 184 31. The i n s u l i n s e c r e t o r y response o f the i s o l a t e d pancreas t o glu c o s e p l u s a GIP g r a d i e n t i n Wistar r a t s t r e a t e d with two types of JIB surgery 186 32. F a s t i n g plasma g l u c o s e , i n s u l i n and GIP c o n c e n t r a t i o n s i n Zucker r a t s t r e a t e d with j e j u n o i l e a l bypass 193 33. The g l u c o s e , i n s u l i n and GIP responses to o r a l glucose o f l e a n JIB Zucker r a t s 195 34. The g l u c o s e , i n s u l i n and GIP responses t o o r a l g l ucose o f obese JIB Zucker r a t s 197 35. The response of the i s o l a t e d pancreas to glucose p l u s a GIP g r a d i e n t o f JIB Zucker r a t s 202 36. The response of the i s o l a t e d pancreas to a glucose g r a d i e n t p l u s GIP i n l e a n JIB Zucker r a t s 204 37. The response of the i s o l a t e d pancreas to a glu c o s e g r a d i e n t p l u s GIP i n obese JIB Zucker r a t s 206 38. The response of the i s o l a t e d pancreas to a glucose g r a d i e n t p l u s GIP i n obese JIB Zucker r a t s compared to a glucose g r a d i e n t alone i n obese c o n t r o l animals 208 39. Endocrine c e l l s c o n t a i n i n g enteroglucagon i n JIB Zucker r a t s 213 40. Endocrine c e l l s c o n t a i n i n g c h o l e c y s t o k i n i n i n JIB Zucker r a t s 215 41. Endocrine c e l l s c o n t a i n i n g n e u r o t e n s i n i n JIB Zucker r a t s 217 42. The e f f e c t o f not f a s t i n g l e a n JIB-NS r a t s one n i g h t per week on the g l u c o s e , i n s u l i n and GIP responses to o r a l glucose ....223 43. The e f f e c t of not f a s t i n g obese JIB-NS r a t s one n i g h t per week on the glu c o s e , i n s u l i n and GIP responses to o r a l g l u c o s e 225 x x i i 44. The i n t e g r a t e d g l u c o s e , i n s u l i n and GIP responses to o r a l g l u c o s e i n JIB r a t s not f a s t e d or f a s t e d one n i g h t per week 227 45. The e f f e c t of not f a s t i n g JIB-NS Zucker r a t s on the response of the i s o l a t e d pancreas to glucose p l u s a GIP g r a d i e n t 229 46. The e f f e c t of d a i l y n u t r i e n t i n f u s i o n i n t o the bypassed loop of JIB-L Zucker r a t s on plasma glucose l e v e l s 235 47. Changes i n plasma g l u c o s e , i n s u l i n and GIP c o n c e n t r a t i o n s f o l l o w i n g a d m i n i s t r a t i o n of glucose v i a the jejunostomy i n Zucker r a t s 237 48. The glucose and i n s u l i n response to o r a l glucose i n l e a n JIB-L r a t s 239 49. The glucose and i n s u l i n response to o r a l glucose i n obese JIB-L r a t s 241 50. The i n t e g r a t e d g lucose and i n s u l i n response to o r a l g lucose i n JIB-L Zucker r a t s 243 51. The response of the i s o l a t e d pancreas to glucose p l u s a GIP g r a d i e n t JIB-L Zucker r a t s 245 52. D a i l y weight l o s s of l e a n and obese Zucker r a t s d e p r i v e d of food 249 53. The e f f e c t of s t a r v a t i o n on the g l u c o s e , i n s u l i n and GIP responses to o r a l g lucose i n l e a n r a t s 251 54. The e f f e c t of s t a r v a t i o n on the g l u c o s e , i n s u l i n and GIP responses to o r a l g lucose i n obese r a t s 253 55. The e f f e c t of s t a r v a t i o n t o on the i n t e g r a t e d g l u c o s e , i n s u l i n and GIP responses to o r a l glucose 255 56. The e f f e c t of s t a r v a t i o n on the response of the i s o l a t e d pancreas to glucose p l u s a GIP g r a d i e n t i n Zucker r a t s 257 57. C h a r a c t e r i z a t i o n of i n s u l i n a n t i s e r a GP7 f o r use i n immunoadsorbent chromatography 293 x x i i i 58. Measurement of antibody t i t r e o f GP7 before and a f t e r c o u p l i n g t o Sepharose 4B-CL with cyanogen bromide 295 59. Te s t of the i n s u l i n immunoadsorbent column using pure p o r c i n e i n s u l i n 297 60. The plasma glucose and GIP responses to o r a l g l ucose i n 6-month-old compared to 2-month-o l d Wistar r a t s 308 61. The i n s u l i n response of the i s o l a t e d pancreas of 6-month-old compared to 2-month-old Wistar r a t s to a glucose g r a d i e n t i n the presence of GIP 310 62. The i n c r e t i n a c t i v i t y of plasma e x t r a c t s from 6-month-old compared to 2-month-old Wistar r a t s 312 63. The i n c r e t i n a c t i v i t y o f plasma e x t r a c t s from Zucker r a t s 318 x x i v ACKNOWLEDGEMENTS I would l i k e to thank my a d v i s o r , Dr. Raymond Pederson for h i s f r i e n d s h i p , p a t i e n c e and encouragement duri n g the past f i v e y e a r s . I a l s o wish to acknowledge the a s s i s t a n c e of Drs. John Brown and A l i s o n Buchan, without whose he l p t h i s t h e s i s would not have been p o s s i b l e . The c o n t i n u i n g f r i e n d s h i p and a s s i s t a n c e of Karen Tubesing was g r e a t l y a p p r e c i a t e d , e s p e c i a l l y her management of the Zucker r a t colo n y . I wish to thank Kerry Ann Green, Dr. Mark Meloche and Jeannie Sharp f o r t h e i r a s s i s t a n c e with v a r i o u s aspects of t h i s t h e s i s . P r e p a r a t i o n of the i l l u s t r a t i o n s by Judy Nairn i s g r a t e f u l l y acknowledged. F i n a n c i a l support i n the form of a Stu d e n t s h i p from the Medical Research C o u n c i l of Canada i s acknowledged. F i n a l l y , I would l i k e to thank my husband, Pat, f o r h i s pa t i e n c e and support d u r i n g the tenure of t h i s r e s e a r c h . xxv GENERAL INTRODUCTION Obesity i s a complex syndrome r e s u l t i n g from prolonged i n g e s t i o n o f c a l o r i e s i n excess of the energy requirement of the i n d i v i d u a l (Bray, 1978) . I t has been d e f i n e d as "an i n c r e a s e i n the percentage c o n t r i b u t i o n o f body f a t to t o t a l body weight" (Bray and York, 1971). Both g e n e t i c background and environmental f a c t o r s may c o n t r i b u t e to i d i o p a t h i c o b e s i t y i n humans. The concept o f " t h r i f t y mutants" has been d i s c u s s e d by C a h i l l (1978) and Coleman (1978). I t was hypothesized by these workers t h a t the s u r v i v a l o f p r i m i t i v e man was enhanced i n i n d i v i d u a l s p ossessing genes coding f o r i n c r e a s e d metabolic e f f i c i e n c y . Thus, i n times o f p l e n t y these " t h r i f t y mutants" would s t o r e more energy than t h e i r g e n e t i c a l l y " l e a n " c ompatriots, whereas i n p e r i o d s of famine these i n d i v i d u a l s would r e q u i r e l e s s food i n t a k e to maintain p o s i t i v e energy balance. In modern i n d u s t r i a l s o c i e t y where food i s p l e n t i f u l and the l i f e s t y l e sedentary, i n d i v i d u a l s with " t h r i f t y " genes tend to become obese. H y p e r i n s u l i n e m i a i s a common denominator i n many cases of o b e s i t y . Clues p o i n t i n g to a b n o r m a l i t i e s i n p a n c r e a t i c f u n c t i o n were observed by many groups p r i o r to a c t u a l measurement of plasma i n s u l i n l e v e l s i n obese s u b j e c t s , but the s i g n i f i c a n c e o f these o b s e r v a t i o n s was not r e a l i z e d . Glucose i n t o l e r a n c e was a s s o c i a t e d with many 1 obese p a t i e n t s ( O g i l v i e , 1935) and a strong c o r r e l a t i o n between o b e s i t y and subsequent development of m a t u r i t y -onset d i a b e t e s was observed (Newburgh and Conn, 1939). Newburgh b e l i e v e d that o b e s i t y - i n d u c e d d i a b e t e s d i f f e r e d from j u v e n i l e - o n s e t d i a b e t e s , s i n c e weight l o s s a l l e v i a t e d the need f o r exogenous i n s u l i n i n the l a t t e r and improved glucose t o l e r a n c e (Newburgh, 1942). In 1944 Conn concluded t h a t e l e v a t e d i n s u l i n l e v e l s caused frequent bouts of hypoglycemia, the e f f e c t s of which were a l l e v i a t e d by i n c r e a s e d food i n t a k e , r e s u l t i n g i n weight g a i n . T h i s hypothesis was c r i t i c i z e d by Rhynearson and Gastineau (1949), who p o i n t e d out t h a t obese p a t i e n t s were r a r e l y hypoglycemic and i n f a c t tended to be normo- or hyperglycemic. Mayer (1953) f u r t h e r d i s c r e d i t e d the importance of " h y p e r i n s u l i n i s m " i n o b e s i t y because at t h a t time h y p e r i n s u l i n i s m was synonymous with hypoglycemia. Thus by d e f i n i t i o n , h y p e r i n s u l i n i s m c o u l d not c o e x i s t with normo- or hyperglycemia. He d i d acknowledge the probable importance of i n s u l i n i n r e g u l a t i o n of energy balance and concluded t h a t the e q u i l i b r i u m between i n s u l i n and the "alpha c e l l s e c r e t i o n " (glucagon) was probably important i n normal carbohydrate metabolism. A secondary r o l e f o r i n s u l i n i n o b e s i t y was p o s t u l a t e d by G e l v i n and McGavack (1957) . They t h e o r i z e d t h a t the i n c r e a s e i n f a t d e p o s i t i o n observed i n o b e s i t y i n c r e a s e d the demand f o r i n s u l i n i n adipose t i s s u e . T h i s would r e s u l t i n a r e l a t i v e i n s u l i n d e f i c i t at other g l u c o s e - m e t a b o l i s i n g 2 t i s s u e s , c r e a t i n g symptoms of d i a b e t e s . F o l l o w i n g the development of a radioimmunoassay s e n s i t i v e enough to determine plasma i n s u l i n c o n c e n t r a t i o n s (Yalow and Berson, 1960) h y p e r i n s u l i n e m i a was demonstrated both b a s a l l y and f o l l o w i n g an intravenous glucose l o a d i n n o n - d i a b e t i c obese p a t i e n t s with normal glucose t o l e r a n c e (Karam et a l , 1963). I t was s p e c u l a t e d t h a t e l e v a t e d i n s u l i n l e v e l s were compensatory f o r p e r i p h e r a l r e s i s t a n c e to endogenous i n s u l i n . I n s u l i n c o n t r o l s the metabolism of both carbohydrates and l i p i d s , p r i m a r i l y by i t s a c t i o n s on adipose and l i v e r c e l l s . Thus, changes 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 or t a r g e t t i s s u e s e n s i t i v i t y c o u l d r e s u l t i n a l t e r a t i o n s i n carbohydrate and l i p i d metabolism. Jeanrenaud (1979) reviewed the r e l a t i o n s h i p between h y p e r i n s u l i n e m i a and i n s u l i n r e s i s t a n c e i n o b e s i t y . E a r l y i n the development of o b e s i t y , h y p e r i n s u l i n e m i a has major e f f e c t s on carbohydrate and f a t metabolism i n the l i v e r and adipose t i s s u e , r e s u l t i n g i n i n c r e a s e d f a t d e p o s i t i o n i n both t i s s u e s . Reduction of c i r c u l a t i n g i n s u l i n l e v e l s r e s u l t s i n p a r t i a l r e v e r s a l of the l i v e r and adipocyte f a t accumulation, i n d i c a t i n g a c a u s a l r e l a t i o n s h i p (Assimacopoulos-Jeannet and Jeanrenaud, 1976). While the e f f e c t s of h y p e r i n s u l i n e m i a on metabolism have been w e l l documented, the cause of e l e v a t e d plasma i n s u l i n l e v e l s i n o b e s i t y has not been e l u c i d a t e d . Four g e n e r a l hypotheses have been pursued. The f i r s t i s t hat a 3 primary p e r i p h e r a l i n s u l i n r e s i s t a n c e may r e s u l t i n compensatory h y p e r s e c r e t i o n of i n s u l i n i n order t o maintain normal glucose uptake. I n s u l i n r e s i s t a n c e has been d e f i n e d as the decreased a b i l i t y o f exogenous or endogenous i n s u l i n to reduce plasma glucose l e v e l s and i s accompanied by b a s a l and/or g l u c o s e - s t i m u l a t e d h y p e r i n s u l i n e m i a . Hyperglycemia may be observed (Salans et a l , 1983) . C e l l u l a r i n s u l i n r e s i s t a n c e may r e s u l t from a decrease i n membrane i n s u l i n r e c e p t o r number or a f f i n i t y (Kahn, 1980; Bar, 1983). Recent evidence suggests that i n s u l i n r e s i s t a n c e i n o b e s i t y a r i s e s as a r e s u l t o f prolonged exposure to e l e v a t e d i n s u l i n l e v e l s . Obese humans d i s p l a y p r o g r e s s i v e i n s u l i n r e s i s t a n c e i n which a receptor d e f e c t i s a s s o c i a t e d with m i l d h y p e r i n s u l i n e m i a but as the obese s t a t e p e r s i s t s and h y p e r i n s u l i n e m i a worsens, a p o s t - r e c e p t o r d e f e c t appears (Ole f s k y , 1981). I n s u l i n s e n s i t i v i t y can be i n c r e a s e d by r e d u c t i o n of i n s u l i n l e v e l s f o l l o w i n g weight r e d u c t i o n (Rabinowitz, 1970; Salans and Cushman, 1978; Salans et a l , 1968; Salans et a l , 1974). Euglycemic i n s u l i n clamp s t u d i e s i n normal i n d i v i d u a l s demonstrated t h a t decreased i n s u l i n responsiveness c o u l d be induced by e l e v a t e d i n s u l i n l e v e l s (Soman and DeFronzo, 1980) . On the other hand, B o l i n d e r et a l (1983) have shown i n non-obese s u b j e c t s t h a t i n s u l i n responsiveness decreases with age due to a decrease i n i n s u l i n r e c e p t o r number t h a t i s not c o r r e l a t e d with 4 changes i n plasma i n s u l i n c o n c e n t r a t i o n . Y k i - J a r v i n e n and K o i v i s t o (1983) were able to c o r r e l a t e i n s u l i n s e n s i t i v i t y d i r e c t l y with muscle mass and i n v e r s e l y with a d i p o s i t y i n normal weight s u b j e c t s . Thus, the r o l e of i n s u l i n r e s i s t a n c e i n development of h y p e r i n s u l i n e m i a remains e q u i v o c a l . H y p e r i n s u l i n e m i a may a l s o be due to a primary d e f e c t of the p a n c r e a t i c i s l e t s . M o r p h o l o g i c a l s t u d i e s have reve a l e d the presence o f enlarged i s l e t s due to h y p e r p l a s i a and hypertrophy of the i n s u l i n - p r o d u c i n g B-c e l l s i n man ( O g i l v i e , 1933) and experimental animals, i n c l u d i n g the obese hyperglycemic mouse ( B l e i s c h , 1952; Gepts et a l , 1960) and the obese Zucker r a t (Shino et a l , 1973; Larsson, 1978). I s l e t s from l e a n mice implanted i n t o obese (ob/ob) l i t t e r m a t e s r e s u l t e d i n n o r m a l i z a t i o n of plasma i n s u l i n and weight l o s s i n ob/ob mice ( S t r a u t z , 1970), l e a d i n g to the hypothesis that obese mice la c k e d an i s l e t f a c t o r e s s e n t i a l to normal metabolism. Since the mice used i n t h i s study were a l r e a d y o v e r t l y obese and h y p e r i n s u l i n e m i c i t c o u l d not be concluded that t h i s d e f e c t was primary. P a n c r e a t i c p o l y p e p t i d e was i m p l i c a t e d as the m i s s i n g f a c t o r i n a second study (Gates and Lazarus, 1977) because both i s l e t implant and d a i l y p a n c r e a t i c p o l y p e p t i d e i n j e c t i o n normalized weight g a i n and plasma i n s u l i n l e v e l s of the New Zealand obese mouse. A primary p a n c r e a t i c l e s i o n to the B - c e l l i t s e l f c o u l d cause h y p e r i n s u l i n e m i a by (a) i n c r e a s e d s e c r e t i o n due to 5 i n c r e a s e d c e l l mass, (b) i n c r e a s e d s e n s i t i v i t y to a g i v e n s t i m u l u s perhaps due to a r e c e p t o r phenomenon or (c) i n c r e a s e d responsiveness to a g i v e n s t i m u l u s at a p o s t -receptor s i t e . These a l t e r n a t i v e s have not been r i g o r o u s l y examined, although i s o l a t e d i s l e t s t u d i e s i n humans (Jahr et a l , 1983) and v a r i o u s animal models, i n c l u d i n g the obese Zucker r a t (Hayek, 1 9 8 0 ; Hayek and Woodside, 1979) and the hyperglycemic obese (ob/ob) mouse ( B e l o f f - C h a i n et a l , 1977) have demonstrated a c o r r e l a t i o n between i n s u l i n s e c r e t i o n and i s l e t s i z e i n some cases and i n c r e a s e d s e n s i t i v i t y to s t i m u l a t i o n i n others i e . a s h i f t to the l e f t o f the dose-response curve. However, these s t u d i e s do not r u l e out the p o s s i b l e i n f l u e n c e of the other i s l e t p e p t i d e s (glucagon, s o m a t o s t a t i n and p a n c r e a t i c p o l y p e p t i d e ) on i n s u l i n r e l e a s e . The pancreas r e c e i v e s i n p u t from the autonomic nervous system (TANS) . In g e n e r a l , i n s u l i n s e c r e t i o n i s p o t e n t i a t e d by c h o l i n e r g i c and b e t a - a d r e n e r g i c inputs and i n h i b i t e d by a l p h a - a d r e n e r g i c s t i m u l i . An i n c r e a s e i n parasympathetic a c t i v i t y or a decrease i n sympathetic a c t i v i t y c o u l d r e s u l t i n an e l e v a t i o n o f i n s u l i n s e c r e t i o n . L e s i o n of the ventromedial hypothalamus i n r a t s r e s u l t s i n r a p i d onset of h y p e r i n s u l i n e m i a which can be e l i m i n a t e d by b i l a t e r a l subdiaphragmatic vagotomy (Berthoud and Jeanrenaud, 1 9 7 9 ). In VMH-lesioned r a t s h y p e r i n s u l i n e m i a and i n c r e a s e d a d i p o s i t y p e r s i s t i n the absence of hyperphagia (Rohner-Jeanrenaud and Jeanrenaud, 6 1980a; Rohner-Jeanrenaud and Jeanrenaud, 1980b). S i m i l a r l y , i t was r e p o r t e d t h a t a t r o p i n e i n f u s i o n p r i o r to intravenous glucose a d m i n i s t r a t i o n i n v i v o , or vagotomy p r i o r to p e r f u s i o n of the i s o l a t e d pancreas, reduced the i n s u l i n response of obese Zucker r a t s to l e v e l s comparable to those o f l e a n c o n t r o l s . E x c e s s i v e s t i m u l a t i o n of the pancreas by a c h o l i n e r g i c mechanism was proposed as c o n t r i b u t i n g to the observed h y p e r s e c r e t i o n of i n s u l i n (Rohner-Jeanrenaud e t a l , 1981). While r e v e r s i n g changes i n p a n c r e a t i c i n s u l i n s e c r e t i o n evoked by VMH-lesions, vagotomy has l i t t l e e f f e c t on plasma i n s u l i n i n normal r a t s . A p a u c i t y of data concerning the r o l e of the ANS i n humans prevents comment on i t s r o l e i n the obese s t a t e . P a t i e n t s with a c c i d e n t a l hypothalamic l e s i o n s have been d e s c r i b e d (Bray and G a l l a g h e r , 1975) but these examples are not r e p r e s e n t a t i v e of the g e n e r a l obese p o p u l a t i o n . L a s t l y , i n s u l i n h y p e r s e c r e t i o n c o u l d be e x p l a i n e d by the presence of an o v e r a c t i v e e n t e r o i n s u l a r a x i s i n obese s u b j e c t s . The term " e n t e r o i n s u l a r a x i s , " i n t r o d u c e d by Unger and E i s e n t r a u t (1969), o r i g i n a l l y r e f e r r e d to the e f f e c t o f gut hormones on p a n c r e a t i c endocrine f u n c t i o n . The d e f i n i t i o n of the term has s i n c e been m o d i f i e d to i n c l u d e the e f f e c t s of n u t r i e n t s , hormones and n e u r a l components ( C r e u t z f e l d t , 1979) . As e a r l y as 1906 (Moore, Edie and Abram, 1906) i t was observed t h a t duodenal-j e j u n a l mucosal e x t r a c t s c o u l d reduce the g l y c o s u r i a of d i a b e t i c p a t i e n t s . Labarre' (1932) p o s t u l a t e d the 7 e x i s t e n c e of a hormone r e l e a s e d from g a s t r i c mucosa by glucose which i n f l u e n c e d i n s u l i n s e c r e t i o n and which was d i s t i n c t from s e c r e t i n . He termed t h i s unknown hormone " i n c r e t i n " . However, the h y p o t h e s i s f e l l from favour when s e v e r e l y c r i t i c i z e d by Loew et a l (1940) who c i t e d the i n c o n s i s t e n c y of r e s u l t s i n the l i t e r a t u r e . M e t h o d o l o g i c a l l i m i t a t i o n s , e s p e c i a l l y l a c k of p u r i f i c a t i o n techniques and an absence of s e n s i t i v e assays f o r small p e p t i d e s , prevented workers from c o n f i r m i n g the e x i s t e n c e of i n c r e t i n f o r t h i r t y years f o l l o w i n g LaBarre's hypothesis. As was the case f o r the d i s c o v e r y of h y p e r i n s u l i n e m i a i n o b e s i t y , the search f o r an i n c r e t i n hormone was r e v i v e d by the development of the radioimmunoassay. E l r i c k et a l (1964) compared the i n s u l i n response obtained i n o r a l compared to intravenous glucose t o l e r a n c e t e s t s and observed that o r a l glucose a d m i n i s t r a t i o n r e s u l t e d i n a l a r g e r and longer i n s u l i n response. The involvement of an i n t e s t i n a l or l i v e r f a c t o r i n i n s u l i n r e l e a s e was suggested. M c l n t y r e et a l (1964) almost s i m u l t a n e o u s l y r e p o r t e d enhanced i n s u l i n s e c r e t i o n f o l l o w i n g i n t r a j e j u n a l glucose i n f u s i o n compared to intravenous a d m i n i s t r a t i o n , d e s p i t e a c h i e v i n g lower plasma glucose l e v e l s v i a the i n t r a j e j u n a l route. The authors supported LaBarre's o r i g i n a l h y p o thesis and suggested t h a t the hormonal f a c t o r was r e l e a s e d from the jejunum dur i n g glucose a b s o r p t i o n . A t h i r d important experiment was performed by Dupre et a l (1964) , which 8 r e v i v e d i n t e r e s t i n the i n s u l i n o t r o p i c a c t i v i t y of mucosal e x t r a c t s . An " i n c r e t i n " can be d e f i n e d as an endocrine t r a n s m i t t e r which s t i m u l a t e s i n s u l i n r e l e a s e . A c c o r d i n g l y , c r i t e r i a have been developed f o r c l a s s i f i c a t i o n of a substance as an i n c r e t i n . The endocrine substance must be (1) r e l e a s e d from the gut by n u t r i e n t s , p a r t i c u l a r l y c a rbohydrates, and (2) s t i m u l a t e i n s u l i n r e l e a s e i n the presence of glucose administered exogenously i n p h y s i o l o g i c a l l y a c h i e v a b l e c o n c e n t r a t i o n s ( C r e u t z f e l d t , 1979). I n i t i a l l y the i n s u l i n - r e l e a s i n g a c t i v i t i e s of known gut hormones were i n v e s t i g a t e d . The primary f u n c t i o n of s e c r e t i n i s the s t i m u l a t i o n of p a n c r e a t i c b i c a r b o n a t e and water s e c r e t i o n f o l l o w i n g a meal. In humans, exogenous s e c r e t i n was shown to i n c r e a s e plasma i n s u l i n l e v e l s when administered with glucose (Dupre et al,1969) but was not r e l e a s e d by glucose i n g e s t i o n (Chisholm et a l , 1969). In f a s t e d dogs, s e c r e t i n induced an i n c r e a s e i n i n s u l i n s e c r e t i o n with no e f f e c t on blood glucose l e v e l s (Unger et a l , 1967). Brown and Otte (1975) reviewed the evidence f o r s e c r e t i n as an i n c r e t i n and concluded t h a t the observed e f f e c t s were probably pharmacologic. More r e c e n t l y , when 99% pure s e c r e t i n was i n f u s e d i n t o the i s o l a t e d r a t pancreas at doses of 1 or 5 ng/ml, no p o t e n t i a t i o n of i n s u l i n r e l e a s e was observed, e i t h e r at b a s a l or e l e v a t e d glucose 9 c o n c e n t r a t i o n s . I t was concluded t h a t s e c r e t i n was not important as an i n c r e t i n (Pederson and Brown, 1979). G a s t r i n i s r e l e a s e d from the antrum and s t i m u l a t e s g a s t r i c a c i d s e c r e t i o n . A crude g a s t r i n e x t r a c t had a t r a n s i e n t s t i m u l a t o r y e f f e c t on i n s u l i n r e l e a s e i n dogs (Unger et a l , 1967). Human s y n t h e t i c g a s t r i n II produced a small r i s e i n plasma i n s u l i n when i n f u s e d s i m u l t a n e o u s l y with glucose i n humans (Dupre* et a l , 1969). However, o r a l glucose i n c r e a s e d plasma g a s t r i n o n l y s l i g h t l y and doses i n excess of p h y s i o l o g i c a l l y a c h i e v a b l e l e v e l s were r e q u i r e d to i n c r e a s e plasma i n s u l i n l e v e l s e i t h e r b a s a l l y or i n the g l u c o s e - s t i m u l a t e d s t a t e (Rehfeld and S t a d i l , 1973). S u p r a p h y s i o l o g i c g a s t r i n l e v e l s were a l s o r e q u i r e d to s t i m u l a t e i n s u l i n r e l e a s e from the i s o l a t e d p e r f u s e d p o r c i n e pancreas (Jensen e t a l , 1980). Cholecystokinin-pancreozymin (CCK) has p h y s i o l o g i c a l l y important e f f e c t s on g a l l bladder emptying and s e c r e t i o n of p a n c r e a t i c enzymes, as w e l l as a p o s s i b l e r o l e as a neuropeptide. In f a s t e d dogs l a r g e doses of " h i g h l y p u r i f i e d " CCK i n c r e a s e d both plasma glucose and i n s u l i n c o n c e n t r a t i o n s . Impure CCK was found to be more potent i n r e l e a s i n g i n s u l i n i n glucose-primed compared to f a s t e d dogs (Meade et a l , 1967) but e f f e c t s were t r a n s i e n t . In man CCK p l u s glucose caused a s u s t a i n e d i n c r e a s e i n i n s u l i n r e l e a s e but the CCK dose was l a r g e enough to cause " e p i g a s t r i c d i s c o m f o r t " (Dupre et a l , 1969). When purer p r e p a r a t i o n s became a v a i l a b l e , Hedner e t a l (1975) 10 observed t h a t o n l y impure p r e p a r a t i o n s of CCK s t i m u l a t e d i n s u l i n r e l e a s e i n f a s t e d humans. Conv e r s e l y , Pederson and Brown (1979) demonstrated i n s u l i n r e l e a s e by CCK i n the perfused r a t pancreas at doses of 1 and 5 ng/ml when glucose l e v e l s were e l e v a t e d above f a s t i n g c o n c e n t r a t i o n s . I t was noted, however, t h a t p h y s i o l o g i c a l l y a c h i e v a b l e plasma l e v e l s of CCK have not been u n e q u i v o c a l l y a s c e r t a i n e d . More r e c e n t l y i t was demonstrated t h a t the o c t a p e p t i d e of CCK (CCK8) p o t e n t i a t e d i n s u l i n r e l e a s e i n the presence of e l e v a t e d g l u c o s e l e v e l s i n the p e r f u s e d r a t pancreas. Doses used were submaximal f o r p a n c r e a t i c exocrine s e c r e t i o n (Muller et a l , 1983). However, the most potent s t i m u l i f o r CCK r e l e a s e f o l l o w i n g a meal are the d i g e s t i o n products of f a t and p r o t e i n (Go, 1978). Thus, none of these p e p t i d e s s a t i s i f i e d the c r i t e r i o n f o r an i n c r e t i n . While CCK and s e c r e t i n may s t i m u l a t e i n s u l i n s e c r e t i o n , t h e i r r e l e a s e i s not s t i m u l a t e d by glucose. Conversely, glucose r e l e a s e s s m a l l amounts of g a s t r i n but g a s t r i n has no e f f e c t on i n s u l i n s e c r e t i o n . None o f the gut hormones known i n 1969 met the c r i t e r i a to q u a l i f y f o r i n c r e t i n s t a t u s . In 1973 Dupre and h i s c o l l e a g u e s i n t r a v e n o u s l y administered a newly-discovered p e p t i d 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 (GIP), s i m u l t a n e o u s l y with intravenous glucose i n human v o l u n t e e r s . An improvement i n glucose t o l e r a n c e and an i n c r e a s e i n i n s u l i n r e l e a s e was observed compared to an i n f u s i o n of glucose alone. 11 The p e p t i d e had no e f f e c t on i n s u l i n s e c r e t i o n i n f a s t e d s u b j e c t s . R a b i n o v i t c h and Dupre (1972) a l s o demonstrated that the i n s u l i n o t r o p i c a c t i o n o f 10% pure CCK disappeared when the CCK p r e p a r a t i o n was f u r t h e r p u r i f i e d , implying that a contaminant of the impure CCK was r e s p o n s i b l e f o r i t s observed i n s u l i n r e l e a s i n g a c t i v i t y . 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 had been i s o l a t e d and i d e n t i f i e d f o l l o w i n g experiments by Brown and h i s c o l l e a g u e s i n which they demonstrated t h a t the i n h i b i t o r y a c t i o n o f 10% pure CCK p r e p a r a t i o n s on g a s t r i c a c i d s e c r e t i o n i n denervated pouches of dog stomach was decreased i n 40% pure p r e p a r a t i o n s (Brown, 1969; Brown and Pederson, 1970). The p u r i f i c a t i o n of t h i s CCK contaminant f r a c t i o n was pursued and r e p o r t e d (Brown et a l , 1970). Subsequently the p e p t i d e was shown to have 43 amino a c i d s (Brown, 1971) and sequencing of the molecule was accomplished (Brown and Dryburgh, 1971). GIP shares sequence homology with s e c r e t i n , glucagon and 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 (Dockray, 1979). The molecular weight was determined to be 5105 d a l t o n s ; a l a r g e r 8000 d a l t o n s p e c i e s was l a t e r observed i n plasma (Brown et a l , 1975) and t i s s u e e x t r a c t s (Dryburgh, 1977) although the f u n c t i o n o f t h i s l a r g e r p e p t i d e remains unknown and does not appear to be a "pro-GIP" (Otte et a l , 1984). A c o r r e c t i o n t o the sequence was r e c e n t l y p u b l i s h e d ( J o r n v a l l et a l , 1981), d e l e t i n g a glutamine r e s i d u e at p o s i t i o n 30. 12 While GIP had been shown to a l t e r both g a s t r i c a c i d and i n s u l i n s e c r e t i o n s , a number o f c r i t e r i a were necessary to e s t a b l i s h GIP as an enterogastrone and/or as an i n c r e t i n . Enterogastrone was d e f i n e d as a hormone r e l e a s e d from the g a s t r o i n t e s t i n a l t r a c t by f a t and f a t d i g e s t i o n products, which i n h i b i t e d g a s t r i c a c i d s e c r e t i o n and delayed g a s t r i c emptying (Kosaka and Lim, 1930). As e a r l i e r mentioned, i n c r e t i n must be r e l e a s e d by glucose and s t i m u l a t e i n s u l i n r e l e a s e i n the presence of hyperglycemia. A radioimmunoassay was developed (Kuzio et a l , 1974) using an antiserum r a i s e d i n guinea p i g a g a i n s t p o r c i n e 125 GIP conjugated to bovine serum albumin, and I - p o r c i n e GIP. P o r c i n e GIP, p u r i f i e d a c c o r d i n g to the method of Brown et a l (1970) was used as standard. In humans the mean f a s t i n g plasma immunoreactive GIP (IR-GIP) l e v e l was 237 pg/ml and rose to 1.2 ng/ml f o l l o w i n g a t e s t meal (Kuzio et a l , 1974). IR-GIP was shown to be r e l e a s e d by o r a l glucose i n man (Cataland et a l , 1974; C l e a t o r and Gourlay, 1975), dog (Pederson e t a l , 1975) and r a t (Pederson et a l , 1982) and by t r i g l y c e r i d e s i n man (Brown, 1973; C r o c k e t t et a l , 1976; C l e a t o r and Gourlay, 1975) and dog (Pederson e t a l , 1975) . IR-GIP r e l e a s e was a l s o r e p o r t e d to be s t i m u l a t e d by mixed amino a c i d s (Thomas et a l , 1976; Thomas et a l , 1978) and h y d r o c h l o r i c a c i d (Ebert et a l , 1979) i n the duodenum. Immunocytochemical s t u d i e s have l o c a l i z e d GIP i n the K-c e l l s of the duodenum and jejunum i n man, dog and p i g 13 (Buffa et a l , 1975; Polak et a l , 1973; S o l c i a et a l , 1975). Reports of GIP appearing i n p a n c r e a t i c A-c e l l s (Smith et a l , 1977) are b e l i e v e d to r e s u l t from c r o s s - r e a c t i v i t y of glucagon with the GIP antibody, s i n c e a r e c e n t l y d e s c r i b e d monoclonal antibody to GIP d i d not s t a i n A - c e l l s (Buchan et a l , 1982). The i n i t i a l experiments concerning the enterogastrone e f f e c t of GIP were performed i n v a g a l l y denervated pouches of dog stomach (Brown and Pederson, 1970; Pederson and Brown, 1971) . I n f u s i o n of exogenous GIP i n man, at doses approximately 3 - f o l d higher than can be achieved p h y s i o l o g i c a l l y , reduced p e n t a g a s t r i n s t i m u l a t e d a c i d s e c r e t i o n by 60 percent (C l e a t o r and Gourlay, 1975). In innervated dog stomach the enterogastrone e f f e c t of GIP was a b o l i s h e d due to the presence of c h o l i n e r g i c s t i m u l a t i o n (Soon-Shiong e t a l , 1984). Maxwell et a l (1979), using s u p r a p h y s i o l o g i c doses of exogenous GIP, d i d not observe s i g n i f i c a n t i n h i b i t i o n o f p e n t a g a s t r i n s t i m u l a t e d g a s t r i c a c i d s e c r e t i o n i n normal human v o l u n t e e r s . Anderson (1981) suggested t h a t GIP may a c t s y n e r g i s t i c a l l y with other p e p t i d e s of the glucagon f a m i l y to i n h i b i t g a s t r i c a c i d s e c r e t i o n although no r e s u l t s from experiments of t h i s type have been r e p o r t e d . Recently a mechanism f o r GIP-mediated i n h i b i t i o n of g a s t r i c a c i d s e c r e t i o n was proposed. Mcintosh et a l (1980) observed that GIP 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 i n the i s o l a t e d 14 a c e t y l c h o l i n e or v a g a l s t i m u l a t i o n . However, the exact r e l a t i o n s h i p between the hormonal and n e u r a l aspects o f the mechanism has not been e l u c i d a t e d . A r n o l d e t a l (1978) have suggested t h a t f a t - s t i m u l a t e d GIP r e l e a s e i s an e v o l u t i o n a r y remnant of a once-important a c i d i n h i b i t o r y mechanism no longer f u n c t i o n a l i n mammals. While the importance of GIP as an enterogastrone remains unresolved, i t s r o l e as an i n c r e t i n has been r e p e a t e d l y demonstrated. The glucose-dependent i n s u l i n o t r o p i c a c t i o n of GIP has been observed i n man when GIP was i n f u s e d i n t r a v e n o u s l y c o n c u r r e n t l y with glucose (Dupre, 1973) and i n glucose clamp s t u d i e s when plasma glucose was maintained at a hyperglycemic l e v e l (Anderson et a l , 1978) . In the dog i t was shown that the i n s u l i n response was augmented by o r a l g l u c o s e , i v glucose p l u s GIP i n f u s i o n , or i v glucose p l u s o r a l f a t compared to i v glucose c o n t r o l s . Plasma GIP l e v e l s a t t a i n e d during i n f u s i o n s were w i t h i n the p h y s i o l o g i c a l range (Pederson et a l , 1975) . The response of the i s o l a t e d p e r f u s e d r a t pancreas to GIP was dependent on the p r e v a i l i n g glucose c o n c e n t r a t i o n . A l s o , at glucose c o n c e n t r a t i o n s sub-t h r e s h o l d f o r i n s u l i n r e l e a s e , GIP augmented a r g i n i n e -s t i m u l a t e d glucagon s e c r e t i o n , suggesting an i n v e r s e r e l a t i o n s h i p between i n s u l i n and glucagon r e l e a s e by GIP (Pederson and Brown, 1976). E l a h i et a l (1979) employed the glucose clamp technique to demonstrate t h a t glucose l e v e l s must r i s e 25 mg/dl above b a s a l f o r the p o t e n t i a t i n g 15 l e v e l s must r i s e 25 mg/dl above b a s a l f o r the p o t e n t i a t i n g e f f e c t of GIP on i n s u l i n r e l e a s e to be observed. S i m i l a r r e s u l t s were obtained i n the i s o l a t e d p e r f u s e d r a t pancreas where the glucose t h r e s h o l d was determined to be 110 mg/dl (Pederson e t a l , 1982). The mechanism of a c t i o n of GIP i n i n s u l i n s e c r e t i o n remains unknown, although the presence of s p e c i f i c high a f f i n i t y b i n d i n g s i t e s on B-c e l l s of hamster insulinomas has r e c e n t l y been d e s c r i b e d ( M a l e t t i et a l , 1984). The a c t i o n s of GIP may be mediated i n t r a c e l l u l a r l y by cAMP s i n c e accumulation of [ H]cAMP incr e a s e d when p a n c r e a t i c i s l e t s were incubated with GIP i n the presence of glucose (Sze*cowka e t a l , 1982) . A r o l e f o r GIP i n the development of h y p e r i n s u l i n e m i a i n o b e s i t y has been p o s t u l a t e d . Some obese s u b j e c t s d i s p l a y e d e l e v a t e d plasma GIP b a s a l l y and f o l l o w i n g n u t r i e n t i n g e s t i o n ( C r e u t z f e l d t et a l , 1978) l e a d i n g to the p r o p o s a l that i n s e n s i t i v i t y of G I P - r e l e a s i n g c e l l s t o p u t a t i v e feedback i n h i b i t i o n by i n s u l i n c o n t r i b u t e d to e l e v a t e d plasma GIP l e v e l s . Weight l o s s f o l l o w i n g d i e t r e s t r i c t i o n r e versed the unresponsiveness of K - c e l l s to i n s u l i n feedback, suggesting t h a t hyperGIPemia i s a consequence, r a t h e r than a cause of h y p e r i n s u l i n e m i a i n o b e s i t y ( C r e u t z f e l d t and E b e r t , 1976). T h i s theory i s supported by s t u d i e s i n the hyperglycemic obese mouse, which i s s e v e r e l y i n s u l i n r e s i s t a n t and h y p e r i n s u l i n e m i c . Plasma GIP l e v e l s were markedly e l e v a t e d i n these animals and were not reduced by a d m i n i s t r a t i o n of exogenous 16 i n s u l i n . The GIP content of the gut was i n c r e a s e d ( F l a t t et a l , 1983). E a r l i e r , Polak e t a l (1975) r e p o r t e d h y p e r p l a s i a of K - c e l l s i n the gut of obese mice. GIP r e l e a s e i n obese VMH-lesioned r a t s has been repo r t e d to be e l e v a t e d d u r i n g i n v i v o i n t e s t i n a l p e r f u s i o n (Takemura et a l , 1984). M a z z a f e r r i et a l (1984) r e p o r t e d t h a t GIP s e c r e t i o n c o u l d be e l e v a t e d by i n c r e a s i n g d i e t a r y carbohydrate i n normal-weight i n d i v i d u a l s , and hypothesized a l t e r a t i o n s i n the i n t e s t i n a l hormone c e l l s . Since many obese s u b j e c t s tend to be hyperphagic, e l e v a t e d GIP s e c r e t i o n may simply be due to i n c r e a s e d c a l o r i c i n t a k e . On the other hand, i n c r e a s e d plasma c o n c e n t r a t i o n of GIP may not be necessary f o r an i n c r e a s e i n the p a n c r e a t i c response, i f i n c r e a s e d s e n s i t i v i t y or responsiveness to GIP e x i s t s at the l e v e l of the i s l e t . Sarson et a l (1981) r e p o r t e d b a s a l hyperGIPemia i n obese p a t i e n t s but no i n c r e a s e i n the i n t e g r a t e d response to a standard t e s t meal, d e s p i t e demonstrable h y p e r i n s u l i n e m i a . In another study, n e i t h e r b a s a l nor g l u c o s e - s t i m u l a t e d plasma GIP c o n c e n t r a t i o n s i n n o n - d i a b e t i c obese s u b j e c t s were found to be s i g n i f i c a n t l y d i f f e r e n t than those o f l e a n c o n t r o l s ( S a l e r a et a l , 1982). Thus, c o n s i d e r a b l e c o n t r o v e r s y e x i s t s r e g a r d i n g the r o l e of the e n t e r o i n s u l a r a x i s i n the development and maintenance of o b e s i t y . The Zucker " f a t t y " ( f a / f a ) r a t has been widely used as a model of o b e s i t y . The f a t t y r a t arose as a spontaneous 17 mutation i n a c r o s s between Merck Stock M and Sherman r a t s (Zucker and Zucker, 1961). The mutation i s monogenic and i s t r a n s m i t t e d i n Mendelian f a s h i o n , such that 25% of the o f f s p r i n g of a heterozygote p a i r w i l l become obese. Zucker f a / f a r a t s become o v e r t l y obese at about 5 weeks of age and t h e r e a f t e r g a i n weight r a p i d l y (Zucker and Zucker, 1963). Increased a d i p o s i t y i s a r e s u l t of both h y p e r p l a s i a and hypertrophy o f f a t c e l l s , a c o n d i t i o n which resembles childhood-onset o b e s i t y i n humans (Johnson et a l , 1978). However, u n c o n t r o l l e d a d ipocyte p r o l i f e r a t i o n i s not b e l i e v e d t o be the primary l e s i o n i n f a t t y r a t s because (1) adipocyte enlargement tends to precede adipocyte p r o l i f e r a t i o n ; (2) r e g e n e r a t i o n of l i p e c t o m i s e d s i t e s does not occur and (3) c u l t u r e d adipocytes from l e a n and f a t t y r a t s grow at s i m i l a r r a t e s (Johnson and G o l d s t e i n , 1981). V a r i a t i o n s of the f a t t y r a t have r e c e n t l y been d e s c r i b e d , i n c l u d i n g the Wistar f a t t y (Ikeda et a l , 1981) , the d i a b e t i c Zucker (Clark et a l , 1983) and the h y p e r t e n s i v e f a t t y (Koletsky, 1973; K o l e t s k y , 1975) . The b e h a v i o u r a l and m e t a b o l i c events c o n t r i b u t i n g to the onset and maintenance of o b e s i t y i n f a / f a r a t s have been the s u b j e c t of much study. E a r l y s t u d i e s (Zucker and Zucker, 1962) emphasized the presence of h y p e r l i p i d e m i a i n a d u l t r a t s , l a t e r shown to be due to a 6 - f o l d i n c r e a s e i n serum t r i g l y c e r i d e (Bremond et a l , 1982). Increased l i p o g e n e s i s by the l i v e r was b e l i e v e d to cause 18 h y p e r l i p i d e m i a , s i n c e the c o n d i t i o n p e r s i s t e d even when f a / f a r a t s were f e d low f a t d i e t s (Zucker and Zucker, 1962). I t was s p e c u l a t e d t h a t an i n c r e a s e i n the i n s u l i n : g l u c a g o n r a t i o c o u l d e x p l a i n e l e v a t e d h e p a t i c l i p o g e n e s i s (Bryce et a l , 1977). Godbole and York (1978) presented evidence suggesting t h a t e l e v a t e d h e p a t i c l i p o g e n e s i s was secondary to onset of h y p e r i n s u l i n e m i a and hyperphagia s i n c e both s t r e p t o z o t o c i n treatment and p a i r feeding to l e a n c o n t r o l s reduced h e p a t i c l i p o g e n e s i s . However, h e p a t i c l i p o g e n i c enzymes do not become e l e v a t e d u n t i l a f t e r weaning (Bazin and Lavau, 1982; Godbole et a l , 1978; Turkenkopf et a l , 1980) whereas e x c e s s i v e energy d e p o s i t i o n i n f a t depots has been r e p o r t e d as e a r l y as 7 (Boulange", Planche and de Gasquet, 1979) or as l a t e as 13 -days ( B e l l and S t e r n , 1977) . E l e v a t i o n of l i p o g e n i c ^ enzyme a c t i v i t y i n adipose t i s s u e a r i s e s p r i o r to weaning (Bazin and Lavau, 1982; de Waziers and Planche, 1983) and p e r s i s t s i n adulthood (Spydevold et a l , 1978; Taketomi et a l , 1975). While an i n c r e a s e i n l i p o g e n i c enzyme a c t i v i t y may account f o r e x c e s s i v e d e p o s i t i o n of t r i g l y c e r i d e i n adipocytes (de Waziers and Planche, 1983) i t i s unclear whether the primary d e f e c t of these animals i s an i n c r e a s e i n adipocyte l i p o g e n i c enzyme a c t i v i t y or whether t h i s phenomenon i s secondary to other m e tabolic changes. Two t h e o r i e s have been advanced to e x p l a i n the o b e s i t y of Zucker r a t s , the "PUSH" and "PULL" t h e o r i e s . I n s u l i n i s c e n t r a l to both. The PUSH hypothesis s t a t e s that 19 h y p e r i n s u l i n e m i a i s the f i r s t m a n i f e s t a t i o n o f the primary d e f e c t to appear. E l e v a t e d i n s u l i n l e v e l s cause hyperphagia due to secondary hypoglycemia as w e l l as inducing c e r t a i n l i p o g e n i c enzymes such as l i p o p r o t e i n l i p a s e . Thus i n c r e a s e d food i n t a k e and f a t d e p o s i t i o n r e s u l t . The PULL theory suggests t h a t the primary d e f e c t i s an i n c r e a s e i n a c t i v i t y o f l i p o g e n i c enzymes such as l i p o p r o t e i n l i p a s e , which i n c r e a s e f a t d e p o s i t i o n and causes r e l a t i v e undernourishment at other t i s s u e s . Hyperphagia i s induced to compensate f o r t h i s f a l s e energy d e f i c i t , r e s u l t i n g i n i n c r e a s e d p a n c r e a t i c i n s u l i n output (Greenwood et a l , 1983). In support of the PULL h y p o t h e s i s , i n c r e a s e s i n l i p o p r o t e i n l i p a s e a c t i v i t y have been r e p o r t e d as e a r l y as 10 days of age i n f a / f a pups (de Waziers and Planche, 1983), when f a / f a pups are not hyperphagic (Godbole et a l , 1981) nor h y p e r i n s u l i n e m i c (Zucker and Antoniades, 1972; Turkenkopf et a l , 1982a). Hyperphagia i s r e p o r t e d to a r i s e when the pups are o l d enough to eat s o l i d food at 16-17 days (Stern and Johnson, 1977). E x c e s s i v e weight g a i n by f a / f a pups i s not apparent at t h i s age (Godbole et a l , 1981; B e l l and S t e r n , 1977). S i m i l a r l y , plasma i n s u l i n l e v e l s o f f a / f a pups are re p o r t e d to i n c r e a s e c o i n c i d e n t a l l y with onset of hyperphagia as the animals approach weaning (York, S h a r g i l l and Godbole, 1981). The PUSH theory i s supported by a study i n which Turkenkopf et a l (1982a) demonstrated i n c r e a s e d i n s u l i n i n the plasma of 20 f e t a l f a / f a pups t h a t disappeared at b i r t h , reappearing by 18 days of age. I t has been s p e c u l a t e d that exaggerated i n s u l i n s e c r e t i o n by f a / f a pups i s suppressed by the high f a t milk d i e t of preweaning pups (Turkenkopf et a l , 1982a) or absence of hyperphagia (Godbole et a l , 1981; B a z i n and Lavau, 1982). However, attempts to d e l a y onset of h y p e r i n s u l i n e m i a by weaning onto a h i g h - f a t d i e t were not s u c c e s s f u l , although the degree of h y p e r i n s u l i n e m i a was somewhat reduced (Turkenkopf et a l , 1982b; B a z i n and Lavau, 1982). While immunocytochemical s t u d i e s have shown p a n c r e a t i c i s l e t s o f a d u l t obese r a t s are enlarged (Larsson et a l , 1975; Shino et a l , 1973) the e a r l i e s t demonstration of t h i s c o n d i t i o n i s 5 weeks (Shino et a l , 1973), c o i n c i d e n t a l with the v i s u a l i d e n t i f i c a t i o n of f a t t y animals. Thus i n s u l i n appears to p l a y an important r o l e i n the development of o b e s i t y i n Zucker r a t s . E l e v a t i o n of i n s u l i n l e v e l s may cause the i n c r e a s e i n l i v e r l i p o g e n i c enzyme a c t i v i t y observed at weaning and c o n t r i b u t e to developing hyperphagia. Reducing plasma i n s u l i n to l e v e l s s i m i l a r to those observed i n l e a n animals by d a i l y exogenous i n s u l i n i n j e c t i o n , f o l l o w i n g i n d u c t i o n of d i a b e t e s by s t r e p t o z o t o c i n , r e s u l t e d i n n o r m a l i z a t i o n of food i n t a k e and weight r e d u c t i o n ( S t o l z and M a r t i n , 1982; Chan and S t e r n , 1981). However, weight l o s s alone i s i n s u f f i c i e n t to reduce i n s u l i n l e v e l s to normal, s i n c e a f t e r 42 days of s t a r v a t i o n , f a t t y r a t s had body weights 21 comparable to those of l e a n c o n t r o l s but s t i l l d i s p l a y e d an obese body composition and e l e v a t e d plasma i n s u l i n c o n c e n t r a t i o n s (Zucker and Antoniades, 1972) . Hyp e r i n s u l i n e m i a appears t o be d i s s o c i a b l e from exaggerated adipose t i s s u e l i p o g e n e s i s s i n c e t h i s c o n d i t i o n was not rev e r s e d by i n s u l i n r e d u c t i o n with s t r e p t o z o t o c i n . In the same study i n j e c t i o n of l i p o p r o t e i n l i p a s e i n h i b i t o r a l s o d i d not reduce adipocyte l i p o g e n e s i s i n 18-21 day o l d f a / f a pups (York, S h a r g i l l and Godbole, 1981). O b e s i t y decreases expected l i f e s p a n and c o n t r i b u t e s to a host o f other p a t h o l o g i c a l c o n d i t i o n s such as c a r d i o v a s c u l a r d i s e a s e and d i a b e t e s m e l l i t u s . C u r rent evidence suggests that h y p e r i n s u l i n e m i a i s not the primary l e s i o n i n obese humans but does have important e f f e c t s on the development and maintenance of the obese s t a t e . The e t i o l o g y o f h y p e r i n s u l i n e m i a remains unresolved, but because GIP i s a potent s t i m u l a n t of i n s u l i n r e l e a s e , i t has been hypothesized that t h i s hormone may c o n t r i b u t e to el e v a t e d i n s u l i n s e c r e t i o n . A r o l e f o r GIP i n human o b e s i t y i s d i f f i c u l t to study because the onset and p r o g r e s s i o n o f the obese s t a t e i n humans cannot be p r e d i c t e d . The evidence accumulated to date i s e q u i v o c a l and c o n s i s t s mainly of plasma GIP measurements i n the f a s t i n g s t a t e , f o l l o w i n g i n g e s t i o n o f v a r i o u s n u t r i e n t s or a f t e r s u r g i c a l m a n i p u l a t i o n s . The s e n s i t i v i t y of the pancreas of obese p a t i e n t s to n u t r i e n t and hormonal 22 s t i m u l a t i o n has not been d i r e c t l y assessed. S i m i l a r l y , e v a l u a t i o n of c i r c u l a t i n g GIP l e v e l s by radioimmunoassay does not g i v e any i n f o r m a t i o n about the b i o l o g i c a l a c t i v i t y of the measured p e p t i d e . The a v a i l a b i l i t y o f an animal model of o b e s i t y , the Zucker f a t t y r a t , p r o v i d e d the impetus f o r the s t u d i e s d e s c r i b e d i n t h i s t h e s i s because the development of h y p e r i n s u l i n e m i a and o b e s i t y i n these animals f o l l o w s a p r e d i c t a b l e t i m e t a b l e . In a d d i t i o n , i n v i t r o experiments employing the p e r f u s e d r a t pancreas permit e v a l u a t i o n of the s e n s i t i v i t y o f the pancreas to d e f i n e d s t i m u l i . The c o n t r i b u t i o n of the e n t e r o i n s u l a r a x i s to the development and maintenance of o b e s i t y i n Zucker r a t s had not p r e v i o u s l y been assessed. T h e r e f o r e , i t was of i n t e r e s t to i n v e s t i g a t e the r o l e of n u t r i e n t , hormonal and n e u r a l components of the e n t e r o i n s u l a r a x i s on the onset and p r o g r e s s i o n of h y p e r i n s u l i n e m i a i n f a t t y r a t s , because t h i s i n f o r m a t i o n may p r o v i d e i n s i g h t i n t o the events t h a t c o n t r o l development of o b e s i t y i n humans. In a d d i t i o n , development of a bioass a y s e n s i t i v e to plasma l e v e l s of GIP that p r o v i d e s a method f o r a s s e s s i n g the i n s u l i n o t r o p i c a c t i v i t y o f i n c r e t i n s i n plasma from normal and p a t h o l o g i c a l s u b j e c t s was pursued. 23 GENERAL METHODOLOGY I. PEPTIDE QUANTIFICATION A. Radioimmunoassay 1. C a l c u l a t i o n s 125 C a l c u l a t i o n s o f % 1-peptide bound were done by computer a c c o r d i n g to the f o l l o w i n g formula: ( C t o t a l C s a m p l e ) ( C t o t a l CNSB ) % Bound = - X 100. C C t o t a l t o t a l where C=counts per minute. The Tracor 1290 gamma counter equipped with a data r e d u c t i o n system was programmed to p l o t the standard tubes as a s t r a i g h t l i n e a c c o r d i n g to the r e l a t i o n s h i p l o g [Y/(100-Y)] = a + b l o g X, where Y = % bound, X= standard c o n c e n t r a t i o n , a = Y - i n t e r c e p t and b = s l o p e . The pepti d e content o f the unknown samples was determined by reading % bound from the standard curve to o b t a i n p e p t i d e c o n c e n t r a t i o n s . 2. I n s u l i n a. I o d i n a t i o n Phosphate b u f f e r was prepared from 0.4 M stock s o l u t i o n (1.0 1 Na 2HP0 4 t i t r a t e d to pH 7.5 with NaH 2P0 4, sto r e d at 4°C) f o r use i n the i n s u l i n i o d i n a t i o n . 24 Pure p o r c i n e i n s u l i n (Novo) was d i s s o l v e d i n 10 p i 0.01 M HC1, then made up to a f i n a l c o n c e n t r a t i o n o f 5 pg/10 p i i n 0.2 M i o d i n a t i o n b u f f e r . The r e a c t a n t s were mixed i n the i o d i n a t i o n v e s s e l a c c o r d i n g to the f o l l o w i n g p r o t o c o l : 1) 10 jal (5 pg) p o r c i n e i n s u l i n 10 p i (1 mCi) 1 2 5 I o d i n e 25 p i c h l o r amine T (4 mg/ml i n 0.2 M phosphate b u f f e r ) ; mixed g e n t l y 10 sec. 2) 100 p i sodium m e t a b i s u l p h i t e (2.4 mg/ml i n 0.2 M phosphate b u f f e r ) ; mixed g e n t l y 45 sec. 3) 50 p i sodium i o d i d e (10 mg/ml i n 0.2 M phosphate b u f f e r ) . The r e a c t i o n mixture was f u r t h e r d i l u t e d with 1.8 ml 0.04 M phosphate b u f f e r . 125 The i n s u l i n - i n c o r p o r a t e d i o d i n e was adsorbed onto 10 mg m i c r o f i n e s i l i c a (QUSO G-32), vortexed thoroughly and c e n t r i f u g e d . The supernatant c o n t a i n e d non-125 i n c o r p o r a t e d i o d i n e . The p e l l e t was washed with water and r e c e n t r i f u g e d . The i o d i n a t e d i n s u l i n was e l u t e d from the s i l i c a using 3 ml a c i d e t h a n o l (1500 ml 95% et h a n o l , 500 ml d i s t i l l e d water, 30 ml co n c e n t r a t e d HC1; s t o r e d at 4°C) and was s t o r e d at -20°C f o l l o w i n g a d d i t i o n o f a f u r t h e r 2 ml a c i d e t h a n o l and 1.5 ml d i s t i l l e d water. 125 The percent i o d i n e i n c o r p o r a t e d was c a l c u l a t e d by 125 counting 10 p i f r a c t i o n s o f the d i l u t e I - i n s u l i n i n 25 a c i d e t h a n o l , the QUSO p e l l e t and the o r i g i n a l i o d i n a t i o n mixture i n QUSO p r i o r to c e n t r i f u g a t i o n ( t o t a l c o u n t s ) . b. Assay b u f f e r D i l u e n t b u f f e r f o r use i n the assay was prepared by d i l u t i o n o f the stock s o l u t i o n p r e v i o u s l y d e s c r i b e d (Section IA2a) to 0.04 M and a d d i t i o n of 5% c h a r c o a l e x t r a c t e d human plasma (CEP), which a s s i s t s a n t i g e n -antibody b i n d i n g . CEP was prepared by f i l t e r i n g outdated blood bank plasma through S i z e 4 f i l t e r paper (Whatman), then mixing with 1% a c t i v a t e d c h a r c o a l (Norit) f o r 1 h, followed by c e n t r i f u g a t i o n at 10,000 rpm f o r 30 min at 4°C. The supernatant was again f i l t e r e d , d i v i d e d and st o r e d at -20°C. c. Antiserum The i n s u l i n antiserum (GP01) was r a i s e d i n guinea p i g s a g a i n s t unconjugated p o r c i n e i n s u l i n i n Freund's s o l u t i o n . The antiserum has been used r o u t i n e l y f o r measurement of i n s u l i n i n p e r f u s a t e and plasma. L y o p h i l i z e d 100 pl f r a c t i o n s of 1:10 d i l u t e d serum were made up to 50 ml i n d i l u e n t b u f f e r and s t o r e d at -20°C as 1 ml f r a c t i o n s . For use i n the assay these f r a c t i o n s were f u r t h e r d i l u t e d 1:20 so th a t the f i n a l d i l u t i o n of antibody i n the assay was 1:1x10 . 26 Zero-binding with t h i s antibody was t y p i c a l l y 45-55% 125 when f r e s h l y prepared I - I n s u l i n was employed. Standard curves obtained using t h i s antibody were s e n s i t i v e i n a range from 5-160 uU/ml. d. Standards Pure r a t i n s u l i n (Novo) was s t o r e d at -20°C as 1 ml f r a c t i o n s c o n t a i n i n g 200 ng i n s u l i n . For use i n the assay one f r a c t i o n was d i l u t e d to 26.6 ml, and r e - f r o z e n as 1 ml f r a c t i o n s c o n t a i n i n g 160 pU/ml. To c o n s t r u c t a standard curve, the f r a c t i o n s were s e r i a l l y d i l u t e d to 80, 40, 20, 10 and 5 pU/ml. Plasma or p e r f u s a t e b u f f e r (100 pi) were added to each standard curve to compensate f o r p o s s i b l e b i n d i n g v a r i a t i o n s . e. C o n t r o l s Every assay c o n t a i n e d 2 d u p l i c a t e s e t s of c o n t r o l tubes i n order to determine i n t r a - and i n t e r a s s a y v a r i a b i l i t y . The c o n t r o l c o n s i s t e d of p e r f u s a t e pooled from a p e r f u s e d r a t pancreas experiment i n which the s t i m u l u s was 300 mg/dl glu c o s e p l u s 10 mM L - a r g i n i n e . The p e r f u s a t e was d i l u t e d so that a c o n s i s t e n t value of 40 pU/ml was obtained, then d i v i d e d as 1.5 ml f r a c t i o n s and s t o r e d at -20°C. Assays i n which the c o n t r o l value d e v i a t e d by +10 pU/ml from 40 pU/ml were c o n s i d e r e d i n v a l i d . C o n t r o l v a l u e s d i d not d e v i a t e over time. 27 f . P r o t o c o l Each assay was composed of t o t a l counts, NSB f z e r o - b i n d i n g and standard tubes i n t r i p l i c a t e and sample and c o n t r o l tubes i n d u p l i c a t e . The composition of standard, sample and c o n t r o l tubes was 700 p l d i l u e n t b u f f e r , 100 p l sample, c o n t r o l or standard and 100 p l antiserum. These were incubated f o r 24 h at 4°C. 1 2 5 I _ I n s u l i n (10,000 cpm/100 p l ) was then added t o a l l tubes and i n c u b a t i o n proceeded f o r an a d d i t i o n a l 24 h. g. S e p a r a t i o n 125 S e p a r a t i o n o f bound from f r e e I - I n s u l i n by the dextran-coated c h a r c o a l method depends on the 125 a d s o r p t i o n o f f r e e or damaged I - I n s u l i n to the dextran matrix and the e x c l u s i o n o f the l a r g e r a n t i b o d y - a n t i g e n complexes from t h i s m a t r i x . D e x t r an-charcoal (50 g Dextran T70 + 50 g a c t i v a t e d c h a r c o a l i n 1.0 1 0.04 M phosphate b u f f e r , pH 7.5, s t i r r e d o v e r n i g h t at 4°C) was added to each assay tube (200 p l ) , c e n t r i f u g e d f o r 30 min at 4°C and the supernatant d i s c a r d e d . The c h a r c o a l p e l l e t 125 c o n t a i n i n g f r e e I - I n s u l i n was counted on a Tracor 1290 gamma counter and r e s u l t s c a l c u l a t e d as d e s c r i b e d ( S e c t i o n IA1) . 28 3. Glucagon a. I o d i n a t e d glucagon 125 Iodinated p a n c r e a t i c glucagon ( I-glucagon f Amersham) was s t o r e d at -20°C as 100 p i f r a c t i o n s c o n t a i n i n g 3.6x10 cpm. For use i n the assay, each f r a c t i o n was d i l u t e d with assay b u f f e r ( S e c t i o n IA3b) so that 100 p i gave 2,000 cpm. b. Assay b u f f e r A stock s o l u t i o n of 0.6 M phosphate b u f f e r pH 7.4 was prepared by t i t r a t i n g 0.6 M Nal^PO^ a g a i n s t 1.0 1 0.6 M Na 2HP0 4. For use i n the assay, NaCl (1.16%, w/v), EDTA (0.36%, w/v), CEP (0.1%, v / v ) , T r a s y l o l (0.1%, v/v) and sodium azide (0.05%, w/v) were added and the b u f f e r c o n c e n t r a t i o n d i l u t e d to 0.06 M with d i s t i l l e d water. F i n a l c o n c e n t r a t i o n s of a d d i t i v e s to the phosphate b u f f e r were g i v e n . c. Antibody Monoclonal a n t i b o d i e s r a i s e d a g a i n s t p o r c i n e p a n c r e a t i c glucagon (6B4) were used i n t h i s assay and were a g i f t o f Dr. M. Gregor, B e r l i n . These a n t i b o d i e s possessed N-terminal s p e c i f i c i t y thus r e c o g n i z i n g both p a n c r e a t i c glucagon and enteroglucagon molecules. These a n t i b o d i e s were shown to have no c r o s s - r e a c t i v i t y with s t r u c t u r a l l y - r e l a t e d p e p t i d e s VIP, PHI, GIP or s e c r e t i n (Gregor and Riecken, 1984). Antibody i n t i s s u e c u l t u r e 29 medium was added to assay b u f f e r so t h a t the f i n a l d i l u t i o n of the antibody i n the assay was 1:50,000. Zero-binding of t h i s antibody was t y p i c a l l y 50-60%. Standard curves obtained using t h i s antibody were s e n s i t i v e i n a range from 1 fM to 100 pM. d. Standards Pure p a n c r e a t i c glucagon (Sigma) was d i s s o l v e d i n assay b u f f e r and s t o r e d at -20°C as f r a c t i o n s c o n t a i n i n g 1 pmole glucagon/100 u l . For use i n the assay these f r a c t i o n s were d i l u t e d with d i l u e n t b u f f e r . e. P r o t o c o l The composition of the assay tubes was as d e s c r i b e d f o r i n s u l i n ( S e c t i o n I A 2 i ) . A l l the assay 125 components p l u s I-glucagon were added i n e q u i l i b r i u m and the assays incubated f o r 48 h before s e p a r a t i o n . f . C h a r c o a l s e p a r a t i o n 125 S e p a r a t i o n of bound from f r e e I-glucagon was accomplished by c h a r c o a l a d s o r p t i o n . The c h a r c o a l mixture contained 10 ml 0.6 M assay b u f f e r pH 7.4, 1.2 g a c t i v a t e d c h a r c o a l , 0.35 g Dextran T-70 and 5% CEP per 100 ml and was s t i r r e d f o r 30 min p r i o r to use i n the assay. A f t e r a d d i t i o n of 400 u l c h a r c o a l / t u b e , the assays were c e n t r i f u g e d 30 min at 3000 rpm and 4°C, the supernatant d i s c a r d e d and the d r i e d p e l l e t counted f o r 2 min. 30 C a l c u l a t i o n o f glucagon c o n c e n t r a t i o n was as d e s c r i b e d (Section IA1). Because the antibody d i d not d i f f e r e n t i a t e between gut and p a n c r e a t i c glucagon, r e s u l t s were expressed as g l u c a g o n - l i k e immunoreactivity (pg/ml). 4. G a s t r i n The g a s t r i n radioimmunoassay was a m o d i f i c a t i o n of the method d e s c r i b e d by Track e t a l (1979) . a. I o d i n a t i o n S y n t h e t i c human g a s t r i n (SHG) was d i s s o l v e d i n 0.2 M NH4HCO-j to a c o n c e n t r a t i o n o f 0.5 p g / p l , d i v i d e d i n t o 10 p i f r a c t i o n s and l y o p h i l i z e d . For i o d i n a t i o n purposes one 10 p i f r a c t i o n was d i s s o l v e d i n 10 p i 0.4 M phosphate b u f f e r ( S e c t i o n IA2a). SHG was then incubated 1 25 f o r 1 min with 0.2 mCi Na I and 10 p i chloramine T (0.5 mg/ml i n 0.04 M phosphate b u f f e r ) . The o x i d a t i o n r e a c t i o n was stopped by a d d i t i o n of 10 p i sodium m e t a b i s u l p h i t e (0.5 mg/ml i n 0.04 M phosphate b u f f e r ) . The r e a c t i o n mixture was f u r t h e r d i l u t e d by a d d i t i o n o f 1 ml 0.05 M NH 4HC0 3. 125 For p u r i f i c a t i o n o f the I-SHG an aminoethyl c e l l u l o s e column was generated as f o l l o w s : 10 ml aminoethyl c e l l u l o s e beads were washed i n d e i o n i z e d water f o r 20 min, fo l l o w e d by 0.5 M HC1 f o r 2 h, then d e i o n i z e d water u n t i l the pH of the bead suspension was 4.0. The beads were then washed with 0.5 M NaOH f o r 2 h, fo l l o w e d 31 by d e i o n i z e d water u n t i l the pH was 8.0. The beads were s t o r e d at 4°C i n 0.1 M NH^OH. For use i n the p u r i f i c a t i o n of 1 2 5 I - S H G the beads were washed with 0.5 M NH 4HC0 3 to pH 8.0, then poured i n t o a 0.9x25 mm column and e q u i l i b r a t e d o v e r n i g h t with 50 mM NH^HCO^. 125 The I-SHG was a p p l i e d to the column at a flow r a t e of 2.5 ml/min and e l u t e d with a g r a d i e n t o f NH^HCO^ from 50 to 200 mM ( t o t a l volume 120 ml) . F r a c t i o n s were c o l l e c t e d at 1 min i n t e r v a l s , counted, and a damage assay performed to determine f r a c t i o n s c o n t a i n i n g monoiodinated 125 I-SHG. Io d i n a t e d g a s t r i n from each f r a c t i o n was d i l u t e d to approximately 5000 cpm and added to 1 ml assay b u f f e r ( S e c t i o n IA4b) and 200 p l c h a r c o a l ( S e c t i o n IA4g). The samples were c e n t r i f u g e d 30 min at 3000 rpm and 4°C, and both the supernatant (non - adsorbed) and the p e l l e t (adsorbed) counted. F r a c t i o n s c o n t a i n i n g the h i g h e s t p r o p o r t i o n of adsorbed counts were s t o r e d at -20°C f o r use i n the assay. b. Assay b u f f e r Assay b u f f e r c o n s i s t e d of 0.02 M sodium b a r b i t a l and 0.5% bovine serum albumin (RIA grade). Concentrated HC1 was used t o a d j u s t the pH to 8.4. c. Antiserum G a s t r i n antiserum (TR5) was a g i f t o f Dr. N. Track, Toronto and recog n i z e d both the 17 and the 34 amino 32 a c i d forms of g a s t r i n . T h i s antibody had no c r o s s r e a c t i v i t y with m o t i l i n , human 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 , CCK, GIP, VIP or glucagon (Track e t a l , 1979). L y o p h i l i z e d antibody was d i s s o l v e d i n 177 ml phosphate b u f f e r e d s a l i n e (0.01 M phosphate b u f f e r , 0.9% NaCl, 0.5% bovine serum albumin, pH c o r r e c t e d to 7.5 with 1 M NaOH) and s t o r e d at -20°C as 1 ml f r a c t i o n s . For use i n the assay one f r a c t i o n was made up to 45 ml i n assay b u f f e r , g i v i n g a f i n a l d i l u t i o n i n the assay of 1:80,000. d. Standards S y n t h e t i c human g a s t r i n was s t o r e d at -20°C i n 200 p l f r a c t i o n s at a c o n c e n t r a t i o n of 100 ng/ml. For use i n the assay one f r a c t i o n was d i l u t e d to 25 ml i n assay b u f f e r , then s e r i a l l y d i l u t e d to c o n c e n t r a t i o n s from 6 to 400 pg/ml. These standards were s t o r e d at -20°C as 1 ml f r a c t i o n s , thawed and used as i s i n the assay standard curve. 33 e. C o n t r o l s Pooled venous e f f l u e n t from an i s o l a t e d perfused stomach experiment was d i l u t e d so t h a t the g a s t r i n c o n c e n t r a t i o n was approximately 100 pg/ml and s t o r e d at -20°C as 1 ml f r a c t i o n s . D u p l i c a t e tubes of c o n t r o l p e r f u s a t e were i n c l u d e d i n each assay to monitor i n t r a - and i n t e r - a s s a y v a r i a b i l i t y . D e v i a t i o n of +50 pg/ml i n v a l i d a t e d the assay. f . P r o t o c o l The composition of the assay tubes was as d e s c r i b e d f o r i n s u l i n ( S e c t i o n I A 2 f ) . 1 2 5 I - S H G (2000 cpm/100 p i ) was added to each tube i n e q u i l i b r i u m and the assays incubated f o r 48 h at 4°C. g. S e p a r a t i o n G a s t r i n c h a r c o a l (12.5 g a c t i v a t e d c h a r c o a l , 2.5 g Dextran T-70 made up to 1.0 1 i n 0.04 M phosphate b u f f e r , pH 6.5 and s t i r r e d o v e r n i g h t ; 5% CEP added 1 h p r i o r to use) was added to each assay tube (200 pl/tube) . The assays were c e n t r i f u g e d at 3000 rpm, 4°C f o r 30 min, decanted and counted f o r 3 min. The g a s t r i n content of each sample was c a l c u l a t e d as d e s c r i b e d ( S e c t i o n IA1). 5. Glucose-dependent i n s u l i n o t r o p i c p o l y p e p t i d e The radioimmunoassay f o r GIP was a m o d i f i c a t i o n of the method d e s c r i b e d by Kuzio e t a l (1974). 34 To prevent a d s o r p t i o n o f GIP to glassware, a l l procedures were c a r r i e d out using s i l i c o n i z e d g l a s s tubes. a. I o d i n a t i o n Porcine GIP (15 pg) was d i s s o l v e d i n 20 p l 0.4 M phosphate b u f f e r , pH 7.5 ( S e c t i o n IA2a) and added to 1 125 mCi Na I and 5 p l chloramine T (0.5 mg/ml) f o r 15 sec. The r e a c t i o n was stopped with 10 p l sodium m e t a b i s u l p h i t e (200 mg/ml). 125 For p u r i f i c a t i o n o f I-GIP a Sephadex G-25 column (0.9x10 cm) was prepared and e q u i l i b r a t e d with 0.2 M a c e t i c a c i d c o n t a i n i n g 0.5% bovine serum albumin (BSA) and 2.0% T r a s y l o l ( A p r o t i n i n ) . The i o d i n a t i o n mixture was a p p l i e d to the column at a flow r a t e o f 24 ml/h and e l u t e d with e q u i l i b r a t i o n b u f f e r . F r a c t i o n s (400 p l ) were c o l l e c t e d and 10 p l f r a c t i o n s counted. The column p r o f i l e was p l o t t e d and a damage assay performed as d e s c r i b e d f o r g a s t r i n ( S e c t i o n IA4a). F r a c t i o n s were d i l u t e d to 5000 cpm/100 p l , then added to 900 p l assay b u f f e r ( S e c t i o n IA5b) before a d s o r p t i o n to c h a r c o a l . The mono-iodinated f r a c t i o n s were pooled and d i l u t e d 1:1 with a c i d ethanol ( S e c t i o n IA2a) to a f i n a l c o n c e n t r a t i o n o f 2.5x10** cpm/100 p l and s t o r e d at -20°C. 35 b. Assay b u f f e r A stock s o l u t i o n of 0.4 M phosphate b u f f e r was prepared from 0.4 M NaH 2P0 4 t i t r a t e d t o pH 6.5 with 0.4 M Na2HPC» 4. For use i n the assay, stock s o l u t i o n was d i l u t e d to 0.04 M c o n t a i n i n g 5% CEP and 1.5% T r a s y l o l . c. Antiserum Antiserum to p o r c i n e GIP (conjugated to BSA and e m u l s i f i e d with Freund's complete adjuvant) was r a i s e d i n r a b b i t s (Go 5, 28/4/75). L y o p h i l i z e d (200 p l ) f r a c t i o n s of GIP antiserum were d i l u t e d i n assay b u f f e r to g i v e a 3 f i n a l d i l u t i o n i n the assay of 1:3x10 . Zero-binding using t h i s antibody was t y p i c a l l y 25-30%. d. Standards P o r c i n e GIP ( p u r i f i e d a c c o r d i n g to the method of Brown et a l , 1970) was l y o p h i l i z e d as 1 pg f r a c t i o n s . For use i n the assay one f r a c t i o n was d i s s o l v e d i n 12.5 ml assay b u f f e r c o n t a i n i n g 0.5% BSA, g i v i n g a c o n c e n t r a t i o n of 80 ng/ml. F u r t h e r d i l u t i o n produced a standard c o n c e n t r a t i o n range from 4.0 to 0.125 ng/ml. 36 e. C o n t r o l s C o n t r o l s f o r the GIP assay were prepared by a c i d e x t r a c t i o n o f r a t jejunum. A r a t was s a c r i f i c e d by a n e s t h e t i c overdose and the smal l bowel removed immediately. J e j u n a l t i s s u e was b o i l e d i n 0.2 M a c e t i c a c i d f o r 5 min, l i q u i f i e d i n a Waring blender f o r 30 sec and c e n t r i f u g e d f o r 15 min at 5000 rpm, 4°C. The supernatant was c o l l e c t e d and d i l u t e d with assay b u f f e r so that a r e l i a b l e value o f 1 ng/ml was obtained on the GIP standard curve. F r a c t i o n s o f t h i s e x t r a c t were s t o r e d at -20°C. D u p l i c a t e c o n t r o l tubes were i n s e r t e d i n each assay as a measure of i n t r a - and i n t e r - a s s a y v a r i a b i l i t y . D e v i a t i o n by +0.25 ng/ml i n v a l i d a t e d the assay. f . P r o t o c o l The p r o t o c o l was as d e s c r i b e d f o r the g a s t r i n radioimmunoassay ( S e c t i o n I A 4 f ) . g. S e p a r a t i o n C h a r c o a l s e p a r a t i o n was as d e s c r i b e d f o r the g a s t r i n radioimmunoassay ( S e c t i o n IA4g). 37 6. Somatostatin Somatostatin was assayed a c c o r d i n g to the method of Mcintosh e t a l (1978). a. I o d i n a t i o n Phosphate b u f f e r f o r i o d i n a t i o n purposes was prepared by t i t r a t i n g 0.5 M NaH 2P0 4 to pH 7.5 with 1 M KOH. S y n t h e t i c Tyr-1 som a t o s t a t i n (Serono) was d i s s o l v e d i n 10 p l d i s t i l l e d water, d i l u t e d with 10 p l 0.5 M 125 phosphate b u f f e r , pH 7.5 and added to 1 mCi Na I and 10 p l Chloramine T (2 mg/ml i n 0.05 M phosphate b u f f e r , pH 7.5 and mixed f o r 30 sec. The r e a c t i o n was stopped by a d d i t i o n of 10 p l sodium m e t a b i s u l p h i t e (5 mg/ml i n 0.05 M phosphate b u f f e r , pH 7.5). The r e a c t i o n mixture was vortexed, added to 1 ml CEP and 20 mg QUSO, vortexed thoroughly, and c e n t r i f u g e d 3 min. The supernatant was d i s c a r d e d and the p e l l e t washed twice with 1 ml d i s t i l l e d water, c e n t r i f u g i n g between washes. The p e l l e t was then resuspended i n 1 ml a c e t i c acid/acetone (100 p l g l a c i a l a c e t i c a c i d , 3.9 ml acetone, 4 ml d i s t i l l e d water) and c e n t r i f u g e d 20 min. The supernatant was d i l u t e d to 500,000 cpm/100 p l with 0.1 M a c e t i c a c i d c o n t a i n i n g 0.5% BSA and d i v i d e d i n t o 100 p l f r a c t i o n s , l y o p h i l i z e d and s t o r e d at -20°C. 125 On the day of the assay, the I-so m a t o s t a t i n was p u r i f i e d on a C M - c e l l u l o s e column (0.9x10 cm) e q u i l i b r a t e d 38 with 0.002 M ammonium a c e t a t e , pH 4.6. L a b e l f r a c t i o n s were thawed and d i l u t e d with e q u i l i b r a t i o n b u f f e r , a p p l i e d to the column at a flow r a t e o f 1 ml/min, and e l u t e d with 30 ml e q u i l i b r a t i o n b u f f e r . The l a b e l was then e l u t e d with 0.2 M ammonium a c e t a t e , pH 4.6. A p p r o p r i a t e f r a c t i o n s were pooled, n e u t r a l i z e d with 1 M NaOH and subsequently d i l u t e d with assay b u f f e r ( S e c t i o n IA6b) to 3000 cpm/100 pi f o r use i n the assay. b. Assay b u f f e r A stock s o l u t i o n c o n s i s t i n g o f 4.9 g sodium b a r b i t a l (23.8 mM), 0.32 g sodium a c e t a t e (3.9 mM), 2.55 g NaCl (43.6 mM) and 0.1 g sodium e t h y l m e r c u r i t h i o s a l i c y c l i c a c i d (0. 247 mM) with pH ad j u s t e d to 7.4 with 1 M HC1 was made up to 1.0 1 with d i s t i l l e d water and s t o r e d at 4°C. For use i n the assay a t e n f o l d d i l u t i o n o f the stock s o l u t i o n supplemented with 0.5% BSA and 0.1% T r a s y l o l was employed. c. Antiserum Rabbit antiserum Go VI, 3.2 was s t o r e d as l y o p h i l i z e d 250 pi f r a c t i o n s . Each f r a c t i o n was added to 25 ml d i s t i l l e d water (1:100) and s t o r e d at -20°C as 1 ml f r a c t i o n s . For use i n the assay each 1 ml f r a c t i o n was f u r t h e r d i l u t e d i n 150 ml assay b u f f e r f o r a f i n a l d i l u t i o n i n the assay of 1:15,000. 39 d. Standards S y n t h e t i c c y c l i c s o m a t o s t a t i n (Peninsula) was d i s s o l v e d i n 5 ml 0.1 M a c e t i c a c i d c o n t a i n i n g 0.05% BSA, l y o p h i l i z e d and s t o r e d at -20°C as 25 u l f r a c t i o n s c o n t a i n i n g 5 ug p e p t i d e . For use i n the assay each 25 p l f r a c t i o n was made up to 500 p l i n d i s t i l l e d water and s e r i a l l y d i l u t e d with assay b u f f e r to f i n a l c o n c e n t r a t i o n s i n the assay of 3.9 to 250 pg/ml. e. Samples P e r f u s a t e samples were mixed with 1% T r a s y l o l before s t o r i n g . f . P r o t o c o l The t o t a l volume of each assay tube was 400 p l and c o n s i s t e d of 100 p l assay b u f f e r , 100 p l standard or sample, 100 p l antibody and 100 p l l a b e l . The assays were incubated at 4°C f o r 48 h before c h a r c o a l s e p a r a t i o n . g. S e p a r a t i o n Somatostatin c h a r c o a l c o n s i s t e d of 0.25 g/100 ml Dextran T70, 1.25 g/100 ml N o r i t and 100 pl/100 ml CEP i n 0.05 M phosphate b u f f e r , pH 7.5, which was s t i r r e d f o r 1 h p r i o r t o a d d i t i o n to the assays. Each tube r e c e i v e d 1 ml c h a r c o a l . The assays were c e n t r i f u g e d f o r 30 min at 3000 rpm and the d r i e d p e l l e t counted f o r 3 min. Peptide content of each sample tube was c a l c u l a t e d as d e s c r i b e d 40 ( S e c t i o n IA1). Somatostatin content of each tube was expressed as s o m a t o s t a t i n - l i k e immunoreactivity ( S L I ) . I I . ANIMAL STUDIES A. I s o l a t e d Perfused Organ P r e p a r a t i o n s I s o l a t e d organ p r e p a r a t i o n s p r o v i d e d an i n v i t r o model i n which the e f f e c t s o f v a r i o u s p h y s i o l o g i c a l and pharmacological agents c o u l d be examined. 1. S o l u t i o n s and Reagents a. Krebs-Ringer b i c a r b o n a t e b u f f e r A c o n c e n t r a t e d stock (lOx) s o l u t i o n of the f o l l o w i n g composition was prepared and s t o r e d at 4°C: 285 ml of 154 mM KC1 243 ml of 102.7 mM C a C l 2 78 ml of 154 mM MgS0 4'7H 20 97 ml o f 154 mM KH 2P0 4. Per f u s a t e b u f f e r was prepared by d i s s o l v i n g dextran and BSA i n 0.9% s a l i n e o v e r n i g h t so t h a t the f i n a l c o n c e n t r a t i o n s of these substances would be 3% and 0.2% r e s p e c t i v e l y . To t h i s were added Krebs c o n c e n t r a t e , NaHCO^ and glucose or other m e t a b o l i t e s to g i v e the f o l l o w i n g f i n a l c o n c e n t r a t i o n s : 4.4 mM KC1 2.5 mM C a C l 2 1.2 mM MgSO *7H-0 41 1.5 mM KH 2P0 4 25 mM NaHC0 3 120 mM NaCl Glucose or other m e t a b o l i t e s as d e s i r e d . b. Sidearm i n f u s i o n s P h y s i o l o g i c a l or p h a r m a c o l o g i c a l agents to be in f u s e d v i a a sidearm attachment were d i l u t e d from stock s o l u t i o n s i n p e r f u s a t e b u f f e r at a c o n c e n t r a t i o n c a l c u l a t e d to d e l i v e r the f i n a l d e s i r e d c o n c e n t r a t i o n , c o n s i d e r i n g an i n f u s i o n d e l i v e r y r a t e o f 0.206 ml/min and a p e r f u s a t e flow r a t e o f 3 (stomach) or 4 (pancreas) ml/min. 2. Procedure a. Apparatus ( F i g 1) The p e r f u s i o n apparatus c o n s i s t e d of a f l a s k connected to a p e r i s t a l t i c pump, which d e l i v e r e d p e r f u s a t e to the i s o l a t e d organ. P e r f u s a t e was maintained at pH 7.4 by continuous g a s s i n g with 95% G"2 and 5% C 0 2 which was s a t u r a t e d with water vapour by bubbling through a water r e s e r v o i r . Temperature of the p e r f u s a t e and organ p r e p a r a t i o n were maintained by servo-feedback c o n t r o l . P e r f u s i o n p r e s s u r e was monitored and maintained between 40-60 mm Hg. P e r f u s a t e was screened and l a r g e bubbles removed by a f i l t e r / b u b b l e t r a p device at the l e v e l of the a r t e r i a l cannula. 42 The c o n c e n t r a t i o n o f p h y s i o l o g i c a l or phar m a c o l o g i c a l agents d e l i v e r e d to the pancreas c o u l d be a l t e r e d i n two ways: 1) Square wave c o n c e n t r a t i o n changes. Two v a r i a t i o n s of t h i s method were employed. For changes i n p e r f u s a t e glucose c o n c e n t r a t i o n , a dual-channel p e r i s t a l t i c pump was used so th a t two f l a s k s of d i f f e r i n g glucose c o n c e n t r a t i o n s c o u l d be c i r c u l a t e d independently. Changes i n glucose c o n c e n t r a t i o n s were achieved by r a p i d s w i t c h i n g o f the in p u t l i n e to the pancreas. For a d d i t i o n of v a r i o u s p h y s i o l o g i c a l or ph a r m a c o l o g i c a l agents superimposed on a background of g l u c o s e - c o n t a i n i n g p e r f u s a t e , a sidearm i n f u s i o n using a Harvard i n f u s i o n pump was employed. 2) L i n e a r g r a d i e n t c o n c e n t r a t i o n changes were produced by connecting two f l a s k s o f equal c r o s s - s e c t i o n (Figure 1) and adding the more conc e n t r a t e d s t i m u l a n t to the d i s t a l f l a s k . Vagal s t i m u l a t i o n o f stomach p r e p a r a t i o n s was accomplished by p l a c i n g d i p o l a r e l e c t r o d e s around the vagal trunks at the l e v e l o f the lower esophageal s p h i n c t e r . S t i m u l a t i o n parameters were 10 Hz, 7 V, 5 ms. In a l l cases venous drainage was c o l l e c t e d using an automated f r a c t i o n c o l l e c t o r . 43 F i g 1. Apparatus f o r the i n v i t r o p e r f u s i o n o f the i s o l a t e d r a t pancreas. P e r f u s a t e was pumped to the pancreas from a p a i r of connected f l a s k s (A and B) . I f a sti m u l a n t was added o n l y to the d i s t a l f l a s k (B) , sti m u l a n t would be d e l i v e r e d t o the pancreas as a l i n e a r g r a d i e n t . The p e r f u s a t e temperature was maintained at 37°C and the p e r f u s i o n p r e s s u r e was maintained at 40-60 mmHg. 44 Apparatus for the in vitro Vascular Perfusion of the Rat Pancreas. 45 b. E q u i l i b r a t i o n Each experiment was preceded by an e q u i l i b r a t i o n p e r i o d (pancreas 10 min; stomach 20 min) to ensure t h a t s e c r e t o r y r a t e s o f p e p t i d e s were not a f f e c t e d by s u r g i c a l trauma. c. S u r g i c a l p r e p a r a t i o n i . A n e s t h e s i a Rats were a n e s t h e t i z e d i n t r a p e r i t o n e a l l y with p e n t o b a r b i t a l (60 mg/kg) f o l l o w i n g an o v e r n i g h t f a s t . The peritoneum was exposed by a m i d l i n e i n c i s i o n from sternum to p e l v i s with p a i r e d l a t e r a l i n c i s i o n s t o the body w a l l . i i . Pancreas The i s o l a t i o n of the pancreas was ac c o r d i n g to a m o d i f i c a t i o n o f the method of Grodsky et a l (1967). The v a s c u l a r i s o l a t i o n o f the pancreas was as f o l l o w s : The descending c o l o n and a s s o c i a t e d a r t e r y were s i n g l y l i g a t e d . A drainage tube was i n s e r t e d i n the duodenum i n f e r i o r to the l e v e l o f the pancreas. The mesenteric arcades were s y s t e m a t i c a l l y doubly l i g a t e d and the c o l o n doubly l i g a t e d caudal t o the cecum and the gut was removed. The esophagus and a s s o c i a t e d a r t e r y was doubly l i g a t e d as f a r cephalad as p o s s i b l e and the c e l i a c branches connecting the stomach and pancreas along the greater c u r v a t u r e were s i n g l y l i g a t e d . The duodenum was doubly l i g a t e d at the p y l o r u s and the stomach was removed. 46 The v a s c u l a r connections between the spleen and the pancreas were s i n g l y l i g a t e d . The spleen was not removed. The r i g h t kidney and a d r e n a l gland were i s o l a t e d by a s i n g l e l i g a t u r e . The l e f t kidney was doubly l i g a t e d to expose the abdominal a o r t a and i n f e r i o r vena cava. Loose l i g a t u r e s were p l a c e d cephalad of the c e l i a c a r t e r y , caudal to the s u p e r i o r mesenteric a r t e r y and 1-2 cm caudal to the r e n a l a r t e r y stump. An a r t e r i a l cannula (PE 160) was i n s e r t e d t o the l e v e l of the s u p e r i o r mesenteric a r t e r y such t h a t p e r f u s i o n of the pancreas o c c u r r e d v i a both the s u p e r i o r mesenteric and c e l i a c a r t e r i e s . H e p a r i n - s a l i n e (200 ul) was i n j e c t e d . The animal was hemisected at the diaphragm and a venous cannula f o r c o l l e c t i o n of e f f l u e n t i n s e r t e d i n the p o r t a l v e i n . i i i . Stomach The v a g i were l o c a t e d s u b d i a p h r a g m a t i c a l l y on the s u r f a c e of the esophagus and marked with a l o o s e l i g a t u r e . The descending c o l o n was doubly l i g a t e d and f r e e d from c o n n e c t i v e t i s s u e to the l e v e l of the s u p e r i o r mesenteric a r t e r y . The s u p e r i o r mesenteric a r t e r y was doubly l i g a t e d . V a s c u l a r connections between the stomach and pancreas and spleen were doubly l i g a t e d , care being taken to p r e s e r v e the r i g h t g a s t r i c and r i g h t g a s t r o e p i p l o i c a r t e r i e s . The duodenum was doubly l i g a t e d caudal to the g a s t r i c venous drainage to the p o r t a l v e i n . A drainage tube was i n s e r t e d i n t o the duodenal remnant and 47 the pancreas, spleen, s m a l l bowel and c o l o n removed. The . r i g h t kidney and adre n a l gland were i s o l a t e d by a s i n g l e l i g a t u r e . The l e f t r e n a l and ad r e n a l a r t e r i e s were doubly l i g a t e d to expose the abdominal a o r t a . Loose l i g a t u r e s were p l a c e d cephalad of the c e l i a c a r t e r y and caudal t o the s u p e r i o r mesenteric a r t e r y stump. An a r t e r i a l cannula (PE 160) was i n s e r t e d t o the l e v e l of the c e l i a c a r t e r y and 200 u l h e p a r i n - s a l i n e was i n j e c t e d . The animal was hemisected at the diaphragm and the upper c a r c a s s d i s c a r d e d . A venous cannula f o r c o l l e c t i o n o f e f f l u e n t was i n s e r t e d i n the p o r t a l v e i n . B. Glucose Tolerance T e s t s 1. O r a l Rats were f a s t e d o v e r n i g h t and 500 u l blood samples were c o l l e c t e d from the t a i l v e i n b a s a l l y and f o l l o w i n g a d m i n i s t r a t i o n o f 1 g/kg 40% glucose by s y r i n g e and feeding tube. C o l l e c t i o n s times were 0, 10, 20, 30 and 60 min. The blood was c e n t r i f u g e d at 9,500 rpm f o r 2 min at 4°C and the plasma s t o r e d at -20°C u n t i l assayed. Separate 50 p l blood samples were ob t a i n e d at each time i n t e r v a l and c e n t r i f u g e d i n a Beckman microfuge f o r measurement of plasma glucose l e v e l s on a Beckman II glucose an a l y z e r (glucose oxidase method). 48 C. I d e n t i f i c a t i o n of Obese Zucker Pups 1. C o l o n i c Temperature Measurement Obese Zucker r a t s were found to have d e f e c t i v e thermogenesis as e a r l y as 16 days (Godbole et a l , 1978) . T h i s o b s e r v a t i o n was used to i d e n t i f y pre-obese s u c k l i n g and weanling r a t pups. A s p e c i a l l y - c o n s t r u c t e d t h e r m i s t o r probe 1 cm long and 1.5 mm i n diameter was connected to a d i g i t a l r e c o r d e r . The c o l o n i c temperature of Zucker pups was measured at 10:00 a.m. f o r three c o n s e c u t i v e days s t a r t i n g at 18 days of age. The pups were separated from t h e i r mother f o r 10 min p r i o r to temperature d e t e r m i n a t i o n . Pups whose average temperature was lower than the average of the whole l i t t e r were p r e d i c t e d to be obese. 2. Pancreas H i s t o l o g y F o l l o w i n g p e r f u s i o n s t u d i e s , the s p l e n i c p o r t i o n of the pancreas was removed and f i x e d i n Bouin's s o l u t i o n (75 ml p i c r i c a c i d , 25 ml formaldehyde, 5 ml g l a c i a l a c e t i c acid) f o r 2 h, then t r a n s f e r r e d to 70% e t h a n o l . The t i s s u e was dehydrated i n graded a l c o h o l using an automatic p r o c e s s o r , imbedded i n p a r a f f i n at 60°C and c u t i n t o 5 pm s e c t i o n s using a Jung microtome. The s e c t i o n s were a i r -d r i e d on s l i d e s o v e r n i g h t , dewaxed i n xylene, and s t a i n e d with haematoxylin and e o s i n according to the f o l l o w i n g p r o t o c o l : Hematoxylin 5 min Rinse i n running water. 49 L i t h i u m carbonate 1 min Rinse 10 min. E o s i n (water-base) 5 min Rinse b r i e f l y . 70% ethanol 1 min 90% ethanol 1 min Absolute e t h a n o l 5 min Xylene 10 min The s l i d e s were c o v e r s l i p p e d with Permamount (Fisher) and the s i z e o f the i s l e t s i n each s e c t i o n were compared under the l i g h t microscope ( Z e i s s , Canada). D. Zucker Rat Colony 1. Breeding O r i g i n a l breeding p a i r s were s u p p l i e d by Dr. P. B e c h t e l , Animal S c i e n c e s L a b o r a t o r y , U n i v e r s i t y o f I l l i n o i s . S i n c e f a t t y females are s t e r i l e , the best breeding combination was f a t t y males ( f a / f a ) p a i r e d with heterozygous, l e a n females (Fa/fa) so t h a t r e s u l t a n t l i t t e r s were approximately 50% obese. A 12 h l i g h t / d a r k c y c l e was maintained. 2. Housing Breeding p a i r s , l a c t a t i n g females and t h e i r l i t t e r s , and newly weaned pups were housed i n p o l y e t h y l e n e cages l i n e d with wood shavings. Other a d u l t r a t s were housed i n wire mesh cages with 4 animals/cage. F o l l o w i n g s u r g i c a l procedures animals were housed i n i n d i v i d u a l wire mesh cages. 50 3. N u t r i t i o n Pups were r o u t i n e l y weaned 21 days a f t e r b i r t h , but had access to the mother's r a t chow and water at a l l times p r i o r to weaning. A f t e r weaning r a t s had ad l i b i t u m access to chow and water. 4 . I d e n t i f i c a t i o n Each l i t t e r was numbered ac c o r d i n g t o date of b i r t h . At weaning each animal was assigned a unique i d e n t i f i c a t i o n number, the l a s t 2 d i g i t s of which were marked on the animal by n i c k i n g the e a r s . I I I . IMMUNOCYTOCHEMISTRY A. R a t i o n a l e Immunocytochemistry allows v i s u a l i z a t i o n of the c e l l u l a r l o c a l i z a t i o n o f antigens i n t i s s u e . T i s s u e i s f i x e d i n such a way t h a t a n t i g e n i c determinants are preserved, then incubated with antibody r a i s e d a g a i n s t the s p e c i f i c a n t i g e n . The c e l l s c o n t a i n i n g s p e c i f i c a n t i g e n can then be i d e n t i f i e d i f a c o l o u r change can be induced, e i t h e r by a p p l i c a t i o n o f f l u o r e s c e n t or enzyme r e a c t i o n techniques. The p e r o x i d a s e - a n t i p e r o x i d a s e (PAP) technique (Sternberger, 1979) i s c u r r e n t l y one of the most s e n s i t i v e methods employed and was used e x c l u s i v e l y i n these s t u d i e s . 51 B. P e r o x i d a s e - a n t i p e r o x i d a s e (PAP) Immunostaining 1. F i x a t i o n of T i s s u e Gut was f r e s h l y o b t a i n e d d u r i n g the s u r g i c a l p r e p a r a t i o n of the animal f o r p e r f u s i o n ( Sections I I A 2 c i i and c i i i ) . Pancreas or stomach was obtained immediately f o l l o w i n g the p e r f u s i o n experiment. T i s s u e samples were f i x e d i n Bouin's s o l u t i o n ( S e c t i o n IID) f o r 2 h, then t r a n s f e r r e d to 70% e t h a n o l . The samples were kept at room temperature to prevent c r y s t a l l i z a t i o n o f p i c r i c a c i d p r i o r t o d e h y d r a t i o n . 2. Dehydration T i s s u e samples were loaded i n t o c a s s e t t e s (Fisher) f o r automatic p r o c e s s i n g . Dehydration was accomplished by i n c r e a s i n g a l c o h o l c o n c e n t r a t i o n (70%, 90%, 99%) f o l l o w e d by a l c o h o l removal with x y l e n e . The dehydrated samples were l e f t t o soak i n p a r a f f i n . 3. Embedding Samples were removed from the processor and embedded i n p a r a f f i n wax at 60°C. The p a r a f f i n b l o c k s were allowed to set at 4°C f o r 2 h, then s t o r e d at -20°C u n t i l c u t . 52 4. C u t t i n g S e c t i o n s For best r e s u l t s p a r a f f i n b l o c k s were c u t when c o l d . M u l t i p l e 5 pm s e c t i o n s were c u t . The s e c t i o n s were f l o a t e d on 50°C water to f l a t t e n , then mounted onto s l i d e s . The s e c t i o n s were d r i e d on the s l i d e s at 37°C f o r 24-48 h. 5. D e p a r a f f i n i z a t i o n S e c t i o n s were dewaxed by soaking i n xylene, then d r i e d with petroleum e t h e r . 6. Immunostaining Procedures a. F i r s t l a y e r antibody (Table I) The f i r s t l a y e r antibody was d i r e c t e d a g a i n s t a s p e c i f i c p e p t i d e a n t i g e n (eg. i n s u l i n , g a s t r i n ) . S l i d e s were p l a c e d i n humid s t a i n i n g chambers. T h i s prevented s l i d e s from d r y i n g out d u r i n g the i n c u b a t i o n . The antibody source, s p e c i f i c i t y and d i l u t i o n s are noted i n Table I. S l i d e s were incubated o v e r n i g h t at 4°C. b. B l o c k i n g The s l i d e s were allowed to warm to room temperature i n t h e i r humid chambers, then washed i n phosphate-buffered s a l i n e (PBS). To prevent n o n - s p e c i f i c s t a i n i n g , s e c t i o n s were "blocked" by i n c u b a t i n g with 1% H 2 ° 2 ^ n P B S ^ o r min. The s e c t i o n s were then washed i n PBS. 53 c. Second l a y e r antibody (Table II) r Second l a y e r a n t i b o d i e s were chosen depending on the source of the f i r s t l a y e r antibody (Table II) . For example, i f the f i r s t l a y e r antibody was r a b b i t a n t i -g a s t r i n , the second l a y e r antibody was goat a n t i - r a b b i t immunoglobulin. These were a p p l i e d f o r 30 min at room temperature, then washed with PBS. d. T h i r d l a y e r antibody (Table II) The t h i r d l a y e r antibody was a peroxidase a n t i p e r o x i d a s e complex t h a t would a l s o b i n d to the second l a y e r antibody eg. r a b b i t PAP. The p e r o x i d a s e -a n t i p e r o x i d a s e complexes were made up f r e s h d a i l y to the a p p r o p r i a t e d i l u t i o n (Table II) and incubated f o r 30 min at room temperature before washing i n PBS. 54 TABLE I . ANTIBODY SOURCES (FIRST LAYER) ANTIBODY/SERA SOURCE SPECIFICITY DILUTION C h o l e c y s t o k i n i n Enteroglucagon* G a s t r i n GIP* Rabbit, Immunonuclear Mouse, Dr. Gregor, B e r l i n Rabbit, Dr. Track, Toronto Mouse, Vancouver N-t e r m i n a l 1:2500 1:100 C- t e r m i n a l 1:5000 1:50000 Glucagon (pa n c r e a t i c ) I n s u l i n N e u r o t e n s i n P a n c r e a t i c p o l y p e p t i d e Somatostatin Rabbit, M i l a b C - t e r m i n a l 1:1000 Guinea p i g , Vancouver Rabbit, Immunonuclear Rabbit, Dr. Buchanan, B e l f a s t Rabbit, Immunonuclear C - t e r m i n a l 1:3000 1:2000 1:1000 1:1000 •Monoclonal antibody 55 TABLE I I . ANTIBODY SOURCES (SECOND AND THIRD LAYERS) ANTIBODY/SERA SOURCE DILUTION Goat a n t i - r a b b i t IgG Cappel 1:300 Rabbit anti-mouse IgG Dr. Levy, Vancouver 1:200 Rabbit a n t i - g u i n e a p i g IgG A r n e l Labs 1:200 Rabbit PAP Cappel 1:300 Mou se PAP Sternberger-Meyer 1:100 Guinea p i g PAP A r n e l Labs 1:100 56 e. Peroxidase r e a c t i o n S e c t i o n s were developed i n diaminobenzidene (25 ug/100 ml PBS) and ^2°2 ( ° ' 0 2 5 % ) - S i n c e diaminobenzidene (DAB) i s a known ca r c i n o g e n t h i s procedure was c a r r i e d out i n the fume hood and extreme c a u t i o n was taken. S l i d e s were developed i n DAB-H2C>2, then washed i n PBS, then d i s t i l l e d water. The s l i d e s were c o u n t e r s t a i n e d i n haematoxylin according t o the p r o t o c o l below, then c o v e r s l i p p e d with Permamount. Hematoxylin 5 min Rinse under running water A c i d e t h a n o l 5 min Rinse 70% ethanol 1 min 90% ethanol 1 min Absolute e t h a n o l 1 min Xylene 10 min 7. Q u a n t i f i c a t i o n P A P - p o s i t i v e c e l l s were q u a n t i f i e d using a computer morphometries program (Videoplan MOP40, Z e i s s , Canada) d i r e c t l y coupled to a re s e a r c h microscope ( Z e i s s , Canada) v i a a videocamera. a. Pancreas 2 The average t o t a l a r e a / i s l e t (mm ) of ten i s l e t s / r a t was measured. In a d d i t i o n , the percentage o f 57 the i s l e t s occupied by each of the endocrine c e l l types ( i n s u l i n , glucagon, s o m a t o s t a t i n , p a n c r e a t i c polypeptide) was c a l c u l a t e d f o r each i s l e t from s p e c i f i c a l l y s t a i n e d s e r i a l s e c t i o n s . b. Antrum F i v e r e p r e s e n t a t i v e areas of a n t r a l mucosa, each 2 c o n s i s t i n g o f about 200 mm , were examined f o r each r a t . The average h e i g h t of the e p i t h e l i u m (pm) i n these areas was c a l c u l a t e d . The number of g a s t r i n and somatostatin cells/mm was determined from s p e c i f i c a l l y s t a i n e d s e r i a l s e c t i o n s as d e s c r i b e d by S t e i n e t a l (1983). c. I n t e s t i n e Ten r e p r e s e n t a t i v e c r y p t / v i l l u s p r o f i l e s were chosen f o r each r a t . For c o n t r o l animals, s e c t i o n s o f jejunum and ileum were examined. For animals t r e a t e d with j e j u n o i l e a l bypass, s e c t i o n s of jejunum and ileum from both the continuous bowel and the bypassed loop were examined. T i s s u e was obtained at l e a s t 1 cm from the anastomosis i n the continuous bowel, and 1-2 cm from the stoma i n the bypassed l o o p . The average h e i g h t of the c r y p t / v i l l u s p r o f i l e s (pm) was c a l c u l a t e d . The number of s p e c i f i c endocrine c e l l s (GIP, CCK, enteroglucagon, n e u r o t e n s i n , and somatostatin) per square m i l l i m e t e r were determined from s p e c i f i c a l l y s t a i n e d s e r i a l s e c t i o n s . To c o r r e c t f o r v a r i a t i o n s i n e p i t h e l i a l h e i g h t , the numerical 58 d e n s i t y of the c e l l s was expressed as the number of c e l l s i n the e p i t h e l i u m o v e r l y i n g a g i v e n l e n g t h of mucosa (cells/mm) as d e s c r i b e d by S t e i n et a l (1983). IV. STATISTICAL INTERPRETATION Without e x c e p t i o n , s t a t i s t i c a l s i g n i f i c a n c e (p<.05) of experimental r e s u l t s vs c o n t r o l s was determined using Student's t - t e s t f o r p a i r e d or unpaired data, as a p p l i c a b l e . 59 APPENDIX TO METHODS-CHEMICAL SOURCES CHEMICAL GRADE SOURCE A c e t i c a c i d A r i s t a r BDH Ammonium a c e t a t e Baker Ammonium bic a r b o n a t e F i s h e r Ammonium hydroxide Baker Ammonium sulphate Baker At r o p i n e sulphate C l i n i c a l BDH B a r b i t a l sodium C-IV C l i n i c a l F i s h e r Bovine serum albumin RIA Sigma Calcium c h l o r i d e F i s h e r Carbon d e c o l o u r i z i n g N e u t r a l (Norit) F i s h e r Chloramine T Eastman Kodak CM-Cellulose Whatman Cyanogen bromide Eastman Kodak DEAE-Sephacel Pharmacia Dextran C l i n i c a l Sigma Dextran T-70 Pharmacia Dimethylformamide Sigma Diaminobenzidene BDH Disodium ethylenediamine t e t r a a c e t a t e (EDTA) F i s h e r Ensure Ross Ether A n a e s t h e s i a M a l l i n c k r o d t Ethanolamine S igma E t h a n o l , 95% Commercial A l c o h o l E o s i n , aqueous BDH Formaldehyde H i s t o l o g i c a l F i s h e r Freund's complete adjuvant Sigma G a s t r i n ( s y n t h e t i c human) Research Plus Glucagon (porcine p a n c r e a t i c ) S igma 125 I-glucagon Amersham Glucose (dextrose) C l i n i c a l Abbott Glucose oxidase reagent Beckman Hematoxylin Mercury-free F i s h e r Heparin sodium L a b o r a t o r y F i s h e r Hemophilus p e r t u s s i s C l i n i c a l Connaught Hydrogen peroxide H i s t o l o g i c a l F i s h e r I n s u l i n (porcine) Novo I n s u l i n (porcine/bovine) C l i n i c a l Connaught I n s u l i n (rat) Novo L-+-Arginine monohydro-c h l o r i d e F i s h e r L a n o l i n Burroughs Wellcome L i t h i u m carbonate F i s h e r L i d o c a i n e C l i n i c a l E l k i n s - S i n n Magnesium sulphate F i s h e r M i n e r a l o i l (Nujol) Plough P a r a f f i n (Paraplast) Monoject 60 Permamount Petroleum ether P r o v i o d i n e t o p i c a l a n a e s t h e t i c P e n t o b a r b i t a l sodium Phenol P i c r i c a c i d Potassium c h l o r i d e Potassium hydroxide Potassium phosphate, monobasic QUSO m i c r o f i n e s i l i c a , G-32 Sephadex G-25 Sepharose CL-4B Sodium azid e Sodium bi c a r b o n a t e Sodium borate Sodium c h l o r i d e Sodium e t h y l m e r c u r i t h i o -s a l i c y c l i c a c i d Sodium hydroxide A n a l y t i c a l C l i n i c a l C l i n i c a l A r i s t a r P r a c t i c a l Sodium i o d i d e 125, Sodium J " i , J i o d i d e i n NaOH Sodium m e t a b i s u l p h i t e Sodium phosphate, d i b a s i c Sodium phosphate, monobasic T r a s y l o l ( p r o t e o l y t i c enzyme i n h i b i t o r , A p r o t i n i n ) T r i s - H C l Trizma base Xylene H i s t o l o g i c a l F i s h e r BDH Rougier MTC Pharmaceuticals A l l i e d Chemical BDH F i s h e r BDH F i s h e r P h i l a d e l p h i a Quartz Pharmacia Pharmacia Baker F i s h e r F i s h e r F i s h e r Eastman Kodak American S c i e n t i f i c and Chemical F i s h e r Amersham F i s h e r F i s h e r F i s h e r M i l e s S igma Sigma F i s h e r 61 APPENDIX TO METHODS—LIST OF ABBREVIATIONS ANS Autonomic nervous system BSA Bovine serum albumin CCK Chole c y s t o k i n i n - p a n c r e o z y m i n CEP C h a r c o a l e x t r a c t e d plasma DAB Diaminobenzidene db/db D i a b e t i c mouse EG Enteroglucagon Fa/- Lean Zucker r a t (hetero- or homozygous Fa/Fa Homozygous l e a n Zucker r a t F a / f a Heterozygous l e a n Zucker r a t f a / f a Obese Zucker r a t Fr F r a c t i o n GIP Glucose-dependent i n s u l i n o t r o p i c p o l y p e p t i d 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 GLI G l u c a g o n - l i k e immunoreactivity 125 T 125 T ,. I Iodine IR-GIP Immunoreactive GIP IRI; I R - I n s u l i n Immunoreactive i n s u l i n JIB J e j u n o i l e a l bypass JIB-L J e j u n o i l e a l bypass with loop p e r f u s i o n JIB-NS J e j u n o i l e a l bypass not f a s t e d NSB N o n - s p e c i f i c b i n d i n g NT Neurotensin ob/ob Obese, hyperglycemic mouse PAP P e r o x i d a s e - a n t i p e r o x i d a s e PBS Phosphate b u f f e r e d s a l i n e PHI Peptide h i s t i d i n e i s o l e u c i n e PP P a n c r e a t i c p o l y p e p t i d e RIA Radioimmunoassay SHG S y n t h e t i c human g a s t r i n SLI S o m a t o s t a t i n - l i k e immunoreactivity Soma Somatostatin Tyr-1 T y r o s i n e at p o s i t i o n 1 VIP 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 VMH Ventro-medial hypothalamus 62 CHAPTER ONE: THE ROLE OF GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE IN THE DEVELOPMENT AND MAINTENANCE OF HYPERINSULINEMIA IN THE OBESE ZUCKER RAT I. INTRODUCTION The term " e n t e r o i n s u l a r a x i s " was coi n e d to d e s c r i b e the r e l a t i o n s h i p between e n t r y of n u t r i e n t s i n t o the gut and the r e l e a s e o f hormones from the endocrine pancreas. Components of the e n t e r o i n s u l a r a x i s i n c l u d e n u t r i e n t s , n e u r a l connections and hormones, such as glucose-dependent i n s u l i n o t r o p i c p o l y p e p t i d e (GIP). The r o l e o f the e n t e r o i n s u l a r a x i s i n the development and maintenance of the h y p e r i n s u l i n e m i a a s s o c i a t e d with o b e s i t y i n humans i s not w e l l understood. Using the g e n e t i c a l l y obese Zucker " f a t t y " r a t as an animal model of o b e s i t y , s t u d i e s were undertaken to i n v e s t i g a t e the r o l e of s p e c i f i c components of the e n t e r o i n s u l a r a x i s i n the onset o f o b e s i t y . Glucose-dependent i n s u l i n o t r o p i c p o l y p e p t i d e i s c u r r e n t l y the b e s t - d e s c r i b e d p e p t i d e with i n c r e t i n c h a r a c t e r i s t i c s i e . s t i m u l a t i o n o f i n s u l i n r e l e a s e i n the presence of hyperglycemia. Measurement of serum l e v e l s o f GIP i n obese humans has produced c o n f l i c t i n g r e s u l t s . C r e u t z f e l d t et a l (1978) d e s c r i b e d e l e v a t i o n o f GIP l e v e l s i n obese p a t i e n t s with p a t h o l o g i c a l glucose t o l e r a n c e , both i n the f a s t i n g s t a t e and f o l l o w i n g n u t r i e n t i n g e s t i o n . The authors proposed t h a t e l e v a t i o n of c i r c u l a t i n g GIP l e v e l s c o u l d c o n t r i b u t e to the h y p e r i n s u l i n e m i a observed i n these p a t i e n t s . However, GIP c o n c e n t r a t i o n s were found to be normal i n e i t h e r the fed or f a s t e d s t a t e i n other s t u d i e s ( S a l e r a et a l , 1982; Amland et a l , 1984) and were e l e v a t e d only a f t e r o v e r n i g h t food d e p r i v a t i o n i n another (Sarson et a l , 1981). Data c o l l e c t e d from morbidly obese i n d i v i d u a l s i s d i f f i c u l t t o i n t e r p r e t , s i n c e the events t h a t p r e c i p i t a t e the onset of o b e s i t y i n humans are unknown and may d i f f e r between i n d i v i d u a l s . A l s o , i f the p a n c r e a t i c response to GIP i s a l t e r e d by other m e t a b o l i c events, i t would not be necessary f o r an obese i n d i v i d u a l to d i s p l a y e l e v a t e d GIP l e v e l s i n order f o r GIP to c o n t r i b u t e to h y p e r i n s u l i n e m i a . G e n e t i c a l l y obese rodents are convenient f o r the study of o b e s i t y , s i n c e the development of the obese syndrome f o l l o w s a p r e d i c t a b l e t i m e t a b l e . Plasma GIP l e v e l s were abnormally high i n both g l u c o s e - i n t o l e r a n t n o n - d i a b e t i c ob/ob mice and o v e r t l y d i a b e t i c db/db mice ( F l a t t et a l , 1983a). E l e v a t e d s e c r e t i o n of GIP i n these mice may be due to h y p e r p l a s i a of K - c e l l s i n the gut r e p o r t e d by Polak e t a l (1975) but the temporal r e l a t i o n s h i p between these phenomena has not been d e s c r i b e d . I t was shown t h a t d i e t r e s t r i c t i o n decreased GIP l e v e l s but not to c o n t r o l l e v e l s i n ob/ob mice ( F l a t t et a l , 1983b) and t h a t GIP s e c r e t i o n was i n s e n s i t i v e to i n h i b i t i o n by exogenous i n s u l i n i n f u s i o n ( F l a t t et al,1983a). The r o l e of GIP i n development and maintenance of h y p e r i n s u l i n e m i a has not been examined i n a second g e n e t i c model of o b e s i t y , the Zucker r a t . Zucker r a t s d i f f e r from 64 the ob/ob mouse i n t h a t they are l e s s i n s u l i n - r e s i s t a n t , normoglycemic and do not become o v e r t l y d i a b e t i c (Bray and York, 1971) and thus may be a more accurate model of obese humans not prone to d i a b e t e s . In the c u r r e n t i n v e s t i g a t i o n s , the p o s s i b l e r o l e of GIP i n the onset and maintenance of h y p e r i n s u l i n e m i a i n obese Zucker r a t s was s t u d i e d . In young a d u l t r a t s the i n v i v o i n s u l i n and GIP response to g l u c o s e was determined. The minimum, or t h r e s h o l d c o n c e n t r a t i o n s of glucose and GIP necessary f o r the i n s u l i n o t r o p i c a c t i o n of GIP were determined i n the i s o l a t e d p e r f u s e d pancreas p r e p a r a t i o n . In a d d i t i o n , t i s s u e samples from pancreas and jejunum were taken f o r immunocytochemical a n a l y s i s to e s t a b l i s h the s i z e and composition of p a n c r e a t i c i s l e t s and to examine the GIP-c o n t a i n i n g c e l l s of the upper i n t e s t i n e . In a second group of s t u d i e s i n v o l v i n g s u c k l i n g and weanling Zucker r a t s , the onset of both b a s a l and g l u c o s e - s t i m u l a t e d h y p e r i n s u l i n e m i a was examined i n v i v o and i n v i t r o because p r e v i o u s s t u d i e s (York, S h a r g i l l , and Godbole, 1981) i n d i c a t e d t h a t the onset of g l u c o s e - s t i m u l a t e d h y p e r i n s u l e m i a may precede e l e v a t i o n of f a s t i n g plasma i n s u l i n l e v e l s . These phenomena may t h e r e f o r e be caused by independent mechanisms. 65 I I . METHODS A. Glucose Tolerance T e s t s 1. Intravenous Rats were s u r g i c a l l y prepared with i n d w e l l i n g venous c a t h e t e r s 3 days p r i o r to intravenous glucose t o l e r a n c e t e s t s . Cannulae (PE 50) were prepared by soaking o v e r n i g h t i n h e p a r i n ( P o l y s c i e n c e s ) . Animals were a n e s t h e t i z e d i . p . with 40 mg/kg p e n t o b a r b i t a l . A n e s t h e s i a was i n c r e a s e d with ether, i f necessary. The s u r g i c a l areas were shaved and cleaned with an i o d i n e s o l u t i o n ( P r o v i o d i n e , Rougier) followed by 70% a l c o h o l . The r i g h t j u g u l a r v e i n was exposed with a 2 cm i n c i s i o n to the r i g h t of m i d l i n e . The e x t e r n a l j u g u l a r v e i n was cannulated with heparin-coated PE 50 t u b i n g . The cannula was guided under the s k i n using a hollow needle to the area between the shoulder b l a d e s . The cannula was attached t o a p o l y e t h y l e n e harness secured under the s k i n between the shoulder b l a d e s . The animals were allowed to recover f o r 3 days be f o r e t e s t i n g , at which time the r a t s were f a s t e d o v e r n i g h t . A b a s a l blood sample was obtained and then the animals were weighed and r e s t r a i n e d (Centrap, F i s h e r ) . Glucose (0.6 mg/kg) was administered v i a the j u g u l a r cannula over a 1 min p e r i o d and washed i n with 0.2 ml h e p a r i n - s a l i n e . Blood samples were obtained and t r e a t e d as d e s c r i b e d f o r the o r a l g l u c o s e t o l e r a n c e t e s t (General Methodology, S e c t i o n IIB1). 66 2. I n t r a p e r i t o n e a l T h i s v a r i a t i o n o f the glucose t o l e r a n c e t e s t was used fo r 3-5 week o l d r a t pups. F o l l o w i n g an o v e r n i g h t f a s t the pups were weighed and admi n i s t e r e d 1 g/kg glucose i . p . F i f t e e n minutes f o l l o w i n g glucose a d m i n i s t r a t i o n the animals were a n e s t h e t i z e d with ether and a 250 p l blood sample c o l l e c t e d from the t a i l a r t e r y . Blood treatment was as e a r l i e r d e s c r i b e d ( S e c t i o n I I B 1 ) . 67 I I I . RESULTS A. Development of H y p e r i n s u l i n e m i a ; The Role of GIP 1. I d e n t i f i c a t i o n of S u c k l i n g and Weanling Rats a. Weight g a i n (Table III) The body weight of f a / f a r a t s d i d not become s i g n i f i c a n t l y higher than t h a t of l e a n l i t t e r m a t e s u n t i l 35 days of age. b. C o l o n i c temperature (Table IV) Average c o l o n i c temperature from 18-20 days of age was c a l c u l a t e d f o r l e a n and obese s u c k l i n g pups and the phenotype deduced from t h i s data. A t o t a l of 238 pups were t e s t e d . Of these, the phenotype of 200 pups (84%) was c o r r e c t l y i d e n t i f i e d . The average c o l o n i c temperature of the l e a n r a t s was 35.0 + 0.08°C compared to 33.7 + 0.1°C f o r obese l i t t e r m a t e s (p<0.001). At t h i s age no s i g n i f i c a n t weight d i f f e r e n c e c o u l d be d e t e c t e d between l e a n (30.2 + 0.6 g) and obese (31.4 + 0.8 g, p>0.05) r a t s . c. P a n c r e a t i c morphology (Figs 2, 3 and 4) The t o t a l area of the p a n c r e a t i c i s l e t s and the p r o p o r t i o n of i s l e t s c o n s i s t i n g of i n s u l i n c e l l s were incr e a s e d i n f a / f a r a t s a t 7 (Fig 2 ) , 15, 21 ( F i g 3) and 28 days of age compared to l e a n l i t t e r m a t e s . The i s l e t s of 1-day o l d f a / f a neonates were not d i s t i n g u i s h a b l e from those of l e a n l i t t e r m a t e s . Lean and f a / f a pups c o u l d not be 68 r e t r o a c t i v e l y i d e n t i f i e d because the r a t pups were s a c r i f i c e d f o r c o l l e c t i o n of the pancreas. T h e r e f o r e , i s l e t s i z e was compared to that of normal Wistar r a t pups at 1, 7 and 15 days of age. The i s l e t s of Wistar pups were of a c o n s i s t e n t s i z e with that observed i n l e a n Zucker pups. When d i s t r i b u t i o n curves were produced f o r the i s l e t area of W i s t a r , l e a n and f a / f a pups, the curves f o r the Wistar and lean pups were i n d i s t i n g u i s h a b l e , whereas the f a / f a pups were c l e a r l y a d i f f e r e n t p o p u l a t i o n . The average s i z e of i s l e t from 7-day-old obese r a t s was twice t h a t of l e a n or Wistar pups ( F i g 4 ) . 69 TABLE I I I . BODY WEIGHT (X" + SEM) OF LEAN AND OBESE ZUCKER RATS AGE GENOTYPE BODY WEIGHT (< 21 days Fa/- 31.1 + 0.90, n=54 f a / f a 29.9 + 1.54, n=25 28 days Fa/- 44.0 + 1.75, n=19 f a / f a 44.5 + 1.76, n=15 35 days Fa/- 51.8 + 2.65, n=25 f a / f a 67.2 + 5.10, n=13* g) p<.05 compared to Fa/- r a t s o f the same age group 70 TABLE IV. IDENTIFICATION OF f a / f a RATS BY COLONIC TEMPERATURE AT 20 DAYS Fa/- f a / f a NUMBER OF ANIMALS 154 84 BODY WEIGHT (g + SEM) 3 0 . 2 + 0 . 6 3 1 . 4 + 0 . 8 COLONIC TEMPERATURE (°C + SEM) 35.0 + 0.08 33.7 + 0.10* *p<.05 compared to Fa/- r a t s of the same age group. 71 F i g 2. P a n c r e a t i c i s l e t s o f 7-day-old Zucker pups s t a i n e d f o r i n s u l i n u s ing the p e r o x i d a s e - a n t i p e r o x i d a s e method ( m a g n i f i c a t i o n x60). A. Lean B. Obese, showing the i n c r e a s e i n B - c e l l number and i s l e t s i z e . 72 F i g 3. P a n c r e a t i c i s l e t s of 21-day-old Zucker pups s t a i n e d f o r i n s u l i n using the p e r o x i d a s e - a n t i p e r o x i d a s e method ( m a g n i f i c a t i o n x60). A. Lean B. Obese, showing the i n c r e a s e i n B - c e l l number and i s l e t s i z e . 74 75 F i g 4. P a n c r e a t i c i s l e t area i n 7-day-old l e a n and obese Zucker pups compared to normal 7-day-old Wistar pups. A. D i s t r i b u t i o n p r o f i l e of i s l e t area i n Wi s t a r , l e a n Zucker and obese Zucker pups. B. Average i s l e t area i n Wistar (n=44), l e a n Zucker (n=67) and obese Zucker (n=67) pups. *p<.05 comparing obese to l e a n Zucker pups. There was no s i g n i f i c a n t d i f f e r e n c e between l e a n Zucker and Wistar pups. 76 NUMBER OF ISLETS 2. H y p e r i n s u l i n e m i a i n S u c k l i n g and Weanling f a / f a  Rats a. F a s t i n g plasma i n s u l i n (Table V) B a s a l i n s u l i n l e v e l s i n f a / f a pups d i d not d i f f e r from those of l e a n l i t t e r m a t e s at 21 (n=15 vs n=ll) or 28 (n=8 vs n=9) days of age. At 35 days f a s t i n g i n s u l i n l e v e l s were s i g n i f i c a n t l y e l e v a t e d i n f a / f a r a t s (n=15) compared to l e a n l i t t e r m a t e s (n=20) (p<.05). b. G l u c o s e - s t i m u l a t e d plasma i n s u l i n  (Table V) F o l l o w i n g s t i m u l a t i o n of i n s u l i n r e l e a s e by i n t r a p e r i t o n e a l g lucose i n j e c t i o n , f a / f a pups at 21 (n=10 vsn=ll) , 28 (n=7 vs n=7) and 35 days of age (n=9 vs n=18) were a l l found to be hyper i n s u l i n e m i c compared to l e a n l i t t e r m a t e s (p<.05). c. F a s t i n g plasma GIP (Table VI) At no time d i d plasma GIP c o n c e n t r a t i o n s i n f a / f a pups d i f f e r s i g n i f i c a n t l y from those measured i n l e a n l i t t e r m a t e s . d. The e f f e c t o f GIP on 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 from the i n v i t r o pancreas ( F i g 5) I n s u l i n s e c r e t i o n from the pancreas was s t i m u l a t e d with 300 mg/dl g l u c o s e . Between 11 and 30 min of the 45 min p e r f u s i o n p e r i o d , 1 ng/ml GIP was i n t r o d u c e d v i a a sidearm 78 i n f u s i o n . The i n s u l i n response of f a / f a 21-day (n=10 vs n=7) and 28-day-old (n=10 vs n=10) obese Zucker r a t s was not s i g n i f i c a n t l y d i f f e r e n t from t h a t of l e a n l i t t e r m a t e s i n response to glu c o s e alone or to glucose p l u s GIP ( F i g 5, A and B) . However, 35-day o l d f a / f a r a t s (n=6) d i d y i e l d a s i g n i f i c a n t l y g r e a t e r i n s u l i n response to both glucose alone and glucose p l u s GIP compared to l e a n l i t t e r m a t e s (n=10) (Fig 5 C, p<.05). e. The e f f e c t o f GIP on i n s u l i n r e l e a s e under  b a s a l glucose c o n d i t i o n s i n the p e r f u s e d  pancreas ( F i g 6) The i s o l a t e d pancreas was p e r f u s e d with 80 mg/dl glucose p e r f u s a t e , which i s r e p r e s e n t a t i v e of f a s t i n g plasma glucose l e v e l s i n the r a t . Between 11 and 30 min 1 ng/ml GIP was i n t r o d u c e d v i a a sidearm i n f u s i o n . Twenty-one (n=6 vs n=8) and 28-day o l d (n=6 vs n=7) f a / f a pups and t h e i r lean l i t t e r m a t e s e x h i b i t e d a t r a n s i e n t i n c r e a s e i n the i n s u l i n response to b a s a l g l u c o s e when 1 ng/ml GIP was in t r o d u c e d . I n s u l i n responses of l e a n versus f a / f a r a t s were not s i g n i f i c a n t l y d i f f e r e n t ( F i g 6, A and B) . The i n s u l i n response of 35-day o l d f a / f a r a t s (n=12) to b a s a l glucose p l u s GIP was s i g n i f i c a n t l y e l e v a t e d compared to l e a n l i t t e r m a t e s (n=9) ( F i g 6 C, p<.05). 79 TABLE V. PLASMA INSULIN CONCENTRATIONS (X + SEM) OF LEAN AND OBESE ZUCKER RATS AGE GENOTYPE PLASMA INSULIN (pU/ml) FASTING GLUCOSE-STIMULATED 21 days Fa/-f a / f a 28 days Fa/-f a / f a 35 days Fa/-f a / f a 10.7 + 2.5, n = l l 13.3 + 3.1, n=15 11.8 + 2.3, n=9 13.3 + 2.3, n=8 8.6 + 1.4, n=20 20.3 + 5.9, n=15* 24.0 + 2.0, n = l l 41.7 + 6.9, n=10* 16.1 + 2.7, n=7 27.2 + 4.6, n=7* 15.2 + 2.0, n=18 43.5 + 5.3, n=9* *p<.05 compared to l e a n r a t s o f the same age group. 80 TABLE VI. PLASMA GIP CONCENTRATIONS (X + SEM) OF LEAN AND OBESE ZUCKER RATS AGE GENOTYPE PLASMA GIP (pg/ml) 21 days Fa/- 493 + 38.6, n=14 f a / f a 580 + 73.5, n=6 28 days Fa/- 529 + 43.4, n=10 f a / f a 488 + 25.1, n=6 35 days Fa/- 517 + 64.7, n=20 f a / f a 613 + 63.3, n=12 81 The response of the i s o l a t e d p e r f u s e d pancreas of l e a n and obese Zucker pups at (A) 21 (n=10 vs n=7), (B) 28 (n=10 vs n=10) and (C) 35 (n=10 vs n=6) days of age to 300 mg/dl glucose p l u s 1 ng/ml GIP. *p<.05 using unpaired Student's t - t e s t , compared to l e a n pups of the same age group. 82 83 The response of the i s o l a t e d p e r f u s e d pancreas of le a n and obese Zucker pups at (A) 21 (n=8 vs n=6) , (B) 28 (n=7 vs n=6) and (C) 35 (n=9 vs n=12) days of age to 80 mg/dl glucose p l u s 1 ng/ml GIP. *p<.05 using unpaired Student's t - t e s t , compared t o l e a n pups of the same age group.. 84 85 3. A d u l t Rats a. P a n c r e a t i c morphology ( F i g 7, Table VII) In a l l cases, the p r o p o r t i o n of i n s u l i n c e l l s per i s l e t and the t o t a l area of the p a n c r e a t i c i s l e t s were g r e a t l y i n c r e a s e d i n f a / f a r a t s as compared with l e a n l i t t e r m a t e s . Many i s l e t s o f f a / f a r a t s d e v i a t e d from the compact s p h e r i c a l shape a s s o c i a t e d with the i s l e t s of l e a n r a t s . The remaining i s l e t c e l l s c o n t a i n i n g glucagon, somatostatin and p a n c r e a t i c p o l y p e p t i d e showed no change i n c e l l numbers with the descending order being glucagon, PP and then s o m a t o s t a t i n . The i n s u l i n c e l l s (Fig 7, A and B) always formed the core of the i s l e t s , with the other c e l l s occupying the p e r i p h e r y (Fig 7, C-H). Q u a n t i t a t i v e analyses of the i s l e t s are shown i n Table V I I . b. Q u a n t i f i c a t i o n of GIP c e l l number ( F i g 8) The number of GIP-immunoreactive c e l l s was s i m i l a r i n both l e a n and obese groups (3 c e l l s / 1 0 v i l l u s / c r y p t p r o f i l e s ) . c. O r a l glucose t o l e r a n c e i n young (8-12 wk old) a d u l t r a t s ( F i g s 9 and 12) F a s t i n g glucose l e v e l s i n f a / f a r a t s were s i g n i f i c a n t l y higher (73 + 4 mg/dl) than i n t h e i r l e a n l i t t e r m a t e s (62 + 5 mg/dl) although s t i l l w i t h i n the normal range f o r the r a t . The peak glucose response to o r a l glucose was not s i g n i f i c a n t l y d i f f e r e n t i n l e a n (n=18) and 86 obese (n=18) r a t s , peaking at 210 mg/dl at 20 min ( F i g 9 A ) . In c o n t r a s t , f a s t i n g I R - i n s u l i n l e v e l s were four times higher i n f a / f a r a t s (n=18) compared with l e a n l i t t e r m a t e s (n=18) (152 + 8 pU/ml vs 39 + 2 uU/ml). Peak i n s u l i n v a l u e s (349 + 12 pU/ml) oc c u r r e d i n f a / f a r a t s at 30 min a f t e r glucose i n g e s t i o n compared with 8 0 + 2 uU/ml f o r l e a n l i t t e r m a t e s ( F i g 9 B). F a s t i n g IR-GIP l e v e l s were not s i g n i f i c a n t l y d i f f e r e n t i n f a t and l e a n animals (830 + 180 pg/ml versus 560 + 120 pg/ml) . Peak l e v e l s o f IR-GIP were a l s o not s i g n i f i c a n t l y a l t e r e d i n the f a t (n=6) as compared with l e a n r a t s (n=6) (1210 +180 pg/ml ve r s u s 1220 + 210 pg/ml) 20 min a f t e r glucose i n g e s t i o n ( F i g 9 C ) . S i m i l a r l y , the i n t e g r a t e d glucose and GIP responses to o r a l glucose s t i m u l a t i o n were not s i g n i f i c a n t l y d i f f e r e n t between l e a n and obese r a t s ( F i g 12 A and C) , whereas the i n t e g r a t e d i n s u l i n response of obese r a t s was 3 - f o l d higher than t h a t of l e a n animals ( F i g 12 B) . d. O r a l glucose t o l e r a n c e i n o l d e r (6-8 month  o l d ) a d u l t r a t s ( F i g 10) The g l u c o s e , i n s u l i n and GIP responses to 1 g/kg o r a l glucose were measured i n l e a n and obese 6-8 month o l d Zucker r a t s . F a s t i n g glucose l e v e l s o f obese r a t s (n=36) were s i g n i f i c a n t l y (p<.05) higher than those of l e a n c o n t r o l s (n=36), but were s t i l l w i t h i n the normal range (<100 mg/dl). F o l l o w i n g glucose i n g e s t i o n , plasma l e v e l s rose to a p l a t e a u of 250 mg/dl at 20 min and d i d not d e c l i n e 87 by 60 min i n obese r a t s . In c o n t r a s t l e a n r a t s achieved a s i g n i f i c a n t l y lower p l a t e a u o f 200 mg/dl at 20 min which d e c l i n e d 20% by 60 min (Fig 10 A) . F a s t i n g i n s u l i n l e v e l s were 7 - f o l d higher i n f a t (n=ll) compared to l e a n r a t s (n=10) and i n c r e a s e d 2 - f o l d to 300 LiU/ml at 10 min f o l l o w i n g glucose a d m i n i s t r a t i o n . G l u c o s e - s t i m u l a t e d i n s u l i n l e v e l s of l e a n r a t s i n c r e a s e d 5 - f o l d to 100 LiU/ml at 20 min, d e c l i n i n g to b a s a l l e v e l s by 60 min. At one hour the i n s u l i n l e v e l s of obese r a t s were s t i l l 50% higher than b a s a l ( F ig 10 B) . F a s t i n g GIP l e v e l s of obese r a t s (n=25) were s i g n i f i c a n t l y higher than those o f l e a n animals (n=23) and achieved a peak of 800 pg/ml at 30 min, whereas l e v e l s i n l e a n r a t s reached a maximum of 400 pg/ml at 20 min (Fig 10 C) . e. Intravenous glucose t o l e r a n c e ( F i g s 11 and 12) A d m i n i s t r a t i o n of 0.6 g/kg glucose v i a the intravenous route r e s u l t e d i n e l e v a t i o n of plasma glucose t o l e v e l s s i m i l a r to those seen f o l l o w i n g o r a l g lucose a d m i n i s t r a t i o n . The maximum glucose response at 10 min approached 200 mg/dl f o r both l e a n (n=9) and obese (n=9) animals ( F i g 11 A) , the same as was observed i n the o r a l glucose t e s t ( F i g 9 A ) . Moreover, the i n t e g r a t e d glucose response was 8-10 g/60 min ( F i g 12 A ), which d i d not d i f f e r from the o r a l g lucose t e s t . The i n s u l i n response of l e a n animals (n=ll) to intr a v e n o u s g l u c o s e was 50% l e s s than t o o r a l g l u c o s e , whereas the i n s u l i n response o f obese r a t s 88 (n=10) to intravenous glucose was reduced 30% ( F i g s 11 B and 12 B) . 89 Immunocytochemical examination of p a n c r e a t i c i s l e t s o f a d u l t and obese Zucker r a t s ( M a g n i f i c a t i o n x60) A. Lean, i n s u l i n B. Lean, glucagon C. Lean, soma t o s t a t i n D. Lean, p a n c r e a t i c p o l y p e p t i d e E. Obese , i n s u l i n F. Obese , glucagon G. Obese , som a t o s t a t i n H. Obese , p a n c r e a t i c p o l y p e p t i d e 90 91 92 TABLE V I I . ISLET SIZE AND COMPOSITION IN 8-12 WEEK OLD ZUCKER RATS GENOTYPE ISLET SIZE ISLET COMPOSITION (% + SEM) (mm2) INSULIN GLUCAGON SOMA PP Fa/- 0. 2 + 0.01 72.6 19.5 4.2 4.7 n=320 +1.0 + 0.8 +0.3 +0.4 f a / f a 0. 3 + 0.01* 81.2 10.5 5.2 3.1 n=350 +0.9 +0.8 + 0.4 +0.5 *p<.05 93 F i g 8. Immunocytochemical l o c a l i z a t i o n o f GIP-containing c e l l s o f the jejunum i n l e a n and obese Zucker r a t s ( c r o s s - s e c t i o n , m a g n i f i c a t i o n x40) A. Lean B. Obese 94 A B 4 95 F i g 9. The plasma (A) glucose (n=18 vs n=18), (B) i n s u l i n (n=18 vs n=18) and (C) GIP (n=6 vs n=6) responses to 1 g/kg o r a l g l ucose i n l e a n and obese young (8-12-week-old) a d u l t Zucker r a t s . ^ rep r e s e n t s the a d m i n i s t r a t i o n of g l u c o s e at 0 minutes. *p<.05 using unpaired t - t e s t , compared to l e a n r a t s . 96 A. 0J . . . . . . 0 10 20 30 40 90 60 MINUTES 97 F i g 10. The plasma (A) glucose (n=36 vs n=36), (B) i n s u l i n (n=10 vs n=ll) and (C) GIP (n=25 vs n=23) responses t o 1 g/kg o r a l g l ucose i n l e a n and obese o l d e r (6-8-month-old) Zucker r a t s , -v- r e p r e s e n t s the a d m i n i s t r a t i o n o f glucose at 0 minutes. *p<.05 using unpaired Student's t - t e s t , compared to l e a n r a t s . 98 I B. C . •o Lean Obese O-L • • ' ' ' • ' 0 20 40 60 MINUTES 99 F i g 11. The plasma (A) glucose (n=9 vs n=9) and (B) i n s u l i n ( n = l l vs n=10) responses to 0.6 g/kg intravenous glucose i n l e a n and Q obese young a d u l t Zucker r a t s . ^ r e p r e s e n t s the a d m i n i s t r a t i o n of glucose at 0 minutes. *p<.05 using unpaired Student's t - t e s t , compared to l e a n r a t s . 100 A. 101 F i g 12. The i n t e g r a t e d (A) g l u c o s e , (B) i n s u l i n and (C) GIP responses to o r a l and intravenous glucose i n l e a n and obese a d u l t Zucker r a t s using the data shown i n F i g s 9 and 11. The formula used f o r c a l c u l a t i o n of the i n t e g r a t e d responses was X T = ( x t - n + x f l ) ( t l - t O ) + ( X t l + X t 2 ) ( t 2 - t l ) . . - t n _ 1 ( X t Q ) 2 2 where X I = i n t e g r a t e d r e l e a s e of the measured parameter X=concentration of the measured parameter at time=t. *p<.05 using the Student's t - t e s t f o r unpaired data, comparing obese to l e a n r a t s . °p<.05, comparing intravenous to o r a l g lucose t o l e r a n c e t e s t s . 102 103 f . The e f f e c t o f a GIP g r a d i e n t on the i n s u l i n response to gl u c o s e of the p e r f u s e d pancreas of l e a n and obese r a t s ( F i g 13) GIP was presented to the pancreas as a g r a d i e n t from 0 to 1 ng/ml i n the presence of 300 mg/dl g l u c o s e . The i n s u l i n response to these s t i m u l i was compared with t h a t produced by 300 mg/dl gl u c o s e alone i n both l e a n and obese Zucker r a t s . The i n s u l i n response to GIP at a dose l e v e l of l e s s than 250-300 pg/ml d i d not d i f f e r from t h a t to glucose alone i n e i t h e r l e a n (n=12 vs n=7) ( F i g 13 A) or obese (n=13 vs n=5) (Fig 13 B) r a t s . The t o t a l i n s u l i n response t o 1 ng/ml GIP was 247 x 10 pU i n obese r a t s compared with 89 x 3 10 uU i n l e a n animals, but the r e l a t i v e i n c r e a s e i n the i n s u l i n response caused by GIP was approximately t h r e e f o l d i n both l e a n and obese r a t s . g• The e f f e c t of GIP on the i n s u l i n response of the p e r f u s e d pancreas of l e a n and  obese r a t s to a gl u c o s e g r a d i e n t ( F i g 14) Glucose was presented to the i s o l a t e d pancreas as a g r a d i e n t from 50 to 150 mg/dl i n the presence or absence of 1 ng/ml GIP. F i g u r e 14 shows the r e s u l t s of these experiments. For l e a n r a t s , the i n s u l i n curves do not di v e r g e at glucose l e v e l s l e s s than 110 mg/dl. Above 110 mg/dl the i n s u l i n response to glucose p l u s GIP was s i g n i f i c a n t l y d i f f e r e n t from t h a t to glucose alone ( F i g 14 A). The maximum i n s u l i n output i n the presence of 150 mg/dl 104 glucose p l u s 1 ng/ml GIP (n=7) i s t h r e e f o l d t h a t to glucose alone (n=10). In obese r a t s , an i n s u l i n response was e l i c i t e d by GIP at the lowest g l u c o s e c o n c e n t r a t i o n employed (50 mg/dl) and the maximum response to 150 mg/dl glucose p l u s 1 ng/ml GIP (n=6) was s e v e n f o l d g r e a t e r than t h a t to glucose alone (n=8) ( F i g 14 B ) . h. The e f f e c t o f GIP on the i n s u l i n response of  the p e r f u s e d pancreas of l e a n and obese r a t s to  b a s a l glucose ( F i g 15) The i s o l a t e d pancreas was p e r f u s e d with a b a s a l glucose c o n c e n t r a t i o n o f 80 mg/dl i n l e a n and obese r a t s . Between times 11 and 30 min, 1 ng/ml GIP was i n f u s e d v i a a sidearm from a Harvard i n f u s i o n pump. There was no s i g n i f i c a n t change from b a s a l i n the i n s u l i n response to glucose p l u s GIP i n l e a n r a t s (n=8) , but obese r a t s (n=8) e x h i b i t e d a f i v e f o l d i n c r e a s e above b a s a l i n the i n s u l i n response to p e r f u s i o n with 1 ng/ml GIP. i . The e f f e c t o f a GIP g r a d i e n t on the i n s u l i n response t o b a s a l glucose o f  the p e r f u s e d pancreas of lea n and obese r a t s  ( F i g 16) The i s o l a t e d pancreas was pe r f u s e d with 80 mg/dl glucose i n the presence of 0-1 ng/ml GIP. In l e a n r a t s (n=7), there was a s l i g h t but t r a n s i e n t i n c r e a s e i n the i n s u l i n response to 80 mg/dl glu c o s e at GIP c o n c e n t r a t i o n s 105 g r e a t e r than 300 pg/ml. In obese r a t s (n=7), there was a s i g n i f i c a n t and s u s t a i n e d augmentation of the i n s u l i n response to b a s a l glucose at GIP l e v e l s g r e a t e r than 300 pg/ml. 106 F i g 13. The i n s u l i n response of the i s o l a t e d p e r f u s e d pancreas of (A) l e a n (n=7 vs n=12) and (B) obese (n=5 vs n=13) a d u l t Zucker r a t s to 300 mg/dl glucose i n the absence or presence of a GIP g r a d i e n t from 0-1 ng/ml. *p<.05 using unpaired Student's t - t e s t , compared to l e a n r a t s . In t h i s and subsequent f i g u r e s of data c o l l e c t e d from i s o l a t e d p e r f u s e d pancreas s t u d i e s i n which e i t h e r g lucose or GIP was i n f u s e d as a g r a d i e n t , the c o n c e n t r a t i o n of the i n f u s a t e was shown to i n c r e a s e i n l i n e a r f a s h i o n , although GIP or glucose c o n c e n t r a t i o n s were not measured f o r each experiment. The apparatus used i n these experiments was p r e v i o u s l y shown to d e l i v e r i n f u s a t e s i n a l i n e a r f a s h i o n (Pederson et a l , 1982). 107 Glucose Control Glucose -+ GIP Gradient 8000 4000 0.5 E c a. 10 20 30 M I N U T E S 108 F i g 14. The i n s u l i n response of the i s o l a t e d p e r f u s e d pancreas of (A) l e a n (n=10 vs n=7) and (B) obese (n=8 vs n=6) a d u l t Zucker r a t s to a glucose g r a d i e n t of 50-150 mg/dl alone or i n the presence of 1 ng/ml GIP. *p<.05 using unpaired Student's t - t e s t , compared to l e a n r a t s . 109 50 - 150 mg/dl Glucose 50-150 mg/dl Glucose + M I N U T E S j 110 F i g 15. The i n s u l i n response of the i s o l a t e d p e r f u s e d pancreas of lea n (n=8) and obese (n=8) a d u l t Zucker r a t s t o 80 mg/dl glucose i n the presence of 1 ng/ml GIP. *p<.05 comparing i n s u l i n output f o r min 11-45 to the average i n s u l i n output f o r the f i r s t 10 min of the p e r f u s i o n (shown by the bar from min 1-10) using the Student's t - t e s t f o r p a i r e d data. I l l 112 F i g 16. The i n s u l i n response of the i s o l a t e d p e r f u s e d pancreas of l e a n (n=7) and obese (n=7) a d u l t Zucker r a t s t o 80 mg/dl glucose i n the presence o f a GIP g r a d i e n t from 0-1 ng/ml. *p<.05 comparing i n s u l i n output f o r min 11-45 to the average i n s u l i n output f o r the f i r s t 10 min of the p e r f u s i o n (shown by the bar from min 1-10) u s i n g the Student's t - t e s t f o r p a i r e d d ata. 113 114 IV. DISCUSSION The present study has shown t h a t 8-12-wk-old Zucker f a t t y r a t s are normoglycemic but h y p e r i n s u l i n e m i c (Fig 9, A and B) . T h i s i n d i c a t e s i n s u l i n r e s i s t a n c e , but not to the extent that r e s u l t e d i n glucose i n t o l e r a n c e , and was i n agreement with e a r l i e r s t u d i e s (Bray, 1977). Measurement of f a s t i n g GIP l e v e l s has shown no s i g n i f i c a n t d i f f e r e n c e between obese and l e a n l i t t e r m a t e s at t h i s age ( F i g 9 C ) , a f i n d i n g c o n s i s t e n t with s i m i l a r numbers of G I P - c o n t a i n i n g c e l l s i n the samples of s m a l l i n t e s t i n e ( F i g 8) . In a d d i t i o n , F i g u r e s 9 C and 11 C show t h a t the GIP response to o r a l glucose i s s i m i l a r i n f a t t y r a t s compared with l e a n . I t has r e c e n t l y been demonstrated i n Wistar r a t s , a f t e r j e j u n o i l e a l bypass surgery, that GIP l e v e l s are c h r o n i c a l l y e l e v a t e d and t h a t t h i s r e s u l t s i n a decreased s e n s i t i v i t y of the pancreas to i n v i t r o s t i m u l a t i o n by GIP (Pederson et a l , 1982). Thus, an i n c r e a s e i n f a s t i n g GIP l e v e l s would be u n l i k e l y to cause the observed i n c r e a s e i n f a s t i n g i n s u l i n l e v e l s i n the obese r a t . T h e r e f o r e , i f the e n t e r o i n s u l a r a x i s i s i n v o l v e d i n the i n v i v o h y p e r i n s u l i n e m i a of the young a d u l t obese Zucker r a t , i t i s l i k e l y t o be a r e s u l t of i n c r e a s e d B - c e l l s e n s i t i v i t y to normal GIP l e v e l s . Older a d u l t (6-8-month-old) obese Zucker r a t s were shown to be glucose i n t o l e r a n t and s e c r e t e d s i g n i f i c a n t l y more GIP than l e a n l i t t e r m a t e s . The peak i n s u l i n response of o l d e r obese r a t s was, however, s i m i l a r to that observed 115 i n younger obese animals (Fig 10) . These r e s u l t s c o u l d be e x p l a i n e d by an i n c r e a s e i n i n s u l i n r e s i s t a n c e with age, causing decreased glucose u t i l i z a t i o n and g l u c o s e i n t o l e r a n c e . GIP s e c r e t i o n has been r e p o r t e d to be r e g u l a t e d by feedback from i n s u l i n , which may be impaired i n o b e s i t y ( C r e u t z f e l d t et a l , .1978) due to K - c e l l i n s u l i n r e s i s t a n c e ( F l a t t et a l , 1983b). Down-regulation of GIP r e c e p t o r s on i n s u l i n - s e c r e t i n g B - c e l l s , h y p o t h e s i z e d by Pederson et a l (1982) would prevent f u r t h e r e l e v a t i o n o f i n s u l i n l e v e l s . These s t u d i e s of g l u c o s e t o l e r a n c e i n a d u l t r a t s suggest that as the obese s t a t e p r o g r e s s e s and i n s u l i n r e s i s t a n c e becomes more severe, g l u c o s e t o l e r a n c e s h i f t s from a normal to p a t h o l o g i c a l c o n d i t i o n . T h i s i s accompanied by GIP h y p e r s e c r e t i o n r e s u l t i n g perhaps from impaired feedback r e g u l a t i o n on K - c e l l s by i n s u l i n . E a r l y i n v e s t i g a t i o n s t h at p r o v i d e d evidence f o r the e x i s t e n c e of the e n t e r o i n s u l a r a x i s demonstrated t h a t glucose g i v e n i n t r a d u o d e n a l l y (Mclntyre et a l , 1964) or o r a l l y ( E l r i c k , 1964) s t i m u l a t e d a g r e a t e r i n s u l i n response than glucose a d m i n i s t e r e d v i a the i n t r a v e n o u s r o u t e . In order to demonstrate an i n t a c t e n t e r o i n s u l a r a x i s i n f a t t y Zucker r a t s , the i n s u l i n responses to o r a l and i n t r a v e n o u s glucose loads were compared. F i g u r e 11 shows the glucose and i n s u l i n responses to 0.6 g/kg i n t r a v e n o u s g l u c o s e . While glucose l e v e l s were not s i g n i f i c a n t l y d i f f e r e n t between l e a n and obese r a t s , the i n s u l i n response of f a t t y r a t s i n c r e a s e d 175% over f a s t i n g l e v e l s , whereas the i n s u l i n 116 response of l e a n r a t s was o n l y e l e v a t e d 100%. As demonstrated i n F i g u r e 12 A, 0.6 g/kg glucose administered i n t r a v e n o u s l y produced an i n t e g r a t e d glucose response e q u i v a l e n t to t h a t evoked by 1 g/kg o r a l l y - g i v e n g l u c o s e . I n s u l i n r e l e a s e was s i g n i f i c a n t l y g r e a t e r i n response to o r a l vs intravenous glucose a d m i n i s t r a t i o n (Figure 12 B). However, i t appeared t h a t l e a n r a t s were s l i g h t l y more s e n s i t i v e to components of the e n t e r o i n s u l a r a x i s , s i n c e the i n s u l i n response to o r a l glucose was about 100% g r e a t e r than to intravenous glucose i n l e a n r a t s compared to 50% g r e a t e r i n obese animals. The combined data of o r a l and intravenous glucose t o l e r a n c e t e s t s i n d i c a t e d t h a t an i n t a c t e n t e r o i n s u l a r a x i s e x i s t s i n obese Zucker r a t s , although the f a t t y animals may have reduced s e n s i t i v i t y to e i t h e r the hormonal or the n e u r a l component of the a x i s . A mechanism f o r t h i s phenomenon w i l l be d i s c u s s e d l a t e r . H y p e r s e n s i t i v i t y to glucose, one of the n u t r i e n t components of the a x i s , p r e v i o u s l y r e p o r t e d by Hayek and Woodside (1979), was confirmed i n t h i s study. S t u d i e s i n the i n v i t r o p e r f u s e d pancreas have been employed to i n v e s t i g a t e p a n c r e a t i c s e n s i t i v i t y to v a r i o u s s t i m u l i . P r e v i o u s work by Seino et a l (1981) showed t h a t the h y p e r i n s u l i n e m i a of the obese r a t p e r s i s t e d i n the p e r f u s e d pancreas p r e p a r a t i o n i n response to an a r g i n i n e s t i m u l u s . The i n s u l i n response of obese r a t s to hyperglycemic c o n d i t i o n s (300 mg/dl glucose) i n the p r e s e n t 117 s t u d i e s was approximately t h r e e f o l d that of l e a n l i t t e r m a t e s (Fig 13). However, the i n v i t r o pancreas of the obese r a t s d i d not appear to be more s e n s i t i v e to GIP. I t has p r e v i o u s l y been shown t h a t the t h r e s h o l d l e v e l of GIP necessary f o r s t i m u l a t i o n of i n s u l i n r e l e a s e from the perfused pancreas of Wistar r a t s under hyperglycemic c o n d i t i o n s was 200 pg/ml (Pederson et a l , 1982). T h i s value i s s i m i l a r to the t h r e s h o l d observed f o r l e a n and obese r a t s i n t h i s study ( F i g 13 A ) . In a d d i t i o n , the presence of GIP i n c r e a s e d the t o t a l i n s u l i n output of obese r a t s to very o high l e v e l s when compared with l e a n animals (247 x 10 ull vs 3 89 x 10 pU) but the r e l a t i v e i n c r e a s e i n i n s u l i n output over t h a t produced by g l u c o s e alone was s i m i l a r i n both l e a n and obese animals ( t h r e e f o l d ) . R e s u l t s obtained from g l u c o s e g r a d i e n t experiments i n d i c a t e d that the l e a n Zucker r a t has a glucose t h r e s h o l d of 110 mg/dl f o r p o t e n t i a t i o n of the i n s u l i n response by GIP (Fig 14 A) , which i s s i m i l a r to r e s u l t s o b t a i n e d f o r Wistar r a t s (Pederson and Brown, 1976). U n l i k e t h e i r l e a n l i t t e r m a t e s , obese Zucker r a t s e x h i b i t e d an exaggerated i n v i t r o i n s u l i n response to 1 ng/ml GIP at glucose c o n c e n t r a t i o n s as low as 50 mg/dl (Fig 14 B). By p e r f u s i n g the pancreas of obese r a t s with a glucose c o n c e n t r a t i o n c orresponding to f a s t i n g l e v e l s i n v i v o (80 mg/dl) and a GIP g r a d i e n t of 0-1 ng/ml, t h i s study has demonstrated that f a s t i n g plasma l e v e l s of GIP (500 pg/ml) were capable of p o t e n t i a t i n g the i n s u l i n response to f a s t i n g glucose l e v e l s . 118 In c o n t r a s t , l e a n r a t s e x h i b i t e d a normal glucose t h r e s h o l d (110 mg/dl) f o r the i n s u l i n o t r o p i c a c t i o n of GIP (Figures 15 and 16). The r e s u l t s of i n v i t r o pancreas p e r f u s i o n s t u d i e s i n d i c a t e d t hat the f a s t i n g h y p e r i n s u l i n e m i a observed i n the a d u l t Zucker f a t t y r a t i n the presence of normal f a s t i n g glucose l e v e l s was at l e a s t p a r t l y due to an i n c r e a s e d s e n s i t i v i t y of the pancreas to f a s t i n g l e v e l s of GIP. These data suggest that there i s an impairment of the p h y s i o l o g i c c o n t r o l mechanisms w i t h i n the pancreas t h a t would normally prevent GIP from 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 i n the absence of hyperglycemia. Immunocytochemical s t u d i e s of the pancreas ( F i g 7, Table VII) have confirmed the presence of h y p e r p l a s t i c , h y p e r t r o p h i c i s l e t s as p r e v i o u s l y r e p o r t e d (Shino et a l , 1973; Larsson et a l , 1977). The r e s u l t s are c o n s i s t e n t with an i n c r e a s e d B - c e l l mass w i t h i n the i s l e t s with unchanged glucagon (A) , s o m a t o s t a t i n (D) and p a n c r e a t i c p o l y p e p t i d e (PP) c e l l s . The presence o f normal p a n c r e a t i c p o l y p e p t i d e c e l l s i n the 12-wk-old Zucker r a t (Table V I I , F i g 7 G and H) i s i n c o n t r a s t to a r e p o r t of the absence of PP c e l l s i n these r a t s (Larsson et a l , 1977) . T h i s d i s c r e p a n c y may be due to d i f f e r e n c e s i n the s p e c i f i c i t y and s e n s i t i v i t y of the PP antibody employed i n the immunocytochemical procedure. In New Zealand obese (NZO) mice, i t has been suggested t h a t the high f r e e f a t t y a c i d and t r i g l y c e r i d e l e v e l s observed may be due to an absence of PP (Gates and Lazarus, 1977) . The demonstration of a p p a r e n t l y normal P P - c e l l s l e s s e n s the 119 l i k e l i h o o d o f h y p e r t r i g l y c e r i d e m i a i n Zucker f a t t y r a t s being a t t r i b u t a b l e to an absence of PP, although r e l e a s e of t h i s p e p t i d e from p a n c r e a t i c i s l e t s of obese r a t s has not been measured. R e s u l t s obtained i n t h i s study do not r e v e a l the primary cause of h y p e r i n s u l i n e m i a i n a d u l t obese Zucker r a t s . Although the i n c r e a s e d s e n s i t i v i t y of the B - c e l l s to f a s t i n g GIP l e v e l s probably c o n t r i b u t e s to t h e i r f a s t i n g h y p e r i n s u l i n e m i a , the cause of t h i s B - c e l l h y p e r s e n s i t i v i t y was not e s t a b l i s h e d i n t h i s study. A p o s s i b l e hypothesis to e x p l a i n t h i s phenomenon may i n v o l v e e i t h e r a d i r e c t m o d i f i c a t i o n of the B - c e l l or a change i n the i n t r a - i s l e t r e g u l a t i o n of i n s u l i n r e l e a s e by other p a n c r e a t i c hormones. The hypothesis i n v o l v i n g r e c e p t o r or p o s t - r e c e p t o r a l t e r a t i o n i n the B - c e l l response to GIP i s supported by the recent d e s c r i p t i o n of s p e c i f i c h i g h - a f f i n i t y GIP b i n d i n g s i t e s on hamster B - c e l l tumours ( M a l e t t i e t a l , 1984). Conversely, the data r e g a r d i n g s e c r e t i o n of the other i s l e t hormones, glucagon and s o m a t o s t a t i n are c o n f l i c t i n g and d i f f i c u l t t o i n t e r p r e t because of v a r i a t i o n s i n methodology. In obese Zucker r a t s , glucagon s e c r e t i o n has been r e p o r t e d as decreased from i s o l a t e d i s l e t s (Hayek and Woodside, 1979), normal i n the i s o l a t e d , p e r f u s e d pancreas (Seino et a l , 1981) or e l e v a t e d i n v i v o (Nishikawa, Ikeda and Matsua, 1981). Somatostatin r e l e a s e has a l s o been d e s c r i b e d as normal i n the i s o l a t e d , p e r f u s e d pancreas (Seino et a l , 1981), decreased i n p o r t a l plasma (Sheppard et a l , 1980) or 120 i n c r e a s e d i n i s o l a t e d i s l e t s (Trimble, Herberg and Renold, 1980) . The s i t e of the abnormal GIP response cannot be concluded from the r e s u l t s of t h i s study, s i n c e GIP has been shown to s t i m u l a t e both glucagon (Pederson and Brown, 1978) and s o m a t o s t a t i n (Ipp et a l , 1977) r e l e a s e from the pancreas. In a second study, the r o l e o f GIP i n the development o f h y p e r i n s u l i n e m i a of pre-obese r a t s was i n v e s t i g a t e d . S t u d i e s were c a r r i e d out to determine the s i g n i f i c a n c e of a lack of a glucose t h r e s h o l d f o r the i n s u l i n o t r o p i c a c t i o n of GIP i n the onset of h y p e r i n s u l i n e m i a i n n e o n a t a l , f a / f a pups, s i n c e the metabolic events c o n t r i b u t i n g to t h i s c o n d i t i o n have not been u n e q u i v o c a l l y e s t a b l i s h e d . F a s t i n g plasma i n s u l i n and GIP l e v e l s were measured i n s u c k l i n g and weanling r a t s and these changes were c o r r e l a t e d with changes i n the response of the i n v i t r o i s o l a t e d p e r f u s e d r a t pancreas to glucose and GIP i n 21-, 28- and 35-day-old l e a n and obese Zucker r a t s . In a d d i t i o n , p a n c r e a t i c t i s s u e samples were obtained from r a t s aged 1 day to 28 days and examined by immunocytochemistry to determine when enlarged p a n c r e a t i c i s l e t s c h a r a c t e r i s t i c of a d u l t obese r a t s , as demonstrated i n t h i s study and o t h e r s (Shino e t a l , 1973; Larsson, 1977), became apparent i n f a / f a pups. To ensure t h a t d i f f e r e n c e s i n plasma GIP c o n c e n t r a t i o n between l e a n and f a / f a pups were not r e s p o n s i b l e f o r changes i n i n s u l i n l e v e l s , the plasma c o n c e n t r a t i o n of GIP was 121 measured i n 21-, 28- and 35-day-old r a t s . V i s u a l i d e n t i f i c a t i o n of phenotype was made r e t r o a c t i v e l y at 6 weeks of age. At no time were any s i g n i f i c a n t d i f f e r e n c e s i n plasma l e v e l s observed between l e a n and obese r a t s (Table VI) . T h i s i s c o n s i s t e n t with r e s u l t s o b t a i n e d i n young (8-12-week old) a d u l t r a t s (Figure 9). Immunocytochemical examination of neonatal Zucker r a t pancreas has demonstrated the e x i s t e n c e of en l a r g e d i s l e t s i n f a / f a pups as e a r l y as 7 days of age (F i g u r e s 2 and 3) . This o b s e r v a t i o n supports the hypothesis of ot h e r s (Godbole et a l , 1981; Turkenkopf, Johnson and Greenwood, 1982) that the p o t e n t i a l f o r i n s u l i n h y p e r s e c r e t i o n i s p r e s e n t e a r l y i n the s u c k l i n g stage, d e s p i t e the f a c t t h at e l e v a t e d plasma i n s u l i n c o n c e n t r a t i o n s cannot be measured u n t i l the t h i r d p o s t - n a t a l week. A demonstrated absence of en l a r g e d i s l e t s i n f a / f a pups i n the f i r s t week of age was supported by a report by Hayek (1982) that showed no i n c r e a s e i n i n s u l i n s e c r e t i o n from i s o l a t e d , c u l t u r e d i s l e t s taken from f a / f a and lean pups from 2-5 days of age. Thus, h y p e r p l a s i a of p a n c r e a t i c i s l e t s i n f a / f a pups appears t o a r i s e concomitantly with the observed i n c r e a s e i n adipose t i s s u e f a t t y a c i d synthetase a c t i v i t y at 9 days (Bazin and Lavau, 1982) and i n c r e a s e d c a l o r i c d e p o s i t i o n i n f a t depots at 7 days (Boulange, Planche and de Gasquet, 1979). Measurement of 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 (Table V) confirms development of h y p e r i n s u l i n e m i a by 21 days i n f a / f a r a t s compared to F a / f a or Fa/Fa l e a n c o n t r o l s 122 (Bazin and Lavau, 1982; Turkenkopf, Johnson and Greenwood, 1982; Rohner-Jeanrenaud et a l , 1983). There has been a s i n g l e r e p o r t (Blonz, S t e r n and Curr y , 1985) o f h y p e r i n s u l i n e m i a appearing i n obese pups by 14 days of age when compared to homozygote Fa/Fa l e a n pups o n l y . Measurement of plasma i n s u l i n f o l l o w i n g a 12-h f a s t r e v e a l e d t h a t b a s a l h y p e r i n s u l i n e m i a d i d not occur u n t i l f a / f a r a t s reached 35 days of age (Table V) . T h e r e f o r e , the mechanisms which c o n t r o l the onset of f a s t i n g versus g l u c o s e - s t i m u l a t e d h y p e r i n s u l i n e m i a i n f a / f a r a t s appear to have important d i f f e r e n c e s . In order to i n v e s t i g a t e the p o s s i b l e r o l e of GIP i n the development of both g l u c o s e - s t i m u l a t e d and f a s t i n g h y p e r i n s u l i n e m i a i n weanling f a / f a r a t s , the i n v i t r o i s o l a t e d p e r f u s e d pancreas was employed. Phenotype was determined by two methods. Because body weight of pre-obese pups was not r e l i a b l y g r e a t e r than t h a t o f l e a n pups u n t i l the 5th week of age (Table III) , both measurement of c o l o n i c temperature (Table IV) and examination o f p a n c r e a t i c i s l e t area (Figs 3 and 4) were performed. As e a r l i e r r e p o r t e d (Godbole, York and Bloxham, 1978) the mean c o l o n i c temperature of f a / f a pups was about 1°C lower than t h a t of lean c o n t r o l s . Secondly, p a n c r e a t i c i s l e t s o f 3 wk o l d pups were s i g n i f i c a n t l y enlarged compared t o those of l e a n l i t t e r m a t e s . Combining these two techniques gave a r e l i a b l e p r e d i c t i o n of " f a t n e s s " i n v i s u a l l y u n i d e n t i f i a b l e pups. 123 The i n s u l i n response to hyperglycemic c o n d i t i o n s (300 mg/dl glucose) p l u s GIP i n these s t u d i e s was not s i g n i f i c a n t l y d i f f e r e n t between l e a n and f a / f a r a t s u n t i l 35 days of age when f a / f a pups e x h i b i t e d a higher i n s u l i n response than t h e i r l e a n l i t t e r m a t e s (Figure 5) . These r e s u l t s suggest t h a t GIP was not important i n development of 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 i n s u c k l i n g f a / f a pups t h a t arose by 21 days of age i n v i v o . F u r t h e r evidence t h a t GIP i s not e s s e n t i a l to g l u c o s e - s t i m u l a t e d h y p e r i n s u l i n e m i a i n s u c k l i n g f a / f a r a t s was g i v e n i n i n v i v o experiments where i t was shown that g l u c o s e a d m i n i s t r a t i o n e i t h e r i n t r a p e r i t o n e a l l y (York, S h a r g i l l and Godbole, 1981; a l s o Table V) or i n t r a v e n o u s l y (Rohner-Jeanrenaud et a l , 1983) induced an e l e v a t e d i n s u l i n response i n f a / f a pups. Abso r p t i o n of glucose by the gut was necessary f o r GIP r e l e a s e (Cataland et a l , 1974; Pederson, Schubert and Brown, 1975; Thomas et a l , 1976). Thus, p o t e n t i a t i o n of i n s u l i n r e l e a s e by GIP when gl u c o s e was administered i n t r a p e r i t o n e a l l y or i n t r a v e n o u s l y was u n l i k e l y . C o n t r a r y to these r e s u l t s , i t was r e p o r t e d t h a t i n s u l i n h y p e r s e c r e t i o n from the i s o l a t e d pancreas was e v i d e n t as e a r l y as 14 days of age i n f a / f a pups compared to Fa/Fa l e a n c o n t r o l s (Blonz, S t e r n and C u r r y , 1985) i n response to 325 mg/dl g l u c o s e . P a n c r e a t i c t i s s u e l e v e l s of i n s u l i n were not e l e v a t e d . The authors concluded t h a t s u b t l e d i f f e r e n c e s i n Fa/Fa l e a n vs F a / f a l e a n pups had b i a s e d e a r l i e r r e p o r t s that h y p e r i n s u l i n e m i a d i d not appear u n t i l the 3rd week. 124 Turkenkopf, Johnson and Greenwood (1982), comparing f a / f a pups with F a / f a c o n t r o l s d i d not observe h y p e r i n s u l i n e m i a u n t i l 21 days of age i n f a / f a pups. These authors a l s o found no d i f f e r e n c e s i n p a n c r e a t i c t i s s u e l e v e l s of i n s u l i n p r i o r to weaning. In the experiments d e s c r i b e d here, a l l pups were a r e s u l t of mating f a / f a males with F a / f a females. Thus, a l l l e a n animals were heterozygous ( F a / f a ) . While the presence of the f a gene i n heterozygote c o n t r o l s may a l t e r i n s u l i n r e l e a s e i n these animals, there i s no e x p l a n a t i o n as to why these t r a i t s disappear i n adulthood. F u r t h e r evidence regarding normal i n s u l i n r e l e a s e comes from comparison of i n s u l i n s e c r e t i o n from the p e r f u s e d pancreas of l e a n Zucker r a t s i n the c u r r e n t study with normal Wistar r a t s i n s i m i l a r s t u d i e s (Pederson et a l , 1982). P r e v i o u s l y , no d i f f e r e n c e s were observed between F a / f a and Fa/Fa a d u l t s (Bray, 1977). P o s s i b l e e x p l a n a t i o n s f o r the d i f f e r e n c e s i n r e s u l t s observed by Blonz's group compared to those of Turkenkopf and the c u r r e n t study i n c l u d e (1) l i m i t a t i o n of l i t t e r s i z e to 8 p u p s / l i t t e r ; (2) use of a high flow r a t e i n pancreas p e r f u s i o n s t u d i e s (2.4 ml/min f o r 14-day-old pups and 4.8 ml/min f o r 4-wk-old pups); (3) d e t e r m i n a t i o n of the f a / f a phenotype by measurement of body f a t content. P r e v i o u s l y , the weight of adipose t i s s u e s compared to t o t a l body weight was found to be a good i n d i c a t o r of impending f a t n e s s , but only a f t e r 16 days of age, as s i g n i f i c a n t o v e r l a p o c c u r r e d p r i o r to t h i s age (Lavau and B a z i n , 1982) . Body weight was a l s o r e p o r t e d by Blonz's group to be s i g n i f i c a n t l y higher i n 125 f a / f a pups compared to Fa/Fa animals by 2 weeks, although t h i s has not been observed elsewhere (Table I I I ; Zucker and Zucker, 1961; Bazin and Lavau, 1982). A s i g n i f i c a n t d i f f e r e n c e i n the i n s u l i n response of f a / f a pups versus l e a n l i t t e r m a t e s at 35 days of age, but not at 21 or 28 days of age (Figure 6) was r e v e a l e d i n these s t u d i e s . In v i t r o i n s u l i n h y p e r s e c r e t i o n to b a s a l g l u c o s e p l u s GIP c o i n c i d e d with development o f f a s t i n g h y p e r i n s u l i n e m i a i n v i v o . These phenomena may be c a u s a l l y r e l a t e d , perhaps r e s u l t i n g from development of a p o s t -receptor d e f e c t i n the B - c e l l response to g l u c o s e at 5 weeks of age i n f a / f a r a t s . I t was p o s t u l a t e d t h a t t h i s d e f e c t would allow GIP to s t i m u l a t e i n s u l i n r e l e a s e i n the absence of hyperglycemia. I t i s p o s s i b l e t h a t development o f f a s t i n g h y p e r i n s u l i n e m i a c o n t r i b u t e s to i n c r e a s i n g hyperphagia and weight g a i n (Zucker and Zucker 1961; Table I I I ) i n these r a t s , s i n c e a l l of these c h a r a c t e r i s t i c s of the f a / f a r a t become o v e r t at the same age. Both weight g a i n and hyperphagia are probably secondary to e l e v a t e d i n s u l i n s e c r e t i o n , s i n c e n o r m a l i z a t i o n of plasma i n s u l i n l e v e l s i n f a / f a r a t s r e s u l t e d i n weight l o s s and reduced food i n t a k e (Chan and S t e r n , 1981; S t o l z and M a r t i n , 1982). The p o s s i b i l i t y t h a t i n s u l i n s e c r e t i o n i s secondary to food i n t a k e and weight g a i n cannot be e l i m i n a t e d without s t u d i e s i n which food i n t a k e and weight g a i n are c o n t r o l l e d i n f a / f a r a t s . 126 Data obtained i n t h i s study suggests t h a t f a s t i n g h y p e r i n s u l i n e m i a may be secondary to the absence of a glucose t h r e s h o l d f o r the i n s u l i n o t r o p i c a c t i o n of GIP appearing by 35 days i n f a / f a weanling pups. T h i s c o n d i t i o n may c o n t r i b u t e to important b e h a v i o r a l changes such as hyperphagia, l e a d i n g to e x c e s s i v e weight g a i n . Moreover, both enlarged i s l e t s and g l u c o s e - s t i m u l a t e d h y p e r i n s u l i n e m i a e x i s t p r i o r to m a n i f e s t a t i o n of f a s t i n g h y p e r i n s u l i n e m i a . Thus, c h r o n i c o v e r - s t i m u l a t i o n of the enlarged i s l e t s by glucose p l u s o t h e r , unknown f a c t o r s may l e a d to a breakdown of B - c e l l r e g u l a t o r y p r o c e s s e s , i n c l u d i n g those which c o n t r o l the a c t i o n of GIP on i n s u l i n s e c r e t i o n . 127 CHAPTER TWO: THE ROLE OF THE CHOLINERGIC NERVOUS SYSTEM IN THE DEVELOPMENT AND MAINTENANCE OF HYPERINSULINEMIA IN OBESE ZUCKER RATS I. INTRODUCTION The autonomic nervous system i s an important component of the e n t e r o i n s u l a r a x i s . Evidence suggests t h a t s p e c i f i c b r a i n n u c l e i have important i n f l u e n c e s on fe e d i n g behaviour and energy balance (Rohner-Jeanrenaud, Ionescu and Jeanrenaud, 1983). D e s t r u c t i o n o f the ventromedial hypothalamic n u c l e i has long been known to r e s u l t i n r a p i d onset of h y p e r i n s u l i n e m i a , hyperphagia and weight g a i n (Bray and G a l l a g h e r , 1975). H y p e r i n s u l i n e m i a i n r a t s with VMH l e s i o n s was reversed by subdiaphragmatic vagotomy (Berthoud and Jeanrenaud, 1979; Inoue and Bray, 1977). A l s o , a t r o p i n e i n f u s i o n normalized i n s u l i n s e c r e t i o n from the i s o l a t e d , p e r f u s e d pancreas of VMH-lesioned r a t s , whereas the c h o l i n e r g i c a g o n i s t methacholine caused h y p e r s e c r e t i o n o f i n s u l i n compared to c o n t r o l s , suggesting involvement of the autonomic nervous system (Rohner-Jeanrenaud and Jeanrenaud, 1981). The r o l e of the parasympathetic, c h o l i n e r g i c nervous system has been i n v e s t i g a t e d i n obese Zucker r a t s but the r e s u l t s are e q u i v o c a l . Opsahl and Powley (1974) r e p o r t e d that h y p e r i n s u l i n e m i a i n o v e r t l y obese Zucker r a t s was not a l l e v i a t e d by vagotomy but Rohner-Jeanrenaud et a l (1983) 128 subsequently demonstrated t h a t both vagotomy and a t r o p i n e i n f u s i o n i n young a d u l t r a t s decreased i n s u l i n s e c r e t i o n i n f a t but not l e a n r a t s . S t i m u l a t i o n of the c e r v i c a l vagus a l s o e l i c i t e d a h y p e r i n s u l i n e m i c response i n young a d u l t f a t t y r a t s . In the i s o l a t e d p e r f u s e d pancreas p r e p a r a t i o n , i n s u l i n r e l e a s e by obese r a t s s t i m u l a t e d by a r g i n i n e was reduced by a d d i t i o n of 25 x 10 -^ M a t r o p i n e to the p e r f u s a t e . A s e r i e s of experiments was undertaken to attempt to c o n f i r m some of the o b s e r v a t i o n s of Rohner-Jeanrenaud et a l (1983), and to i n v e s t i g a t e the e f f e c t s of m u s c a r i n i c and n i c o t i n i c antagonism on 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 glucose and GIP, i n order to examine the p o s s i b i l i t y of i n t e r a c t i o n between a l l components of the e n t e r o i n s u l a r a x i s i n the s t i m u l a t i o n of i n s u l i n r e l e a s e i n obese Zucker r a t s . A second hormone whose r e l e a s e i s under the p a r t i a l c o n t r o l of the vagus nerve i s g a s t r i n r e l e a s e from the stomach (Grossman, 1976). In experiments employing the i s o l a t e d p e r f u s e d r a t stomach i t was demonstrated t h a t g a s t r i n r e l e a s e i s s t i m u l a t e d by c h o l i n e r g i c a g o n i s t s (Pederson et a l , 1984) and e l e c t r i c a l s t i m u l a t i o n of the v a g a l trunks (Pederson et a l , 1981). In animals made hypergastrinemic by vagotomy, a t r o p i n e reduced g a s t r i n s e c r e t i o n i n the i s o l a t e d pancreas to c o n t r o l l e v e l s (Pederson et a l , 1984). I t was suggested by these authors and others (Debas et a l , 1984) t h a t the stomach was i n f l u e n c e d by both s t i m u l a t o r y and i n h i b i t o r y v a g a l inputs 129 and that n o n - s e l e c t i v e vagotomy removed mostly the i n h i b i t o r y i n f l u e n c e . In the c u r r e n t i n v e s t i g a t i o n , g a s t r i n s e c r e t i o n i n v i v o and i n v i t r o i n Zucker r a t s was i n v e s t i g a t e d to see i f evidence e x i s t e d f o r an imbalance i n the s t i m u l a t o r y / i n h i b i t o r y e f f e c t s o f the vagus on g a s t r i n r e l e a s e . I t was hypothesized t h a t an i n c r e a s e i n the c h o l i n e r g i c s t i m u l a t o r y component of the vagus c o u l d r e s u l t i n h y pergastrinemia i n these animals. 130 I I . METHODS A l l methods were as d e s c r i b e d i n General Methodology. I I I . RESULTS A. Weanling Rats 1. The E f f e c t of A c e t y l c h o l i n e on I n s u l i n  S e c r e t i o n of S u c k l i n g r a t s under  Hyperglycemic C o n d i t i o n s ( F i g 17) I n s u l i n s e c r e t i o n from the pancreata of 3-week-old s u c k l i n g r a t s was s t i m u l a t e d with 300 mg/dl gl u c o s e . Between 11 and 30 min of the 45 min p e r f u s i o n p e r i o d , 100 pM a c e t y l c h o l i n e was i n t r o d u c e d v i a a sidearm i n f u s i o n . The i n s u l i n response of 21-day-old f a / f a Zucker r a t s (n=7) was not s i g n i f i c a n t l y d i f f e r e n t from that of l e a n c o n t r o l s (n=6). 131 F i g 17. The response of the i s o l a t e d p e r f u s e d pancreas of l e a n (n=6) and obese (n=7) Zucker pups at 21 days of age to 300 mg/dl - 4 glucose p l u s 10 M a c e t y l c h o l i n e . *p<.05 using unpaired Student's t - t e s t . 132 133 B. A d u l t Rats 1. I n s u l i n S e c r e t i o n from the I s o l a t e d Perfused  Pancreas a. The e f f e c t of a t r o p i n e on 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 of le a n and obese r a t s ( F i g 18) The pancreata o f 4-month-old l e a n and obese r a t s were per f u s e d with 300 mg/dl g l u c o s e . A t r o p i n e sulphate — 5 (10 M) was i n f u s e d from 26-35 min of the 50 min experiment. In t h i s and other experiments i n v o l v i n g the r o l e of the c h o l i n e r g i c nervous system i n the pancreas, r e s u l t s were expressed as the average i n s u l i n output per 5 min p e r i o d . P r e - a t r o p i n e i n s u l i n l e v e l s i n l e a n r a t s (n=6) approached mean l e v e l s of 300 pU/min and rose to 400 pU/min during and 650 pU/min a f t e r a t r o p i n e i n f u s i o n ( F i g 18 A) . In obese r a t s (n=6) the mean i n s u l i n r e l e a s e was 550 juU/min, d i d not change d u r i n g a t r o p i n e i n f u s i o n , and rose t o 1150 pU/min f o l l o w i n g a t r o p i n e ( F i g 18 B). b. The e f f e c t of hexamethonium on 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 of l e a n and obese  r a t s ( F i g 19) The pancreata of 4-month-old l e a n and obese r a t s were p e r f u s e d with 300 mg/dl g l u c o s e . Hexamethonium bromide ( 4 x l 0 - ^ M) was in t r o d u c e d as d e s c r i b e d f o r a t r o p i n e ( S e c t i o n I I I B 1 ) . I n s u l i n s e c r e t i o n i n response t o glu c o s e alone was 200 pU/min i n lea n r a t s (n=4) . I n t r o d u c t i o n of hexamethonium caused a s l i g h t i n c r e a s e t o 300 pU/min. A f t e r 134 hexamethonium was removed, i n s u l i n l e v e l s c ontinued to inc r e a s e to 500 pU/min ( F i g 19 A) . In obese r a t s (n=4) , ba s a l i n s u l i n r e l e a s e was 500 pU/min, r i s i n g to 700 uU/min during hexamethonium i n f u s i o n , and 1300 pU/min a f t e r the i n f u s i o n was stopped ( F i g 19 B). c. The e f f e c t of a t r o p i n e on 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 L - a r g i n i n e ( F i g 20) The p e r f u s e d pancreata of 4-month-old l e a n and obese r a t s were p e r f u s e d with 10 mM L - a r g i n i n e . No glucose was present i n the p e r f u s a t e . In l e a n r a t s (n=4) i n s u l i n r e l e a s e was l e s s than 10 pU/min p r i o r to a t r o p i n e i n f u s i o n . When a t r o p i n e was i n t r o d u c e d as d e s c r i b e d e a r l i e r ( S e c t i o n I I I B 1 ) , i n s u l i n l e v e l s rose s i g n i f i c a n t l y , peaking at 50 pU/min i n the 5-min p e r i o d a f t e r the a t r o p i n e was removed before slowly d e c l i n i n g ( F i g 20 A ) . The i n s u l i n s e c r e t o r y p a t t e r n was s i m i l a r i n obese r a t s (n=4) but b a s a l l e v e l s were t h r e e f o l d higher than i n l e a n r a t s and rose to 130 pU/min during a t r o p i n e i n f u s i o n and 180 pU/min a f t e r the i n f u s i o n was d i s c o n t i n u e d ( F i g 20 B). d. The e f f e c t of a t r o p i n e on i n s u l i n r e l e a s e of l e a n and obese r a t s s t i m u l a t e d by glucose p l u s GIP ( Fig 21) The i s o l a t e d pancreata of 4-month-old l e a n and obese r a t s were p e r f u s e d with 300 mg/dl g l u c o s e . Between 16 and 40 min 1 ng/ml GIP was i n f u s e d v i a a sidearm. At 26 min 135 a t r o p i n e (10~ M) was i n t r o d u c e d f o r 10 min. Glucose-s t i m u l a t e d i n s u l i n s e c r e t i o n was t h r e e f o l d higher i n obese (n=4) compared to l e a n animals (n=4). T h i s r e l a t i o n s h i p p e r s i s t e d i n the presence of 1 ng/ml GIP. A t r o p i n e had no e f f e c t on i n s u l i n s e c r e t i o n s t i m u l a t e d by GIP i n e i t h e r l e a n or obese r a t s ( F i g 21, A and B). e. The e f f e c t of hexamethonium on i n s u l i n r e l e a s e  of l e a n and obese r a t s s t i m u l a t e d by glu c o s e p l u s GIP ( F i g 22) The pancreata of 4-month-old l e a n and obese r a t s were p e r f u s e d with 300 mg/dl g l u c o s e . Between 16-40 min of the 50 min experiment 1 ng/ml GIP was i n f u s e d . Glucose-s t i m u l a t e d i n s u l i n l e v e l s of l e a n r a t s (n=4) were l e s s than 400 pU/min and rose to 1000 pU min f o l l o w i n g GIP i n f u s i o n . Hexamethonium i n f u s e d between 26 and 35 min had no e f f e c t , although a t r a n s i e n t i n c r e a s e t o 1600 pU/min oc c u r r e d i n the 5 min p e r i o d f o l l o w i n g hexamethonium i n f u s i o n ( F i g 22 A ) . A s i m i l a r p a t t e r n of r e l e a s e was observed f o r obese r a t s (n=4) but at p r o p o r t i o n a t e l y higher s e c r e t o r y r a t e (basal 900 pU/min, p l u s GIP and hexamethonium 2500 pU/min) ( F i g 22 B). 136 F i g 18. The i n s u l i n response of the i s o l a t e d p e r f u s e d pancreas of (A) l e a n (n=6) and (B) obese (n=6) a d u l t Zucker r a t s t o 300 — 5 mg/dl glu c o s e i n the presence o f 10 M a t r o p i n e s u l p h a t e , expressed as the mean i n s u l i n output per 5-min p e r i o d . I n s u l i n output during and subsequent t o a t r o p i n e i n f u s i o n was compared t o the mean i n s u l i n output of the 2nd to 5th 5-min p e r i o d . *p<.05 using Student's t - t e s t f o r p a i r e d data. 137 80 300 mg/dl GLUCOSE ^ IO'BM ATROPINE I 1 A. 600-400 -200 -0 B . 1200 -1000-800-600 -4 0 0 -200 -0 -* , T _T_ 1 2 3 4 5 6 7 8 9 10 FIVE-MINUTE PERIODS 138 F i g 19. The i n s u l i n response o f the i s o l a t e d p e r f u s e d pancreas o f (A) l e a n (n=4) and (B) obese (n=4) a d u l t Zucker r a t s to 300 mg/dl glucose i n the presence o f 4 x 10"^ M hexamethonium bromide, expressed as the mean i n s u l i n output per 5-min p e r i o d . I n s u l i n output d u r i n g and subsequent to hexamethonium i n f u s i o n was compared to the mean i n s u l i n output o f the 2nd to 5th 5-min p e r i o d . *p<.05 using Student's t - t e s t f o r p a i r e d d ata. 139 80 300 mg/dl GLUCOSE I 4xlO _ 4 M HEXAMETHONIUM 1 1 B. JL 2 3 4 5 6 7 8 9 FIVE MINUTE PERIODS 10 140 F i g 20. The i n s u l i n response of the i s o l a t e d p e r f u s e d pancreas o f (A) l e a n (n=4) and (B) obese (n=4) a d u l t Zucker r a t s t o 10 -5 mM L - a r g i n i n e i n the presence o f 10 M a t r o p i n e sulphate, expressed as the mean i n s u l i n output per 5-min p e r i o d . I n s u l i n output during and subsequent to a t r o p i n e i n f u s i o n was compared to the mean i n s u l i n output of the 2nd to 5th 5-min p e r i o d . *p<.05 using Student's t - t e s t f o r p a i r e d data. 141 A. 10 mM L-ARGININE I0"BM ATROPINE 1 I 60-1 40 20 0 1 t-JL B. 160" 120-80-40 O x T I I 2 3 4 5 6 7 8 9 10 FIVE MINUTE PERIODS 142 F i g 21. The i n s u l i n response of the i s o l a t e d p e r f u s e d pancreas o f (A) l e a n (n=4) and (B) obese (n=4) a d u l t Zucker r a t s to 300 — 5 mg/dl glucose p l u s 1 ng/ml GIP i n the presence of 10 M at r o p i n e s u l p h a t e , expressed as the mean i n s u l i n output per 5-min p e r i o d . I n s u l i n output d u r i n g and subsequent to a t r o p i n e i n f u s i o n was compared to the mean i n s u l i n output of the 4th and 5th 5-min p e r i o d . *p<.05 using Student's t - t e s t f o r p a i r e d data. 143 80 300 mg/dl GLUCOSE r r I ng/ml GIP J 10 " B M ATROPIN^ 2000 1000 0-L c E 3 or 8000 =^ 6000 3 CO 4000 2000 B . o i c T JL _T_ 1 2 3 4 5 6 7 8 9 10 FIVE MINUTE PERIODS 144 The i n s u l i n response o f the i s o l a t e d p e r f u s e d pancreas o f (A) l e a n (n=4) and (B) obese (n=4) a d u l t Zucker r a t s t o 300 -4 mg/dl glucose p l u s 1 ng/ml GIP i n the prsence o f 4 x 10 M hexamethonium bromide, expressed as the mean i n s u l i n output per 5-min p e r i o d . I n s u l i n output d u r i n g and subsequent to hexamethonium i n f u s i o n was compared to the mean i n s u l i n output of the 4th and 5th 5-min p e r i o d . *p<.05 using the Student's t - t e s t f o r p a i r e d d a t a . 145 80 r r 300 mg/dl GLUCOSE I ng/ml GIP 4 « I 0 " 4 M HEXAMETHONIUM 1 1 2000 -1000 • 0-B. 3000 -2000 • 1000 - • T _L I 2 3 4 5 6 7 8 9 10 FIVE MINUTE PERIODS 146 2. G a s t r i n S e c r e t i o n i n Lean and Obese Rats;  C h o l i n e r g i c I n f l u e n c e a. F a s t i n g plasma g a s t r i n (Table VIII) Lean and obese Zucker r a t s i n three age groups were f a s t e d o v e r n i g h t and a b a s a l blood sample ob t a i n e d f o r I R - G a s t r i n (IRG) measurement. The age groups were 6-8 months, 3-4 months and 3 weeks. Blood samples were c o l l e c t e d from c o n s c i o u s u n r e s t r a i n e d animals i n the case of the a d u l t r a t s . Three week o l d pups were a n e s t h e t i z e d with ether p r i o r to b l o o d c o l l e c t i o n . Phenotype of 3-week-old r a t s was determined by examination of p a n c r e a t i c i s l e t s (Chapter One, S e c t i o n IID2). Obese 6-8-month-old r a t s (n=9) had f a s t i n g IRG l e v e l s 5-f o l d higher than those of l e a n l i t t e r m a t e s (n=10) (263 + 36.6 pg/ml vs 54 + 5.4 pg/ml). Younger (3-4-month-old) obese r a t s (n=8) d i s p l a y e d f a s t i n g g a s t r i n l e v e l s 3 - f o l d higher than those of l e a n l i t t e r m a t e s (n=9) (138 + 23.0 pg/ml vs 45.3 + 4.9 pg/ml) but 3-week-old weanling obese r a t s (n=15) were not hypergastr inemic compared to l e a n l i t t e r m a t e s (n=14) (104.6 + 15.3 pg/ml vs 88.1 + 10.7 pg/ml). 147 TABLE V I I I . FASTING PLASMA GASTRIN CONCENTRATIONS (X + SEM) IN LEAN AND OBESE ZUCKER RATS AGE GENOTYPE IR-GASTRIN (pg/ml) 3 weeks Fa/-, n=14 f a / f a , n=15 88.1 + 10.7 105 + 15.3 3--4 months Fa/-, n=9 f a / f a , n=8 45.3 + 4.9 138 + 23.0* 6 months Fa/-, n=10 f a / f a , n=9 53.8 + 5.4 263 + 36.6* *p<.05 comparing f a / f a r a t s to Fa/- r a t s of the same age group. 148 b. In v i t r o g a s t r i n r e l e a s e from the i s o l a t e d  p e r f u s e d stomach Six-month-old male Zucker r a t s were used i n these experiments. i . B a s a l g a s t r i n r e l e a s e of l e a n and  obese r a t s ( F i g 23) The i s o l a t e d stomachs of l e a n (n=7) and obese (n=5) Zucker r a t s were pe r f u s e d with b u f f e r c o n t a i n i n g 80 mg/dl g l u c o s e . B a s a l g a s t r i n r e l e a s e of obese r a t s averaged 550 pg/ml compared to 150 pg/ml i n l e a n animals. i i . The e f f e c t of a t r o p i n e on the b a s a l g a s t r i n  r e l e a s e of l e a n and obese Zucker r a t s ( F i g 24) The i n v i t r o i s o l a t e d stomachs of 6-month-old le a n (n=7) and obese (n=8) Zucker r a t s were per f u s e d under b a s a l c o n d i t i o n s ( S e c t i o n I I I B 2 b i ) . From 11-30 min of the 40 min experiment, a t r o p i n e (10~ 5 M) was i n f u s e d v i a a sidearm. The a d d i t i o n o f a t r o p i n e had no e f f e c t on b a s a l g a s t r i n s e c r e t i o n i n l e a n or obese r a t s ( F i g 24). i i i . The e f f e c t o f a t r o p i n e on v a g a l l y s t i m u l a t e d  g a s t r i n r e l e a s e of l e a n and obese Zucker  r a t s ( F i g 25) F o l l o w i n g a 10 min of p e r f u s i o n of the i s o l a t e d stomach under b a s a l c o n d i t i o n s , the v a g a l trunks 149 were e l e c t r i c a l l y s t i m u l a t e d (7 v o l t s , 10 Hz f 5 ms duration) from 11-30 min. Between 21 and 30 min an i n f u s i o n of 10 ^ M a t r o p i n e was superimposed on the v a g a l s t i m u l a t i o n . G a s t r i n s e c r e t i o n was reduced to b a s a l s e c r e t o r y l e v e l s by a t r o p i n e treatment i n both l e a n (n=4) and obese (n=6) r a t s . 150 F i g 23. G a s t r i n s e c r e t i o n from the i s o l a t e d stomach of l e a n (n=7) and obese (n=5) Zucker r a t s under b a s a l c o n d i t i o n s . *p<.05 usi n g unpaired Student's t - t e s t . 151 152 F i g 24. — 5 The e f f e c t o f 10 M a t r o p i n e sulphate on b a s a l g a s t r i n s e c r e t i o n from the i s o l a t e d stomach of l e a n (n=7) and obese (n=8) Zucker r a t s . *p<.05 using p a i r e d Student's t - t e s t and comparing g a s t r i n r e l e a s e d u r i n g and subsequent to i n f u s i o n to the average b a s a l g a s t r i n r e l e a s e from min 1-10. The average b a s a l g a s t r i n r e l e a s e i s represented by the bar from min 1-10. 153 M I N U T E S F i g 25. — 5 The e f f e c t o f 10 M a t r o p i n e sulphate on g a s t r i n s e c r e t i o n e l i c i t e d by s t i m u l a t i o n of the v a g a l trunks (7 V, 10 Hz, 5 ms) from the i s o l a t e d stomach of l e a n (n=4) and obese (n=6) Zucker r a t s . *p<.05 using p a i r e d Student's t - t e s t and comparing g a s t r i n r e l e a s e d u r i n g and subsequent to v a g a l s t i m u l a t i o n to the average b a s a l g a s t r i n r e l e a s e from min 1-10. The average b a s a l g a s t r i n r e l e a s e i s represented by the bar from min 1-10. 155 156 .i c. B a s a l s o m a t o s t a t i n r e l e a s e from the i s o l a t e d  stomach of l e a n and obese Zucker r a t s (Fig 26) Somatostatin r e l e a s e was measured under b a s a l c o n d i t i o n s i n the i s o l a t e d p e r f u s e d stomach of l e a n (n=4) and obese (n=4) r a t s . No d i f f e r e n c e i n the s o m a t o s t a t i n response was observed between l e a n and obese r a t s . Somatostatin l e v e l s i n p o r t a l p e r f u s a t e averaged 100 pg/ml. 157 F i g 26. Somatostatin r e l e a s e from (n=4) and obese (n=4) c o n d i t i o n s . No s i g n i f i c a n t l e a n and obese animals. the i s o l a t e d stomach of l e a n Zucker r a t s under b a s a l d i f f e r e n c e was observed between 158 159 d. A n t r a l c e l l morphology (Table IX,  F i g s 27 and 28) F o l l o w i n g p e r f u s i o n , a n t r a l t i s s u e samples were obtained from a d u l t l e a n and obese r a t s (6-months-old). Three-week-old Zucker pups were s a c r i f i c e d f o l l o w i n g an o v e r n i g h t f a s t and a n t r a l t i s s u e c o l l e c t e d . The t i s s u e was f i x e d i n Bouin's s o l u t i o n and examined by immunocytochemistry f o r g a s t r i n and som a t o s t a t i n c e l l number (General Methodology, S e c t i o n I I C ) . G a s t r i n c e l l number was markedly i n c r e a s e d i n 6-month-o l d obese (n=7) Zucker r a t s compared to l e a n l i t t e r m a t e s (n=10) (40.3 + 7.4 vs 24.6 + 4.9 cells/mm). In s u c k l i n g r a t s no s i g n i f i c a n t d i f f e r e n c e i n the a n t r a l G - c e l l p o p u l a t i o n was noted between l e a n (n=4) and obese (n=9) animals (11.8 + 4.0 vs 18.2 + 4.9 cells/mm) (Table IX, F i g 27) . No d i f f e r e n c e i n a n t r a l D - c e l l p o p u l a t i o n was observed between le a n and obese r a t s at 6 months or 3 weeks of age (Table IX, F i g 28). 160 TABLE IX. ENDOCRINE CELL QUANTIFICATION OF THE ANTRUM (X + SEM) IN LEAN AND OBESE ZUCKER RATS AGE GENOTYPE ENDOCRINE CELL NUMBER (cells/mm) GASTRIN SOMATOSTATIN 3 weeks Fa/-, n=4 11.8 + 4.0 6.3 + 2.0 f a / f a , n=9 18.2 + 4.9 6.8 + 1.4 6 months Fa/-, n=10 24.6 + 4.9 6.9 + 1.2 f a / f a , n=7 40.3 + 7.4* 5.1 + 1.1 *p<.05 161 F i g 27. Immunocytochemical examination of a n t r a l endocrine c e l l s c o n t a i n i n g g a s t r i n i n l e a n and obese Zucker r a t s ( c r o s s -s e c t i o n , m a g n i f i c a t i o n x60). A. Lean, 6 months o l d B. Obese, 6 months o l d , showing the i n c r e a s e i n G - c e l l number compared to l e a n animals of the same age C. Lean, 21 days o l d D. Obese, 21 days o l d , showing no d i f f e r e n c e i n the G-c e l l number compared to l e a n animals of the same age. 162 163 Fig 28. Immunocytochemical examination of a n t r a l endocrine c e l l s c o n t a i n i n g s o m a t o s t a t i n i n l e a n and obese Zucker r a t s ( c r o s s - s e c t i o n , m a g n i f i c a t i o n x60). A. Lean B. Obese, showing a s i m i l a r number of D - c e l l s compared to l e a n c o n t r o l s . 164 165 IV. DISCUSSION Fi g u r e s 18-22 show the r e s u l t s of s t u d i e s i n which i n s u l i n r e l e a s e i n 6-month-old l e a n and obese Zucker r a t s was examined i n the presence of v a r i o u s secretagogues and c h o l i n e r g i c a n t a g o n i s t s . Compared to l e a n l i t t e r m a t e s , obese Zucker r a t s had an e l e v a t e d i n s u l i n response to a l l secretagogues t e s t e d , i n c l u d i n g the n u t r i e n t s glucose and a r g i n i n e and the hormone GIP. I n s u l i n s e c r e t i o n s t i m u l a t e d by 300 mg/dl glucose alone or i n the presence of 1 ng/ml GIP — 5 was not i n h i b i t e d by e i t h e r 10 M a t r o p i n e , a m u s c a r i n i c -4 a n t a g o n i s t (Figs 18 and 21) or 4 x 10 M hexamethonium, a n i c o t i n i c , g a n g l i o n i c a n t a g o n i s t ( F i g s 19 and 22) . These s t u d i e s i n d i c a t e d t h a t obese Zucker r a t s had normal responses to c h o l i n e r g i c blockade compared to l e a n l i t t e r m a t e s . To t r y to c o n f i r m the s t u d i e s of Rohner-Jeanrenaud et a l (1983), 10 ^ M a t r o p i n e was i n f u s e d i n the presence of 10 mM L - a r g i n i n e ( F i g 20) . C o n t r a r y to the p r e v i o u s l y reported r e s u l t s , no i n h i b i t i o n o f a r g i n i n 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 was observed. In f a c t , a d d i t i o n of a t r o p i n e to the p e r f u s i o n medium r e s u l t e d i n a p e r s i s t e n t i n c r e a s e i n i n s u l i n r e l e a s e by both l e a n and obese animals. While the a t r o p i n e c o n c e n t r a t i o n employed i n by Rohner-Jeanrenaud was 2 . 5 - f o l d g r e a t e r than t h a t used i n the p r e s e n t s t u d i e s , g a s t r i n r e l e a s e from the i s o l a t e d p e r f u s e d stomach 166 p r e p a r a t i o n was p r e v i o u s l y shown to be i n h i b i t e d by 10 M a t r o p i n e (Pederson et a l , 1984). Rohner-Jeanrenaud e t a l (1983) a l s o r e p o r t e d t h a t i n v i v o a t r o p i n e i n f u s i o n i n 3-week-old h y p e r i n s u l i n e m i c Zucker r a t s reduced c i r c u l a t i n g i n s u l i n l e v e l s to those of l e a n c o n t r o l s , suggesting a r o l e f o r the c h o l i n e r g i c nervous system i n the onset of h y p e r i n s u l i n e m i a i n these animals. To t e s t t h i s h y p o thesis i n a c o n t r o l l e d , i n v i t r o experiment, the pancreata of l e a n and obese 3-week-old Zucker pups, known to d i s p l a y h y p e r i n s u l i n e m i a s t i m u l a t e d by a d m i n i s t r a t i o n of i n t r a p e r i t o n e a l glucose (Table V) , were perfused with 100 pM a c e t y l c h o l i n e i n the presence of 300 mg/dl glucose (Fig 17). No d i f f e r e n c e i n i n s u l i n s e c r e t i o n was observed between l e a n and obese s u c k l i n g pups to these s t i m u l i . These r e s u l t s suggest t h a t p r e v i o u s l y observed e f f e c t s of the vagus on p a n c r e a t i c i n s u l i n s e c r e t i o n i n weanling animals may i n v o l v e the presence of other, as yet unknown, f a c t o r s which are not e a s i l y i d e n t i f i e d i n i n v i v o experiments. Thus, the r e s u l t s of these s t u d i e s do not suggest a r o l e f o r the c h o l i n e r g i c nervous system as a c a u s a l f a c t o r i n the h y p e r i n s u l i n e m i a observed i n obese Zucker r a t s . Neither an a n t a g o n i s t that b l o c k s c h o l i n e r g i c a c t i v i t y at p o s t - g a n g l i o n i c s i t e s (atropine) nor a g a n g l i o n i c b l o c k e r (hexamethonium) had i n h i b i t o r y e f f e c t s on i n s u l i n s e c r e t i o n i n l e a n or obese r a t s . S i m i l a r l y , a c e t y l c h o l i n e , a c h o l i n e r g i c a g o n i s t , d i d not cause pre-oBese Zucker r a t s to 167 hypersecrete i n s u l i n from the i s o l a t e d p e r f u s e d pancreas. These data support the c o n c l u s i o n s of Opsahl and Powley (1974) that the o b e s i t y of the Zucker r a t has important d i f f e r e n c e s compared to the o b e s i t y of r a t s t r e a t e d with l e s i o n s to the ventromedial hypothalamus. S e v e r a l f a c t o r s may account f o r the d i s c r e p a n c i e s observed between the r e s u l t s observed by Rohner-Jeanrenaud et a l (1983) and the data obtained i n the c u r r e n t i n v e s t i g a t i o n . F i r s t l y , i n v i v o experiments i n v o l v i n g i n f u s i o n of a t r o p i n e or s t i m u l a t i o n of the c e r v i c a l vagus are not o r g a n - s p e c i f i c and may a f f e c t many organ systems r e c e i v i n g p o s t - g a n g l i o n i c c h o l i n e r g i c i n n e r v a t i o n . Thus, the observed e f f e c t s of a t r o p i n e - a t t e n u a t i o n or v a g a l s t i m u l a t i o n of h y p e r i n s u l i n e m i a may be i n d i r e c t . A l t e r n a t i v e l y , i n c r e a s e d i n s u l i n r e l e a s e by obese r a t s i n response to s t i m u l a t i o n of the c e r v i c a l vagus may o n l y r e f l e c t the e f f e c t of s t i m u l a t i n g the enlarged B - c e l l p o p u l a t i o n of the p a n c r e a t i c i s l e t s , r a t h e r than an i n c r e a s e d s e n s i t i v i t y of i n d i v i d u a l i s l e t s to such s t i m u l a t i o n or h y p e r a c t i v i t y of the c h o l i n e r g i c nervous system. Secondly, the r a t s used by Rohner-Jeanrenaud i n the p e r f u s e d pancreas s t u d i e s with a r g i n i n e and a t r o p i n e were 6-9 weeks of age, compared to 6 months of age i n the c u r r e n t s t u d i e s . L a s t l y , s t u d i e s demonstrating the e f f e c t s of vagotomy on i n s u l i n s e c r e t i o n were acute i n nature and p e r s i s t e n c e of the c o n d i t i o n beyond 70 minutes was not demonstrated. 168 Despite the negative r e s u l t s obtained i n s t u d i e s on the r o l e of the c h o l i n e r g i c nervous system on i n s u l i n h y p e r s e c r e t i o n i n obese Zucker r a t s , a second set of experiments was undertaken to f u r t h e r examine by i n d i r e c t means the p o s s i b i l i t y of a h y p e r a c t i v e nervous component of the e n t e r o i n s u l a r a x i s . F i g u r e s 23-28 and Tables V I I I and IX show the r e s u l t s of these s t u d i e s . B a s a l g a s t r i n l e v e l s i n v i v o (Table VIII) and i n v i t r o ( F i g 23) were e l e v a t e d 3-4 f o l d i n a d u l t (6-month-old) obese Zucker r a t s . The c o n d i t i o n appeared to be p r o g r e s s i v e , with 3-week-old pups not demonstrably hypergastrinemic, 3-month-old a d u l t s moderately hypergastrinemic and o l d e r a d u l t (6-month-old) r a t s s e v e r e l y h y p e r g a s t r i n e m i c . I t was a l s o demonstrated that a t r o p i n e had no e f f e c t on i n v i t r o b a s a l g a s t r i n r e l e a s e i n e i t h e r l e a n or obese r a t s ( F i g 24). Thus obese Zucker r a t s d i d not resemble r a t s made hyp e r g a s t r i n e m i c by vagotomy (Pederson et a l , 1984) and suggested t h a t b a s a l h y p e r s e c r e t i o n of g a s t r i n i n obese r a t s was not mediated by p o s t - g a n g l i o n i c c h o l i n e r g i c i n p u t s . In a second set of experiments, the e f f e c t of a t r o p i n e on g a s t r i n r e l e a s e e l i c i t e d by e l e c t r i c a l s t i m u l a t i o n of the vagal trunks was examined. Vagal s t i m u l a t i o n i n c r e a s e d g a s t r i n r e l e a s e from the i s o l a t e d stomach 2 - 3 - f o l d i n both le a n and obese animals. S i m i l a r l y , a t r o p i n e reduced s t i m u l a t e d g a s t r i n s e c r e t i o n to b a s a l l e v e l s i n both groups of animals ( F i g 25). These experiments pr o v i d e d f u r t h e r 169 evidence that the G - c e l l s of Zucker f a t t y r a t s remain under normal c h o l i n e r g i c i n f l u e n c e . I t was hypothesized t h a t i n c r e a s e d g a s t r i n r e l e a s e c o u l d a l s o r e s u l t from a decrease i n s o m a t o s t a t i n s e c r e t i o n , s i n c e s o m a t o s t a t i n has been shown to i n h i b i t g a s t r i n r e l e a s e (Bloom et a l , 1974; Hayes et a l , 1975). Measurement of somatostatin r e l e a s e was c a r r i e d out under b a s a l c o n d i t i o n s and found not to d i f f e r between l e a n and obese animals (Fig 26) . Thus, these s t u d i e s d i d not support a r o l e f o r somatostatin i n the abnormal g a s t r i n s e c r e t i o n observed i n obese Zucker r a t s under f a s t i n g c o n d i t i o n s . T h i s c o n c l u s i o n was supported by q u a n t i f i c a t i o n of s o m a t o s t a t i n - c o n t a i n i n g D - c e l l s i n the g a s t r i c antrum of l e a n and obese Zucker r a t s (Table IX and F i g 28) . No d i f f e r e n c e i n D - c e l l number was observed between l e a n and obese animals at e i t h e r 3 weeks or 6 months of age. Another p o s s i b l e e x p l a n a t i o n f o r i n c r e a s e d g a s t r i n s e c r e t i o n was h y p e r p l a s i a of a n t r a l G - c e l l s , s i m i l a r to the i n c r e a s e d p o p u l a t i o n of B - c e l l s observed i n the p a n c r e a t i c i s l e t s . A n t r a l t i s s u e was f i x e d and examined by immunocytochemistry. S p e c i f i c s t a i n i n g f o r g a s t r i n -c o n t a i n i n g G - c e l l s r e v e a l e d an i n c r e a s e i n number i n a d u l t obese r a t s but not i n 3-week o l d pre-obese s u c k l i n g pups compared to l e a n l i t t e r m a t e s (Table IX and F i g 27), demonstrating a strong c o r r e l a t i o n between c e l l number and plasma g a s t r i n l e v e l s . 170 These o b s e r v a t i o n s l e d to the f o l l o w i n g c o n c l u s i o n s : (1) that h y p e r g a s t r i n e m i a i n obese Zucker r a t s i s a p r o g r e s s i v e c o n d i t i o n not p r e s e n t at 3 weeks of age but a r i s i n g some time b e f o r e 3 months of age. I n d i r e c t evidence suggests t h a t these developments may be r e l a t e d to developing hyperphagia, s i n c e G - c e l l number has been reported to be decreased d u r i n g f a s t i n g i n r a t s and to p r o l i f e r a t e r a p i d l y on r e f e e d i n g (Bertrand and Willems, 1980) . Obese Zucker r a t s are not hyperphagic i n the s u c k l i n g s t a t e , but develop t h i s c h a r a c t e r i s t i c subsequent to weaning (Godbole et a l , 1981); (2) t h a t h y p e r g a s t r i nemia i s not due to o v e r - a c t i v i t y of the p o s t - g a n g l i o n i c c h o l i n e r g i c component of the vagus nerve, s i n c e obese r a t s responded i n a normal f a s h i o n to both v a g a l s t i m u l a t i o n and i n h i b i t i o n of g a s t r i n r e l e a s e by c h o l i n e r g i c a n t a g o n i s t s i n the i s o l a t e d p e r f u s e d stomach. Nor i s t h i s c o n d i t i o n l i k e l y to be caused by reduced feedback of a c i d on the G - c e l l s , s i n c e g a s t r i c a c i d s e c r e t i o n was r e p o r t e d to be normal i n obese Zucker r a t s (Opsahl and Powley, 1974) ; (3) t h a t h ypergastrinemia i s l i k e l y due to h y p e r p l a s i a of the g a s t r i n - c o n t a i n i n g G - c e l l s t h a t a r i s e s sometime a f t e r weaning. These data, i n d i c a t i n g t h a t Zucker r a t s are not s u b j e c t to abnormal c h o l i n e r g i c nervous a c t i v i t y e i t h e r e n t e r i c a l l y or v i a the vagus nerve, concur with data obtained i n s t u d i e s of the c h o l i n e r g i c nervous system i n the endocrine pancreas and t h e r e f o r e support a hypothesis t h a t the c h o l i n e r g i c nervous system i s not an important f a c t o r i n 171 the onset or maintenance of the h y p e r i n s u l i n e m i c s t a t e i n obese Zucker r a t s . Moreover, hypergastrinemia i s probably a consequence of normal c o n t r o l mechanisms a c t i n g on an i n c r e a s e d p o p u l a t i o n of G - c e l l s , r a t h e r than of e x c e s s i v e v a g a l s t i m u l a t i o n . Further to t h i s study, the p h y s i o l o g i c s i g n i f i c a n c e of hypergastrinemia i n o b e s i t y i n unknown. E l e v a t e d g a s t r i n s e c r e t i o n has been r e p o r t e d i n young ob/ob mice t h a t p e r s i s t e d under p a i r - f e e d i n g c o n d i t i o n s (Morton, Hanson and Dockray, 1985) and disappeared by 30 weeks of age. Hypergastrinemia was accompanied by reduced g a s t r i c a c i d s e c r e t i o n and m a l f u n c t i o n of the a c i d feedback system on g a s t r i n s e c r e t i o n was proposed. Hypergastrinemia independent of hyperphagia was a l s o observed i n obese, d i a b e t i c (db/db) mice t h a t had a 30% i n c r e a s e i n G - c e l l number i n the g a s t r i c antrum (Lichtenberger and Ramaswamy, 1979) . G a s t r i c a c i d s e c r e t i o n was not measured. In a t h i r d study, b a s a l and s t i m u l a t e d g a s t r i n r e l e a s e was e l e v a t e d i n normal Wistar r a t s t r e a t e d with VMH l e s i o n s . T h i s hypergastrinemia was not r e v e r s e d by p a i r - f e e d i n g the VMH-l e s i o n e d animals with i d e n t i c a l c a l o r i c i n t a k e as unoperated c o n t r o l s and the authors s p e c u l a t e d involvement of the autonomic nervous system (Chikamori e t a l , 1982). In humans there are c o n f l i c t i n g data r e g a r d i n g plasma g a s t r i n l e v e l s . E l e v a t e d serum g a s t r i n i n obese p a t i e n t s was r e p o r t e d by Schrumpf e t a l (1981), compared to r e p o r t s of normal g a s t r i n l e v e l s by A t k i n s o n et a l (1979) and Sarson et a l (1981) . 172 Serum g a s t r i n l e v e l s or g a s t r i n r e l e a s e from the i s o l a t e d , p erfused stomach have not p r e v i o u s l y been r e p o r t e d i n obese Zucker r a t s , but normal g a s t r i c a c i d s e c r e t i o n was noted i n one r e p o r t (Opsahl and Powley, 1974). In c o n c l u s i o n , f u r t h e r s t u d i e s are r e q u i r e d to determine the st i m u l u s f o r G - c e l l h y p e r p l a s i a and g a s t r i n h y p e r s e c r e t i o n i n obese Zucker r a t s . The mechanism f o r these phenomena may i n v o l v e f e e d i n g behaviour; non-mu s c a r i n i c v a g a l i n p u t s ; i n f l u e n c e s of the i n t r i n s i c nervous system such as bombesin, a known s t i m u l a n t of g a s t r i n s e c r e t i o n p r e s e n t i n e n t e r i c neurons (DuVal e t a l , 1981); or i n t r i n s i c d e f e c t s i n g a s t r i c a c i d s e c r e t i o n t h a t r e s u l t i n a decrease i n feedback i n h i b i t i o n of g a s t r i n r e l e a s e . 173 • CHAPTER THREE: THE EFFECT ON THE ENTEROINSULAR AXIS OF JEJUNOILEAL BYPASS SURGERY IN ZUCKER RATS I. INTRODUCTION Although young a d u l t (8-12-week old) obese animals have normal glucose t o l e r a n c e p r o f i l e s ( F i g 9), o l d e r (6-8-month) f a t t y r a t s become glucose i n t o l e r a n t and have an e l e v a t e d GIP response to o r a l glucose ( F i g 10). E l e v a t e d GIP responses to an o r a l c h a l l e n g e have a l s o been observed i n g l u c o s e - i n t o l e r a n t obese human s u b j e c t s ( C r e u t z f e l d t et a l , 1978) and the hyperglycemic obese (ob/ob) mouse ( F l a t t et a l , 1983) . Hy p e r i n s u l i n e m i a r e l a t e d to o b e s i t y can be p a r t i a l l y reduced by weight l o s s i n both man and experimental animals (Jeanrenaud, 1979) . J e j u n o i l e a l bypass (JIB) surgery has been used to induce weight l o s s and decrease i n s u l i n e m i a (Sarson, Scopinaro and Bloom, 1981) i n morbidly obese humans. In obese Zucker r a t s JIB has been shown to decrease plasma i n s u l i n c o n c e n t r a t i o n s , although not to c o n t r o l l e v e l s (Greenwood e t a l , 1982). The i n s u l i n response of the pancreas i n an environment o f reduced s t i m u l a t i o n from the gut can be s t u d i e d by bypassing much of the smal l bowel, which e f f e c t i v e l y r e s u l t s i n e l i m i n a t i o n o f the e n t e r o i n s u l a r a x i s from l u m i n a l s t i m u l a t i o n . Authors of many p r e v i o u s s t u d i e s i n normal r a t s e x t r a p o l a t e d t h e i r c o n c l u s i o n s to i n c l u d e the obese s t a t e , on the assumption 174 t h a t adaptive changes were s i m i l a r i n l e a n and obese r a t s . The p r e s e n t i n v e s t i g a t i o n i n t o the r o l e of the e n t e r o i n s u l a r a x i s i n J I B - t r e a t e d Zucker r a t s was undertaken because weight l o s s p a t t e r n s ( S c l a f a n i , 1980) and changes i n hormone and m e t a b o l i t e l e v e l s (Greenwood et a l , 1982) f o l l o w i n g JIB were found to d i f f e r i n obese Zucker r a t s compared to le a n l i t t e r m a t e s . The o b j e c t i v e of j e j u n o i l e a l bypass experiments was to determine the e f f e c t o f removing 70-80% of the small i n t e s t i n e from c o n t i n u i t y and hence from s t i m u l a t i o n by n u t r i e n t s i n the lumen. To accomplish t h i s o b j e c t i v e , a r e v i s e d s u r g i c a l procedure was developed and t e s t e d i n Wistar r a t s . I t was hoped t h a t t h i s procedure, which e x t e r i o r i z e d the j e j u n a l and i l e a l ends of the b l i n d loop to the body w a l l , would i n c r e a s e the s u r v i v a l o f obese r a t s . The technique a l s o allowed comparison of the outcome of JIB when the bypassed loop was p e r f u s e d or not per f u s e d with n u t r i e n t s on a d a i l y b a s i s . In a d d i t i o n , temporal changes i n f a s t i n g plasma glucose and hormone l e v e l s were examined on a weekly b a s i s . As a c o n t r o l a g a i n s t the e f f e c t s o f f a s t i n g and c o l l e c t i n g a blood sample from the JIB animals once a week, another s e t of animals was s t u d i e d which were allowed to recover without experimental manipulations f o r the 5 week experimental p e r i o d (designated JIB-NS). Development of the JIB with jejunostomy/ileostomy o f the bypassed loop p r o v i d e d the o p p o r t u n i t y to study the e f f e c t s on the e n t e r o i n s u l a r a x i s o f continued n u t r i e n t 175 s t i m u l a t i o n of the b l i n d loop i n JIB animals (designated J I B - L ) . I t was s p e c u l a t e d that d a i l y p e r f u s i o n of the bypassed loop with n u t r i e n t s c o u l d reverse or modify the adaptive responses to JIB. J e j u n o i l e a l bypass can r e s u l t i n marked weight l o s s . To ensure t h a t the observed e f f e c t s of JIB on the e n t e r o i n s u l a r a x i s were not s o l e l y due to l o s s of weight, a group of l e a n and obese Zucker r a t s was f a s t e d u n t i l weight l o s s mimicked t h a t observed i n JIB. The i n v i v o and i n v i t r o responses of the pancreas to s t i m u l a t i o n by glucose and GIP were then measured. 176 I I . METHODS A. J e j u n o i l e a l Bypass Surgery 1. "Conventional" S u r g i c a l Technique F o l l o w i n g a 48 h f a s t the r a t s were a n e s t h e t i z e d with 40 mg/kg p e n t o b a r b i t a l . The s u r g i c a l area was shaved and washed with an i o d i n e s o l u t i o n (Proviodine) f o l l o w e d by 70% a l c o h o l . The m i d l i n e area of the abdomen was l o c a l l y a n e s t h e t i z e d with l i d o c a i n e p r i o r to c r e a t i o n of a 2-3 cm m i d l i n e i n c i s i o n through the s k i n and muscle l a y e r . The ligament of T r e i t z was l o c a t e d and the bowel s e c t i o n e d 3 cm d i s t a l l y . The ileum was s e c t i o n e d 3-4 cm proximal to the cecum and the bowel was reconnected v i a an end-to-end anastomosis. T h i s segment of bowel was r e f e r r e d to as the continuous bowel. The proximal end of the b l i n d l oop was c l o s e d , while the d i s t a l end of the loop was anastomosed end-to-side with the t r a n s v e r s e c o l o n (Fig 29 A) . The peritoneum was c l o s e d using s t e r i l e s t a i n l e s s s t e e l wound c l i p s . Access to water was p e r m i t t e d as soon as consciousness was regained. The animals were fed r e g u l a r r a t chow 48 h f o l l o w i n g surgery. 2. A Revised S u r g i c a l Approach to J e j u n o i l e a l Bypass In an attempt to improve s u r v i v a l i n obese Zucker r a t s f o l l o w i n g j e j u n o i l e a l bypass surgery, a r e v i s e d s u r g i c a l technique was developed. The surgery proceeded as d e s c r i b e d above ( S e c t i o n I I A l ) except t h a t the b l i n d loop was e x t e r i o r i z e d to the body w a l l both p r o x i m a l l y and 177 d i s t a l l y by c r e a t i o n of a jejunostomy-ileostomy (Fig 29 B). 178 F i g 29. Two methods of j e j u n o i l e a l bypass surgery. A) "Conventional" technique i n which the b l i n d loop was c l o s e d at the proximal end and anastomosed end-to-side with the t r a n s v e r s e c o l o n a t the d i s t a l end. B) Revised technique i n which the b l i n d loop was e x t e r i o r i z e d to the body w a l l by c r e a t i o n of a j ej unostomy/ileostomy. 179 A) JEJUNOILEAL BYPASS 180 B. Glucose Tolerance T e s t s 1. I n t r a j e j u n a l T h i s technique was employed i n Zucker r a t s with j e j u n o i l e a l bypass i n which the b l i n d l oop was e x t e r i o r i z e d to the body w a l l as a jejunostomy-ileostomy ( S e c t i o n IIA2). F o l l o w i n g an o v e r n i g h t f a s t the animals were weighed and a b a s a l blood sample ob t a i n e d . Glucose (0.5 mg/kg, 20% s o l u t i o n ) was administered by i n s e r t i n g a s t e r i l e s t a i n l e s s s t e e l tube 1-2 cm i n t o the jejunum v i a the jejunostomy. Blood samples were c o l l e c t e d and t r e a t e d as p r e v i o u s l y d e s c r i b e d (General Methodology, S e c t i o n IIB1). 181 I I I . RESULTS A. The e f f e c t of j e j u n o i l e a l bypass on the e n t e r o i n s u l a r  a x i s of l e a n versus obese r a t s 1. A comparison of two methods of j e j u n o i l e a l bypass  surgery In an attempt to reduce m o r t a l i t y f o l l o w i n g j e j u n o i l e a l bypass surgery, a novel s u r g i c a l procedure was developed and t e s t e d i n normal Wistar r a t s . Two groups of 250 g Wistar r a t s were prepared with a j e j u n o i l e a l bypass. The f i r s t group (JIB Group 1) underwent " c o n v e n t i o n a l " surgery where the shortened bowel was j o i n e d by end-to-end anastomosis. The bypassed loop was c l o s e d at the proximal end and j o i n e d to the t r a n s v e r s e c o l o n by an end-to-side anastomosis at the d i s t a l end. The second group of r a t s (JIB Group 2) was prepared with end-to-end anastomosis of the continuous sho r t gut but both the proximal and d i s t a l ends of the bypassed loop were e x t e r i o r i z e d to the body w a l l , c r e a t i n g a jejunostomy/ileostomy. To ensure that the r e v i s e d s u r g i c a l techniques had no unexpected e f f e c t s on the experimental parameters t h a t would be s t u d i e d i n Zucker r a t s , the two s u r g i c a l groups were compared to each other and to a group of sham-operated c o n t r o l r a t s f i v e weeks f o l l o w i n g surgery. O r a l glucose t o l e r a n c e t e s t s and p e r f u s i o n of the i s o l a t e d pancreas were performed on a l l groups. 182 a. O r a l glucose t o l e r a n c e ( F i g 30) The r e s u l t s o f a d m i n i s t e r i n g 1 g/kg o r a l glucose on plasma g l u c o s e , i n s u l i n and GIP l e v e l s are shown i n F i g 30. Plasma glucose l e v e l s ( F i g 30 A) of both JIB Group 1 (n=6) and Group 2 (n=8) were reduced compared to c o n t r o l s (n=3) but d i d not d i f f e r from each o t h e r . S i m i l a r r e s u l t s were observed f o r plasma i n s u l i n ( F i g 30 B) and GIP (Fig 30 C) l e v e l s over a 60 min t e s t p e r i o d . B a s a l l e v e l s of these hormones were not a f f e c t e d by JIB. b. In v i t r o 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 glucose and a g r a d i e n t of GIP ( F i g 31) The pancreata of r a t s i n both JIB groups were i s o l a t e d and p e r f u s e d with 300 mg/dl glucose i n the presence of a GIP g r a d i e n t from 0-1 ng/ml. The r e s u l t s were compared to those of c o n t r o l animals ( F i g 31). There was no s i g n i f i c a n t d i f f e r e n c e i n the i n s u l i n response o f Group 1 (n=6) vs Type 2 (n=8) JIB r a t s . Both groups d i s p l a y e d s i g n i f i c a n t l y reduced i n s u l i n output to these s i m u l i when compared to sham-operated c o n t r o l s (n=4) . 183 A comparison of the (A) glu c o s e , (B) i n s u l i n and (C) GIP responses to 1 g/kg o r a l g l ucose i n Wistar r a t s t r e a t e d with c o n v e n t i o n a l j e j u n o i l e a l bypass surgery (Group 1, n=6) , j e j u n o i l e a l bypass surgery with the bypassed loop e x t e r i o r i z e d to the body w a l l v i a a jejunostomy/ileostomy (Group 2, n=8) and sham-operated c o n t r o l s (n=3). ^ represents the a d m i n i s t r a t i o n of glucose at 0 minutes. *p<.05 f o r Group 1 and ^p<.05 f o r Group 2, using the unpaired t - t e s t , comparing each group to c o n t r o l s . No s i g n i f i c a n t d i f f e r e n c e s were observed between Groups 1 and 2. 184 185 F i g 31. A comparison of the i n s u l i n s e c r e t o r y response of the i s o l a t e d p e r f u s e d pancreas to 300 mg/dl glucose p l u s a GIP g r a d i e n t o f 0-1 ng/ml i n Wistar r a t s t r e a t e d with c o n v e n t i o n a l j e j u n o i l e a l bypass surgery (Group 1, n=6), j e j u n o i l e a l bypass surgery with the bypassed loop e x t e r i o r i z e d to the body w a l l v i a a jejunostomy/ileostomy (Group 2, n=8) and sham-operated c o n t r o l s (n=4). *p<.05 using the unpaired t - t e s t f o r Groups 1 and 2 compared to c o n t r o l s . No s i g n i f i c a n t d i f f e r e n c e s were noted between Groups 1 and 2. 186 187 2. The e f f e c t o f JIB i n l e a n versus obese Zucker  r a t s Lean and obese Zucker r a t s were t r e a t e d with JIB using the jejunostomy/ileostomy technique, as d e s c r i b e d f o r Group 2 Wistar r a t s . In order to monitor temporal changes i n plasma glucose and hormone l e v e l s , the animals were f a s t e d o v e r n i g h t one n i g h t per week f o r 5 weeks f o l l o w i n g surgery. The f o l l o w i n g morning the animals were weighed, and a blood sample obtained f o r measurement of glucose, i n s u l i n and GIP. R e s u l t s were compared to sham-operated l e a n and obese c o n t r o l s . a. Weight change f o l l o w i n g JIB surgery (Table X) Lean c o n t r o l r a t s (n=9) l o s t about 3% of t h e i r o r i g i n a l body weight while on the once weekly o v e r n i g h t s t a r v a t i o n regimen. Lean JIB r a t s (n=13) l o s t 14 + 2.5% of t h e i r o r i g i n a l body weight by the second p o s t - s u r g i c a l week but were g a i n i n g weight at the end of the 5 week study (weight l o s s 11.8 + 4%). Obese c o n t r o l r a t s (n=10) l o s t 1.7 + 1.6% of t h e i r o r i g i n a l body weight whereas obese JIB r a t s (n=16) had l o s t 22.8% + 2.9% of t h e i r o r i g i n a l body weight at 5 weeks and were s t i l l l o s i n g weight a t t h i s time. b. Weekly plasma measurements of g l u c o s e , i n s u l i n and GIP ( F i g 32) F a s t i n g plasma l e v e l s o f g l u c o s e , i n s u l i n and GIP were measured on a weekly b a s i s i n l e a n (n=13) and obese 1 8 8 (n=16) JIB r a t s . R e s u l t s were expressed as a percentage of c o n t r o l (n=9 and n=10) valu e s to account f o r weekly v a r i a t i o n i n c o n t r o l g lucose and hormone l e v e l s ( F i g 32) . F i g 32 A showed t h a t plasma glucose l e v e l s v a r i e d between 80 and 100% of c o n t r o l l e v e l s over the 5 week p e r i o d f o r both lean and obese JIB r a t s but were not s i g n i f i c a n t l y a l t e r e d at any time i n e i t h e r group. The i n s u l i n c o n c e n t r a t i o n i n plasma of lea n r a t s was s i g n i f i c a n t l y reduced o n l y at week 4, whereas a f t e r the f i r s t week, plasma i n s u l i n l e v e l s of obese JIB r a t s d e c l i n e d s t e a d i l y and s i g n i f i c a n t l y (p<.05) to 40% of c o n t r o l v a l u e s (Fi g 32 B). Plasma GIP measurement i n l e a n JIB r a t s demonstrated an i n i t i a l s i g n i f i c a n t decrease to 50% of c o n t r o l v a l u e s followed by an i n c r e a s e i n the f i n a l 2 weeks to 120% of c o n t r o l v a l u e s . Conversely, GIP l e v e l s o f obese JIB r a t s d e c l i n e d s l o w l y over the 5 week p e r i o d to 60% of obese c o n t r o l l e v e l s (p<.05 at 5 weeks) ( F i g 32 C ) . c. O r a l glucose t o l e r a n c e ( F i g 33, 34 and 44) F i v e weeks f o l l o w i n g bypass surgery, JIB animals were gi v e n a 1 g/kg o r a l glucose l o a d and the glucose, i n s u l i n and GIP responses compared to those of sham-operated c o n t r o l s . F i g 33 shows the responses of le a n JIB (n=13) and c o n t r o l (n=9) animals. There were no d i f f e r e n c e s i n glucose l e v e l s o f JIB vs c o n t r o l animals except at 60 min, at which time the glucose l e v e l s of l e a n 189 JIB r a t s were s i g n i f i c a n t l y reduced compared to those o f c o n t r o l s (162 +26.2 mg/dl vs 210 +14.0 mg/dl, p<.05) (Fig 33 A) . S i m i l a r l y , f a s t i n g and g l u c o s e - s t i m u l a t e d i n s u l i n l e v e l s were not a l t e r e d except at 60 min (JIB: 17.2 + 4.2 pU/ml vs c o n t r o l : 26.3 + 3.6 uU/ml, p<.05) (Fig 33 B) . No d i f f e r e n c e was observed i n the i n t e g r a t e d response of glucose or i n s u l i n ( F i g 44, A and B) . However, the GIP response o f l e a n JIB animals was impaired compared to c o n t r o l s at 20, 30 and 60 min (p<.05). F a s t i n g GIP l e v e l s tended to be higher than c o n t r o l s but t h i s trend d i d not achieve s t a t i s t i c a l s i g n i f i c a n c e ( F i g 33 C ) . A s i g n i f i c a n t r e d u c t i o n i n the i n t e g r a t e d GIP response was observed ( F i g 44 C) . F i g 34 shows the r e s u l t s o f OGTT i n obese JIB (n=16) and c o n t r o l (n=10) r a t s . Plasma glucose l e v e l s of obese JIB r a t s were not d i f f e r e n t from those o f c o n t r o l s except at 20 min (205 + 16.9 mg/dl vs 242 + 7.0 mg/dl, p<.05) ( F i g 33 A). There was no d i f f e r e n c e i n the i n t e g r a t e d response ( F i g 44 A) . F a s t i n g i n s u l i n l e v e l s o f obese r a t s were s i g n i f i c a n t l y reduced by JIB surgery, and the t o t a l i n s u l i n response was a l s o s i g n i f i c a n t l y reduced (Figs 34 B and 44 B ). F a s t i n g GIP l e v e l s were reduced i n obese JIB r a t s compared to obese c o n t r o l s (236 + 37.4 pg/ml vs 402 + 86.7 pg/ml, p<.05) and the t o t a l GIP response o f obese JIB r a t s was 50% of th a t of c o n t r o l s (Figs 34 C and 44 C ) . 190 d. Plasma Glucagon (Table XI) A f a s t i n g blood sample was obtained at 5 weeks f o l l o w i n g bypass surgery and the g l u c a g o n - l i k e immunoreactivity (GLI) of the plasma determined. Plasma GLI of obese c o n t r o l r a t s (n=ll) was 75% reduced compared to lean c o n t r o l r a t s (n=ll) but i n c r e a s e d a f t e r bypass (n=13) to l e v e l s comparable to those of le a n c o n t r o l s . There was no s i g n i f i c a n t d i f f e r e n c e i n GLI l e v e l s a f t e r bypass i n l e a n r a t s (n=12) compared to l e a n c o n t r o l s . 191 TABLE X. BODY WEIGHT CHANGES IN LEAN AND OBESE ZUCKER RATS FOLLOWING JEJUNOILEAL BYPASS SURGERY INITIAL BODY FINAL BODY £\WEIGHT WEIGHT (g) WEIGHT (g) (% + SEM) (X + SEM) (X + SEM) LEAN C o n t r o l , n=9 321 + 29.1 312 + 28. 9 -3.2 + 0.88 J I B a , n=13 286 + 20.3 250 + 17. 1 -11.8 + 4.0* J I B - L b , n=6 205 + 4.5 189 + 10. 4 -8.1 + 4.0 JIB-NS C, n=5 248 + 35.6 195 + 26. 7 -20.3 + 3.8* OBESE C o n t r o l , n=10 514 + 21.4 506 + 24. 7 -1.7 + 1.6 JIB, n=16 539 + 44.0 385 + 19. 6 -22.8 + 2.9* JIB-L, n=4 363 + 9.0 284 + 14. 9 -21.3 + 5.5* JIB-NS, n=5 439 + 41.1 338 + 31. 4 -23.0 + 1.8* *p<.05 compared t o c o n t r o l s a. animals s t a r v e d o v e r n i g h t on a weekly b a s i s b. animals with d a i l y i n t r a j e j u n a l n u t r i e n t i n f u s i o n c. animals not st a r v e d o v e r n i g h t on a weekly b a s i s 1 9 2 Measurement of f a s t i n g plasma (A) glucose, (B) i n s u l i n and (C) GIP c o n c e n t r a t i o n s as a percentage of le a n (n=9) or obese (n=10) c o n t r o l s i n le a n (n=13) and obese (n=16) r a t s t r e a t e d with j e j u n o i l e a l bypass. Plasma samples were obtained from week 1 through 5 f o l l o w i n g surgery. *p<.05 using Student's t - t e s t f o r unpaired data, comparing l e a n and obese JIB r a t s with t h e i r r e s p e c t i v e c o n t r o l s . 193 O Lean Bypass 0 Obese Bypass 150 1 2 3 4 5 1 2 3 4 5 WEEKS AFTER SURGERY 194 F i g 33. The (A) glucose, (B) i n s u l i n and (C) GIP responses to 1 g/kg o r a l g lucose of l e a n Zucker r a t s 5 weeks f o l l o w i n g j e j u n o i l e a l bypass surgery (n=13) compared to sham-operated c o n t r o l s (n=9). rep r e s e n t s the a d m i n i s t r a t i o n of glucose at 0 min. *p<.05 using unpaired Student's t - t e s t , comparing l e a n JIB r a t s to sham-operated l e a n c o n t r o l s . 195 MINUTES The (A) gl u c o s e , (B) i n s u l i n and (C) GIP responses to 1 g/kg o r a l g lucose of obese Zucker r a t s 5 weeks f o l l o w i n g j e j u n o i l e a l bypass surgery (n=16) compared to sham-operated c o n t r o l s (n=10) . r e p r e s e n t s the a d m i n i s t r a t i o n of glucose at 0 min. *p<.05 using unpaired t - t e s t , comparing obese JIB r a t s to sham-operated obese c o n t r o l s . 197 198 TABLE XI. GLUCAGON-LIKE IMMUNOREACTIVITY (GLI, X + SEM) OF LEAN AND OBESE RATS FIVE WEEKS FOLLOWING JEJUNOILEAL BYPASS SURGERY GENOTYPE GLI (pg/ml) CONTROL Fa/-, n = l l 143 + 35.5 f a / f a , n = l l 30.5 + 9.0* JIB Fa/-, n=12 174 +57.3 f a / f a , n=13 96.5 + 25.1+ *p<.05 compared t o l e a n c o n t r o l s +p<.05 compared to obese c o n t r o l s 199 e. In v i t r o i n s u l i n r e l e a s e i n response to glucose  and a GIP g r a d i e n t ( F i g 35) Lean and obese JIB r a t s and t h e i r r e s p e c t i v e c o n t r o l s were s a c r i f i c e d at 5 weeks f o l l o w i n g surgery and t h e i r pancreata i s o l a t e d and p e r f u s e d with 300 mg/dl glucose p l u s a GIP g r a d i e n t of 0-1 ng/ml. F i g 35 A shows the r e s u l t s o f these experiments i n l e a n r a t s . No s i g n i f i c a n t d i f f e r e n c e i n the i n s u l i n response of JIB (n=5) vs c o n t r o l r a t s (n=13) was observed. S i m i l a r l y , i n s u l i n output of the i s o l a t e d pancreata of obese r a t s was not a f f e c t e d by JIB surgery (n=8) compared to c o n t r o l s (n=9) ( F i g 35 B). f . The i n v i t r o i n s u l i n response to a glucose  g r a d i e n t p l u s GIP (Figs 36, 37 and 38) F i v e weeks f o l l o w i n g surgery the pancreata of l e a n and obese JIB and c o n t r o l r a t s were p e r f u s e d with a 50-150 mg/dl g r a d i e n t of glucose i n the presence of 1 ng/ml GIP. Lean JIB r a t s (n=7) ( F i g 36) showed no change i n t h e i r i n s u l i n response to graded glucose p l u s GIP compared to l e a n c o n t r o l s (n=15) . Obese JIB r a t s (n=7) (Fig 37) d i d have a s i g n i f i c a n t l y reduced i n s u l i n response to these s t i m u l i compared to c o n t r o l s (n=7). The i n s u l i n response of these animals was a l s o compared to t h a t e l i c i t e d by a 50-150 mg/dl g r a d i e n t of glucose i n the absence of GIP (n=12) (Fig 38) . These experiments demonstrated that while i n s u l i n s e c r e t i o n of obese JIB r a t s was reduced under those c o n d i t i o n s , i t was 200 not as low as the response to glucose alone obtained i n c o n t r o l r a t s . 201 : F i g 35. The e f f e c t o f JIB on the response of the i s o l a t e d p e r f u s e d pancreas to 300 mg/dl glucose p l u s a GIP g r a d i e n t of 0-1 ng/ml i n (A) l e a n (n=5) and (B) obese (n=8) Zucker r a t s compared to l e a n (n=13) and obese (n=9) c o n t r o l s r e s p e c t i v e l y . *p<.05 using unpaired Student's t - t e s t , comparing le a n and obese JIB r a t s to t h e i r r e s p e c t i v e c o n t r o l s . 202 203 F i g 36. The e f f e c t of JIB on the response of the i s o l a t e d p e r f u s e d pancreas to a g l u c o s e g r a d i e n t of 50-150 mg/dl p l u s 1 ng/ml GIP i n l e a n (n=7) Zucker r a t s compared to sham-operated c o n t r o l s (n=15). *p<.05 using unpaired Student's t - t e s t , comparing l e a n JIB r a t s to c o n t r o l s . 204 300 Lean Bypass M I N U T E S 205 F i g 37. The e f f e c t of JIB on the response of the i s o l a t e d p e r f u s e d pancreas to a glucose g r a d i e n t of 50-150 mg/dl p l u s 1 ng/ml GIP i n obese (n=7) Zucker r a t s compared to sham-operated c o n t r o l s (n=7). *p<.05 using unpaired Student's t - t e s t , comparing obese JIB r a t s t o c o n t r o l s . 206 207 F i g 38. The e f f e c t of JIB on the response of the i s o l a t e d p erfused pancreas to a glucose g r a d i e n t of 50-150 mg/dl p l u s 1 ng/ml GIP i n obese (n=7) JIB Zucker r a t s compared to a glucose g r a d i e n t alone i n obese c o n t r o l (n=12) animals. *p<.05 using unpaired Student's t - t e s t . 208 OBESE BYPASS 10 20 30 40 M I N U T E S 209 g. Ada p t a t i o n of the gut f o l l o w i n g JIB i n l e a n and  obese r a t s i . E p i t h e l i a l h e i g h t (Table XII) I n t e s t i n a l t i s s u e samples were obtained at s a c r i f i c e 5 weeks a f t e r surgery. The c r y p t / v i l l u s p r o f i l e s i n the continuous bowel and the bypassed loop were measured and compared to those of sham-operated c o n t r o l s . The e p i t h e l i a l h e i g h t of obese c o n t r o l s (n=8) was not d i f f e r e n t from that of l e a n c o n t r o l s (n=8) i n e i t h e r the jejunum or ileum. A f t e r JIB the e p i t h e l i a l h e i g h t of the continuous ileum was i n c r e a s e d by 60-70% f o r both l e a n (n=8) and obese (n=9) animals, whereas c r y p t / v i l l u s p r o f i l e s o f the continuous jejunum i n c r e a s e d 20-30%. There were no s i g n i f i c a n t changes i n e p i t h e l i a l h e i g h t of the ileum or jejunum i n the bypassed loop i n e i t h e r l e a n or f a t JIB r a t s . i i . Endocrine c e l l q u a n t i f i c a t i o n (Tables XIII and  XIV, F i g s 39, 40 and 41) I n t e s t i n a l samples obtained at s a c r i f i c e 5 weeks f o l l o w i n g JIB were examined by immunocytochemistry f o r GIP, somatostatin (soma), CCK and enteroglucagon (EG) c e l l s i n the jejunum (Table XIII) and these p l u s n e u r o t e n s i n (NT) c e l l s i n the ileum (Table XIV). A s i g n i f i c a n t d i f f e r e n c e between l e a n (n=8) and obese (n=8) c o n t r o l r a t s o c c u r r e d o n l y i n the number of EG c e l l s i n the jejunum ( l e a n : 11.3 + 1.2 cells/mm vs f a t : 4.9 + 1.1 cells/mm, p<.05) ( F i g 39, A and B). 2 1 0 F o l l o w i n g bypass l e a n animals (n=8) had no c e l l p o p u l a t i o n changes i n the continuous ileum, but a 40% d e c l i n e i n EG c e l l number was noted i n the continuous jejunum. In the bypassed loop, l e a n r a t s had a 40% i n c r e a s e i n C C K - c e l l number i n the jejunum and a 100% i n c r e a s e i n NT c e l l number i n the bypassed ileum. In c o n t r a s t , obese animals (n=9) showed i n c r e a s e s i n both CCK (40%) ( F i g 40 B and C) and EG (100%) ( F i g 39 B and C) c e l l number i n the continuous jejunum and a 40% i n c r e a s e i n EG c e l l s i n the continuous ileum. In the j e j u n a l segment of the bypassed loop of obese JIB r a t s there was an i n c r e a s e i n GIP (130%) , CCK (30%) and EG (130%) c o n t a i n i n g c e l l s . There were no changes i n NT (Fig 41) or other endocrine c e l l number i n the i l e a l segment of the bypassed l o o p . 211 TABLE XII. EPITHELIAL HEIGHT (X + SEM) IN LEAN AND OBESE RATS TREATED WITH JEJUNOILEAL BYPASS SURGERY GENOTYPE EPITHELIAL HEIGHT (mm) JEJUNUM ILEUM CONTROLS Fa/-, n=8 f a / f a , n=8 0.69+0.04 0.73+0.01 0.47+0.03 0.54+0.02 JIB Continuous gut Fa/-, n=8 f a / f a , n=9 Bypassed loop Fa/-, n=8 f a / f a , n=9 0.91+0.07* 0.86+0.04+ 0.66+0.04 0.66+0.05 0.80+0.06* 0.87+0.03+ 0.56+0.05 0.50+0.01 *p<.05 compared to l e a n c o n t r o l s +p<.05 compared to obese c o n t r o l s 212 F i g 39. Immunocytochemical examination of j e j u n a l endocrine c e l l s c o n t a i n i n g enteroglucagon i n the bowel remaining i n c o n t i n u i t y o f le a n and obese Zucker r a t s 5 weeks f o l l o w i n g JIB compared t o sham operated c o n t r o l s ( c r o s s - s e c t i o n , m a g n i f i c a t i o n x40). A. Lean c o n t r o l B. Obese c o n t r o l s showing the decreased number of EG-p o s i t i v e c e l l s C. Obese JIB showing an i n c r e a s e i n E G - c e l l number to l e v e l s comparable t o t h a t of l e a n c o n t r o l s . S i m i l a r r e s u l t s were observed i n the j e j u n a l r e g i o n of the bypassed loop of these animals. EG c e l l number was a l s o s i g n i f i c a n t l y i n c r e a s e d i n the continuous ileum of obese r a t s . Lean bypassed r a t s d i s p l a y e d a 50% d e c l i n e i n the number of j e j u n a l EG c e l l s i n the continuous bowel, but no changes i n other r egions (not shown). 213 A 4 214 F i g 40. Immunocytochemical examination of j e j u n a l endocrine c e l l s c o n t a i n i n g c h o l e c y s t o k i n i n i n the s m a l l bowel remaining i n c o n t i n u i t y of obese Zucker r a t s 5 weeks f o l l o w i n g JIB compared to sham-operated c o n t r o l s ( c r o s s - s e c t i o n , m a g n f i c a t i o n x40). A. Lean c o n t r o l B. Obese c o n t r o l , showing no d i f f e r e n c e i n C C K - c e l l number compared to c o n t r o l s . C. Obese JIB showing the i n c r e a s e i n c e l l number i n the continuous gut. S i m i l a r r e s u l t s were observed i n the bypassed loop of these animals. CCK c e l l number was a l s o increased i n the bypassed loop of l e a n r a t s , but was not a l t e r e d i n any other r e g i o n (not shown). 215 216 F i g 41. Immunocytochemical examination of i l e a l e ndocrine c e l l s c o n t a i n i n g n e u r o t e n s i n i n the small bowel remaining i n c o n t i n u i t y of l e a n and obese Zucker r a t s 5 weeks f o l l o w i n g JIB compared to sham-operated c o n t r o l s . A. Lean c o n t r o l B. Obese c o n t r o l , showing no d i f f e r e n c e i n N T - c e l l number compared to c o n t r o l s C. Obese JIB showing no change i n N T - c e l l number. S i m i l a r l y no s i g n i f i c a n t changes i n N T - c e l l number were observed i n l e a n JIB animals (not shown). 217 218 TABLE X I I I . QUANTIFICATION 3 OF ENDOCRINE CELLS (X + SEM) IN THE JEJUNUM OF LEAN AND OBESE J I B RATS GENOTYPE ENDOCRINE CELL NUMBER (cells/mm) GIP SOMA CCK EG CONTROLS F a / - , n=8 6.2+1.1 5.7+1.0 29.8+4.1 11.3+1.2 f a / f a , n=8 4.3+1.5 4.0+1.2 30.9+3.3 4.9+1.1* J I B C o n t i n u o u s g u t F a / - , n=8 4.1+1.2 4.2+1.2 27.1+6.1 6.1+1.2* f a / f a , n=9 4.9+1.3 2.3+0.9 42.5+2.6*+ 10.0+1.5+ B y p a s s e d l o o p F a / - , n=8 7.0+1.0 7.3+1.0 42.4+2.4* 12.9+2.8 f a / f a , n=9 10.0 + 1.8+ 5.4+1.3 4.1.6 + 3.8*+ 11.1+1.5 + *p<.05 compared t o l e a n c o n t r o l s +p<.05 compared t o o b e s e c o n t r o l s a. E i g h t t o t e n c r y p t / v i l l u s p r o f i l e s f r o m e a c h o f two non-s e r i a l s e c t i o n s were q u a n t i f i e d f o r e a c h c e l l t y p e f o r each r a t . 219 TABLE XIV. QUANTIFICATION 3 OF ENDOCRINE CELLS (X + SEM) IN THE ILEUM OF LEAN AND OBESE J I B RATS GENOTYPE ENDOCRINE CELL NUMBER (cells/mm) GIP SOMA CCK NT EG CONTROLS F a / - 1.4+1.4 2.3+1.6 30.3+2.3 4.9+1.4 23.3+2.7 n=8 f a / f a 0.6+0.6 4.9+1.3 28.0+1.8 7.8+1.4 26.7+2.4 n=8 J I B C o n t i n u o u s g u t F a / - 0 2.2+0.58 30.9+3.4 8.8+2.8 29.3+3.9 n=8 f a / f a 0.8+0.5 2.6+0.92 39.5+6.4 12.4+2.4 38.8+4.3+ n=7 B y p a s s e d l o o p F a / - 1.7+1.1 4.4+1.8 24.4+3.9 11.4+1.6* 27.6+5.6 n=8 f a / f a 2.4+1.5 5.2+1.1 25.9+3.5 11.8+3.0 30.9+3.4 n=9 *p<.05 compared t o l e a n c o n t r o l s +p<.05 compared t o o b e s e c o n t r o l s a. E i g h t t o t e n c r y p t / v i l l u s p r o f i l e s f r o m e a c h o f two non-s e r i a l s e c t i o n s were q u a n t i f i e d f o r e a c h c e l l t y p e f o r each r a t . 220 3. The e f f e c t of JIB on the e n t e r o i n s u l a r a x i s i n  animals not f a s t e d o v e r n i g h t one n i g h t per week A second group of JIB animals (JIB-NS) was prepared to t e s t the e f f e c t t h a t the one n i g h t per week o v e r n i g h t f a s t i n g regimen (for the purpose of s t u d y i n g changes i n f a s t i n g l e v e l s of glucose and hormones over time) may have had on o r a l glucose t o l e r a n c e and response of the i s o l a t e d pancreas to s t i m u l a t i o n by glucose and GIP. These animals were not f a s t e d o v e r n i g h t one n i g h t per week, nor was a blood sample c o l l e c t e d . At f i v e weeks f o l l o w i n g JIB surgery, OGTT and pancreas p e r f u s i o n were performed. a. Weight change (Table X) Lean JIB-NS r a t s (n=5) l o s t 20.3% of t h e i r o r i g i n a l body weight by 5 weeks, twice the weight l o s s of lean JIB r a t s (n=13) i n the temporal study of g l u c o s e and hormones su b j e c t e d to f a s t i n g one n i g h t per week. The weight l o s s of obese JIB-NS r a t s (n=5) d i d not d i f f e r s i g n i f i c a n t l y from JIB obese r a t s (n=16) f a s t e d one n i g h t per week. b. O r a l glucose t o l e r a n c e (Figs 42, 43 and 44) In obese JIB-NS r a t s (n=4), f a s t i n g g lucose and GIP l e v e l s were not d i f f e r e n t from obese JIB animals (n=15) (Fig 43, A and C ) , but f a s t i n g plasma i n s u l i n c o n c e n t r a t i o n s were s i g n i f i c a n t l y (p<.05) higher i n the JIB-NS grouo (Fig 43 B). F o l l o w i n g a d m i n i s t r a t i o n of o r a l g l u c o s e the 221 i n t e g r a t e d glucose response of JIB-NS r a t s was s i g n i f i c a n t l y -decreased (Fig 44 A), whereas the i n t e g r a t e d i n s u l i n response was markedly e l e v a t e d compared to JIB animals, so th a t there was no s i g n i f i c a n t r e d u c t i o n from c o n t r o l i n s u l i n output ( F i g 44 B). There was no d i f f e r e n c e i n GIP output of the two groups ( F i g 44 C ) . In lean r a t s , JIB-NS r a t s (n-3) showed no d i f f e r e n c e i n basal i n s u l i n or GIP l e v e l s ( F i g 42, B and C) although plasma glucose c o n c e n t r a t i o n s were somewhat reduced compared to JIB r a t s (n=12) (Fig 42 A). F o l l o w i n g o r a l g l u c o s e there was no d i f f e r e n c e i n the GIP or i n s u l i n response ( F i g 44, B and C), d e s p i t e a s i g n i f i c a n t r e d u c t i o n i n the t o t a l g lucose response (Fig 44 A) . c. In v i t r o i n s u l i n response to gl u c o s e p l u s GIP  (F i g 45) The i s o l a t e d pancreata of l e a n and obese JIB-NS r a t s were perfused with 300 mg/dl gl u c o s e p l u s 0-1 ng/ml GIP and the response compared to t h a t of sham-operated c o n t r o l s . I n s u l i n output of l e a n JIB-NS r a t s (n=6) was s i g n i f i c a n t l y reduced compared to c o n t r o l s (n=13) to glucose p l u s GIP but not to glucose alone ( F i g 45 A) . The i n s u l i n output of JIB-NS obese r a t s (n=6) was not s i g n i f i c a n t l y reduced compared to c o n t r o l s (n=9) to e i t h e r s t i m u l u s ( F i g 45 B) . 222 F i g 42. The e f f e c t of not f a s t i n g lean JIB-NS r a t s (n=3) o v e r n i g h t one night per week on the (A) glu c o s e , (B) i n s u l i n and (C) GIP responses to 1 g/kg o r a l glucose compared to lea n JIB Q r a t s that had been st a r v e d o v e r n i g h t one n i g h t per week. represents the a d m i n i s t r a t i o n of glu c o s e at 0 minutes. *p<.05 using unpaired Student's t - t e s t . 223 224 F i g 43. The e f f e c t of not s t a r v i n g obese JIB-NS (n=4) r a t s o v e r n i g h t one n i g h t per week on the (A) glu c o s e , (B) i n s u l i n and (C) GIP responses to 1 g/kg o r a l g lucose compared to obese JIB ra t s that had been f a s t e d o v e r n i g h t one n i g h t per week (n=15). m- r e p r e s e n t s the a d m i n i s t r a t i o n o f glu c o s e at 0 minutes. *p<.05 using unpaired Student's t - t e s t . 225 226 F i g 44. The i n t e g r a t e d (A) glucose, (B) i n s u l i n and (C) GIP responses t o o r a l glucose i n JIB and JIB-NS r a t s compared to sham-operated c o n t r o l s using data shown i n F i g u r e s 25, 26, 41 and 42. *p<.05 compared to c o n t r o l s using unpaired Student's t - t e s t . The formula f o r c a l c u l a t i n g i n t e g r a t e d responses was as shown i n F i g 12. 227 228 F i g 45. The e f f e c t o f not f a s t i n g (A) l e a n (n=6) and (B) obese (n=6) JIB-NS Zucker r a t s on the response of the i s o l a t e d p e r f u s e d pancreas to 300 mg/dl glucose p l u s a GIP g r a d i e n t of 0-1 ng/ml compared to sham-operated l e a n (n=13) and obese (n=9) c o n t r o l s . *p<.05 using unpaired t - t e s t . 229 A 8000 MINUTES 230 4. The e f f e c t on t h e e n t e r o i n s u l a r a x i s o f p e r f u s i n g  t h e b y p a s s e d l o o p o f l e a n and o b e s e J I B r a t s w i t h  n u t r i e n t s The s u r g i c a l p r o t o c o l c r e a t i n g t h e j e j u n o s t o m y / i l e o s t o m y w i t h t h e b y p a s s e d g u t a l l o w e d i n v e s t i g a t i o n o f t h e e f f e c t s o f i n t e r m i t t e n t l u m i n a l s t i m u l a t i o n o f t h e b y p a s s e d l o o p on t h e a d a p t i v e r e s p o n s e o f t h e p a n c r e a s t o j e j u n o i l e a l b y p a s s s u r g e r y . F o u r o b e s e and s i x l e a n a n i m a l s t r e a t e d w i t h J I B were g i v e n 1 m i l l i l i t e r o f l i q u i d d i e t ( E n s u r e , Ross L a b o r a t o r i e s ) d a i l y i n t o t h e j e j u n a l end o f t h e b y p a s s e d l o o p v i a a s t e r i l e s t a i n l e s s s t e e l t u b e a t t a c h e d t o a 1 ml s y r i n g e . One ml o f E n s u r e c o n t a i n s 1.06 k i l o c a l o r i e s , a b o u t 2% t h e number o f c a l o r i e s e s t i m a t e d as t h e d a i l y i n t a k e o f a r a t ( H a r v a r d c a t a l o g u e ) . T hese a n i m a l s were d e s i g n a t e d J I B - L . a. W e i g h t change ( T a b l e X) The J I B - L r a t s and t h e i r c o n t r o l s were s t a r v e d o v e r n i g h t b e f o r e w e i g h i n g . L e a n r a t s (n=6) l o s t a maximum o f 14% o f t h e i r o r i g i n a l body w e i g h t by t h e 2nd week f o l l o w i n g s u r g e r y and t h e r e a f t e r s l o w l y g a i n e d w e i g h t t o a f i n a l w e i g h t l o s s o f o n l y 8.1%. T h i s was s i m i l a r t o r e s u l t s o b t a i n e d f o r J I B l e a n r a t s n o t g i v e n t h e d a i l y n u t r i e n t i n f u s i o n i n t o t h e b y p a s s e d l o o p (n=13) ( T a b l e X ) . Obese J I B - L r a t s (n=4) had l o s t 2.1% o f t h e i r o r i g i n a l body w e i g h t by t h e 5 t h week a f t e r s u r g e r y and were s t i l l l o s i n g w e i g h t a t t h i s t i m e . T h e s e r e s u l t s a l s o were s i m i l a r 231 to those obtained f o r JIB obese r a t s not g i v e n d a i l y n u t r i e n t i n f u s i o n i n t o the bypassed loop (n=16) (Table X) . b. Weekly plasma glucose measurement ( F i g 46) A f t e r o v e r n i g h t s t a r v a t i o n a b a s a l blood sample was obtained from l e a n and obese JIB-L r a t s . Due to t e c h n i c a l d i f f i c u l t i e s encountered d u r i n g blood c o l l e c t i o n , plasma i n s u l i n and GIP l e v e l s were not measured. Plasma glucose l e v e l s of both l e a n (n=6) (Fig 46 A) and obese (n=4) (Fig 46 B) JIB-L r a t s were 30% lower than those of c o n t r o l s (n=9 and n=13) 1 week a f t e r s u r g e r y , although s t i l l w i t h i n the normal range (JIB-L: 80 mg/dl vs c o n t r o l s : 110 mg/dl). D a i l y Ensure i n f u s i o n s were s t a r t e d at t h i s time. A f t e r commencement of d a i l y n u t r i e n t i n f u s i o n s f a s t i n g plasma glucose l e v e l s d e c l i n e d a f u r t h e r 30% of c o n t r o l v a l u e s (<50 mg/dl vs 100-120 mg/dl) u n t i l the 5th week f o l l o w i n g surgery, when plasma glucose l e v e l s were once again found to be normal. c. Weekly i n t r a j e j u n a l glucose s t i m u l a t i o n  ( F i g 47) A f t e r a b a s a l blood sample was o b t a i n e d , 0.5 g/kg glucose was administered as a 20% s o l u t i o n i n t o the proximal end of the bypassed loop using a s t e r i l e s t a i n l e s s s t e e l feeding tube and a 3 ml s y r i n g e . Blood samples were obtained at 10, 20, 30 and 60 min f o r g l u c o s e , i n s u l i n and GIP d e t e r m i n a t i o n . The data f o r 3 c o n s e c u t i v e weeks was 232 pooled and shown i n F i g 47, demonstrating t h a t a d m i n i s t r a t i o n of i n t r a j e j u n a l n u t r i e n t s i n t o the b l i n d loop can cause changes i n plasma glucose and v a r i o u s hormones. F i g 47 A shows an i n c r e a s e i n plasma g l u c o s e from 50 mg/dl to 120 mg/dl i n le a n JIB-L r a t s (n=23) and a s i m i l a r change i n obese JIB-L animals (n=14) from 50 to 140 mg/dl f o l l o w i n g i n t r a j e j u n a l glucose a d m i n i s t r a t i o n . Glucose l e v e l s remained e l e v a t e d 60 min f o l l o w i n g the g l u c o s e dose. Plasma i n s u l i n l e v e l s (Fig 47 B) were not s i g n i f i c a n t l y i n c r e a s e d i n le a n r a t s (n=9) (14+3.2 uU/ml b a s a l l y vs 21 + 6.3 pU/ml at 30 min), whereas obese JIB-L r a t s (n=5) had a small r i s e i n plasma i n s u l i n l e v e l s (77 + 19.5 /iTj/ml b a s a l l y vs 116 ± 17 pU/ml at 10 min, p<.05). Both l e a n (n=17) and obese (n=10) JIB-L r a t s demonstrated an approximately 100% i n c r e a s e i n plasma IR-GIP c o n c e n t r a t i o n s at 10 min, d e c l i n i n g to b a s a l l e v e l s at 60 min (Fig 47 C ) . d. O r a l glucose t o l e r a n c e ( F i g 48, 49 and 50) The e f f e c t o f d a i l y n u t r i e n t p e r f u s i o n i n t o the bypassed loops of JIB-L r a t s on o r a l g l u c o s e t o l e r a n c e was examined. The glucose reponse of l e a n JIB-L r a t s (n=5) was not s i g n i f i c a n t l y d i f f e r e n t from l e a n c o n t r o l s (n=9) ( F i g s 48 A and 50 A) but the i n t e g r a t e d i n s u l i n response to 1 g/kg o r a l glucose was markedly reduced (Figs 48 B and 50 B) . S i m i l a r r e s u l t s were observed f o r obese JIB-L r a t s (n=4) compared to obese c o n t r o l s (n=13) (Figs 49 and 50). 233 e. In v i t r o i n s u l i n s e c r e t i o n i n response to  glucose p l u s a GIP g r a d i e n t ( F i g 51) Fi v e weeks f o l l o w i n g JIB surgery and 4 weeks f o l l o w i n g commencement of d a i l y i n t r a j e j u n a l n u t r i e n t i n f u s i o n i n t o the bypassed loop, the pancreata of le a n and obese JIB-L r a t s were i s o l a t e d and p e r f u s e d . The s t i m u l u s for i n s u l i n s e c r e t i o n was 300 mg/dl gl u c o s e i n the presence of a GIP g r a d i e n t from 0-1 ng/ml. F i g 51 A shows the r e s u l t s of these experiments i n lean JIB-L animals (n=6) compared to c o n t r o l s (n=13). The i n s u l i n response of these r a t s was reduced s i g n i f i c a n t l y to 60% of the c o n t r o l response. S i m i l a r r e s u l t s were observed f o r obese JIB-L animals (n=4) compared to sham-operated obese l i t t e r m a t e s (n=9) (Fig 51 B) . 234 F i g 46. The e f f e c t of d a i l y n u t r i e n t p e r f u s i o n of the bypassed loop of (A) l e a n (n=6) and (B) obese (n=4) JIB-L Zucker r a t s on f a s t i n g plasma glucose l e v e l s measured on a weekly b a s i s compared to le a n (n=9) and obese (n=13) c o n t r o l s . *p<.05 using unpaired Student's t - t e s t , comparing le a n and obese JIB-L r a t s to t h e i r r e s p e c t i v e c o n t r o l s . 235 A. 0 — 0 Control O 4- 1 1 1 1 — i 0 1 2 3 4 5 WEEKS AFTER SURGERY 236 F i g 47. Changes i n plasma (A) glucose (n=23 vs n=14), (B) i n s u l i n (n=9 vs n=5) and (C) GIP (n=17 vs n=10) c o n c e n t r a t i o n s f o l l o w i n g a d m i n i s t r a t i o n of 0.5 g/kg g l u c o s e i n t r a j e j u n a l l y v i a the jejunostomy c r e a t e d during JIB surgery i n l e a n and obese JIB-L Zucker r a t s . m- r e p r e s e n t s the a d m i n i s t r a t i o n of glucose at 0 min. Values at 10, 20, 30 and 60 min were compared to b a s a l values to determine s i g n i f i c a n c e . * (lean) and 0 (obese): p<.05 using p a i r e d Student's t - t e s t , compared to r e s p e c t i v e c o n t r o l s . 237 238 F i g 48. The (A) glucose and (B) i n s u l i n responses t o o r a l glucose i n lean JIB-L r a t s f i v e weeks f o l l o w i n g JIB surgery and four weeks f o l l o w i n g commencement of d a i l y n u t r i e n t i n f u s i o n i n t o the bypassed loop (n=5) compared to sham-operated c o n t r o l s (n=9) . r e p r e s e n t s the a d m i n i s t r a t i o n o f gl u c o s e at 0 minutes. *p<.05 using unpaired Student's t - t e s t , compared to lean c o n t r o l s . 239 QJ , . ^ — . . • r -0 10 20 30 40 50 60 MINUTES 240 F i g 49. The (A) glucose and (B) i n s u l i n responses t o o r a l g lucose i n obese JIB-L r a t s f i v e weeks f o l l o w i n g JIB surgery and four weeks f o l l o w i n g commencement of d a i l y n u t r i e n t i n f u s i o n i n t o the bypassed loop (n=4) compared to sham-operated c o n t r o l s (n=13). r e p r e s e n t s the a d m i n i s t r a t i o n of gl u c o s e at 0 minutes. *p<.05 using unpaired Student's t - t e s t , compared to obese c o n t r o l s . 241 A. 300 0 J , , , , , , , 0 10 20 30 40 50 60 MINUTES 242 F i g 50. The i n t e g r a t e d (A) glucose and glucose i n lean and obese JIB-L and 49. *p<.05 using unpaired c o n t r o l s . The formula used was (B) i n s u l i n responses to o r a l r a t s u s ing data from F i g s 48 Student's t - t e s t compared to shown i n F i g 12. 243 F i g 51. The response of the i s o l a t e d p e r f u s e d pancreas to 300 mg/dl glucose p l u s a GIP g r a d i e n t o f 0-1 ng/ml of (A) l e a n (n=6) and (B) obese (n=4) JIB-L r a t s f i v e weeks f o l l o w i n g surgery compared to l e a n (n=13) and obese (n=9) c o n t r o l s . *p<.05 using unpaired Student's t - t e s t , comparing le a n and obese JIB-L animals with t h e i r r e s p e c t i v e c o n t r o l s . 245 MINUTES 246 5. The e f f e c t on the e n t e r o i n s u l a r a x i s of weight l o s s by s t a r v a t i o n of l e a n and obese Zucker r a t s Because some of the e f f e c t s on the e n t e r o i n s u l a r a x i s of JIB may be due to weight l o s s r a t h e r than a d i r e c t e f f e c t o f changes i n hormone s e c r e t i o n , the e f f e c t s of s t a r v a t i o n to 80% o f the o r i g i n a l body weight were i n v e s t i g a t e d i n 6 month o l d l e a n and obese r a t s . a. Weight change ( F i g 52) Lean (n=4) and obese (n=4) Zucker r a t s were denied access to food u n t i l 20% of t h e i r o r i g i n a l body weight was l o s t . T h i s weight l o s s mimicked that observed i n JIB r a t s . D a i l y weight l o s s averaged 20 g/day f o r the f i r s t day and d e c l i n e d to 5-10 g/day t h e r e a f t e r f o r both l e a n and obese r a t s ( F i g 52 A). Lean r a t s l o s t an average of 20% of t h e i r o r i g i n a l body weight by the 6th day of s t a r v a t i o n , whereas t h i s d i d not occur i n obese r a t s u n t i l the 11th day (Fig 52 B) . b. O r a l g lucose t o l e r a n c e ( F i g s 53, 54 and 55) Lean and obese r a t s s t a r v e d to 80% of t h e i r o r i g i n a l body weight were compared to o v e r n i g h t - s t a r v e d c o n t r o l s . S t a r v e d l e a n animals (n=4) showed no changes i n b a s a l c o n c e n t r a t i o n s of plasma g l u c o s e , i n s u l i n or GIP compared to c o n t r o l r a t s (n=4). In obese s t a r v e d animals (n=8) there was no d i f f e r e n c e i n b a s a l plasma glucose or i n s u l i n l e v e l s but plasma GIP c o n c e n t r a t i o n s were 247 s i g n i f i c a n t l y reduced compared to c o n t r o l s (n=6) (265 + 53.7 pg/ml vs 446 + 53.2 pg/ml, p<.05). F o l l o w i n g the a d m i n i s t r a t i o n of o r a l g l u c o s e , there was no s i g n i f i c a n t a l t e r a t i o n i n the glucose or GIP responses of s t a r v e d l e a n r a t s compared to t h e i r c o n t r o l s ( F i g s 53 and 55, A and C) , but a marked r e d u c t i o n i n i n s u l i n output was observed (Figs 53 and 55, B). Obese s t a r v e d r a t s d i s p l a y e d no s i g n i f i c a n t changes i n t h e i r g l u c o s e , i n s u l i n or GIP response t o o r a l g l u c o s e ( F i g s 54 and 55, A, B and C). c. In v i t r o i n s u l i n response to glucose p l u s a GIP  g r a d i e n t ( F i g 56) The i s o l a t e d pancreata of s t a r v e d l e a n (n=4) and obese (n=8) r a t s were p e r f u s e d with 300 mg/dl glucose i n the presence of a GIP g r a d i e n t from 0-1 ng/ml. The i n s u l i n response was compared to t h a t of l e a n (n=4) and obese (n=6) o v e r n i g h t - s t a r v e d c o n t r o l s r e s p e c t i v e l y , and the data expressed as a percentage of the c o n t r o l v a l u e s ( F i g 56). The i n s u l i n output of l e a n s t a r v e d r a t s was 70-80% reduced i n response to s t i m u l a t i o n by glucose and GIP, whereas there was no change i n the i n s u l i n response of obese s t a r v e d r a t s compared to c o n t r o l s to these s t i m u l i . 248 F i g 52. D a i l y weight l o s s o f l e a n (n=4) and obese (n=4) Zucker r a t s d eprived o f food expressed as (A) a c t u a l weight l o s s and (B) percentage weight l o s s . The data demonstrated t h a t obese r a t s l o s t a s i m i l a r amount of weight when expressed as g/day, r e s u l t i n g i n a s i g n i f i c a n t l y reduced percentage weight l o s s compared to l e a n animals. The experiment was terminated when animals reached a t a r g e t weight l o s s of 20% of the o r i g i n a l body weight (Day 5 f o r l e a n animals vs Day 11 f o r obese r a t s ) . *p<.05 comparing le a n vs obese animals using unpaired Student's t - t e s t . 249 A. 5 CO CO O I UJ 25 20 15 10 1 ~ 0 J s [] Lean [\] Obese I X LL \ I a. ft \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ o cc o L L o to CO o X LU > r-< _1 ID 5 a B . 20 16 12 I I I • \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ s N \ \ \ S \ \ \ \ s \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ s \ s 1 10 II DAYS 250 F i g 53. The e f f e c t of f a s t i n g to 80% of the o r i g i n a l body weight on the plasma (A) glucose, (B) i n s u l i n and (C) GIP responses to 1 g/kg o r a l glucose i n l e a n f a s t e d r a t s (n=4) compared to Q o v e r n i g h t - s t a r v e d c o n t r o l s (n=4). r e p r e s e n t s the a d m i n i s t r a t i o n o f glucose at 0 minutes. *p<.05 using unpaired Student's t - t e s t , compared to l e a n c o n t r o l s . 251 A. oJ i 1 • 1 1 • 1 0 10 20 30 40 50 60 MINUTES 252 F i g 54. The e f f e c t of s t a r v a t i o n to 80% of the o r i g i n a l body weight on the (A) g l u c o s e , (B) i n s u l i n and (C) GIP responses to 1 g/kg o r a l glucose i n obese s t a r v e d r a t s (n=8) compared to o v e r n i g h t - s t a r v e d c o n t r o l s (n=6) . r e p r e s e n t s the a d m i n i s t r a t i o n of glucose at 0 minutes. *p<.05 using unpaired Student's t - t e s t , compared to obese c o n t r o l s . 253 254 F i g 55. The e f f e c t o f s t a r v a t i o n to 80% of the o r i g i n a l body weight on the i n t e g r a t e d (A) g l u c o s e , (B) i n s u l i n and (C) GIP responses to o r a l glucose using data shown i n F i g s 52 and 53. The formula used to c a l c u l a t e the i n t e g r a t e d responses was shown i n F i g 12. *p<.05 compared to o v e r n i g h t s t a r v e d c o n t r o l s using the unpaired Student's t - t e s t , compared to o v e r n i g h t - s t a r v e d c o n t r o l s . 255 F i g 56. The e f f e c t of s t a r v a t i o n to 80% of the o r i g i n a l body weight on the response of the i s o l a t e d p e r f u s e d pancreas to 300 mg/dl glucose p l u s a GIP g r a d i e n t of 0-1 ng/ml i n l e a n (n=4) and obese (n=8) f a s t e d r a t s compared to l e a n (n=4) and obese (n=6) c o n t r o l s . I n s u l i n s e c r e t i o n data f o r s t a r v e d groups was expressed as a percentage of t h e i r r e s p e c t i v e c o n t r o l s . *p<.05 using unpaired Student's t - t e s t f compared to o v e r n i g h t - f a s t e d c o n t r o l s . 257 Lean Starved 20 30 4 0 MINUTES 258 IV. DISCUSSION I n i t i a l l y , a r e v i s e d s u r g i c a l procedure was developed i n Wistar r a t s i n an attempt to improve s u r v i v a l r a t e i n obese r a t s because Zucker f a t t i e s were r e p o r t e d to be l e s s t o l e r a n t o f surgery than l e a n c o n t r o l s ( G r o s f e l d et a l , 1976). Conventional JIB procedure i n v o l v e d end-to-end or end-to-side anastomosis of the jejunum and the ileum. T y p i c a l l y the b l i n d l oop was sutured c l o s e d at the proximal end with drainage v i a an end-to-side anastomosis of the d i s t a l end to the t r a n s v e r s e c o l o n . In the procedure used i n the c u r r e n t s t u d i e s , the end-to-end anastomosis of the continuous jejunum and ileum was c a r r i e d out normally. The b l i n d loop was e x t e r i o r i z e d to the body w a l l by c r e a t i o n of a jejunostomy/ileostomy. I t was t h e o r i z e d that t h i s m a n i p u l a t i o n would reduce the danger of anastomotic leakage. To ensure that the novel JIB s u r g i c a l method had no e f f e c t on experimental parameters, three groups of normal Wistar r a t s were prepared: a group s u b j e c t e d to c o n v e n t i o n a l bypass surgery (Group 1) , a group t r e a t e d with the jejunostomy/ileostomy procedure (Group 2) , and a sham-operated c o n t r o l group. F i v e weeks f o l l o w i n g surgery, a l l groups of animals were gi v e n an o r a l glucose t o l e r a n c e t e s t with plasma samples c o l l e c t e d f o r g l u c o s e , i n s u l i n and GIP measurement. The animals were then s a c r i f i c e d and the i s o l a t e d pancreas p e r f u s e d f o r measurement of p a n c r e a t i c s e n s i t i v i t y to s t i m u l a t i o n by gl u c o s e and GIP. 259 The r e s u l t s of these experiments are shown i n F i g u r e s 30 and 31. I t was demonstrated t h a t no s i g n i f i c a n t d i f f e r e n c e s i n g l u c o s e , i n s u l i n or GIP l e v e l s e x i s t e d between Group 1 and Group 2 bypass animals ( F i g 30) . S i m i l a r l y , the response of the i s o l a t e d pancreas to 300 mg/dl glucose p l u s a GIP g r a d i e n t of 0-1 ng/ml r e s u l t e d i n a 75% r e d u c t i o n i n the i n s u l i n response of both groups compared to c o n t r o l s ( F i g 31) . These r e s u l t s were s i m i l a r to those observed by Pederson et a l (1982) and confirmed t h a t the novel s u r g i c a l method had no e f f e c t on o r a l glucose t o l e r a n c e or i n v i t r o i n s u l i n r e l e a s e compared to c o n v e n t i o n a l s u r g i c a l procedure. Thus, t h i s procedure was a p p l i e d to Zucker obese and l e a n animals f o r study. F i v e - to 8-month-old g l u c o s e - i n t o l e r a n t obese Zucker r a t s with e l e v a t e d plasma GIP l e v e l s and t h e i r l e a n l i t t e r m a t e s were t r e a t e d with JIB surgery. J e j u n o i l e a l bypass animals were d i v i d e d i n t o three groups. A n a l y s i s of weight l o s s f o r the three bypass groups (JIB, JIB-NS and JIB-L) i n d i c a t e d t h a t obese animals l o s t a gre a t e r percentage of t h e i r body weight over the f i v e week study p e r i o d than d i d l e a n bypass animals. There was no d i f f e r e n c e i n the t o t a l weight l o s s of the three groups of obese r a t s t r e a t e d with j e j u n o i l e a l bypass (Table X). Thus i t appears that o v e r n i g h t s t a r v a t i o n i s not d e t r i m e n t a l to the recovery of r a t s t r e a t e d with j e j u n o i l e a l bypass. These o b s e r v a t i o n s are i n agreement with those of others ( K i s s i l e f f , Nakashima and Stunkard, 1979; Madura et a l , 260 1975; S c l a f a n i et a l , 1978) who r e p o r t e d g r e a t e r weight l o s s i n obese bypass r a t s than i n l e a n bypass animals. Weekly measurement of f a s t i n g plasma c o n c e n t r a t i o n s of g l u c o s e , i n s u l i n and GIP r e v e a l e d some d i f f e r e n c e s between lea n and obese JIB r a t s . B a s a l g l u c o s e c o n c e n t r a t i o n s d i d not d i f f e r s i g n i f i c a n t l y from plasma glucose l e v e l s of c o n t r o l r a t s (Fig 32 A) . T h i s trend was a l s o e v i d e n t i n o r a l glucose t o l e r a n c e t e s t data ( F i g s 33, 34 and 44) and concurred with p r e v i o u s r e p o r t s (Greenwood e t a l , 1982) where no s i g n i f i c a n t d i f f e r e n c e s were observed i n ambient glucose l e v e l s of l e a n or obese bypassed r a t s compared to t h e i r r e s p e c t i v e c o n t r o l s 10 months f o l l o w i n g surgery. Thus JIB d i d not appear to a f f e c t the a b i l i t y of the r a t s to r e g u l a t e f a s t i n g glucose l e v e l s , nor was the a b s o r p t i o n of glucose impaired. Measurement of e p i t h e l i a l h e i g h t from the continuous and bypassed sma l l bowel of both l e a n and obese JIB r a t s i n d i c a t e d t h a t c r y p t / v i l l u s p r o f i l e i n c r e a s e d about 25% i n the jejunum and up to 70% i n the ileum, thus i n c r e a s i n g the a b s o r p t i v e s u r f a c e of the continuous s m a l l bowel. No changes i n the e p i t h e l i a l h e i g h t of the bypassed small bowel were noted i n e i t h e r l e a n or obese animals (Table X I I ) . I t was l i k e l y t h a t the l a r g e i n c r e a s e i n a b s o r p t i v e s u r f a c e of the s m a l l bowel remaining i n c o n t i n u i t y i n c r e a s e d glucose a b s o r p t i o n to near normal l e v e l s . Plasma i n s u l i n l e v e l s of obese JIB r a t s became markedly reduced by the 2nd p o s t - s u r g i c a l week and remained at 40-60% 261 of c o n t r o l v a l u e s f o r the d u r a t i o n of the study ( F i g 32 B). However, f a s t i n g i n s u l i n l e v e l s i n these animals were s t i l l higher than those of l e a n c o n t r o l s . Greenwood et a l (1982) s i m i l a r l y r e p o r t e d a 30% decrease i n ambient i n s u l i n l e v e l s of obese bypass r a t s compared to sham-operated c o n t r o l s a f t e r 10 months. These i n s u l i n c o n c e n t r a t i o n s were l i k e w i s e s i g n i f i c a n t l y e l e v a t e d compared to l e a n c o n t r o l s . No d i f f e r e n c e s were noted between l e a n bypass and l e a n c o n t r o l s i n these s t u d i e s or those of Greenwood et a l (1982). F a s t i n g GIP l e v e l s were i n i t i a l l y decreased i n l e a n JIB animals but i n c r e a s e d to c o n t r o l l e v e l s by the 5th week of the study. C o n v e r s e l y , GIP c o n c e n t r a t i o n s measured i n plasma of obese JIB r a t s were not s i g n i f i c a n t l y a l t e r e d u n t i l the 5th week, where they were reduced to 60% of c o n t r o l l e v e l s (Fig 32 C) . These r e s u l t s c o n t r a s t with those of Pederson et a l (1982), who r e p o r t e d e l e v a t i o n of f a s t i n g GIP l e v e l s i n normal Wistar r a t s with j e j u n o i l e a l bypass 5 weeks f o l l o w i n g surgery. GIP l e v e l s have not p r e v i o u s l y been measured i n Zucker r a t s f o l l o w i n g JIB, but the r i s i n g t rend of f a s t i n g GIP l e v e l s i n l e a n bypass animals i n weeks 4 and 5 may i n d i c a t e t h a t s i g n i f i c a n t e l e v a t i o n of b a s a l GIP may have been observed i f the experiments had been continued f o r a longer d u r a t i o n , whereas f a s t i n g GIP l e v e l s of obese r a t s appear to be f a l l i n g at t h i s time. Another f a c t o r may be the age of the r a t s used, s i n c e Zucker r a t s employed here were 5-8 months of age, whereas the r a t s of Pederson's study (1982) were 2-3 262 months of age. Conversely, these d i f f e r e n c e s may r e f l e c t d i f f e r e n c e s i n the a d a p t a t i o n of the pancreas of the Wistar vs Zucker s t r a i n of r a t s to J I B . O r a l glucose t o l e r a n c e t e s t s i n d i c a t e d t h a t l e a n JIB r a t s e x h i b i t e d normal i n s u l i n s e c r e t i o n , d e s p i t e a s i g n i f i c a n t l y reduced GIP output (Fig 33, B and C) . These data are i n c o n t r a s t to those r e p o r t e d f o r normal Wistar r a t s given JIB surgery (Pederson et a l , 1982; Buchan et a l , 1983). I t was presumed by these authors t h a t the r e d u c t i o n i n both the GIP and i n s u l i n response to o r a l g lucose was due to the removal of approximately 70% of the GIP c e l l p o p u l a t i o n from c o n t a c t with o r a l l y i n g e s t e d n u t r i e n t s . The c u r r e n t study i n d i c a t e d t hat w hile decreased GIP r e l e a s e may indeed be due to reduced c o n t a c t with n u t r i e n t s , i t appeared that the pancreas of Zucker r a t s was able to compensate f o r the r e d u c t i o n i n GIP s e c r e t i o n and m a i n t a i n normal i n s u l i n output. In obese r a t s t r e a t e d with JIB, both the i n s u l i n and GIP responses to o r a l glucose were reduced by 50% compared to sham-operated c o n t r o l s ( F i g 34, B and C) . Viewed i n i s o l a t i o n , these r e s u l t s p o i n t to a c a u s a l r e l a t i o n s h i p between decreased GIP r e l e a s e and a b l u n t e d i n s u l i n response, as has been noted elsewhere (Sarson, Scopinaro and Bloom, 1981). S i n c e data from l e a n JIB r a t s c o n t r a d i c t s these, d i f f e r e n c e s between the i n i t i a l s t a t e of i n s u l i n e m i a may account f o r the d i f f e r e n c e s i n end r e s u l t noted between obese and l e a n animals. T h i s i s analogous to the 263 n o r m a l i z a t i o n of i n s u l i n l e v e l s f o l l o w i n g vagotomy i n VMH-l e s i o n e d r a t s , although vagotomy i n normal l e a n r a t s had no e f f e c t on plasma i n s u l i n ( M i l l e r , 1981). To f u r t h e r i n v e s t i g a t e the e f f e c t of reduced GIP s e c r e t i o n on i n s u l i n r e l e a s e , the pancreata of l e a n and obese JIB animals were i s o l a t e d and p e r f u s e d . In the f i r s t set of experiments, the s t i m u l u s f o r i n s u l i n s e c r e t i o n was 300 mg/dl glucose i n the presence of a GIP g r a d i e n t from 0-1 ng/ml. As shown i n F i g u r e 35, no d i f f e r e n c e i n the i n s u l i n s e c r e t o r y response to these s t i m u l i was noted i n e i t h e r l e a n or obese JIB r a t s compared to t h e i r r e s p e c t i v e c o n t r o l s . T h i s i n d i c a t e d that the pancreas of JIB r a t s had r e t a i n e d normal s e n s i t i v i t y to s t i m u l a t i o n by GIP, d e s p i t e the r e d u c t i o n i n s t i m u l a t e d GIP l e v e l s i n v i v o . These data c o n t r a s t with those observed by Pederson et a l (1982). These authors rep o r t e d a r e d u c t i o n i n the i n s u l i n response to 300 mg/dl glucose p l u s 0-1 ng/ml GIP i n normal Wistar r a t s t r e a t e d with JIB. The e s s e n t i a l d i f f e r e n c e i n the r e s u l t s between these two s t u d i e s was the presence of e l e v a t e d f a s t i n g GIP l e v e l s i n v i v o i n the Wistar JIB animals compared to sham-operated c o n t r o l s . The authors sp e c u l a t e d t h a t c h r o n i c a l l y e l e v a t e d b a s a l GIP c o n c e n t r a t i o n s c o u l d r e s u l t i n down-regulation of GIP r e c e p t o r s on i n s u l i n c e l l s , t h e r e f o r e d e c r e a s i n g p a n c r e a t i c s e n s i t i v i t y to GIP. T h i s h y p othesis was supported by a second study i n which bypassed Wistar r a t s were compared to animals t r e a t e d with s m a l l bowel r e s e c t i o n . GIP 264 c o n c e n t r a t i o n s and the response of the i s o l a t e d , p e r f u s e d pancreas to s t i m u l a t i o n by g l u c o s e p l u s GIP were normal i n r e s e c t e d Wistar r a t s (Buchan et a l , 1983). A c c o r d i n g to the above theory, because b a s a l GIP l e v e l s were normal i n l e a n JIB Zucker r a t s and s l i g h t l y decreased i n obese JIB Zucker r a t s , no decrease i n p a n c r e a t i c s e n s i t i v i t y to t h i s hormone should have been observed. Thus, these r e s u l t s , while demonstrating d i f f e r e n c e s i n the outcome of JIB i n Wistar vs Zucker r a t s , i n d i r e c t l y support the theory t h a t f a s t i n g GIP c o n c e n t r a t i o n s can e f f e c t p a n c r e a t i c s e n s i t i v i t y to s t i m u l a t i o n by GIP. As a c o n t r o l f o r the e f f e c t s of f a s t i n g JIB animals one n i g h t per week p r i o r to plasma sampling, a second group of animals (JIB-NS) were prepared and l e f t to recover u n t i l 5 weeks f o l l o w i n g surgery. R e s u l t s from these s t u d i e s i n d i c a t e t h a t the weekly f a s t i n g regimen may have a l t e r e d the measured responses. In the case of l e a n JIB vs l e a n JIB-NS animals ( F i g 42), there was a r e d u c t i o n i n the plasma glucose response to a d m i n i s t r a t i o n of o r a l g l u c o s e , but the i n s u l i n and GIP responses were not s i g n i f i c a n t l y d i f f e r e n t ; i . e . i n s u l i n was unchanged compared to sham-operated c o n t r o l s , whereas GIP was markedly reduced. Obese JIB-NS r a t s a l s o d i s p l a y e d a reduced glucose response compared to both JIB and c o n t r o l r a t s (Fig 43 A) . As f o r JIB animals, the GIP response to o r a l g l u c o s e was b l u n t e d i n JIB-NS r a t s , although f a s t i n g l e v e l s were not s i g n i f i c a n t l y a l t e r e d ( F i g 43 C) . The most n o t i c e a b l e r e s u l t was that the i n s u l i n 265 response to o r a l g l u c o s e of JIB-NS obese r a t s was s i g n i f i c a n t l y higher than that of JIB obese animals (Fig 43 B) , so that the i n t e g r a t e d response was not s i g n i f i c a n t l y d i f f e r e n t from t h a t of unoperated c o n t r o l s ( F i g 44 B). Thus, the r e s u l t s of these i n v i v o s t u d i e s suggest t h a t glucose a b s o r p t i o n i s improved by the weekly s t a r v a t i o n regimen i n bypassed r a t s . However, the mechanism f o r t h i s o b s e r v a t i o n i s u n c l e a r , s i n c e the e p i t h e l i a l h e i g h t of the continuous s m a l l bowel of JIB-NS r a t s was not measured. The second important d i f f e r e n c e between JIB-NS and JIB r a t s occurred i n obese animals o n l y ; t h i s was the f a i l u r e of j e j u n o i l e a l bypass to reduce 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 unless accompanied by o v e r n i g h t f a s t i n g one n i g h t per week. S i n c e weight l o s s d i d not d i f f e r between these two groups (Table X) over the f i v e weeks and changes i n the GIP response to o r a l g l u c o s e were s i m i l a r , the cause of t h i s d i s c r e p a n c y can o n l y be s p e c u l a t e d . B a c t e r i a l overgrowth of the bypassed loop occurs i n JIB r a t s and humans and may c o n t r i b u t e to the changes observed f o l l o w i n g JIB (McGouran et a l , 1981). In one r e p o r t (Nygaard, 1967) 24-hour s t a r v a t i o n reduced b a c t e r i a l overgrowth. I t i s p o s s i b l e t h a t r e g u l a r o v e r n i g h t f a s t i n g a l t e r e d the e f f e c t of i n t e s t i n a l f l o r a on JIB compared to JIB-NS animals. While a decrease i n f a s t i n g h y p e r i n s u l i n e m i a may r e f l e c t a decrease i n i n s u l i n r e s i s t a n c e , no improvement i n glucose t o l e r a n c e was observed i n obese JIB r a t s that would support t h i s h y p o t h e s i s . 266 I n v e s t i g a t i o n of the response of the p e r f u s e d pancreas of,JIB-NS r a t s t o 300 mg/dl gl u c o s e i n the presence of a GIP g r a d i e n t from 0-1 ng/ml showed that the p a n c r e a t i c i n s u l i n response of obese JIB-NS r a t s was not a l t e r e d from that of c o n t r o l s , whereas the response of l e a n JIB-NS was somewhat lower than that of l e a n c o n t r o l s ( F i g 45) . R e c a l l i n g t h a t there was no d i f f e r e n c e between the response of e i t h e r l e a n or obese JIB r a t s and t h e i r r e s p e c t i v e c o n t r o l s , i t can be concluded t h a t the weekly s t a r v a t i o n regiment a f f e c t e d the p a n c r e a t i c response of l e a n but not obese animals. A summary of the i n v i v o and i n v i t r o responses of l e a n and obese JIB and JIB-NS animals i s presented i n Table XV. Comparison between these two groups i n d i c a t e s t h a t , of the parameters measured, the major d i f f e r e n c e i n l e a n animals i s glucose a b s o r p t i o n . JIB-NS l e a n r a t s with a reduced glucose response to a d m i n i s t r a t i o n of o r a l glucose a l s o e x h i b i t e d a reduced response of the i s o l a t e d p e r f u s e d pancreas to s t i m u l a t i o n by glucose p l u s GIP. On the other hand, numerous d i f f e r e n c e s are noted between JIB and JIB-NS obese r a t s but no change i n the i n s u l i n response of the i s o l a t e d pancreas was noted i n e i t h e r group compared to c o n t r o l s . While the mechanisms f o r these a l t e r a t i o n s are were not e s t a b l i s h e d , changes i n b a c t e r i a l overgrowth or hypertrophy of the continuous bowel may have c o n t r i b u t e d . A second important o b s e r v a t i o n of these s t u d i e s i s t h a t obese animals do not respond to j e j u n o i l e a l bypass i n a manner i d e n t i c a l to t hat of l e a n r a t s . T h e r e f o r e , care must be taken when 267 .conclusions about a d a p t a t i o n to from experiments i n l e a n animals. JIB i n o b e s i t y are drawn 268 TABLE XV. A SUMMARY OF THE EFFECTS ON THE ENTEROINSULAR AXIS OF JIB, JIB-NS, JIB-L AND FASTING TO 80% OF THE ORIGINAL BODY WEIGHT COMPARED TO CONTROLS IN LEAN AND OBESE ZUCKER RATS JIB JIB-NS JIB-L FASTED LEAN B a s a l glucose — — Ba s a l i n s u l i n — — — Ba s a l GIP — — NA OGTT glucose — — OGTT i n s u l i n — — OGTT GIP • • NA Perfused pancreas — i n s u l i n OBESE Ba s a l glucose B a s a l i n s u l i n B a s a l GIP OGTT glucose OGTT i n s u l i n OGTT GIP • * NA Perfused pancreas — — — i n s u l i n — No change compared to c o n t r o l s • Response reduced compared t o c o n t r o l s NA Data not a v a i l a b l e 269 Zucker f a t t y r a t s were p r e v i o u s l y shown to la c k a glucose t h r e s h o l d f o r the i n s u l i n o t r o p i c a c t i v i t y of GIP (Chapter One). I t was hypothesized that r e s t o r a t i o n of a glucose t h r e s h o l d f o r GIP f o l l o w i n g JIB c o u l d cause the r e d u c t i o n i n f a s t i n g plasma i n s u l i n observed i n obese JIB r a t s . F i v e weeks f o l l o w i n g bypass surgery, the pancreata of l e a n and obese JIB r a t s were p e r f u s e d with a glucose g r a d i e n t of 50-150 mg/dl glucose i n the presence of 1 ng/ml GIP. F i g 36 shows no change i n the p a n c r e a t i c i n s u l i n response to these s t i m u l i i n l e a n JIB r a t s compared to unoperated c o n t r o l s . A normal glucose t h r e s h o l d of 110-120 mg/dl glucose was observed. I n s u l i n s e c r e t i o n from the i s o l a t e d pancreas of obese JIB r a t s to 50-150 mg/dl gl u c o s e p l u s 1 ng/ml GIP was compared to c o n t r o l s i n F i g 37. I t was demonstrated t h a t the i n s u l i n response of obese JIB r a t s to these s t i m u l i was s i g n i f i c a n t l y reduced compared to c o n t r o l s . To examine the p o s s i b i l i t y t h a t the glucose t h r e s h o l d had been r e s t o r e d i n these animals, the same data was compared to a second s e t of c o n t r o l s i n which the i s o l a t e d pancreas was p e r f u s e d with a glucose g r a d i e n t (50-150 mg/dl) i n the absence of GIP ( F i g 38). However, these experiments showed that i n s u l i n s e c r e t i o n was s t i l l s i g n i f i c a n t l y e l e v a t e d compared to c o n t r o l s , even a t glucose c o n c e n t r a t i o n s as low as 50 mg/dl, i n d i c a t i n g t h at the l a c k of a glucose t h r e s h o l d i n these animals i s a r e l a t i v e l y permanent l e s i o n and cannot be r e c t i f i e d by r e d u c t i o n of plasma i n s u l i n l e v e l s and 270 depressed GIP s e c r e t i o n , such as was observed i n obese JIB r a t s . Moreover, p e r s i s t e n c e of the absence of a glucose t h r e s h o l d may e x p l a i n the continued e l e v a t i o n of f a s t i n g plasma i n s u l i n l e v e l s above l e a n c o n t r o l l e v e l s observed here at 5 weeks and up to 10 months f o l l o w i n g bypass surgery i n other s t u d i e s (Greenwood et a l , 1982). P r e v i o u s s t u d i e s i n d i c a t e t hat JIB has a g r e a t e r a n o r e x i c e f f e c t on obese animals than l e a n ( S c l a f a n i et a l , 1978; K i s s i l e f f , Nakashima and Stunkard, 1979; S c l a f a n i , 1980), thus r e s u l t i n g i n g r e a t e r weight l o s s i n obese animals. However, the c a u s a l f a c t o r s f o r prolonged r e d u c t i o n of food i n t a k e were not c l e a r . One p r o p o s a l i n v o l v e d m o d i f i c a t i o n of the endocrine response of the gut to n u t r i e n t i n g e s t i o n . In order to i n v e s t i g a t e t h i s p o s s i b i l i t y , j e j u n a l and i l e a l t i s s u e from the continuous s m a l l bowel and from the bypassed loop were obtained and examined by immunocytochemistry. In a d d i t i o n , plasma glucagon (GLI) l e v e l s were measured by radioimmunoassay using an antibody t h a t recognized both p a n c r e a t i c glucagon and enteroglucagon (Gregor and Riecken, 1984). S i g n i f i c a n t d i f f e r e n c e s i n c e l l number occu r r e d i n s p e c i f i c endocrine c e l l types between l e a n and obese c o n t r o l animals. In p a r t i c u l a r , enteroglucagon (EG) c e l l number was markedly decreased i n the jejunum but normal i n the ileum of obese r a t s compared to l e a n c o n t r o l s . Obese c o n t r o l r a t s d i s p l a y e d s i g n i f i c a n t l y lower plasma GLI c o n c e n t r a t i o n s than t h e i r l e a n l i t t e r m a t e s which corresponded to the lower c e l l 271 number observed i n these r a t s (Table X I I I , F i g 39) . There were no a l t e r a t i o n s i n GIP, som a t o s t a t i n (Soma), c h o l e c y s t o k i n i n (CCK) or n e u r o t e n s i n (NT) c e l l number between le a n and obese c o n t r o l r a t s (Tables XIII and XIV, F i g 39-41) . F i v e weeks f o l l o w i n g bypass, enteroglucagon c e l l number was unchanged i n the continuous jejunum of l e a n r a t s but in c r e a s e d s i g n i f i c a n t l y i n both the continuous jejunum and ileum o f obese r a t s ( F i g 39 C) . EG c e l l number was a l s o i n c r e a s e d i n the bypassed j e j u n a l segment. These changes i n EG c e l l number were r e f l e c t e d by changes i n c i r c u l a t i n g GLI l e v e l s (Table XI) . F o l l o w i n g bypass, there was no change i n GLI of l e a n r a t s but obese JIB animals demonstrated a 3-f o l d i n c r e a s e compared t o c o n t r o l l e v e l s , so t h a t plasma c o n c e n t r a t i o n s of GLI were no longer d i f f e r e n t than those of le a n c o n t r o l s . Enteroglucagon may have important e f f e c t s on the p r o l i f e r a t i v e c a p a c i t y of the gut. The r e s u l t s of the c u r r e n t i n v e s t i g a t i o n have demonstrated t h a t f a s t i n g plasma GLI c o r r e l a t e s c l o s e l y with EG c e l l p o p u l a t i o n , and t h a t changes i n EG c e l l number are induced by j e j u n o i l e a l bypass. However, the changes that occur may depend on genotype, s i n c e c e l l number was i n c r e a s e d i n obese JIB r a t s but somewhat decreased i n l e a n JIB r a t s . S i n c e d e l i v e r y of n u t r i e n t - r i c h chyme to the d i s t a l s m a l l bowel i s presumably i n c r e a s e d i n both l e a n and obese JIB r a t s , t h i s cannot account f o r the d i f f e r e n t i a l r e s u l t s observed. In a d d i t i o n , EG c e l l number was i n c r e a s e d i n the bypassed loop o f obese 272 JIB r a t s . Changes that occur i n the bypassed loop must be mediated v i a e i t h e r a n e u r a l or c i r c u l a t o r y mechanism, because the bypassed loop r e c e i v e s no n u t r i e n t s t i m u l a t i o n v i a the o r a l route. I t would be o f i n t e r e s t to examine the e f f e c t of r e s e c t i n g the bypassed loop of l e a n and obese Zucker r a t s on plasma GLI l e v e l s , to gauge the c o n t r i b u t i o n of the EG c e l l s i n the bypassed loop to t o t a l GLI s e c r e t i o n . Previous s t u d i e s i n bypassed (Koopmans et a l , 1984) or r e s e c t e d (Gornacz et a l , 1984) Wistar r a t s and obese humans t r e a t e d with JIB (Sarson et a l , 1981) noted i n c r e a s e s i n plasma enteroglucagon l e v e l s , i n both the f a s t i n g s t a t e and f o l l o w i n g i n g e s t i o n of a meal. I t was concluded that i n c r e a s e d d e l i v e r y of e n r i c h e d chyme to the d i s t a l s m a l l bowel r e s u l t e d i n i n c r e a s e d s e c r e t i o n of t h i s hormone (Sarson et a l , 1981; Koopmans et a l , 1984), s i n c e EG c e l l s are p r i m a r i l y c o n f i n e d to the lower small bowel (SjSlund et a l , 1983). These authors d i d not q u a n t i f y the E G - c e l l p o p u l a t i o n , but i n c r e a s e d EG s e c r e t i o n may r e s u l t from EG-c e l l p r o l i f e r a t i o n s t i m u l a t e d by i n c r e a s e d n u t r i e n t d e l i v e r y . C h o l e c y s t o k i n i n c e l l number was normal i n obese c o n t r o l r a t s compared to l e a n l i t t e r m a t e s . F o l l o w i n g JIB, i l e a l CCK c e l l number was unchanged i n both l e a n and obese r a t s . In the continuous jejunum of obese r a t s the number of CCK-c o n t a i n i n g c e l l s i n c r e a s e d 30% while i n l e a n animals the number was unchanged. CCK c e l l number i n c r e a s e d 30% i n the j e j u n a l p o r t i o n of the bypassed loop i n both l e a n and obese 273 JIB r a t s (Fig 40). C h o l e c y s t o k i n i n has been proposed as an i n t e s t i n a l mediator of s a t i e t y (Smith and Gibbs, 1984). Decreased s e n s i t i v i t y to CCK i n obese Zucker r a t s has been hypothesized to c o n t r i b u t e to hyperphagia i n these animals (McLaughlin and B a i l e , 1980). T h e r e f o r e , changes i n the s e c r e t o r y c a p a c i t y of CCK may a f f e c t f e e d i n g behaviour i n obese Zucker r a t s f o l l o w i n g bypass surgery. While c i r c u l a t i n g l e v e l s of CCK were not measured i n the c u r r e n t study, i n c r e a s e s i n the number of endocrine c e l l s c o n t a i n i n g immunoreactive-CCK suggest t h a t e l e v a t i o n of plasma CCK i s p o s s i b l e and may c o n t r i b u t e to the anorexic e f f e c t s of JIB. However, i n a study of obese human p a t i e n t s t r e a t e d with JIB the authors r e p o r t e d no d i f f e r e n c e s i n c i r c u l a t i n g CCK l e v e l s p r e - or p o s t - o p e r a t i v e l y , or compared to non-obese c o n t r o l s (Burhol et a l , 1984). I t was concluded from these r e s u l t s t hat CCK i s not important as a s a t i e t y s i g n a l i n obese humans. However, t a r g e t - s i t e s e n s i t i v i t y may have been a l t e r e d i n o b e s i t y , so t h a t plasma l e v e l s of CCK d i d not r e f l e c t f u n c t i o n a l a c t i v i t y . C h o l e c y s t o k i n i n - c e l l p o p u l a t i o n was not determined. The s i g n i f i c a n c e of a l t e r a t i o n s i n CCK c e l l number observed i n the c u r r e n t i n v e s t i g a t i o n s thus awaits f u r t h e r s t u d i e s i n v o l v i n g measurement of plasma l e v e l s of CCK and t a r g e t - o r g a n s e n s i t i v i t y i n l e a n and obese r a t s . Neurotensin has been a s c r i b e d a f u n c t i o n i n the r e g u l a t i o n of i n s u l i n s e c r e t i o n and was found to i n c r e a s e b a s a l h y p e r i n s u l i n e m i a i n ob/ob mice ( B a i l e y et a l , 1984). 274 Normal or i n c r e a s e d n e u r o t e n s i n l e v e l s f o l l o w i n g bypass surgery i n obese r a t s may h e l p m a i n t a i n f a s t i n g h y p e r i n s u l i n e m i a . In the c u r r e n t study, no changes i n n e u r o t e n s i n c e l l number were noted i n the ileum remaining i n c o n t i n u i t y of l e a n or obese JIB r a t s (Fig 41) . Plasma l e v e l s of t h i s hormone were not measured. In a study of obese humans, f a s t i n g n e u r o t e n s i n plasma l e v e l s were s i g n i f i c a n t l y e l e v a t e d i n obese compared to normal i n d i v i d u a l s . F o l l o w i n g j e j u n o i l e a l bypass, b a s a l l e v e l s d e c l i n e d to normal c o n t r o l v a l u e s . However, n e u r o t e n s i n r e l e a s e f o l l o w i n g i n g e s t i o n of a meal was markedly i n c r e a s e d , presumably due to i n c r e a s e d d e l i v e r y of n u t r i e n t s to the d i s t a l s m a l l bowel (Sarson, Scopinaro and Bloom, 1981). Due to the r e p o r t e d i n h i b i t o r y e f f e c t of n e u r o t e n s i n on gut m o t i l i t y (Andersson et a l , 1977) i t was proposed t h a t i n c r e a s e d n e u r o t e n s i n r e l e a s e may c o n t r i b u t e t o r e d u c t i o n of food i n t a k e by d e l a y i n g g a s t r i c emptying and c r e a t i n g the s e n s a t i o n of s a t i e t y (Sarson, Scopinaro and Bloom, 1981). C o n f i r m a t i o n of these s t u d i e s i n the obese Zucker r a t await measurement of plasma n e u r o t e n s i n l e v e l s . GIP c e l l number was unchanged a f t e r bypass i n l e a n and obese r a t s except i n the j e j u n a l segment of the bypassed loop i n obese r a t s , where c e l l number was more than doubled compared to c o n t r o l s . However, as e a r l i e r d i s c u s s e d , there was no s i g n i f i c a n t a l t e r a t i o n i n c i r c u l a t i n g l e v e l s of t h i s hormone. These r e s u l t s d i f f e r from those p r e v i o u s l y r e p o r t e d (Pederson et a l , 1982; Buchan et a l , 1983). These 275 authors observed an i n c r e a s e i n f a s t i n g GIP l e v e l s d e s p i t e a blunted response to glucose i n normal Wistar r a t s t r e a t e d with JIB. The number of GIP c e l l s was i n c r e a s e d i n the bypassed loop of these animals and were a p p a r e n t l y s e c r e t i n g hormone, d e s p i t e the absence of l u m i n a l s t i m u l a t i o n , s i n c e r e s e c t i o n of the bypassed loop r e s u l t e d i n n o r m a l i z a t i o n of c i r c u l a t i n g GIP l e v e l s . However, r e s u l t s of other s t u d i e s i n normal Wistar r a t s (Koopmans e t a l , 1984) and obese humans (Sarson, Scopinaro and Bloom, 1981) i n d i c a t e d no change i n f a s t i n g GIP l e v e l s compared to unoperated c o n t r o l s of comparable weight. The decrease i n GIP r e l e a s e s t i m u l a t e d by glucose or a meal observed i n t h i s study and by others (Klapdor, 1982; Sarson, Scopinaro and Bloom, 1981; Koopmans et a l , 1984; Pederson et a l , 1982) c o u l d be accounted f o r by the e x c l u s i o n of a l a r g e number of GIP-producing c e l l s from l u m i n a l s t i m u l a t i o n by n u t r i e n t s . S ince GIP i s a potent s t i m u l a n t of i n s u l i n r e l e a s e i n the presence of hyperglycemia, a decrease i n the amount of g l u c o s e - or meal-stimulated GIP r e l e a s e would be expected to r e s u l t i n d i m i n u t i o n of the i n s u l i n response to these s t i m u l i . In the c u r r e n t i n v e s t i g a t i o n , GIP s e c r e t i o n i n response to glucose was depressed by 60% i n both l e a n and obese r a t s with JIB, but i n s u l i n s e c r e t i o n was decreased only i n obese JIB r a t s . These r e s u l t s i n d i c a t e t h a t other f a c t o r s , p a r t i c u l a r l y genotype c o n t r i b u t e to the f i n a l outcome of i n s u l i n s e c r e t i o n . 276 The s u r g i c a l p r e p a r a t i o n employed i n these s t u d i e s , with the b l i n d loop e x t e r i o r i z e d to the body w a l l v i a the jejunostomy/ileostomy, f a c i l i t a t e d a study i n which the e f f e c t of i n t e r m i t t e n t l u m i n a l s t i m u l a t i o n of endocrine c e l l s i n the bypassed gut on the outcome of j e j u n o i l e a l bypass c o u l d be examined, compared to JIB animals whose bypassed loops were not p e r f u s e d ( d i s c u s s e d above) and unoperated c o n t r o l s . The parameters measured i n t h i s study i n c l u d e d weight l o s s , weekly f a s t i n g plasma glucose l e v e l s , o r a l glucose t o l e r a n c e at 5 weeks and the s e n s i t i v i t y of the i n v i t r o pancreas to s t i m u l a t i o n by glucose and GIP. Animals t r e a t e d with d a i l y n u t r i e n t i n f u s i o n were r e f e r r e d to as JIB-L. In these s t u d i e s i t was necessary to demonstrate t h a t i n t r a j e j u n a l l y - a d m i n i s t e r e d n u t r i e n t was absorbed and c o u l d r e s u l t i n changes i n plasma hormone l e v e l s . I n t r a j e j u n a l i n f u s i o n of a s m a l l volume of g l u c o s e f o l l o w i n g an o v e r n i g h t f a s t r e s u l t e d i n an i n c r e a s e i n plasma glucose l e v e l s i n both l e a n and obese r a t s (Fig 47 A) that i n d i c a t e d glucose a b s o r p t i o n was o c c u r r i n g i n the bypassed lo o p . Moreover, s i g n i f i c a n t r e l e a s e of GIP was a l s o observed i n both l e a n and obese r a t s ( F i g 47 C ) , a demonstration t h a t bypassed GIP c e l l s were f u n c t i o n a l l y i n t a c t . Plasma i n s u l i n l e v e l s were not s i g n i f i c a n t l y i n c r e a s e d i n l e a n JIB-L r a t s d e s p i t e the i n c r e a s e i n glucose and GIP. However, maximal glucose c o n c e n t r a t i o n s reached were about 120 mg/dl, which has been shown to be the minimum t h r e s h o l d f o r the i n s u l i n o t r o p i c 277 a c t i v i t y of GIP (Pederson and Brown, 1976) . In obese JIB-L r a t s , i n s u l i n s e c r e t i o n doubled f o l l o w i n g i n t r a j e j u n a l glucose (Fig 47 B) . T h i s change may be a t t r i b u t e d to the s l i g h t l y higher peak glucose c o n c e n t r a t i o n reached i n obese animals (145 mg/dl) and the absence of a g lucose t h r e s h o l d f o r the i n s u l i n o t r o p i c a c t i v i t y of GIP i n these animals. Weight l o s s over the 5 week experimental p e r i o d was compared i n JIB-L and JIB r a t s (Table X) . Weight l o s s was not s i g n i f i c a n t l y d i f f e r e n t between the two groups i n e i t h e r l e a n or obese r a t s . Thus, the s m a l l amount of c a l o r i e s g iven i n t r a j e j u n a l l y had no e f f e c t on weight i n JIB-L r a t s and d i f f e r e n c e s i n the outcome of j e j u n o i l e a l bypass under the two regimens were t h e r e f o r e not r e l a t e d to changes i n weight g a i n . Although food i n t a k e was not measured, these data suggest that i n t r a j e j u n a l n u t r i e n t i n f u s i o n d i d not a f f e c t s a t i e t y . I n t r a j e j u n a l n u t r i e n t i n f u s i o n d i d have a profound e f f e c t on f a s t i n g plasma glucose l e v e l s (Fig 46) . One week f o l l o w i n g surgery, b a s a l g lucose l e v e l s of both l e a n and obese JIB-L r a t s were depressed compared to c o n t r o l s but were s t i l l w i t h i n the normal range (80 mg/dl). D a i l y n u t r i e n t i n f u s i o n was commenced at t h i s time. Two, three and four weeks f o l l o w i n g surgery, plasma glucose l e v e l s were s e v e r e l y depressed i n both l e a n and obese JIB-L r a t s (<50 mg/dl) but recovered to c o n t r o l l e v e l s by the 5th week a f t e r surgery. F a s t i n g g lucose l e v e l s of JIB r a t s not g i v e n d a i l y loop i n f u s i o n of n u t r i e n t s d i d not f l u c t u a t e s i g n i f i c a n t l y 278 ( F i g 32, A and B) . D e s p i t e severe hypoglycemia f o r three weeks of the t e s t , the animals remained a l e r t and h e a l t h y i n appearance. D a i l y i n f u s i o n of n u t r i e n t s i n t o the bypassed loop of these r a t s appeared to impair the a b i l i t y of these animals to m a i n t a i n normal f a s t i n g glucose l e v e l s . The cause of t h i s phenomenon was not r e a d i l y apparent because g l u c o r e g u l a t i o n i s a complex process i n v o l v i n g the i n t e g r a t i o n of many i n p u t s . S i n c e r e l e a s e of GIP by i n t r a l u m i n a l glucose was demonstrated i t i s p o s s i b l e t h a t the n u t r i e n t s o l u t i o n g i v e n (Ensure), which c o n s i s t e d of a mixture of carbohydrate, f a t and p r o t e i n , c o u l d have s t i m u l a t e d the r e l e a s e of many gut hormones, i n a d d i t i o n to e f f e c t s mediated v i a the e n t e r i c nervous system. Moreover, i t appears t h a t a d a p t a t i v e mechanisms e x i s t t h a t r e s t o r e d plasma glucose to normal l e v e l s by the 5th week of the experiment. O r a l glucose t o l e r a n c e t e s t s were performed 5 weeks f o l l o w i n g bypass surgery. Glucose l e v e l s a t t a i n e d were not d i f f e r e n t from c o n t r o l s i n e i t h e r l e a n ( F i g 48 A) or obese (Fig 49 A) JIB-L r a t s compared to sham-operated c o n t r o l s , nor were any changes noted i n the t o t a l i n t e g r a t e d response (Fig 50 A) . I n t e g r a t e d i n s u l i n s e c r e t i o n was markedly reduced i n both l e a n and obese JIB-L animals ( F i g 50 B) , although s i g n i f i c a n c e was not apparent on a p e r i o d - t o - p e r i o d b a s i s (Fig 48 B and 49 B) . Due to t e c h n i c a l d i f f i c u l t i e s , data on GIP s e c r e t i o n was u n a v a i l a b l e . 279 The s e n s i t i v i t y o f the pancreas to s t i m u l a t i o n by glucose (300 mg/dl) and GIP (0-1 ng/ml) was s t u d i e d i n v i t r o i n the p e r f u s e d pancreas of l e a n and obese JIB-L r a t s compared to c o n t r o l s ( F i g 51). I n s u l i n s e c r e t i o n was reduced by 60% i n l e a n JIB-L r a t s and 40% i n obese JIB-L r a t s compared to r e s p e c t i v e c o n t r o l s . P a n c r e a t i c s e n s i t i v i t y to both glucose and GIP s t i m u l a t i o n appeared to be impaired, s i n c e i n s u l i n s e c r e t i o n was decreased both below and above the GIP t h r e s h o l d f o r i n s u l i n o t r o p i c a c t i o n (250 pg/ml). The r e s u l t s of o r a l g lucose t o l e r a n c e t e s t s and i n v i t r o pancreas p e r f u s i o n experiments suggest that d a i l y n u t r i e n t i n f u s i o n i n t o the b l i n d l oop of animals t r e a t e d with j e j u n o i l e a l bypass surgery had important e f f e c t s on the outcome of such surgery, as r e l a t e d to the changes i n the e n t e r o i n s u l a r a x i s . The changes observed are summarized i n Table XV. Whereas JIB had no e f f e c t on e i t h e r plasma i n s u l i n or i n v i t r o i n s u l i n s e c r e t i o n i n l e a n r a t s , JIB-L r e s u l t e d i n r e d u c t i o n of both parameters. In obese animals plasma i n s u l i n was reduced by both procedures, but the i n v i t r o i n s u l i n response to glucose and GIP was decreased o n l y by JIB-L. I t i s unfortunate t h a t i n v i v o f a s t i n g and s t i m u l a t e d GIP l e v e l s were u n a v a i l a b l e from these experiments, s i n c e these data may have g i v e n important c l u e s about changes o c c u r r i n g i n the e n t e r o i n s u l a r a x i s i n JIB-L animals, e s p e c i a l l y i n l i g h t o f the o b s e r v a t i o n o f others that changes i n f a s t i n g GIP l e v e l s (Pederson e t a l , 1982; 280 Buchan et a l , 1983) or the d i u r n a l p a t t e r n of GIP r e l e a s e (Pederson et a l , 1985) can have important e f f e c t s on p a n c r e a t i c s e n s i t i v i t y to t h i s hormone. While j e j u n o i l e a l bypass i s a good model f o r the study of the e n t e r o i n s u l a r a x i s , changes t h a t occur i n i n s u l i n s e c r e t o r y l e v e l s may be complicated by the e f f e c t s of weight l o s s , s i n c e weight r e d u c t i o n by d i e t a r y means has been shown to reduce h y p e r i n s u l i n e m i a (Jeanrenaud, 1979). The f o l l o w i n g experiments were designed to compare changes i n the i n s u l i n s e c r e t o r y response f o l l o w i n g weight l o s s induced by f a s t i n g compared to the changes that o c c u r r e d f o l l o w i n g JIB, which a l s o caused s i g n i f i c a n t weight l o s s . Weight l o s s of the food-deprived animals i s shown i n F i g u r e 52. A c t u a l weight l o s s per day of l e a n vs obese r a t s was not s i g n i f i c a n t l y d i f f e r e n t , but when expressed as a percentage of the o r i g i n a l weight was found to be s i g n i f i c a n t l y g r e a t e r i n l e a n r a t s . Over 20 g was l o s t by both groups o v e r n i g h t but d e c l i n e d to approximately 5-10 g/day t h e r e a f t e r . Thus the t a r g e t weight r e d u c t i o n of 20%, designed to mimic the weight l o s s observed i n JIB r a t s (Table X) , was achieved by the 5th day of food d e p r i v a t i o n i n l e a n r a t s but r e q u i r e d 11 days i n obese animals. Once a weight l o s s of 20% had been achieved, the animals were giv e n an o r a l glucose t o l e r a n c e t e s t and t h e i r pancreata pe r f u s e d t o measure s e n s i t i v i t y t o s t i m u l a t i o n by glucose and GIP. R e s u l t s of the o r a l glucose t o l e r a n c e t e s t r e v e a l e d no change i n the glucose or GIP response to o r a l glucose 281 a d m i n i s t r a t i o n i n l e a n r a t s f a s t e d to 80% of t h e i r o r i g i n a l body weight compared to o v e r n i g h t s t a r v e d c o n t r o l s (Fig 53, A and C; F i g 55, A and C) . F a s t i n g i n s u l i n l e v e l s were unchanged, but r e l e a s e i n response to o r a l glucose was s e v e r e l y reduced (Fig 53 B and F i g 55 B) . In f a s t e d obese r a t s weighing 80% of t h e i r o r i g i n a l body weight, no d i f f e r e n c e s were noted i n g l u c o s e , i n s u l i n or GIP responses compared to o v e r n i g h t - f a s t e d c o n t r o l s (Figs 54 and 55). When the i s o l a t e d p a n c r e a t a of these animals were perfused with 300 mg/dl gl u c o s e i n the presence of a GIP g r a d i e n t of 0-1 ng/ml, i n s u l i n s e c r e t i o n of l e a n f a s t e d r a t s was about 20% that of l e a n c o n t r o l s , whereas there was no r e d u c t i o n i n the i n s u l i n response of f a s t e d obese r a t s to these s t i m u l i ( F i g 54) . From these experiments i t was concluded t h a t (1) i n food-deprived r a t s , plasma i n s u l i n l e v e l s a c c u r a t e l y p r e d i c t e d the s e c r e t o r y response of the p e r f u s e d pancreas of these animals. These r e s u l t s d i f f e r e d from those observed f o l l o w i n g j e j u n o i l e a l bypass, where plasma i n s u l i n l e v e l s f r e q u e n t l y had no c o r r e l a t i o n with the response of the pancreas to s t i m u l a t i o n by glucose and GIP (Table XV) . Thus, the a l t e r a t i o n s i n p a n c r e a t i c response observed a f t e r j e j u n o i l e a l bypass c o u l d not be s o l e l y a t t r i b u t a b l e to weight l o s s ; (2) whereas s t a r v e d l e a n r a t s had a s e v e r e l y impaired i n s u l i n response to glucose and GIP, 20% weight l o s s i n obese r a t s had no e f f e c t on i n s u l i n e m i a or i n v i t r o p a n c r e a t i c s e n s i t i v i t y . In obese Zucker r a t s , prolonged 282 f a s t i n g f o r up to 80 days r e s u l t e d i n r e d u c t i o n of plasma i n s u l i n and g lucose c o n c e n t r a t i o n s . I n s u l i n l e v e l s f e l l to non-detectable l e v e l s a f t e r a 2 day f a s t i n l e a n animals but were p e r s i s t e n t l y e l e v a t e d i n obese r a t s f o r up to 42 days of food d e p r i v a t i o n (Zucker and Antoniades, 1972). In normal r a t s s t a r v e d f o r 1-3 days, B - c e l l s e n s i t i v i t y to s t i m u l a t i o n by glucose p l u s amino a c i d s was impaired but s e n s i t i v i t y of B - c e l l s of obese r a t s a f t e r s t a r v a t i o n was not i n v e s t i g a t e d (Matschinsky et a l , 1980). The s t u d i e s d e s c r i b e d i n t h i s t h e s i s c o n f i r m these r e s u l t s i n l e a n r a t s . An absence of changes i n the i n v i v o or i n v i t r o responses of the pancreas to glucose i n obese Zucker r a t s may be r e l a t e d to the e x c e s s i v e c a p a c i t y of these animals f o r l i p i d m o b i l i z a t i o n , s i n c e obese r a t s 14 weeks of age had 3 - f o l d more body l i p i d than l e a n l i t t e r m a t e s (Zucker and Antoniades, 1972). 283 CHAPTER FOUR: INCRETINS IN PLASMA-CHARACTERIZATION BY IMMUNOREACTIVITY AND BIOACTIVITY I. INTRODUCTION An i n c r e t i n can be d e f i n e d as an i n s u l i n o t r o p i c e n t i t y r e l e a s e d from the i n t e s t i n a l mucosa i n t o the blood. To date, GIP i s the onl y w e l l - c h a r a c t e r i z e d i n c r e t i n . The presence o f i n c r e t i n s other than GIP i n plasma has not been demonstrated. The o b j e c t i v e of the f o l l o w i n g s t u d i e s was to compare the i n c r e t i n b i o a c t i v i t y o f plasma e x t r a c t s from r a t s with the GIP content of the e x t r a c t s measured by radioimmunoassay. From these data, the presence o f other i n s u l i n o t r o p i c hormones i n plasma c o u l d be determined. Some evidence suggests that other i n c r e t i n s may e x i s t (Ebert, I l l m e r and C r e u t z f e l d t , 1979; E b e r t , Unger and C r e u t z f e l d t , 1983) but these have not been i s o l a t e d and p u r i f i e d , so the i d e n t i t y o f these p u t a t i v e i n s u l i n o t r o p i c hormones i s unknown. In a d d i t i o n , i n s t a t e s of p a t h o l o g i c a l glucose t o l e r a n c e t hat are re p o r t e d to occur i n normal aging Wistar (Narimiya et a l , 1984) and obese r a t s (Fig 10), the IR-GIP s e c r e t e d may have abnormal b i o l o g i c a l a c t i v i t y which a f f e c t s i n s u l i n s e c r e t i o n . The o b j e c t i v e o f the f o l l o w i n g s t u d i e s was t o compare b i o a s s a y a b l e i n c r e t i n with radioimmunoassayable GIP i n order to (1) examine evidence f o r i n s u l i n o t r o p i c hormones other than GIP i n plasma and (2) 284 assess p o s s i b l e a l t e r a t i o n s i n the b i o l o g i c a l a c t i v i t y of GIP i n aging i n Wistar r a t s and o b e s i t y i n Zucker r a t s . 285 I I . METHODS A. Immunoadsorbent A f f i n i t y Chromatography 1. R a t i o n a l e The p r i n c i p l e of a f f i n i t y chromatography i n v o l v e s the presence of a s p e c i f i c l i g a n d coupled to an i n e r t m atrix, to which s p e c i f i c substances can be r e v e r s i b l y bound and, hence, separated from a heterogeneous m i l i e u . In the case of immunoadsorbent a f f i n i t y chromatography a s p e c i f i c antibody i s c o v a l e n t l y l i n k e d to the i n e r t matrix so that s p e c i f i c a n t i g e n can be p r e f e r e n t i a l l y removed from crude e x t r a c t s . S u c c e s s f u l immunoadsorbent chromatography i s dependent on three important f a c t o r s : 1) Chemical i n e r t n e s s of the column ma t r i x . T h i s prevents n o n - s p e c i f i c b i n d i n g of the a n t i g e n of other sample components. 2) S p e c i f i c i t y of antibody f o r the a n t i g e n . 3) R e v e r s i b l e b i n d i n g of a n t i g e n to the antibody. 2. I n s u l i n a. Antibody Guinea p i g a n t i - i n s u l i n immunoglobulin was r a i s e d according to the method of Makulu and Wright (1971). The inoculum c o n s i s t e d o f a w a t e r - i n - o i l emulsion of unconjugated bovine and p o r c i n e i n s u l i n (Connaught, Toronto) which prevented r a p i d a b s o r p t i o n of i n s u l i n and thus, hypoglycemia. A suspension of b a c t e r i a l adjuvant was added 286 to improve the immunogenicity of the emulsion. The composition of the emulsion i s p r e s e n t e d : O i l y phase - 10 ml m i n e r a l o i l (Nujol) heated and s t i r r e d with 5 ml l a n o l i n , then c o o l e d to room temperature. Aqueous phase - 3.75 ml (100 U/ml) b o v i n e / p o r c i n e i n s u l i n d i l u t e d with 6.25 ml 0.3% a c i d i f i e d phenol pH 2.3 added to 1 ml Hemophilus p e r t u s s i s d i l u t e d with 3.3 ml a c i d i f i e d phenol. The guinea p i g s (700 g) were i n o c u l a t e d subcutaneously with 2 ml inoculum at s e v e r a l s i t e s on the abdomen f o r 3 c o n s e c u t i v e days. A 2 ml booster dose was g i v e n at 21 days from the i n i t i a l i n o c u l a t i o n date, then the animals were bl e d (5 ml) by c a r d i a c puncture 40 days from the i n i t i a l i n o c u l a t i o n date. The animals continued to be b l e d and boosted every 40 days f o r a 1 yr p e r i o d . A f t e r 1 y r the serum of a guinea p i g with an antibody t i t r e o f 2.5 x 10 i n the i n s u l i n RIA (Chapter One, S e c t i o n IIA) was chosen f o r use i n a f f i n i t y chromatography. b. P r e p a r a t i o n of column i . G e l matrix The g e l chosen f o r the column matrix was Sepharose CL-4B (Pharmacia), an agarose g e l with an e x c l u s i o n l i m i t of 20x10^. The beads were washed with 0.05 M sodium borate b u f f e r pH 8.0 (38.1 g Na 2B 40 7'10H 2O, 0.65 g NaN_ up to 2 1, pH a d j u s t e d to 8.0 with 5M HCl) over 287 s u c t i o n , then added to an equal volume of d i s t i l l e d water and s t i r r e d on i c e . i i . C o upling r e a c t i o n The c o u p l i n g r e a c t i o n was c a r r i e d out i n two st e p s . F i r s t l y , the Sepharose g e l was a c t i v a t e d by d e r i v a t i z a t i o n o f hyd r o x y l groups on agarose sugars. Cyanogen bromide (CNBr) was d i s s o l v e d i n dimethylformamide (250 mg/ml) and added dropwise to the agarose beads to a f i n a l c o n c e n t r a t i o n of 16 mg/ml. The pH of the mixture was maintained betweeen 10.5-11.0 f o r 15 min with 4 M NaOH. The a c t i v a t e d beads were washed on a g l a s s f u n n e l with 250 ml i c e water f o l l o w e d by 500 ml i c e d 0.05 M borate b u f f e r pH 8.0. In the second stage of the c o u p l i n g r e a c t i o n the a c t i v a t e d beads were added to the i n s u l i n antiserum d i l u t e d with borate b u f f e r so th a t the absorbance at 280 nm was 1.6 (approximately 1 mg/ml). The beads were g e n t l y rocked o v e r n i g h t . During t h i s stage i s o - u r e a bonds are formed between a c t i v a t e d h y d r o x y l groups and the immunoglobulin l i g a n d . The excess b i n d i n g s i t e s were i n a c t i v a t e d by i n c u b a t i o n with 25 ml 0.16 M ethanolamine f o r 2 h at 4°C. The column was s t o r e d i n 10 mM T r i s b u f f e r , pH 8.0 at 4°C. c. E l u t i o n b u f f e r s T r i s - H C l b u f f e r , pH 7.5 was prepared as a lOx concentrated stock s o l u t i o n (41.3 ml conc e n t r a t e d HC1, 77.5 288 g T r i s and d i s t i l l e d water to 2 1 ) , and d i l u t e d with d i s t i l l e d water p r i o r to use. Sodium a z i d e (0.01%) was added t o the b u f f e r f o r column s t o r a g e . S p e c i f i c l i g a n d was e l u t e d from the column using 0.1 M HC1. d. D e t e c t i o n of e l u t e d m a t e r i a l M a t e r i a l e l u t e d from the column was de t e c t e d by measurement of the absorbance of each f r a c t i o n at 280 nm. 3. Rat Plasma P r e p a r a t i o n Six-month-old r a t s were a n e s t h e t i z e d (60 mg/kg p e n t o b a r b i t a l ) and b l e d v i a a c a r o t i d a r t e r i a l cannula 20 min a f t e r a d m i n i s t r a t i o n of 3 g/kg o r a l glucose by s y r i n g e and f e e d i n g tube. The blood was pooled and c e n t r i f u g e d at 12,000 rpm, 4°C f o r 15 min. The r e s u l t a n t plasma was heated i n a b o i l i n g water bath f o r 6 min to denature p r o t e o l y t i c enzymes, c o o l e d on i c e , and c e n t r i f u g e d at 12,000 rpm, 4°C f o r 1 h. The de p r o t e i n a t e d plasma was then a p p l i e d t o the i n s u l i n immunoadsorbent column ( S e c t i o n IIB2) and e l u t e d with 0.032 M T r i s HC1, pH 7.5 at a flow r a t e o f 2.5 ml/min. Plasma components bound to the i n s u l i n immunoadsorbent column were e l u t e d with 0.1 M HC1 and d i s c a r d e d . T r i s HC1 f r a c t i o n s were pooled, c o n c e n t r a t e d by e v a p o r a t i o n and s t o r e d at 4°C. 289 4. Bioassay The i s o l a t e d p e r f u s e d r a t pancreas p r e p a r a t i o n (General Methodology, S e c t i o n IIA) was used to assay the i n c r e t i n a c t i v i t y of plasma e x t r a c t s from r a t s . T h i s p r e p a r a t i o n has been found t o be s e n s i t i v e to pure p o r c i n e GIP c o n c e n t r a t i o n s as low as 250-300 pg/ml (Pederson et a l , 1982) . Two v a r i a t i o n s o f the bi o a s s a y were employed. In some experiments the plasma e x t r a c t was presented t o the pancreas as a g r a d i e n t s t a r t i n g at 0 i n c r e t i n a c t i v i t y and proceeding to a maximum i n c r e t i n c o n c e n t r a t i o n a t the end of the 45 min experiment. In a second group of experiments the pancreas r e c e i v e d a constant background s t i m u l a t i o n o f 300 mg/dl glucose and a constant i n f u s i o n of i n c r e t i n between 16-23 min of the 35 min p e r f u s i o n experiment. 290 I I I . RESULTS A. P r e p a r a t i o n of the I n s u l i n Immunoadsorbent Column 1. C h a r a c t e r i z a t i o n of I n s u l i n Antibody (Fig 57) The antibody chosen f o r use i n a f f i n i t y chromatography was GP7. One year a f t e r i n s u l i n immunization commenced, the t i t r e o f GP7 antiserum was determined to be 1:250,000; i e . a 1:250,000 f i n a l d i l u t i o n of the serum gave 50% zero b i n d i n g i n a radioimmunoassay (Fig 57 A) . The antiserum was a l s o t e s t e d i n a standard curve from 0-160 juU 125 i n s u l i n / m l to measure the displacement of I - i n s u l i n by c o l d p e p t i d e (Fig 57 B) . In t h i s t e s t z e r o - b i n d i n g at an antibody d i l u t i o n of 1:400,000 was 26.4% and b i n d i n g at 160 pU/ml was 14.7%, a displacement of 12%. 2. C a p a c i t y of the I n s u l i n Immunoadsorbent Column  (Figs 58 and 59, Table XVI) F o l l o w i n g c o u p l i n g of GP7 antiserum to the Sepharose 4B-CL beads, the b i n d i n g c a p a c i t y of the column was assessed. The borate supernatant was reserved a f t e r r o c k i n g the beads o v e r n i g h t and c e n t r i f u g a t i o n ( S e c t i o n I I A 2 b i i ) . The supernatant was d i l u t e d i n a range from 3 4 1:25x10 to 1:40x10 and z e r o - b i n d i n g measured f o r each d i l u t i o n . The p r e - c o u p l i n g antibody s o l u t i o n was a l s o d i l u t e d and assayed. The r e s u l t s are presented i n F i g 57. 3 At a d i l u t i o n of 25 x 10 p o s t - c o u p l i n g antibody bound 40% 125 of the 10,000 cpm I - I n s u l i n added. E q u i v a l e n t b i n d i n g of 291 the p r e - c o u p l i n g antiserum was 2 x 10 , i n d i c a t i n g t h a t about 80% of the antibody had been bound to the Sepharose beads. To assess the c a p a c i t y of the column to bi n d i n s u l i n , pure p o r c i n e i n s u l i n was added to c h a r c o a l - e x t r a c t e d plasma at a c o n c e n t r a t i o n of 400 ug/ml and s e r i a l l y d i l u t e d t o 203 pg/ml, 101 pg/ml, 51 pg/ml and 25 pg/ml with 0.032 M T r i s -HCl, pH 7.5. The i n s u l i n immunoadsorbent column was e q u i l i b r a t e d with 0.032 M T r i s - H C l , pH 7.5 and each of the i n s u l i n d i l u t i o n s was a p p l i e d to the column at a flow r a t e of 2.5 ml/min and e l u t e d with e q u i l i b r a t i n g b u f f e r , f o l l o w e d by a d i s t i l l e d water wash and then the bound m a t e r i a l was e l u t e d with 0.1 M HC1. The p r o t e i n content of each f r a c t i o n was measured s p e c t r o p h o m e t r i c a l l y at 280 nm and the i n s u l i n c o n c e n t r a t i o n of f r a c t i o n s a c r o s s the T r i s - H C l p r o t e i n peak, the water wash and the pooled HC1 p r o t e i n peak was determined by RIA. Rep r e s e n t a t i v e r e s u l t s of the e l u t i o n p r o f i l e and i n s u l i n c o n c e n t r a t i o n a c r o s s the peaks are shown i n F i g 59. I n s u l i n c o n c e n t r a t i o n s i n the T r i s - H C l e l u a n t and d i s t i l l e d water wash were very low (<1 m U / f r a c t i o n ) . The pooled HC1 f r a c t i o n had a t o t a l i n s u l i n content of 390 mil (15 pg) , which corresponded to 62% of the o r i g i n a l amount of i n s u l i n a p p l i e d to the column. Data f o r a l l i n s u l i n c o n c e n t r a t i o n s a p p l i e d are shown i n Table XVI. 292 F i g 57. C h a r a c t e r i z a t i o n o f i n s u l i n a n t i s e r a GP7 f o r use i n immunoadsorbent chromatography. A. Measurement of antibody t i t r e 125 B. Displacement of I - I n s u l i n by c o l d p e p t i d e i n a standard curve. 29 3 294 F i g 58. Measurement of antibody t i t r e of GP7 before and a f t e r c o u p l i n g to Sepharose 4B-CL with cyanogen bromide. 295 296 F i g 59. Tes t o f the i n s u l i n immunoadsorbent column using pure p o r c i n e i n s u l i n i n c h a r c o a l e x t r a c t e d plasma at a c o n c e n t r a t i o n of 25 pU/ml showing the e l u t i o n p r o f i l e of the p r o t e i n from the column and d e t e c t a b l e I R - I n s u l i n i n the T r i s - H C l , d i s t i l l e d water and HC1 f r a c t i o n s . 297 0/332M Tris-HCl pH 7.5 Distilled Water 0.1 M HCI FRACTION NUMBER 298 TABLE XVI. INSULIN BINDING TO AN INSULIN IMMUNOADSORBENT COLUMN INSULIN APPLIED (mU) IR-INSULIN TRIS-HC1 and WATER DETECTABLE ELUANTS (mU) HCI ELUANT 625 < l / f r a c t i o n 390 1275 < l / f r a c t i o n 580 2525 < l / f r a c t i o n 1410 5075 < l / f r a c t i o n 2080 299 C. Measurement of I n c r e t i n A c t i v i t y i n Rat Plasma  E x t r a c t s 1. C o n t r o l s a. The e f f e c t o f i n c r e a s i n g p r o t e i n on p e r f u s a t e  o s m o l a r i t y and i n s u l i n output from the i n v i t r o  pancreas (Table XVII) To assess the e f f e c t t h a t adding co n c e n t r a t e d r a t serum albumin present i n plasma e x t r a c t s to p e r f u s a t e might have on the i n s u l i n response of the p e r f u s e d pancreas, bi o a s s a y , the BSA c o n c e n t r a t i o n of p e r f u s a t e was i n c r e a s e d i n a g r a d i e n t from 0-1%. O s m o l a r i t y of the p e r f u s a t e i n c r e a s e d from 273 mOs/1 to 277 mOs over t h i s range. A d d i t i o n o f r a t plasma d e r i v e d from 35 ml plasma to p e r f u s a t e c o n t a i n i n g no BSA r e s u l t e d i n an o s m o l a r i t y of 270 mOs/1 (Table XVII). The o s m o l a r i t y of normal r a t plasma i s co n s i d e r e d to be 270 mOs/1. The i s o l a t e d pancreas b i o a s s a y p r e p a r a t i o n was p e r f u s e d with p e r f u s a t e c o n t a i n i n g 300 mg/dl gl u c o s e , 0-1 ng/ml GIP and a g r a d i e n t of 0.2 - 1% BSA. As a c o n t r o l , another pancreas was p e r f u s e d with 300 mg/dl gl u c o s e , 0-1 ng/ml GIP and 0.2% BSA, the BSA c o n c e n t r a t i o n r o u t i n e l y used i n p e r f u s i o n experiments. The t o t a l i n s u l i n output of the pancreas was 268.3 mU f o r the c o n t r o l and 238.3 mil f o r the BSA g r a d i e n t experiment, which was 89% of the c o n t r o l output (Table XVII). 300 b. The e f f e c t of T r i s - H C l b u f f e r on i n s u l i n output  from the i s o l a t e d p e r f u s e d pancreas  (Table XVIII) Because the plasma e x t r a c t p r e p a r a t i o n s were not d e s a l t e d , 50 ml T r i s - H C l b u f f e r , pH 7.5 was evaporated, then r e c o n s t i t u t e d i n p e r f u s a t e b u f f e r t o mimic the r e c o n s t i t u t i o n o f plasma e x t r a c t s e l u t e d from the i n s u l i n immunoadsorbent column with T r i s - H C l . The p e r f u s a t e a l s o c o n t a i n e d 300 mg/dl glucose and a GIP g r a d i e n t of 0-1 ng/ml. The i n s u l i n output of an i s o l a t e d pancreas p e r f u s e d with T r i s - H C l c o n t a i n i n g p e r f u s a t e was compared to a c o n t r o l experiment using p e r f u s a t e c o n t a i n i n g 300 mg/dl glucose p l u s 0-1 ng/ml GIP. The t o t a l i n s u l i n output of the c o n t r o l experiment was 151.7 mU compared to 152.7 mU f o r the T r i s -HCl experiment (Table X V I I ) . 301 TABLE XVII. THE EFFECT OF PERFUSATE OSMOLARITY ON INSULIN OUTPUT FROM THE ISOLATED, PERFUSED RAT PANCREAS PERFUSATE OSMOLARITY (mOs) INSULIN OUTPUT (mU) % OF CONTROL No BSA added 273 0.2% BSA 276 1.0% BSA 277 Lean plasma 270 e x t r a c t 268.3 238.3 88.8 Obese plasma 270 e x t r a c t 302 TABLE XVIII. THE EFFECT OF TRIS-HC1 ON INSULIN OUTPUT FROM THE ISOLATED, PERFUSED RAT PANCREAS PERFUSATE INSULIN OUTPUT(mU) % OF CONTROL C o n t r o l 151.7 100 T r i s - H C l 152.7 101 303 2. The E f f e c t of Age on the I n c r e t i n A c t i v i t y of  Wistar Rat Plasma a. O r a l glucose t o l e r a n c e (Fig 60, Table XIX) Wistar r a t s 2 months o l d (n=9) and 6 months o l d (n=9) , weighing 250 g and 375 g r e s p e c t i v e l y (Table XIX) , were g i v e n 1 g/kg o r a l g lucose a f t e r o b t a i n i n g a b a s a l blood sample. Glucose and IR-GIP l e v e l s were measured over a 1 h p e r i o d , whereas i n s u l i n was determined o n l y f o r the b a s a l sample. F a s t i n g glucose l e v e l s were s i g n i f i c a n t l y higher i n o l d (82+8.1 mg/dl) compared to younger r a t s (58+6.2 mg/dl, p<.05). There were no s i g n i f i c a n t d i f f e r e n c e s i n b a s a l i n s u l i n or GIP c o n c e n t r a t i o n s (Table XIX). Plasma glucose and GIP l e v e l s f o l l o w i n g a d m i n i s t r a t i o n of o r a l glucose were not d i f f e r e n t i n o l d compared to young r a t s , except f o r GIP l e v e l s a t 60 min ( F i g 60). b. In v i t r o i n s u l i n response of o l d and young  r a t s t o a glucose g r a d i e n t p l u s GIP (Fig 61) The i s o l a t e d p ancreata of o l d (n=9) and young (n=9) r a t s were per f u s e d with a g l u c o s e g r a d i e n t of 80-150 mg/dl i n the presence of 1 ng/ml GIP. While the i n s u l i n response of o l d e r r a t s tended to be higher than t h a t of younger r a t s , the d i f f e r e n c e between the two groups was not s i g n i f i c a n t . 304 c. I n c r e t i n a c t i v i t y of plasma from o l d and  young r a t s Blood was obtained from nine 6-month-old and nine 2-month-old r a t s f o l l o w i n g a d m i n i s t r a t i o n of 3 g/kg o r a l g l u cose, y i e l d i n g t o t a l plasma volumes o f 51 and 53 ml r e s p e c t i v e l y . The plasma was e x t r a c t e d and the i n s u l i n removed by immunoadsorbent chromatography. F o l l o w i n g e v a p o r a t i o n of the e l u t i o n b u f f e r the e x t r a c t s were d i s s o l v e d i n 5.5 ml s a l i n e and i n f u s e d i n t o a p e r f u s e d pancreas bioas s a y p r e p a r a t i o n between 16-23 min of a 30 min experiment. The i n s u l i n r e l e a s i n g a c t i v i t y of the e x t r a c t s was compared to standard doses of p o r c i n e GIP i n f u s e d at r a t e s c a l c u l a t e d to g i v e 0.25, 0.5, 0.75 and 1.0 ng/ml f i n a l c o n c e n t r a t i o n . The background p e r f u s a t e c o n t a i n e d 300 mg/dl g l u c o s e . A sample of the e x t r a c t i n f u s a t e was reserved f o r IR-GIP and I R - i n s u l i n d e t e r m i n a t i o n . i . Standard curve f o r known p o r c i n e GIP  c o n c e n t r a t i o n s ( F i g 62, Table XX) A standard curve of i n t e g r a t e d i n s u l i n output vs GIP dose was c o n s t r u c t e d . For each GIP dose, i n t e g r a t e d i n s u l i n response from min 16-30 was c a l c u l a t e d a c cording to the formula: I n t e g r a t e d i n s u l i n response = S t i m u l a t e d - B a s a l = T o t a l i n s u l i n (16-30) - T o t a l i n s u l i n (1-15). The curve was l i n e a r over the range t e s t e d ( F i g 62 B, Table XX) . 305 i i . Rat plasma e x t r a c t s ( F i g 62, Table XX) The i n f u s a t e was d i v i d e d e q u a l l y i n t o 3 f r a c t i o n s so t h a t experimental r e p l i c a t e s c o u l d be performed. The i n t e g r a t e d i n s u l i n output s t i m u l a t e d by young r a t plasma e x t r a c t was e q u i v a l e n t to a GIP dose l e s s than 250 pg/ml, the t h r e s h o l d c o n c e n t r a t i o n f o r the i n s u l i n o t r o p i c a c t i v i t y of GIP. T h i s was r e f l e c t e d i n the r e l e a s e p a t t e r n , which showed o n l y a t r a n s i e n t i n c r e a s e i n i n s u l i n output ( F i g 62 A) . Measurement of IR-GIP i n the o r i g i n a l i n f u s a t e gave 1800 pg/ml which corresponds to <250 pg/ml f i n a l c o n c e n t r a t i o n i n the p e r f u s a t e e n t e r i n g the pancreas. No I R - i n s u l i n was d e t e c t a b l e i n the plasma e x t r a c t d i s s o l v e d i n s a l i n e p r i o r to p e r f u s i o n of the pancreas. S i m i l a r l y , o l d e r r a t plasma s t i m u l a t e d an i n t e g r a t e d i n s u l i n output e q u i v a l e n t to l e s s than 250 pg/ml p o r c i n e GIP. The c o n c e n t r a t i o n of IR-GIP i n the o r i g i n a l i n f u s a t e was 1600 pg/ml, corresponding to 82 pg/ml f i n a l c o n c e n t r a t i o n (Table XX). 306 TABLE XIX. A COMPARISON OF FASTED YOUNG (2 MONTH) AND OLD (6 MONTH) WISTAR RATS AGE 2 MONTHS (n=9) 6 MONTHS (n=9) WEIGHT (g) 256.8 372.6 PLASMA GLUCOSE(mg/dl) 58 + 6.2 82 + 8.1* IR-INSULIN (pU/ml) 56.8 + 7.6 57.1 + 13.8 IR-GIP (pg/ml) 550 + 69.6 659 + 126 *p<.05 compared t o l e a n animals. 307 F i g 60. The plasma (A) glucose and (B) GIP responses t o 1 g/kg o r a l glucose i n 6-month-old (n=9) compared to 2-month-old (n=9) Wistar r a t s . • r e p r e s e n t s the a d m i n i s t r a t i o n of glucose at 0 minutes. *p<.05 using unpaired Student's t - t e s t , comparing o l d to young r a t s . 308 309 F i g 61. The i n s u l i n response of the i s o l a t e d p e r f u s e d pancreas of 6-month-old (n=9) compared to 2-month-old (n=9) Wistar r a t s to a glucose g r a d i e n t of 80-150 mg/dl i n the presence of 1 ng/ml GIP. No s i g n i f i c a n t d i f f e r e n c e was noted i n the i n s u l i n response of the two groups. 310 6 0 0 -M I N U T E S 311 F i g 62. The i n c r e t i n a c t i v i t y of plasma e x t r a c t s from 6-month-old compared to 2-month-old Wistar r a t s . A. The i n s u l i n response of the i s o l a t e d p e r f u s e d r a t pancreas bioassay to plasma e x t r a c t s from 6-month-old (n=3) to 2-month-old (n=3) r a t s . Plasma e x t r a c t s were i n f u s e d from min 16-23 i n the presence of a 300 mg/dl glucose background. B. The average i n t e g r a t e d response to plasma e x t r a c t s using data from F i g u r e 62A, compared to a standard curve of the i n s u l i n response to known amounts of p o r c i n e GIP d e l i v e r e d as d e s c r i b e d above (Table XX). 312 313 TABLE XX. DETERMINATION OF THE INCRETIN ACTIVITY OF RAT PLASMA EXTRACTS INSULIN OUTPUT (pU) TOTAL STIMULATED STANDARD CURVE GIP DOSE (ng/ml) 0.25 9161 3101 0.50 11056 5026 0.75 17730 9240 1.00 20605 11395 PLASMA EXTRACTS Young Wistar 7415 1650 Old Wistar 13536 2505 314 4. I n c r e t i n A c t i v i t y of Plasma E x t r a c t s from Lean and Obese Zucker Rats GIP s e c r e t i o n i n response to o r a l glucose was found to be e l e v a t e d i n o l d e r (6-8 month old) obese Zucker r a t s compared to t h e i r l e a n l i t t e r m a t e s (Fig 10 C) . These animals were a l s o glucose i n t o l e r a n t (Fig 10A). In these s t u d i e s the b i o l o g i c a l a c t i v i t y of GIP i n these o l d e r obese r a t s was i n v e s t i g a t e d . a. I n c r e t i n a c t i v i t y of plasma e x t r a c t s (Table XXI, F i g 63) Lean and obese Zucker r a t s were administered 3 g/kg o r a l g lucose, then a n e s t h e t i z e d and b l e d . In t o t a l , 23 l e a n and 23 obese r a t s were b l e d i n 3 batches, g i v i n g t o t a l plasma volumes of 168 and 148 ml r e s p e c t i v e l y . The mean plasma IR-GIP c o n c e n t r a t i o n was 617 pg/ml ( t o t a l : 104 ng) i n l e a n and 1460 pg/ml ( t o t a l : 216 ng) i n obese animals (Table XXI). Plasma from each batch was t r e a t e d s e p a r a t e l y and presented to the i s o l a t e d pancreas bioass a y i n g r a d i e n t form i n the presence of 300 mg/dl g l u c o s e . Measurement of IR-i n s u l i n i n the e x t r a c t d i s s o l v e d i n p e r f u s a t e r e v e a l e d t h a t no I R - i n s u l i n was d e t e c t a b l e . C o n t r o l s c o n s i s t e d of perfused pancreas experiments i n which e i t h e r 300 mg/dl glucose alone or i n the presence of a p o r c i n e GIP g r a d i e n t was i n f u s e d . To c a l c u l a t e the i n s u l i n o t r o p i c a c t i v i t y of the plasma e x t r a c t s , r e s u l t s were compared with t h a t of a known g r a d i e n t of p o r c i n e GIP. When i n s u l i n s e c r e t i o n i n response 315 to glucose p l u s the plasma e x t r a c t became c o n s i s t e n t l y higher than the i n s u l i n response to glucose alone, the " t h r e s h o l d " was reached and was c o n s i d e r e d e q u i v a l e n t to a c o n c e n t r a t i o n of 250 ng/ml GIP. S i n c e the t h r e s h o l d was not always e a s i l y a s c e r t a i n e d , maximum i n s u l i n output/min at 45 min was a l s o c o n s i d e r e d . The r e s u l t s of these experiments demonstrated t h a t the plasma e x t r a c t s from l e a n r a t s gave an i n s u l i n response e q u i v a l e n t to t h a t of 0-560 pg/ml, whereas obese r a t e x t r a c t r e s u l t e d i n an i n s u l i n response of approximately 0-800 pg/ml (Fig 63) . When the volume of the p e r f u s i o n experiment was accounted f o r , these data were c o n s i s t e n t with t o t a l GIP i n the o r i g i n a l plasma of 168 and 240 ng r e s p e c t i v e l y (Table XXI). T h e r e f o r e , when t o t a l b i o a s s a y a b l e i n c r e t i n i n plasma e x t r a c t s was compared to the t o t a l IR-GIP measured i n the o r i g i n a l plasma, i t was observed t h a t the plasma e x t r a c t s of l e a n r a t s e x h i b i t e d 60% g r e a t e r i n c r e t i n a c t i v i t y than c o u l d be accounted f o r by IR-GIP, whereas the plasma e x t r a c t s of obese r a t s e x h i b i t e d o n l y 10% g r e a t e r i n c r e t i n a c t i v i t y than c o u l d be accounted f o r by IR-GIP (Table XXI). 316 TABLE XXI. PLASMA EXTRACTS FROM LEAN AND OBESE ZUCKER RATS: BIOASSAYABLE INCRETIN vs RADIOIMMUNOASSAYABLE GIP (IR-GIP) GENOTYPE Fa/- f a / f a Plasma IR-GIP (pg/ml) 617 1460 Plasma volume (ml) 168 148 T o t a l IR-GIP (ng) 104 216 Bi o a s s a y a b l e I n c r e t i n (pg/ml) 560 800 Volume of bioa s s a y s (ml) 300 300 T o t a l b i o a s s a y a b l e i n c r e t i n (ng) 168 240 Bio a s s a y a b l e i n c r e t i n : I R - G I P 1.6 1.1 317 F i g 63. The i n c r e t i n a c t i v i t y o f g r a d i e n t s of plasma e x t r a c t s i n the presence of 300 mg/dl gl u c o s e from l e a n (n=3) and obese (n=3) Zucker r a t s compared to the i n s u l i n response t o a p o r c i n e GIP g r a d i e n t of 0-1 ng/ml p l u s 300 mg/dl gl u c o s e , or glucose alone. 318 0 10 2 0 3 0 4 0 MINUTES 319 IV. DISCUSSION Development of an immunoadsorbent column coupled with s p e c i f i c i n s u l i n a n t i b o d i e s allowed removal of endogenous i n s u l i n and the i n v e s t i g a t i o n of the p o s s i b i l i t y of the presence of i n c r e t i n s other than GIP i n the plasma of normal and obese r a t s . In a d d i t i o n , the b i o l o g i c a l a c t i v i t y of immunologically i n t a c t GIP i n obese r a t s c o u l d be compared to t h a t of l e a n c o n t r o l s . Because plasma c o n t a i n s endogenous i n s u l i n , an immunoadsorbent column was prepared with i n s u l i n a n t i b o d i e s . The i n s u l i n a n t i s e r a was r a i s e d a g a i n s t a mixture of bovine and p o r c i n e i n s u l i n i n guinea p i g s and a f t e r one year was found to have a t i t r e o f 1:250,000 (Pig 56 A ) . Displacement 125 of I - i n s u l i n from the antibody by c o l d i n s u l i n (0-160 pU/ml) was 10% (Fig 56 B) . E i g h t y percent of the antibody used was coupled to the Sepharose 4B-CL during the cyanogen bromide c o u p l i n g procedure ( F i g 57). I t was found t h a t t h i s column bound about 50% of pure p o r c i n e i n s u l i n a p p l i e d over a range of 0.6-5.0 U ( F i g 58 and Table XVI). However, at i n s u l i n c o n c e n t r a t i o n s t y p i c a l l y found i n plasma (<100 juU/ml) , the i n s u l i n immunoadsorbent column removed 100% of the i n s u l i n and was t h e r e f o r e s u i t a b l e f o r use i n p r e p a r a t i o n of plasma e x t r a c t s . Further c o n t r o l s t u d i e s were performed to ensure that the plasma e x t r a c t i o n procedure would not a f f e c t subsequent bioassay r e s u l t s . A d d i t i o n of plasma e x t r a c t s to p e r f u s a t e 320 c o n t a i n i n g zero BSA gave normal p e r f u s a t e o s m o l a r i t y . I t was a l s o demonstrated t h a t i n c r e a s i n g BSA c o n c e n t r a t i o n as high as 1% had no e f f e c t on the response of the p e r f u s e d pancreas b i o a s s a y to 300 mg/dl glucose p l u s 0-1 ng/ml GIP (Table XVII). I t was a l s o demonstrated t h a t the presence of T r i s - H C l s a l t s i n the p e r f u s a t e b u f f e r had no e f f e c t on subsequent b i o a s s a y r e s u l t s (Table X V I I I ) . In an i n i t i a l study, the i n c r e t i n a c t i v i t y of plasma e x t r a c t s from o l d (6 months) and young (2 months) Wistar r a t s was compared. The o l d Wistar r a t s were hyperglycemic i n the f a s t i n g s t a t e compared to younger animals, but were not glucose i n t o l e r a n t ( F i g 59 A, Table XIX). GIP s e c r e t i o n i n response to o r a l g lucose was s i m i l a r i n o l d vs young r a t s . Other s t u d i e s r e p o r t e d t h a t Wistar r a t s d i s p l a y e d a r e d u c t i o n i n i n s u l i n - s t i m u l a t e d glucose u t i l i z a t i o n t h a t arose between 6 weeks and 4 months of age and was probably secondary to the weight g a i n observed at t h i s time (Narimiya et a l , 1984) . The o l d r a t s used i n the c u r r e n t study weighed 50% more than the young animals (Table XIX). Older r a t s may a l s o be somewhat h y p e r i n s u l i n e m i c (Bracho-Romero and Reaven, 1977; Reaven and Reaven, 1981) although t h i s was not observed i n the f a s t i n g s t a t e i n t h i s study (Table XIX). In v i v o GIP l e v e l s were unchanged ( F i g 59 B) , as was the response of the i s o l a t e d pancreas of o l d vs young r a t s to s t i m u l a t i o n by glucose and GIP ( F i g 60). However, i t was p o s t u l a t e d t hat other i n c r e t i n s i n plasma might be i n v o l v e d 321 i n the i n c r e a s e i n plasma i n s u l i n l e v e l s r e p o r t e d by others (Bracho-Romera and Reaven, 1977; Reaven and Reaven, 1981). I t was observed that graded doses of p o r c i n e GIP produced a l i n e a r i n c r e a s e i n the i n s u l i n response i n the presence of a background g l u c o s e c o n c e n t r a t i o n of 300 mg/dl (Fig 61 B) . The i n s u l i n response of the p e r f u s e d pancreas bioassay to the plasma e x t r a c t s t r e a t e d on the i n s u l i n immunoadsorbent column of o l d and young Wistar r a t s ( F i g 61 A) was e q u i v a l e n t to GIP c o n c e n t r a t i o n s l e s s than the e s t a b l i s h e d t h r e s h o l d f o r i n s u l i n o t r o p i c a c t i v i t y of 250 pg/ml. While the plasma e x t r a c t from o l d r a t s appeared to have somewhat more i n c r e t i n a c t i v i t y than e x t r a c t s of young r a t plasma based on the i n t e g r a t e d i n s u l i n response, these data are not c o n c l u s i v e because IR-GIP c o n c e n t r a t i o n s were below the t h r e s h o l d f o r the i n s u l i n o t r o p i c a c t i o n of GIP. Although the b i o l o g i c a l a c t i v i t y measured was somewhat higher than the maximum radioimmunoassayable c o n c e n t r a t i o n of GIP, the presence of other i n c r e t i n s i n plasma cannot be concluded from these s t u d i e s . The sequence of r a t GIP i s not known. However, Moody (1984) has shown that human GIP d i f f e r s from the p o r c i n e at two amino a c i d p o s i t i o n s . I f r a t GIP a l s o d i s p l a y s a s l i g h t l y d i f f e r e n t amino a c i d sequence than p o r c i n e GIP, b i o a c t i v i t y o f the two s p e c i e s may not be i d e n t i c a l . I t would then be necessary to compare the i n c r e t i n a c t i v i t y of plasma e x t r a c t s from r a t s to the b i o a c t i v i t y o f pure r a t GIP. The c u r r e n t i n v e s t i g a t i o n d i d r e v e a l that i n s u l i n h y p e r s e c r e t i o n i n o l d e r r a t s i s not due 322 to i n c r e a s e d p a n c r e a t i c s e n s i t i v i t y to GIP, e l e v a t e d r e l e a s e of GIP, or the presence of other i n c r e t i n s i n plasma. Obese Zucker r a t s were found to become glucose i n t o l e r a n t compared to l e a n l i t t e r m a t e s by 6 months of age (Fig 10 A) . In a d d i t i o n , IR-GIP s e c r e t i o n i n both the f a s t i n g and g l u c o s e - s t i m u l a t e d s t a t e was e l e v a t e d compared to l e a n c o n t r o l s (Fig 10 C ) . H y p e r i n s u l i n e m i a was observed (Fig 10 B) . Plasma was c o l l e c t e d from l e a n and obese 6-month-old Zucker r a t s to determine whether the GIP s e c r e t e d from obese r a t s had normal b i o l o g i c a l a c t i v i t y , and to look f o r evidence of other i n c r e t i n s i n these r a t s . The r e s u l t s of these s t u d i e s are shown i n F i g 62 and Table XXI. Plasma e x t r a c t s of both l e a n and obese r a t s c o n t a i n e d s i g n i f i c a n t amounts of i n c r e t i n ( F i g 62) with e x t r a c t s from obese r a t s r e l e a s i n g about twice as much i n s u l i n as e x t r a c t s from l e a n animals. Most of the i n c r e a s e i n i n c r e t i n a c t i v i t y of obese r a t s c o u l d be accounted f o r by the observed e l e v a t i o n of GIP l e v e l s i n the plasma ( F i g 10 C, Table XXI). There was o n l y 10% g r e a t e r i n c r e t i n a c t i v i t y i n the plasma e x t r a c t s o f obese r a t s than c o u l d be accounted f o r by radioimmunoassayable GIP, whereas a 60% d i s c r e p a n c y was observed i n the b i o a s s a y vs radioimmunoassay data of l e a n r a t s . The s u b j e c t i v e nature of determining at what time the i n s u l i n response became e l e v a t e d compared to glucose c o n t r o l s p r e c l u d e s a c o n c l u s i v e i n t e r p r e t a t i o n of the data. I t can be s a i d t h a t p r e l i m i n a r y evidence suggests the e x i s t e n c e of an i n c r e t i n i n the plasma of l e a n r a t s t h a t i s 323 not IR-GIP. Secondly, i f obese Zucker r a t s do i n f a c t l a c k a second i n c r e t i n t h a t e x i s t s i n l e a n animals, t h i s may e x p l a i n the reduced e n t e r o i n s u l a r a x i s observed when r e s u l t s of o r a l vs intravenous glucose t o l e r a n c e t e s t s were compared (F i g 12) . The r e s u l t s of s t u d i e s on plasma e x t r a c t s of o l d and young Wistar r a t s and l e a n and obese Zucker r a t s demonstrated the f o l l o w i n g : (a) t h a t the p e r f u s e d pancreas bioassay was s e n s i t i v e to i n c r e t i n s i n plasma e x t r a c t s . However, r a t s are not a p r a c t i c a l source of blood; (b) t h a t there i s no evidence f o r s t r u c t u r a l changes i n GIP s e c r e t e d from obese Zucker r a t s t h a t c o u l d cause a l t e r a t i o n s i n i n s u l i n s e c r e t i o n , s i n c e b i o l o g i c a l a c t i v i t y corresponded w e l l with immunoreactivity. Even s l i g h t a l t e r a t i o n s i n the GIP molecule have been shown to r e s u l t i n reduced b i o l o g i c a l a c t i v i t y ( J S r n v a l l et a l , 1981; Brown et a l , 1981). No amino a c i d s u b s t i t u t i o n s have been r e p o r t e d that i n c r e a s e the i n s u l i n o t r o p i c a c t i v i t y of GIP. The c u r r e n t study d i d not r u l e out s t r u c t u r a l a l t e r a t i o n s i n GIP i n obese r a t s t h a t d i d not cause changes i n i n s u l i n s e c r e t i o n ; (c) t h a t , while evidence was not c o n c l u s i v e , i n c r e t i n s other than GIP may e x i s t i n the plasma of l e a n r a t s i n which i n c r e t i n r e l e a s e was s t i m u l a t e d by g l u c o s e , but not i n plasma of obese l i t t e r m a t e s , perhaps accounting f o r the reduced e f f e c t of o r a l compared to i ntravenous glucose on i n s u l i n s e c r e t i o n noted i n obese r a t s . E b e r t , I l l m e r and C r e u t z f e l d t (1979) p o s t u l a t e d the e x i s t e n c e of an i n s u l i n - r e l e a s i n g hormone, 324 the r e l e a s e of which was s t i m u l a t e d by a c i d i f i c a t i o n of the duodenum i n r a t s . The i n c r e t i n a c t i v i t y was not a b o l i s h e d by i n f u s i o n of s p e c i f i c GIP a n t i b o d i e s . Subsequently, i t was r e p o r t e d t h a t e x t r a c t s from the s m a l l bowel of r a t r e t a i n e d c o n s i d e r a b l e i n s u l i n o t r o p i c a c t i v i t y a f t e r removal of IR-GIP by immunoadsorbent chromatography (Ebert, Unger and C r e u t z f e l d t , 1983). However, 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 of t h i s o b s e r v a t i o n has y e t to be e s t a b l i s h e d , because i f the substance i s not r e l e a s e d from the gut i n t o the plasma by o r a l g l u c o s e then i t cannot be c l a s s i f i e d as an i n c r e t i n . In a d d i t i o n , these experiments employed the same GIP antibody on the immunoadsorbent column and i n the radioimmunoassay. Thus, r a t GIP t h a t may not have bound to the column a l s o would not be d e t e c t a b l e by RIA. I t c o u l d not, t h e r e f o r e , be concluded t h a t a l l the GIP had been removed from the gut e x t r a c t s . The s t u d i e s d e s c r i b e d i n t h i s t h e s i s p r o v i d e evidence t h a t other i n s u l i n o t r o p i c p e p t i d e s may indeed e x i s t i n plasma, at l e a s t i n l e a n Zucker r a t s . C o n f i r m a t i o n of these s t u d i e s would r e q u i r e removal of the GIP from the plasma and demonstration of i n s u l i n o t r o p i c a c t i v i t y of GIP-free e x t r a c t s . F u r t h e r s t r u c t u r a l s t u d i e s would be needed to c o n c l u s i v e l y show t h a t the i n c r e t i n was not GIP or a G I P - l i k e p e p t i d e . In summary, many t e c h n i c a l d i f f i c u l t i e s prevented the perfused pancreas b i o a s s a y from being a u s e f u l method of measuring i n c r e t i n l e v e l s i n the plasma of r a t s . Some of the r e s u l t s suggested the presence of i n c r e t i n s other than 325 GIP i n r a t plasma, but the sm a l l q u a n t i t y of i n c r e t i n present i n the plasma volumes a v a i l a b l e made f u r t h e r c h a r a c t e r i z a t i o n of p u t a t i v e i n c r e t i n s i m p o s s i b l e . Further experiments, perhaps employing r e f i n e d e x t r a c t i o n techniques, are r e q u i r e d . 326 SUMMARY AND CONCLUSIONS The s t u d i e s d e s c r i b e d i n t h i s t h e s i s were designed to i l l u m i n a t e v a r i o u s aspects of the e n t e r o i n s u l a r a x i s i n the development and maintenance of h y p e r i n s u l i n e m i a . H y p e r s e c r e t i o n of i n s u l i n i n response to n u t r i e n t s , p a r t i c u l a r l y g lucose and a r g i n i n e was observed i n obese Zucker r a t s , and confirmed p r e v i o u s r e p o r t s . E x c e s s i v e i n s u l i n r e l e a s e due to d i r e c t s t i m u l a t i o n by n u t r i e n t s i s probably due to the enlarged B - c e l l mass of p a n c r e a t i c i s l e t s observed here and by o t h e r s . The involvement of the n e u r a l component of the e n t e r o i n s u l a r a x i s was a l s o i n v e s t i g a t e d . No evidence was shown f o r h y p e r a c t i v i t y of the i n t r i n s i c c h o l i n e r g i c nervous system or vagus nerve. D i f f e r e n c e s between these and other p u b l i s h e d data may be r e l a t e d to the age of r a t used i n the two s t u d i e s , or other methodological c o n s i d e r a t i o n s . However, f u r t h e r evidence that the c h o l i n e r g i c nervous system was not h y p e r a c t i v e i n obese r a t s was shown by data which demonstrated t h a t g a s t r i n s e c r e t i o n from the stomach was under normal c h o l i n e r g i c c o n t r o l mechanisms. While obese Zucker r a t s were shown to be h y p e r g a s t r i n e m i c , t h i s c o n d i t i o n was probably due to the enlarged G - c e l l p o p u l a t i o n of the g a s t r i c antrum. The p h y s i o l o g i c s i g n i f i c a n c e of h y p e r g a s t r i n e m i a i n o b e s i t y remains unknown. Involvement of the i n s u l i n o t r o p i c hormone GIP i n the h y p e r i n s u l i n e m i a of Zucker f a t t y r a t s was s t u d i e d from 327 weaning to 5-8 months of age. Obese r a t s appeared to develop enlarged p a n c r e a t i c i s l e t s p r i o r to onset of h y p e r i n s u l i n e m i a , s u p p o r t i n g the hypothesis t h a t the p o t e n t i a l e x i s t s f o r i n s u l i n h y p e r s e c r e t i o n before i t s a c t u a l onset. Development of g l u c o s e - s t i m u l a t e d h y p e r i n s u l i n e m i a preceded onset of f a s t i n g h y p e r i n s u l i n e m i a i n v i v o , i n d i c a t i n g t h a t these phenomena were the r e s u l t o f separate metabolic events. G l u c o s e - s t i m u l a t e d h y p e r i n s u l i n e m i a i n v i v o c o u l d not be r e p l i c a t e d i n v i t r o , suggesting t h a t t h i s c o n d i t i o n was caused by i n d i r e c t e f f e c t s o f glucose on the pancreas. However, n e i t h e r a c e t y l c h o l i n e nor GIP were mediators of i n s u l i n h y p e r s e c r e t i o n i n v i v o i n the presence of hyperglycemia. In c o n t r a s t , f a s t i n g h y p e r i n s u l i n e m i a and a breakdown i n the glucose t h r e s h o l d f o r the i n s u l i n o t r o p i c a c t i o n of GIP arose concomitantly at 5 weeks of age and thus may be c a u s a l l y r e l a t e d . The me t a b o l i c events t h a t r e s u l t e d i n t h i s p a n c r e a t i c l e s i o n c o u l d not be determined. I t i s s p e c u l a t e d that l o s s o f the glucose-dependent nature of GIP-stimulated i n s u l i n r e l e a s e , causing f a s t i n g h y p e r i n s u l i n e m i a , c o u l d c o n t r i b u t e to the pronounced hyperphagia and weight g a i n r e p o r t e d t o a r i s e at t h i s time. The degree of h y p e r i n s u l i n e m i a progressed with age up to 3 months, but GIP l e v e l s were normal. Onset of hyperglycemia and glucose i n t o l e r a n c e by s i x months was c o r r e l a t e d with an i n c r e a s e i n plasma l e v e l s o f GIP. I t was p r e v i o u s l y hypothesized t h a t e l e v a t e d l e v e l s o f GIP i n o b e s i t y might r e f l e c t impaired 328 feedback by i n s u l i n on G I P - s e c r e t i n g c e l l s ( C r e u t z f e l d t et a l , 1978) . In t h i s study the obese p a t i e n t s with an e l e v a t e d GIP response tended to d i s p l a y p a t h o l o g i c a l glucose t o l e r a n c e . T h i s theory gained f u r t h e r support i n s t u d i e s which demonstrated t h a t exogenous i n s u l i n i n f u s i o n f a i l e d to suppress GIP r e l e a s e i n g l u c o s e - i n t o l e r a n t ob/ob mice ( F l a t t e t a l , 1983a). Thus, i n the e a r l y stages of o b e s i t y GIP may have important e f f e c t s on f a s t i n g i n s u l i n l e v e l s without s i g n i f i c a n t a l t e r a t i o n i n s e c r e t o r y l e v e l s . These e f f e c t s would be achieved by a l t e r a t i o n s of the response of the p a n c r e a t i c B - c e l l to ambient GIP l e v e l s , e i t h e r at a receptor or p o s t - r e c e p t o r s i t e . As the obese s t a t e p r o g r e s s e s , with i n c r e a s i n g i n s u l i n r e s i s t a n c e , hyperglycemia and hyperGIPemia become apparent. E l e v a t e d GIP s e c r e t i o n would be secondary to development of i n s u l i n r e s i s t a n c e at the l e v e l of the G I P - s e c r e t i n g c e l l . These events would r e i n f o r c e i n s u l i n h y p e r s e c r e t i o n and a s s i s t i n m a i n t a i n i n g the h y p e r i n s u l i n e m i c s t a t e . J e j u n o i l e a l bypass p r o v i d e s an experimental s i t u a t i o n i n which a l a r g e s e c t i o n of the s m a l l bowel i s excluded from l u m i n a l s t i m u l a t i o n . A novel s u r g i c a l method was developed i n an attempt to i n c r e a s e the s u r v i v a l r a t e o f obese r a t s . There have been many s t u d i e s of hormonal changes i n l e a n r a t s t r e a t e d with JIB, but evidence suggests that obese r a t s adapt d i f f e r e n t l y to the s u r g i c a l procedure than l e a n animals ( K i s s i l e f f et a l , 1979; S c l a f a n i et a l , 1978; Greenwood et a l , 1982). S t u d i e s d e s c r i b e d i n t h i s 329 t h e s i s confirmed t h a t weight l o s s p a t t e r n s d i f f e r between lea n and obese r a t s . In a d d i t i o n , a l t e r a t i o n s i n i n s u l i n , GIP and enteroglucagon plasma l e v e l s were re p o r t e d i n t h i s study. C o n t r a r y t o p r e v i o u s r e p o r t s i n Wistar r a t s (Pederson et a l , 1982; Buchan e t a l , 1983), f a s t i n g GIP l e v e l s were not e l e v a t e d , and there was no r e d u c t i o n i n p a n c r e a t i c s e n s i t i v i t y to glu c o s e and GIP e i t h e r i n l e a n or obese r a t s f o l l o w i n g J I B . I t was s p e c u l a t e d t h a t age or species d i f f e r e n c e s may account f o r d i s c r e p a n c i e s i n r e s u l t s . I t was a l s o concluded that the f a / f a genotype has important e f f e c t s on the adapt i v e changes to JIB, s i n c e many d i f f e r e n c e s were noted between l e a n and obese r a t s . However, these changes were not due s o l e l y t o d i f f e r e n c e s i n weight l o s s , s i n c e r e s u l t s of s t a r v a t i o n s t u d i e s i n l e a n and obese r a t s d i d not p a r a l l e l those obtained as a r e s u l t o f JIB. Some aspects may be accounted f o r by changes i n gut hormone s e c r e t o r y p a t t e r n s . Endocrine c e l l numbers, p a r t i c u l a r l y enteroglucagon and c h o l e c y s t o k i n i n , were in c r e a s e d by JIB i n obese r a t s and may e l i c i t changes i n weight l o s s , s a t i e t y , and i n s u l i n s e c r e t i o n . However, i n t e r p r e t a t i o n o f bypass data must be made with c a u t i o n , because sma l l a l t e r a t i o n s i n methods (eg. weekly o v e r n i g h t f a s t i n g ) c o u l d s i g n i f i c a n t l y a l t e r the r e s u l t s . I t was hypothesized t h a t the r e d u c t i o n i n f a s t i n g i n s u l i n l e v e l s observed a f t e r JIB i n obese r a t s might be due to a m e l i o r a t i o n o f the p a n c r e a t i c l e s i o n causing l o s s of the glucose t h r e s h o l d f o r the i n s u l i n o t r o p i c a c t i v i t y o f GIP. 330 However, t h i s was found not to be the case, as i n s u l i n s e c r e t i o n i n the presence of GIP was s t i l l s i g n i f i c a n t l y e l e v a t e d a f t e r JIB, even at f a s t i n g glucose l e v e l s . The cause of reduced b a s a l i n s u l i n c o n c e n t r a t i o n s i n obese r a t s was not e v i d e n t from these s t u d i e s , but d i d not appear to be due to weight l o s s , s i n c e plasma i n s u l i n l e v e l s of obese r a t s were unchanged a f t e r 20% weight l o s s by f a s t i n g . D a i l y n u t r i e n t i n f u s i o n i n t o the b l i n d loop of JIB-L animals was shown to be capable of s t i m u l a t i n g r e l e a s e of GIP. T h i s was accompanied by i n s u l i n r e l e a s e i f the glucose c o n c e n t r a t i o n was e l e v a t e d s u f f i c i e n t l y . D a i l y n u t r i e n t i n f u s i o n appeared to have important consequences on the adaptive response to JIB, s i n c e p a n c r e a t i c s e n s i t i v i t y to GIP was reduced by t h i s treatment. However, immunocytochemical data was u n a v a i l a b l e and so i t i s not known i f changes i n endocrine c e l l number can be c o r r e l a t e d with the a l t e r a t i o n s observed. The s u r g i c a l technique developed i n t h i s study p r o v i d e s a means of studying the e f f e c t s of continued l u m i n a l s t i m u l a t i o n of the bypassed loo p . Doubt about the p h y s i o l o g i c s t a t u s of the i n c r e t i n e f f e c t of GIP (Sarson et a l , 1984) and s t u d i e s which demonstrated continued p o t e n t i a t i o n of i n s u l i n r e l e a s e i n the absence of GIP (Ebert, I l l m e r and C r e u t z f e l d t , 1979; E b e r t , Unger and C r e u t z f e l d t , 1983) have spurred i n t e r e s t i n the search f o r new i n c r e t i n s . In t h i s i n v e s t i g a t i o n , a b i o a s s a y s e n s i t i v e to c i r c u l a t i n g l e v e l s of GIP was employed 331 to measure i n c r e t i n a c t i v i t y i n plasma of r a t s . In r a t s , measurable IR-GIP c o r r e l a t e d w e l l with b i o a s s a y a b l e i n c r e t i n i n plasma, but some evidence suggested other i n c r e t i n s may be present i n the plasma of l e a n but not obese Zucker r a t s . I t was hypothesized t h a t t h i s may account f o r the reduced r e l a t i v e i n c r e a s e i n the i n s u l i n response to o r a l vs intravenous glucose compared to l e a n r a t s . In c o n c l u s i o n , n u t r i e n t and hormonal elements of the e n t e r o i n s u l a r a x i s have a r o l e i n the development and maintenance of o b e s i t y i n Zucker r a t s . P r e l i m i n a r y i n v e s t i g a t i o n s i n d i c a t e d the presence of i n c r e t i n s other than GIP i n the plasma of r a t s . The importance o f these o b s e r v a t i o n s to o b e s i t y i n humans awaits f u r t h e r i n v e s t i g a t i o n . 332 REFERENCES CITED 1. Amland, P.F., Jorde, R., B u r h o l , P.G. and G i e r c k s k y , K.-E. (1984): S i m i l a r plasma GIP responses i n obese and l e a n s u b j e c t s a f t e r an o r a l t e s t meal and a f t e r i n t r a d u d e n a l s t i m u l a t i o n with f a t and g l u c o s e . I n t e r . J . 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