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The study of the chemical characterization of gastric inhibitory polypeptide (GIP) and the role of GIP… Kwauk, Sam Tsung-Ming 1982

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THE STUDY OF THE CHEMICAL CHARACTERIZATION OF GASTRIC INHIBITORY POLYPEPTIDE (GIP) AND THE ROLE OF GIP IN THE ENTEROINSULAR AXIS BY SAM TSUNG-MING KWAUK B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In THE FACULTY OF GRADUATE STUDIES THE DEPARTMENT OF PHYSIOLOGY We accept t h i s t h e s i s as conforming to the r e q u i r e d standard The U n i v e r s i t y of B r i t i s h Columbia 1 982 ( T ) SAM TSUNG-MING KWAUK In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) A b s t r a c t The dual o b j e c t i v e s of t h i s t h e s i s were to study the p h y s i o l o g i c a l r o l e of GIP i n the e n t e r o i n s u l a r a x i s and to c h e m i c a l l y c h a r a c t e r i z e a s i d e f r a c t i o n i d e n t i f i e d i n the p u r i f i c a t i o n of GIP. In the p h y s i o l o g i c a l s t u d i e s , the dependency of the i n s u l i n o t r o p i c a c t i o n of GIP on the p r e v a i l i n g s t a t e of glycaemia was confirmed in dogs using a system of steady s t a t e hyperglycaemia. GIP, from both exogenous and endogenous sources, was demonstrated to p o t e n t i a t e i n s u l i n r e l e a s e i n the presence of moderate hyperglycaemia. Both glucose and f a t a d m i n i s t e r e d e n t e r a l l y r e l e a s e d immunoreactive-GIP (IR-GIP) and p o t e n t i a t e d immunoreactive i n s u l i n (IRI) r e l e a s e during moderate hyperglycaemia (150 mg% above b a s a l ) . Intravenous a d m i n i s t r a t i o n of GIP at 2.0 nq/kq.h was a l s o capable of e l i c i t i n g i n s u l i n o t r o p i c a c t i o n d u r i n g moderate hyperglycaemia. A mixture of ten amino a c i d s was demonstrated to p o t e n t i a t e i n s u l i n r e l e a s e with m i l d hyperglycaemia (40 mg% above f a s t i n g ) r e g a r d l e s s of routes ( i n t r a v e n o u s , i n t r a d u o d e n a l and o r a l ) of a d m i n i s t r a t i o n . However, the r e l e a s e of IR-GIP was not demonstrated 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 the amino a c i d mixture. A r g i n i n e and a l a n i n e i n f u s e d i n d i v i d u a l l y d i d not p o t e n t i a t e i n s u l i n r e l e a s e in the presence of m i l d hyperglycaemia. Intraduodenal h y d r o c h l o r i c a c i d i n f u s i o n was a l s o demonstrated not to r e l e a s e IR-GIP i n the presence of m i l d hyperglycaemia. The i n t e r a c t i o n s of GIP with t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s and pyruvate were s t u d i e d i n euglycaemic c o n d i t i o n s i n dogs. Intravenous a d m i n i s t r a t i o n of i n d i v i d u a l m e t a b o l i t e s ( a - k e t o g l u t a r i c a c i d , s u c c i n a t e and pyruvate) on an equimolar b a s i s were shown not to be i n s u l i n o t r o p i c i n the presence or the absence of concurrent GIP i n f u s i o n (0.4 /xg/kg.h). The presence of a minor peptide component in the stage III GIP was i n i t i a l l y i d e n t i f i e d by t h i n l a y e r chromatography. Co n f i r m a t i o n of the presence of t h i s minor component was obtained from p o l y a c r y l a m i d e - u r e a g e l . These techniques were u n s u i t a b l e f o r p r e p a r a t i v e s e p a r a t i o n , as were c o n v e n t i o n a l g e l f i l t r a t i o n and ion exchange s e p a r a t i o n . Further p u r i f i c a t i o n of GIP on h i g h pressure l i q u i d chromatography i n d i c a t e d the presence of a 5% minor peptide component which was e v e n t u a l l y shown i v to c o n t a i n two l e s s amino a c i d r e s i d u e s ( t y r o s i n e and a l a n i n e ) than GIP. The amino a c i d sequence of GIP III i n d i c a t e d the presence of a peptide component with an amino a c i d sequence d i f f e r e n t from GIP i n that the f i r s t two amino a c i d r e s i d u e s of the N-terminal p o r t i o n of the molecule ( t y r o s i n e and a l a n i n e ) were m i s s i n g . The l a c k of i n h i b i t o r y a c t i v i t y to 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 s y n t h e t i c GIP l e d to a r e i n v e s t i g a t i o n of the amino, a c i d sequence of the molecule. The work i n c o l l a b o r a t i o n with J o r n v a l l (Sweden) i n d i c a t e d an e r r o r , i n that the o r i g i n a l sequence i n c l u d e d a second glutamine i n p o s i t i o n 30. V ACKNOWLEDGEMENTS I wish to thank my s u p e r v i s o r Dr. John Brown f o r h i s pa t i e n c e , guidance and f r i e n d s h i p d u r i n g my graduate s t u d i e s which made the l a s t f i v e years an experience to be remembered. I would a l s o l i k e to thank Mrs. S. Otte, Ms. J . F r o s t , Ms. D. Ching and Ms. C. Wang f o r rende r i n g t h e i r t e c h n i c a l e x p e r t i s e s . I would l i k e to express my a p p r e c i a t i o n to Drs. Mcintosh and Pederson f o r t h e i r advice i n regard to the t h e s i s . The work of high pressure l i q u i d chromatography and chemical m o d i f i c a t i o n of GIP by Dr. Mcintosh i s g r a t e f u l l y acknowledged. The t h i n l a y e r chromatography was c a r r i e d out by Mrs. S. Otte . The amino a c i d sequence work was c a r r i e d out by Dr. J o r n v a l l of Sweden. I would l i k e to thank v a r i o u s members of the l a b o r a t o r y throughout the l a s t f i v e years f o r g i v i n g me a s s i s t a n c e s , c o n s t r u c t i v e c r i t i c i s m s and r e f e r e n c e s to d i f f e r e n t techniques. The p r e p a r a t i o n of the f i g u r e s and photographs i n t h i s t h e s i s by Mr. K. Henze and Mr. R. Assinna are a p p r e c i a t e d . E x c e l l e n t animal care by Mr. Tay and Mr. Russel are acknowledged. F i n a l l y , not the l e a s t , I would thank the members of v i my f a m i l y f o r t h e i r understanding and encouragement to me d u r i n g my graduate s t u d i e s . F i n a n c i a l support from Canadian Medical Research C o u n c i l i s g r a t e f u l l y acknowledged. TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGEMENTS v LIST OF FIGURES x i v LIST OF TABLES x ix INTRODUCTION 1 METHODS 26 I. PHYSIOLOGICAL STUDIES ..26 A. . G a s t r i c A c i d I n h i b i t o r y S t u d i e s 27 1. S u r g i c a l Procedures 27 2. C o l l e c t i o n and A n a l y s i s of G a s t r i c A c i d 28 B. Glucose Homeostatic S t u d i e s 31 1. Glucose Clamp 31 a) I n t e r a c t i o n of Amino A c i d Mixture with Hyperglycaemia 37 b) I n t e r a c t i o n of E n t e r a l l y Administered N u t r i e n t s and Secretogogues with Hyperglycaemia 37 c) I n t e r a c t i o n of Exogenous GIP with Hyperglycaemia 40 2. I n t e r a c t i o n of GIP with Intermediates of T r i c a r b o x y l i c A c i d C y c l e and Pyruvate 40 v i i i C. Radioimmunoassay 43 1 . GIP 43 a) P r e p a r a t i o n of B u f f e r , L a b e l l e d GIP and A n t i s e r a 43 b) Assay Procedures 45 2. I n s u l i n ... 46 a) P r e p a r a t i o n of B u f f e r , L a b e l l e d I n s u l i n , A n t i s e r a and Standards 46 b) Assay Procedures 47 I I . PEPTIDE PURIFICATION AND CHARACTERIZATION STUDIES 48 A. P u r i f i c a t i o n Methodology 48 1. Column Chromatography 48 a) Ion Exchange Chromatography 48 b) Gel F i l t r a t i o n Chromatography 49 2. High Pressure L i q u i d Chromatography 50 B. C h a r a c t e r i z a t i o n of Peptides 52 1. Gel E l e c t r o p h o r e s i s 52 a) Polyacrylamide Gel ......52 i ) Johns Polyacrylamide Gel 55 i i ) Panyim C h a l k l e y Gel 55 i i i ) D i s c o n t i n o u s Sodium Dodecyl-Sulphate Gel 56 iv ) Sodium Dodecyl-Sulphate Urea Gel 58 ix b) S t a i n i n g Procedures 59 i ) Coomassie Blue with T r i c h l o r o a c e t i c A c i d P r e c i p i t a t i o n 59 i i ) Coomassie Blue with Methanol and A c e t i c A c i d P r e c i p i t a t i o n 60 2. Thin Layer Chromatography 60 C. Amino A c i d Composition and Sequence A n a l y s i s ....61 1. Enzymatic Degradation 61 a) T r y p s i n and Chymotrypsin 62 2. Chemical Cleavage and M o d i f i c a t i o n 63 a) O x i d a t i o n 63 i ) Hydrogen Peroxide 63 i i ) Performic A c i d 64 3. I s o l a t i o n of Peptide Fragments 64 a) Paper Chromatography 64 b) High V o l t a g e E l e c t r o p h o r e s i s ...66 4. Amino A c i d A n a l y s i s ..69 5. N-terminus Determination 74 I I I . ANALYSIS OF DATA ..76 RESULTS 77 I. PHYSIOLOGICAL STUDIES 77 A. Glucose Clamp Experiments 77 1. Exogenously Administered GIP on IRI Release i n S t a t e s of Euglycaemia and X Hyperglycaemia 82 a) Euglycaemia 82 b) Steady State Hyperglycaemia Maintained at 40 mg/dl Above Basal L e v e l (G+40) 82 c) Steady State Hyperglycaemia Maintained at 100 mg/dl Above Basal L e v e l (G+100) 87 d) Steady • State Hyperglycaemia Maintained at 150 mg/dl Above Basal L e v e l (G+150) . . 92 2. I n s u i i n o t r o p i c A c t i o n of I n t r a d u o d e n a l l y and O r a l l y Administered Glucose During Hyperglycaemia 104 a) Euglycaemia 104 b) Hyperglycaemia at G+40 110 c) Hyperglycaemia at G+100 116 3. I n s u i i n o t r o p i c A c t i o n of O r a l l y , I n t r a d u o d e n a l l y and In t r a v e n o u s l y Administered Amino A c i d Mixture 121 a) Euglycaemia ....121 i) Plasma Glucose 121 i i ) Plasma IRI 122 i i i ) Plasma IR-GIP 129 x i b) Hyperglycaemia at G+40 130 i ) Plasma IRI 130 i i ) Glucose I n f u s i o n 139 i i i ) Plasma IR-GIP 140 4. O r a l A d m i n i s t r a t i o n of a Commercial P r e p a r a t i o n of Amino A c i d Mixture 140 5. I n s u l i n o t r o p i c ' A c t i o n of O r a l l y A c t i o n of O r a l l y A c t i o n of O r a l l y a) Plasma IRI 149 b) Glucose I n f u s i o n 152 c) Plasma IR-GIP 152 8. I n s u l i n o t r o p i c A c t i o n of I n t r a d u o d e n a l l y Administered H y d r o c h l o r i c A c i d 155 B. I n t e r a c t i o n of GIP with Intermediates of T r i c a r b o x y l i c A c i d Cycle . 160 I I . PEPTIDE PURIFICATION AND CHARACTERIZATION STUDIES 168 A. P u r i f i c a t i o n of GIP 168 1. F r a c t i o n a t i o n of EG Stage I on CM-C e l l u l o s e Column 168 2. F r a c t i o n a t i o n of EG Stage II on Sephadex G-25 Column . 1 68 3. F r a c t i o n a t i o n of GIP on CM-Sephadex C25 Column i 171 a) P h y s i o l o g i c a l A c t i o n of GIP Post CM-Sephadex C25 F r a c t i o n s 171 b) Peptide A n a l y s i s of the F r a c t i o n s of GIP Post CM-Sephadex C25 Column 176 4. P u r i f i c a t i o n of GIP on High Pressure L i q u i d Chromatography 177 B. Peptide A n a l y s i s 189 1. Poly a c r y l a m i d e Gel E l e c t r o p h o r e s i s ....189 a) Johns Method 189 b) Panyim C h a l k l e y Method 189 2. Amino A c i d Composition 194 C. Amino A c i d Sequence A n a l y s i s 194 1. Cyanogen Bromide Cleavage 194 2. T r y p t i c D i g e s t 198 D. Thin Layer Chromatography 199 I I I . ACID INHIBITORY EFFECTS OF DIFFERENT PREPARATIONS OF GIP 199 A. Chemical M o d i f i c a t i o n of GIP 199 B. S y n t h e t i c GIP 205 1. Peptide A n a l y s i s 205 x i i i 2. P h y s i o l o g i c a l A c t i v i t i e s 211 C. Side F r a c t i o n s Of EG I I I F r . A 211 1. Peptide A n a l y s i s 216 2. P h y s i o l o g i c a l S t u d i e s 221 DISCUSSION 222 BIBLIOGRAPHY ...264 x i v LIST OF FIGURES  F i g u r e Page 1. Chronic Dog P r e p a r a t i o n 30 2. G a s t r i c A c i d I n h i b i t i o n by GIP 33 3. O r a l Glucose T o l e r a n c e Test of Dogs Used in the Study 36 4. 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 I n f u s i o n at 1.0 /xg/kg.h i n Euglycaemic State 84 5. 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 I n f u s i o n at 2.0 Mg/kg.h i n Euglycaemic State 86 6. Hyperglycaemic Clamp Maintained at G+40 89 7. E f f e c t of 1.0 Mg/kg.h GIP During Hyperglycaemia Maintained at G+40 91 8. Hyperglycaemic Clamp Maintained at G+100 94 9. E f f e c t of 1.0 Mg/kg.h GIP During Hyperglycaemia Maintained at G+100 9.6 10. Hyperglycaemic Clamp Maintained at G+150 99 11. E f f e c t of 1.0 Mg/kg.h GIP During Hyperglycaemia Maintained at G+150 101 12. E f f e c t of 2.0 Mg/kg.h GIP During Hyperglycaemia Maintained at G+150 103 13. O r a l Glucose Load 106 14. Intraduodenal Glucose Load 108 15. O r a l Glucose Load During Hyperglycaemia XV Maintained at G+40 112 16. Intraduodenal Glucose Load During Hyperglycaemia Maintained at G+40 114 17. O r a l Glucose Load During Hyperglycaemia Maintained at G+100 118 18. Intraduodenal- Glucose Load During Hyperglycaemia Maintained at G+100 120 19. I n s u l i n o t r o p i c A c t i o n of an O r a l Administered Amino A c i d Mixture 124 20. I n s u l i n o t r o p i c A c t i o n of an Intraduodenal Administered Amino A c i d Mixture 126 21. I n s u l i n o t r o p i c A c t i o n of an In t r a v e n o u s l y Administered Amino A c i d Mixture 128 22. I n s u l i n o t r o p i c A c t i o n of an Intravenous Amino A c i d Mixture f o r 20 min During Hyperglycaemia Maintained at G+40 132 23. I n s u l i n o t r o p i c A c t i o n Of an Intravenous Amino A c i d Mixture f o r 40 min During Hyperglycaemia Maintained at G+40 134 24. I n s u l i n o t r o p i c A c t i o n of an Intraduodenal Amino A c i d Mixture During Hyperglycaemia Maintained at G+40 136 25. I n s u l i n o t r o p i c A c t i o n of an O r a l Amino A c i d Mixture During Hyperglycaemia Maintained at x v i G+40 I 33 26. I n s u l i n o t r o p i c A c t i o n of a Commercially A v a i l a b l e Amino A c i d Mixture 142 27. I n s u l i n o t r o p i c A c t i o n of 5 g of O r a l A l a n i n e During Hyperglycaemia Maintained at G+40 .' ...145 28. I n s u l i n o t r o p i c A c t i o n of 5 g of O r a l A r g i n i n e During Hyperglycaemia Maintained at G+40 i 48 29. E f f e c t of O r a l Lipomul on IRI S e c r e t i o n During Hyperglycaemia Maintained at G+40 151 30. E f f e c t of Or a l Lipomul on IRI S e c r e t i o n During Hyperglycaemia Maintained at G+150 154 31. E f f e c t of H y d r o c h l o r i c A c i d on IRI S e c r e t i o n in Euglycaemic S t a t e ...157 32. E f f e c t of H y d r o c h l o r i c A c i d on IRI S e c r e t i o n During Hyperglycaemia Maintained at G+40 160 33. E f f e c t of I n f u s i n g Intermediates of T r i c a r b o x y l i c A c i d C y c l e on Plasma IR-GIP ....,163 34. E f f e c t of I n f u s i n g Intermediates of T r i c a r b o x y l i c A c i d Cycle on Plasma Glucose ....165 35. E f f e c t of I n f u s i n g Intermediates of T r i c a r b o x y l i c A c i d Cycle on Plasma IRI 167 x v i i 36. Absorbance P r o f i l e of EG I on CM-C e l l u l o s e 11 170 37. Absorbance P r o f i l e of EG II on Sephadex G-25 .173 38. Absorbance P r o f i l e of GIP on CM-Sephadex C25 175 39. High Voltage E l e c t r o p h o r e s i s of T r y p t i c Fragments of F r a c t i o n s of GIP Post CM-Sephadex C25 .- 179 40. High Pressure L i q u i d Chromatography P u r i f i c a t i o n of GIP i n Ammonium Aceta t e and A c e t o n i t r i l e System 181 41. High Pressure L i q u i d Chromatography P u r i f i c a t i o n of GIP i n Ammonium Acetate and Ethanol System 183 42. High Pressure L i q u i d Chromatography P u r i f i c a t i o n of GIP i n A c e t o n i t r i l e , Water, Phosphoric A c i d and T r i e t h y l a m i n e System 186 43. High Pressure L i q u i d Chromatography P u r i f i c a t i o n of GIP i n T r i f l u o r o a c e t i c A c i d , Water and A c e t o n i t r i l e System 188 44. Comparison of GIP with I t s Side F r a c t i o n s on Po l y a c r y l a m i d e Gel 191 45. Polyac r y l a m i d e - u r e a Gel E l e c t r o p h o r e s i s of xv i i i O x i d i z e d GIP 1 93 46. Absorbance P r o f i l e of Cyanogen Bromide Treated GIP on CM-1 1 197 47. High Voltage E l e c t r o p h o r e s i s of T r y p t i c Fragments of Wuensch S y n t h e t i c GIP 208 48. Comparison of Amino A c i d Composition of Chymotrypsin Tr e a t e d N-terminal Fragment of Wuensch S y n t h e t i c GIP with Porcine GIP 210 49. G a s t r i c A c i d I n h i b i t o r y A c t i v i t y of S y n t h e t i c GIP (Yanaihara) 213 50. G a s t r i c A c i d I n h i b i t o r y A c i t i v i t y of S y n t h e t i c GIP (Wuensch) 215 51. Absorbance P r o f i l e of Desamido II on Sephadex G-25 Column 218 52. Absorbance P r o f i l e of Desamido II on Sephadex G-50 Column 220 53. Amino A c i d Sequence of GIP 232 x i x LIST OF TABLES Table Page I. , The Composition of the 30 g Amino A c i d Mixture 39 I I . The Composition of the S o l u t i o n of T r i c a r b o x y l i c A c i d Intermediates Used 42 I I I . The B u f f e r Composition and the Program f o r the Amino A c i d A n a l y s i s 73 IV. Summary of the E v a l u a t i o n s of Glucose Clamp Experiments 79 V. Summary of the Mean Glucose I n f u s i o n Rates and Mean Plasma IRI During Glucose Clamp Experiments 81 VI E l e c t r o p h o r e t i c M o b i l i t y on High V o l t a g e E l e c t r o p h o r e s i s and Rf Values of GIP T r y p t i c Fragments on the Thin Layer Chromatography 201 V I I . G a s t r i c A c i d I n h i b i t o r y A c t i v i t y of M o d i f i e d Porcine GIP, Side F r a c t i o n s of GIP and S y n t h e t i c GIP 204 1 INTRODUCTION Regu l a t i o n of serum glucose occurs as a r e s u l t of a set of complex i n t e r a c t i o n s i n v o l v i n g the c i r c u l a t i n g n u t r i e n t s , endocrines and nervous system. The p r e c i s e endocrine r o l e of the pancreas was recog n i z e d with the d i s c o v e r y of i n s u l i n by Banting and Best (1920) and glucagon by M u r l i n (1923). E x t r a - p a n c r e a t i c glucose homeostatic mechanisms i n v o l v e p i t u i t a r y glands, gonads, adren a l medulla and the autonomic nervous system. Most of these c o n t r o l systems e x e r t t h e i r i n f l u e n c e s both d i r e c t l y and i n d i r e c t l y through the r e l e a s e of i n s u l i n . The main r e g u l a t o r s of i n s u l i n s e c r e t i o n are the metabolic f u e l s , of which glucose i s the prime determinant ( G e r i c h et a l . (1976)). Glucose causes a m u l t i p h a s i c 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 and monophasic i n h i b i t i o n of glucagon i n the r a t i s o l a t e d pancreas p r e p a r a t i o n ( G e r i c h et a l . (1976)). However, the comparatively h i g h t h r e s h o l d f o r 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 ir\ v i t r o suggests that glucose alone does not r e g u l a t e the f a s t i n g i_n v i v o i n s u l i n l e v e l . In c o n t r a s t to glucose, amino a c i d s have been demonstrated to s t i m u l a t e the r e l e a s e of both i n s u l i n and 2 glucagon. In the absence of glucose, a r g i n i n e and other amino a c i d s have been shown to s t i m u l a t e monophasic i n s u l i n r e l e a s e ( G e r i c h et a l . (1974)), while the a d d i t i o n of a small amount of glucose r e s u l t s i n m u l t i p h a s i c s e c r e t i o n of i n s u l i n . The i n s u l i n o t r o p i c a c t i o n of the i n d i v i d u a l amino a c i d s i s v a r i a b l e . In dogs (Rocha et a l . (1972)), tryptophan, l e u c i n e , a s p a r t i c a c i d and i s o l e u c i n e are the most potent s t i m u l a n t s f o r i n s u l i n r e l e a s e , whereas asparagine, g l y c i n e , and p h e n y l a l a n i n e are the most e f f e c t i v e f o r glucagon s e c r e t i o n . In i n  v i t r o p r e p a r a t i o n s , the amino a c i d s at p h y s i o l o g i c a l c o n c e n t r a t i o n s have weak i n s u l i n o t r o p i c e f f e c t s but i n i t i a t e a potent r e l e a s e of glucagon. However, the presence of glucose w i l l augment i n s u l i n r e l e a s e to amino a c i d s while i n h i b i t i n g r e l e a s e of glucagon (Ohneda et a l . (1968)., P a g l i a r a et a l . (1974)). T h e r e f o r e , the presence of glucose i s important in d i f f e r e n t i a t i n g between the r e l e a s e of both p a n c r e a t i c hormones to amino a c i d s . Other metabolic f u e l s such as f a t t y a c i d s , 0-h y d r o x y l b u t y r a t e and a c e t o a c e t a t e a f f e c t i n s u l i n r e l e a s e (Pelkonen et a l . (1968)), and t h e i r r o l e s are s t i l l u n c l e a r . S e y f f r e t et a l . (1967) has proposed that t h e i r 3 r o l e s are important d u r i n g s t a r v a t i o n to ensure i n s u l i n r e l e a s e to prevent k e t o a c i d o s i s . The n e u r a l 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 can be d i v i d e d i n t o a d r e n e r g i c , c h o l i n e r g i c and the p u t a t i v e p e p t i d e r g i c / p u r i n e r g i c mechanisms (Larsson (1980), Woods and Porte (1974)). The sympathetic nervous system can a f f e c t the 0 - c e l l a c t i v i t y n e u r o n a l l y or by the r e l e a s e of catecholamines v i a the a d r e n a l medulla. Ep i n e p h r i n e , the major adrenomedullary hormone w i l l i n h i b i t g l u c o s e -mediated i n s u l i n r e l e a s e ( M a l a i s s e et a l . (1967)). Edwards et a l . (1980) have demonstrated, in conscious adrenalectomized c a l v e s , that s t i m u l a t i o n of the s p l a n c h n i c nerves w i l l i n h i b i t i n s u l i n s e c r e t i o n and e l e v a t e plasma glucose. There i s a r i s e of i n s u l i n s e c r e t i o n on c e s s a t i o n of s t i m u l a t i o n . I n f u s i o n of a c e t y l c h o l i n e w i l l s t i m u l a t e i n s u l i n and glucagon r e l e a s e i n p e r f u s e d canine pancreas (Iversen (1973)). These e f f e c t s can be a b o l i s h e d by the a d m i n i s t r a t i o n of a t r o p i n e suggesting the involvement of m u s c a r i n i c r e c e p t o r s . However, a f t e r c h r o n i c vagotomy, ba s a l i n s u l i n and glucagon l e v e l s are not a p p r e c i a b l y a f f e c t e d , but the i n s u l i n response to o r a l or i . v . glucose i s d i m i n i s h e d (Hakanson et a l . (1971)). 4 An i n t e r r e l a t i o n s h i p between the g a s t r o i n t e s t i n a l t r a c t and the pancreas i n glucose homeostasis has long been recognized (Laughton and MaCallum (1923), C r e u t z f e l d t (1979)). Claude Bernard (1877) noted s u p e r i o r h a n d l i n g of glucose when admi n i s t e r e d v i a the o r a l as opposed to the intravenous route. He a t t r i b u t e d the reduced degree of hyperglycaemia as due to h e p a t i c uptake of most of the glucose d u r i n g the f i r s t passage i n the p o r t a l c i r c u l a t i o n . T h i r t y years l a t e r , Moore, Edie and Abram (1906) suggested that the duodenum r e l e a s e d a substance which s t i m u l a t e d the " i n t e r n a l s e c r e t i o n " of the pancreas, analogous to s e c r e t i n which had been shown to r e g u l a t e the " e x t e r n a l s e c r e t i o n " . Dixon and Wadia (1926) demonstrated hypoglycaemia i n r a b b i t s by i n j e c t i n g a duodenal mucosal e x t r a c t , prepared by b o i l i n g , f o l l o w e d by f i l t r a t i o n . They s p e c u l a t e d that the a c t i v e substance i n the e x t r a c t was not s e c r e t i n , s i n c e b o i l i n g the e x t r a c t i n d i l u t e a c i d destroyed s e c r e t i n and d i d not a b o l i s h the hypoglycaemic a c t i v i t y . Zunz and Labarre (1929) performed c r o s s c i r c u l a t i o n experiments i n dogs and demonstrated a r o l e f o r the pancreas i n producing hypoglycaemia f o l l o w i n g the i n f u s i o n of the crude s e c r e t i n p r e p a r a t i o n . Three years l a t e r , Labarre and 5 S t i l l (1930) p o s t u l a t e d the e x i s t e n c e of " i n c r e t i n " , a substance which s t i m u l a t e d the i n t e r n a l s e c r e t i o n of the pancreas. In the next t h i r t y years, attempts were made to i s o l a t e t h i s g a s t r o i n t e s t i n a l i n s u i i n o t r o p i c f a c t o r but with l i t t l e success. Some of the f a i l u r e s were merely due to lack of exactness concerning experimental c o n d i t i o n s e.g. t e s t i n g the i n t e s t i n a l e x t r a c t s only i n the f a s t i n g normoglycaemic s t a t e w h i l s t o m i t t i n g the t e s t in the hyperglycaemic s t a t e (Loew et a l . (1940)). The development of the technique of radioimmunoassay f o r i n s u l i n gave new impetus to the c h a r a c t e r i z a t i o n of the hypoglycaemic f a c t o r of e n t e r i c o r i g i n . E l r i c k , Stimmler, Hlad and A r a i (1964), compared the i n s u l i n responses 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 an i d e n t i c a l l o a d of glucose v i a o r a l and intravenous route to the same p a t i e n t s on two separate o c c a s i o n s . The gr e a t e r degree of in c r e a s e i n plasma i n s u l i n f o l l o w i n g o r a l a d m i n i s t r a t i o n l e d the authors to suggest t h a t , e i t h e r a g a s t r o i n t e s t i n a l f a c t o r was r e l e a s e d by the presence of glucose i n the stomach or upper i n t e s t i n e , or a l i v e r f a c t o r was t r i g g e r e d by the i n c r e a s e d glucose c o n c e n t r a t i o n in the p o r t a l v e i n . In the same year, 6 M c l n t y r e , Holdsworth and Turner (1964) observed a g r e a t e r i n s u l i n response in man to i n t r a j e j u n a l than to intravenous i n f u s i o n of g l u c o s e . They concluded that a humoral substance was r e l e a s e d from the j e j u n a l w a l l d u r i n g the glucose a b s o r p t i o n and t h i s agent acted along with the r i s e i n plasma glucose to f u r t h e r s t i m u l a t e i n s u l i n r e l e a s e . Support f o r t h i s concept came from P e r l e y and K i p n i s (1967) who observed a g r e a t e r i n s u l i n response to o r a l than to an intravenous glucose l o a d i n p a t i e n t s with d i v e r t e d p o r t a l c i r c u l a t i o n . They estimated that 50-60% of the i n s u l i n response to o r a l glucose l o a d was due to the a l i m e n t a r y mechanism. The concept of the " e n t e r o i n s u l a r a x i s " was proposed by Unger and E i s e n t r a u t (1969). They proposed a r e g u l a t o r y system i n which hormones from the g a s t r o i n t e s t i n a l t r a c t were r e l e a s e d when ing e s t e d s u b s t r a t e s act on the gut mucosa and these hormones exert a p a r t i a l i n f l u e n c e on s e c r e t i o n from the p a n c r e a t i c i s l e t s . The f u n c t i o n of the e n t e r o i n s u l a r a x i s i s to augment and to a c c e l e r a t e the r e l e a s e of i n s u l i n , glucagon or both to absorbed s u b s t r a t e s i n the c i r c u l a t i o n , so that the p a n c r e a t i c responses w i l l be g r e a t e r and more r a p i d than by the a r t e r i a l c o n c e n t r a t i o n 7 of absorbed s u b s t r a t e s alone. So f a r glucose has been demonstrated u n e q u i v o c a l l y to e l i c i t the e n t e r o i n s u l a r a x i s i n man (Dupre, Ross et a l . (1973)), in dogs (Pederson, Schubert and Brown (1975)) and i n r a t s (Pederson and Brown (1976)), as i n d i c a t e d by the g r e a t e r i n s u l i n response to o r a l than intravenous a d m i n i s t r a t i o n of g l u c o s e . Although l e s s c o n v i n c i n g l y than f o r glucose, the i n s u l i n and glucagon responses to hyperaminoacidaemia i s g r e a t e r f o l l o w i n g duodenal a d m i n i s t r a t i o n than v i a the intravenous route (Thomas et a l . (1976)). The search f o r the mediator of the e n t e r o i n s u l a r a x i s has focussed mainly on humoral f a c t o r ( s ) but not on the n e u r a l i n f l u e n c e (Woods and Porte J r . (1974)). The reasons f o r t h i s are numerous: f i r s t l y e a r l y experiments i n d i c a t e d that denervation of the pancreas had l i t t l e or no e f f e c t On blood glucose r e g u l a t i o n ; secondly, i n s u l i n s e c r e t i o n observed i n the i_n v i t r o p r e p a r a t i o n s appeared to be analogous to that _in_ v i v o ; t h i r d l y the t r a n s p l a n t e d pancreas was r e p o r t e d to maintain blood glucose w i t h i n the normal range. However, the development of immunofluorescence techniques combined with e l e c t r o n microscopy, has shown the presence of i n t e s t i n a l p e p t i d e s in both the c e n t r a l nervous system and gut. T h i s has 8 prompted a new a p p r a i s a l of the r o l e of n e u r a l elements i n the e n t e r o i n s u l a r a x i s . Some of these i n t e s t i n a l p e p t i d e s have been found not only i n endocrine c e l l s but a l s o i n nerve t e r m i n a l s , which suggested a p o s s i b l e r o l e as n e u r o t r a n s m i t t e r substances i n a p e p t i d e r g i c nervous system (D a n i e l (1978)). In a d d i t i o n , p e p t i d e r g i c nerves have been i d e n t i f i e d i n hamster, cat and p i g pancreas (Larsson (1980)). I f the e n t e r o i n s u l a r a x i s was mediated by the p e p t i d e r g i c nervous system with the peptides as the n e u r o t r a n s m i t t e r substances, then changes i n the plasma l e v e l of the peptides might not be great enough to be d e t e c t e d . Evidence s u p p o r t i n g a ne u r a l r o l e came from the o r t h o t o p i c t r a n s p l a n t a t i o n of the pancreas (denervated) which i n d i c a t e d impairment of the i n s u l i n response to o r a l glucose while not a f f e c t i n g the response to intravenous glucose (Jakob, L a r g i a d e r and Froesch (1970)). However, c o n f l i c t i n g o b s e r v a t i o n s were made i n pi g s with a h e t e r o t o p i c a l l y t r a n s p l a n t e d pancreas. The i n s u l i n responses to both o r a l and intravenous glucose a d m i n i s t r 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 from normal p i g s (Jensen, N i e l s e n and Ruhl (1976)). P h y l o g e n e t i c a l l y , there i s a c l o s e anatomical a s s o c i a t i o n between the endocrine producing c e l l s of the 9 g a s t r o i n t e s t i n a l t r a c t and the i n s u l i n ( 0 - c e l l s ) and glucagon ( a - c e l l s ) producing c e l l s (Van Noorden and Falkmer (1980)). In an i n v e r t e b r a t e such as the mollusc, a - c e l l s and /3 - c e l l s are l o c a t e d randomly amongst c e l l s l i n i n g the gut lumen and l a c k a separate i s l e t organ. U l t r a s t r u c t u r a l s t u d i e s showed that a l l the endocrine producing c e l l s possessed g r a n u l a t i o n at the b a s a l p o l e , while the a p i c a l pole p r o j e c t s i n t o the lu m i n a l s u r f a c e . The h a g f i s h , an animal at a higher e v o l u t i o n a r y developmental s t a t e , has a separate i s l e t organ l o c a l i z e d at the j u n c t i o n of the common b i l e duct and the gut. In the mammal, s e p a r a t i o n of the i s l e t organ from the gut i s completed and the p a n c r e a t i c i s l e t i s exposed to the a r t e r i a l supply and ne u r a l i n f l u e n c e s , while the endocrine producing c e l l s of the g a s t r o i n t e s t i n a l t r a c t are s t i l l exposed to n u t r i e n t s i n the lumen. S e v e r a l c r i t e r i a have to be s a t i s f i e d i n order f o r a g a s t r o i n t e s t i n a l hormone to f u l f i l l the r o l e of i n c r e t i n . F i r s t l y , the hormone must be r e l e a s e d by n u t r i e n t , e s p e c i a l l y g l u c o s e . Secondly, i n the presence of glucose, the amount of exogenous hormone 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 s e c r e t i o n must achieve c i r c u l a t i n g l e v e l s comparable to those achieved f o l l o w i n g food i n g e s t i o n . 10 T h i r d l y , the hormone p r e p a r a t i o n must be pure. Most of s t u d i e s with the gut hormones have not s a t i s f i e d a l l of these- c r i t e r i a . For example, the p u r i t y of c h o l e c y s t o k i n i n - p a n c r e o z y m i n (CCK-PZ) was not w e l l e s t a b l i s h e d , and the a c c u r a t e d e t e r m i n a t i o n of the plasma l e v e l s of both s e c r e t i n and CCK-PZ remained c o n t r o v e r s i a l . Dupre (1964) demonstrated that i n j e c t i o n of a crude s e c r e t i n p r e p a r a t i o n , f o l l o w i n g the intravenous a d m i n i s t r a t i o n of glucose, s i g n i f i c a n t l y i n c r e a s e d the r a t e at which the glucose was removed from plasma. Chisholm et. a l . (1969) demonstrated the i n s u l i n o t r o p i c a c t i o n of h i g h l y p u r i f i e d s e c r e t i n i n man, and proposed that the h y p e r i n s u l i n a e m i a observed a f t e r duodenal a c i d i f i c a t i o n was mediated by s e c r e t i n . Contrary to these i n v i v o s t u d i e s , Schatz et a l . (1974) i n d i c a t e d s e c r e t i n d i d not s t i m u l a t e i n s u l i n r e l e a s e nor the b i o s y n t h e s i s of p r o i n s u l i n i n i s o l a t e d mouse i s l e t p r e p a r a t i o n s . Dupre et a l . (1969) r e p o r t e d enhancement of i n s u l i n r e l e a s e by s e c r e t i n i n the presence of hyperglycaemia. A l a r g e r i n s u l i n r e l e a s e to intravenous a r g i n i n e i n the presence of exogenous s e c r e t i n has a l s o been demonstrated. However, the i n s u l i n response was not s u s t a i n e d throughout the s e c r e t i n i n f u s i o n p e r i o d . The authors concluded that s e c r e t i n c o u l d not be regarded as a p o t e n t i a t o r of i n s u l i n s e c r e t i o n in response to hyperglycaemia. Lerner and Porte (1972) demonstrated that small s e c r e t i n p u l s e s (15 U) and small glucose p u l s e s (5 g) s t i m u l a t e d the r a p i d i n s u l i n r e l e a s e . Repeated i n j e c t i o n s of l a r g e s e c r e t i n p u l s e s (15 U) given i n r a p i d s u c c e s s i o n caused a p r o g r e s s i v e l y d i m i n i s h i n g i n s u l i n response, yet the subsequent glucose pulse e l i c i t e d a gr e a t e r i n s u l i n response than the c o n t r o l . Porte et a l . (1969) made an e a r l i e r o b s e r v a t i o n that d u r i n g a short . glucose i n f u s i o n , a glucose pulse e l i c i t e d a decreased i n s u l i n response to that obtained when the puls e was given without a glucose background, but a s e c r e t i n p u l s e (15 U) i n c r e a s e d the i n s u l i n response compared to the p r e i n f u s i o n c o n t r o l . The authors concluded that glucose and s e c r e t i n s t i m u l a t e d two separate f u n c t i o n a l p o ols of r e a d i l y a v a i l a b l e i n s u l i n . Buchanan et a l . (1968) r e p o r t e d the i n a b i l i t y of a constant i n f u s i o n of s e c r e t i n to e l i c i t i n s u l i n s e c r e t i o n i n a n a e s t h e t i z e d dogs when doses s u f f i c i e n t to produce a copious flow of p a n c r e a t i c j u i c e were used. The absence of endogenous r e l e a s e of serum I R - s e c r e t i n f o l l o w i n g 12 i n g e s t i o n of a mixed meal i n man (Bloom, Byrant and Cochrane (1975)) or i n g e s t i o n of glucose, amino a c i d s and f a t t y a c i d s i n dogs (Boden, Essa, Owen and F r e d e r i c k (1974), Boden, Essa and Owen (1975)) was a l s o non sup p o r t i v e of a r o l e f o r s e c r e t i n in the p h y s i o l o g i c a l 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 . Recently, Fahrenkrug et a l . (1978) demonstrated i n man that i n t r a d u o d e n a l glucose d i d not s t i m u l a t e the r e l e a s e of s e c r e t i n . In the hyperglycaemic s t a t e , i n f u s i o n of HC1 i n t r a d u o d e n a l l y has been a s s o c i a t e d with a l o n g - l i v e d augmentation of i n s u l i n s e c r e t i o n . 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 completely d i f f e r e n t from that observed d u r i n g exogenous i n f u s i o n of s e c r e t i n , at pharmacological doses. Furthermore, i n f u s i o n of s e c r e t i n at a dose which mimicked the serum c o n c e n t r a t i o n a t t a i n e d d u r i n g intraduodenal a c i d i f i c a t i o n , had no e f f e c t on 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 . The absence of a c l e a r demonstration that s e c r e t i n can be r e l e a s e d by n u t r i e n t s and the lack of an i n s u l i n o t r o p i c a c t i o n of exogenous s e c r e t i n at p h y s i o l o g i c a l c o n c e n t r a t i o n s are c o n t r a d i c t o r y to a r o l e for s e c r e t i n as a mediator i n the e n t e r o i n s u l a r a x i s . linger et a l . (1967) demonstrated that intravenous i n j e c t i o n of a g a s t r i n - c o n t a i n i n g p o r c i n e a n t r a l e x t r a c t 1 3 i n t o dogs during a euglycaemic s t a t e r e s u l t e d in a monophasic r e l e a s e of i n s u l i n . During hyperglycaemia, s y n t h e t i c human g a s t r i n II i n c r e a s e d the r e l e a s e of immunoreactive i n s u l i n (IRI) and furthermore, t h i s was a s s o c i a t e d with an a c c e l e r a t e d d i s p o s a l of glucose i n man (Dupre et a l . (1969)). However, Buchanan et a l . (1969), were unable to show an i n s u i i n o t r o p i c a c t i o n f o r g a s t r i n in the i s o l a t e d r a t i s l e t p r e p a r a t i o n when both high and low glucose c o n c e n t r a t i o n s were used in the medium. In man, i n g e s t i o n of glucose e l i c i t e d a small e l e v a t i o n i n plasma immunoreactive g a s t r i n ( I R - g a s t r i n ) (Rehfeld and S t a d i l (1975)) and a p r o t e i n r i c h meal e l i c i t e d a g r e a t e r I R - g a s t r i n r e l e a s e (Rehfeld and S t a d i l (1973)) i n which the predominant molecular s p e c i e s was i d e n t i f i e d as " b i g g a s t r i n " i . e . G34 (Walsh (1975)). A l s o i n man (Walsh (1975)), i t was shown that exogenous a d m i n i s t r a t i o n of g a s t r i n , e i t h e r as a s i n g l e i n j e c t i o n or a prolonged i n f u s i o n , to achieve p h y s i o l o g i c a l c o n c e n t r a t i o n s e q u i v a l e n t to those obtained f o l l o w i n g i n g e s t i o n of a p r o t e i n meal, s t i m u l a t e d a r a p i d but s h o r t - l i v e d r e l e a s e of i n s u l i n (Rehfeld and S t a d i l (1973)). In s p i t e of the i n c r e a s e d s e c r e t i o n of i n s u l i n induced by exogenous g a s t r i n (Rehfeld, H o i s t and Kuhl (1978)), serum glucose l e v e l s were e i t h e r unchanged or i n c r e a s e d . The plasma 1 4 c o n c e n t r a t i o n of g a s t r i n achieved f o l l o w i n g glucose i n g e s t i o n probably c o n t r i b u t e d l i t t l e i f any to i n s u l i n r e g u l a t i o n . However, g a s t r i n may play a r o l e in i n s u l i n s e c r e t i o n during i n g e s t i o n of a p r o t e i n meal. T h e r e f o r e , the c r i t e r i a f o r g a s t r i n as a mediator i n the e n t e r i n s u l a r a x i s have not been f u l f i l l e d . Intravenous i n f u s i o n of i n t e s t i n a l e x t r a c t s c o n t a i n i n g CCK-PZ b i o l o g i c a l a c t i v i t y has been demonstrated to enhance intravenous glucose s t i m u l a t e d IRI r e l e a s e in man (Dupre and Beck (1966)). In dogs, i n f u s i o n of p a r t i a l l y p u r i f i e d CCK-PZ was capable of e l i c i t i n g both i n s u l i n and glucagon r e l e a s e (Buchanan et a l . (1968)) and i n the i s o l a t e d r a t pancreas p r e p a r a t i o n s , CCK-PZ was shown to r e l e a s e i n s u l i n , and p o s s i b l y glucagon i n d i r e c t l y (Fussganger et a l . (1969)). linger and E i s e n t r a u t (1969) have repo r t e d that exogenous CCK-PZ augmented the response of i n s u l i n and glucagon to hyperaminoac idaemia. Most of the e a r l i e r s t u d i e s on the i n s u l i n o t r o p i c a c t i o n of CCK-PZ u t i l i z e d a p a r t i a l l y p u r i f i e d CCK-PZ prepared by Jorpes and Mutt ( C r e u t z f e l d t (1979)). I t was from t h i s p r e p a r a t i o n that another i n s u l i n o t r o p i c hormone, 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), was 15 i s o l a t e d (Brown, Mutt and Pederson (1969)). Experiments using p u r i f i e d CCK-PZ demonstrated no i n s u i i n o t r o p i c or gl u c a g o n o t r o p i c a c t i o n i n ijn v i t r o r a t pancreas p r e p a r a t i o n s , and _in v i v o a n a e s t h e t i z e d r a t s and man (Ra b i n o v i t c h and Dupre (1972)). On the other hand, Dan i e l s s o n et a l . (1974) demonstrated an i n s u i i n o t r o p i c a c t i o n i n mouse i s o l a t e d pancreas. D e t e c t i o n of CCK-PZ i n the c i r c u l a t i o n has been attempted using the techniques of radioimmunoassay (Harvey et a l . (1973)) and bi o a s s a y . Major problems a s s o c i a t e d with the radioimmunoassay f o r CCK-PZ have been the s c a r c i t y of pure p o r c i n e CCK-PZ, d i f f i c u l t y i n i o d i n a t i o n because of the sulphated t y r o s i n e residue in the molecule and the r e l a t i v e l y low immunogenic potency of CCK-PZ (Go and R e i l l y (1975)). T h e r e f o r e , to date i n t e r p r e t a t i o n of the radioimmunoassay r e s u l t s has been d i f f i c u l t . In c o n t r a s t , the bioassay r e l i e s upon the d i r e c t measurement of p a n c r e a t i c enzyme s e c r e t i o n or g a l l b l a d d e r c o n t r a c t i o n which are s t i m u l a t e d by CCK-PZ. Bioassay s t u d i e s using dogs with an a u t o t r a n s p l a n t e d pancreas have demonstrated the r e l e a s e of CCK-PZ by the presence of p r o t e i n s , amino a c i d mixtures, f a t and soap i n the duodenum. Intraduodenal a d m i n i s t r a t i o n of a 16 carbohydrate and intravenous i n f u s i o n of an amino a c i d mixture, however, d i d not s t i m u l a t e the r e l e a s e of CCK-PZ (Wang and Grossman (1951)). Go et a l . (1970) demonstrated an i n c r e a s e i n CCK-PZ b i o a c t i v i t y i n man f o l l o w i n g i n t r a d u o d e n a l a d m i n i s t r a t i o n of a mixture of e s s e n t i a l amino a c i d or f a t but not g l u c o s e . Meyer (1975) f u r t h e r e l u c i d a t e d the i n s u l i n o t r o p i c a c t i o n of i n d i v i d u a l amino a c i d s and found that p h e n y l a l a n i n e and tryptophan were the. most potent, whether administered as f r e e amino a c i d s or when present i n small p e p t i d e s . The region of the i n t e s t i n e r e s p o n s i b l e f o r the r e l e a s e of CCK-PZ by the amino a c i d s was found to extend beyond the duodenum, but the a b i l i t y to r e l e a s e CCK-PZ decreased with the d i s t a n c e from the p y l o r u s (Konturek, T a s l e r and ObtuLowicz (1972)) . 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 was i s o l a t e d from a crude p r e p a r a t i o n of CCK-PZ, i n i t i a l l y f o r i t s a c i d i n h i b i t o r y a c t i v i t y to 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 i n the v a g a l l y denervated canine g a s t r i c pouch (Brown, Mutt and Pederson (1970)). I t was l a t e r shown to have a p o t e n t i a t i n g e f f e c t 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 i n man (Crockett et a l . (1976)), i n i s o l a t e d r a t p e r f u s e d pancreas (Pederson and Brown (1976)) and i n dogs 1 7 (Pederson, Schubert and Brown (1975)). Exogenous i n f u s i o n of p u r i f i e d p o r c i n e GIP i n the presence of intravenous glucose i n f u s i o n caused a g r e a t e r i n s u l i n r e l e a s e than intravenous i n f u s i o n of glucose alone (Dupre et a l . (1973)). The degree of 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 depended upon the p r e v a i l i n g l e v e l of glycaemia and the dosage of GIP i n f u s e d , t h i s prompted the concept that GIP was a 'glucose d e p e n d e n t - 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 ' (Brown and Pederson (1976)). T h i s concept was f u r t h e r s u b s t a n t i a t e d by experiments i n man i n which the glycaemia was c o n t r o l l e d (Andersen, E l a h i , Brown, Debas et a l . (1978)). A steady s t a t e plasma glucose c o n c e n t r a t i o n was maintained throughout the experiment. Exogenous GIP i n f u s i o n was shown to have no i n s u i i n o t r o p i c a c t i o n i n the euglycaemic s i t u a t i o n , l i t t l e augmentation at m i l d hyperglycaemia, and marked e l e v a t i o n at modest hyperglycaemia. Glucose u t i l i z a t i o n d u r i n g GIP i n f u s i o n s i n d i c a t e d a s i g n i f i c a n t i n c r e a s e d u r i n g hyperglycaemia, a moderate i n c r e a s e i n m i l d glycaemic s t a t e and no i n c r e a s e d u r i n g euglycaemia. In both man and i s o l a t e d r a t pancreas models (Andersen, E l a h i , Brown, Tobin et a l . (1978), Pederson and Brown (1976)), the i n s u i i n o t r o p i c a c t i o n of GIP i n 18 the presence of hyperglycaemia i n c r e a s e d both the f i r s t and the second phase of i n s u l i n r e l e a s e . C e s s a t i o n of the i n s u l i n o t r o p i c a c t i o n o c c u r r e d on t e r m i n a t i o n of the GIP i n f u s i o n . The plasma l e v e l of immunoreactive GIP (IR-GIP) achieved i n dogs (Pederson, Schubert and Brown (1975)) durin g the exogenous i n f u s i o n of p o r c i n e GIP was comparable to p h y s i o l o g i c a l l e v e l s of IR-GIP" obtained duri n g o r a l glucose i n f u s i o n . O r a l i n g e s t i o n of glucose, amino a c i d s , p r o t e i n s , f a t and p o s s i b l y intraduodenal i n s t i l l a t i o n of h y d r o c h l o r i c a c i d i n some sp e c i e s have been shown to s t i m u l a t e endogenous GIP r e l e a s e ( C r e u t z f e l d t (1979)). In man, peak plasma l e v e l s of IR-GIP were achieved w i t h i n 45 minutes a f t e r the i n g e s t i o n of glucose and preceeding the peak of IRI r e l e a s e (Brown, Dryburgh, Ross and Dupre (1975)). In dogs, o r a l i n g e s t i o n of glucose e l e v a t e d the plasma IR-GIP f o r 90 minutes before r e t u r n i n g to the p r e i n g e s t i o n l e v e l . A dose dependent r e l a t i o n s h i p e x i s t e d between the o r a l glucose l o a d and the peak e l e v a t i o n of IR-GIP (Pederson, Schubert and Brown (1975)), whereas intravenous glucose i n f u s i o n d i d not e l e v a t e the plasma IR-GIP. The degree of e l e v a t i o n of IR-GIP and subsequent 19 i n s u l i n r e l e a s e to i n t r a d u o d e n a l glucose i n f u s i o n can be reduced i n man, by the intravenous i n f u s i o n of a t r o p i n e (Larrimer et a l . (1978)). However, a t r o p i n e i n f u s i o n d i d not a l t e r the duodenal a b s o r p t i o n of d-xylose suggesting that a t r o p i n e had a d i r e c t e f f e c t on GIP producing c e l l s , as opposed to an e f f e c t mediated through the i n h i b i t i o n of glucose a b s o r p t i o n . S t u d i e s on the r e l a t i o n s h i p between the r e l e a s e of IR-GIP in dogs and the s t r u c t u r e of carbohydrates ( S i r i n e k , O ' D o r i s i o et a l . (1979)) i n d i c a t e d that the i n c r e a s e i n IR-GIP were s i m i l a r f o r D-g l u c o s e , aD-glucose and |3D-glucose, whereas s o r b i t o l , g l u c u r o n i c . a c i d and g l y c e r a l d e h y d e d i d not r e l e a s e IR-GIP. The r e s u l t s suggested that the a l t e r a t i o n in the c o n f i g u r a t i o n of D-glucose has l i t t l e e f f e c t on the c a p a c i t y to s t i m u l a t e GIP r e l e a s e . In a d d i t i o n to the humoral r e l e a s e of GIP, luminal r e l e a s e of GIP was demonstrated by in v i t r o p e r f u s i o n of hamster small i n t e s t i n e with 10% glucose s o l u t i o n (Lewis et a l . (1979)). 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 i s not apparent. A g r e a t e r degree of i n s u l i n r e l e a s e i n response to a mixture of ten amino a c i d s v i a the intraduodenal rather than the intravenous route has been observed i n man (77±9 20 vs 43±6 MU/ml) (Thomas et a l . (1978)). Simultaneously, an e l e v a t i o n of plasma IR-GIP was observed d u r i n g i n t r a d u o d e n a l i n f u s i o n , with the peak l e v e l preceeding the i n s u l i n peak by 15 minutes and no i n c r e a s e i n IR-GIP f o l l o w i n g the intravenous route of i n f u s i o n . Furthermore, in t r a d u o d e n a l i n f u s i o n of methionine, p h e n y l a l a n i n e , tryptophan and v a l i n e which were b e l i e v e d to cause CCK-PZ r e l e a s e , d i d not e l i c i t an e l e v a t i o n of IR-GIP and IRI r e l e a s e . However, a mixture of a r g i n i n e , h i s t i d i n e , i s o l e u c i n e , l e u c i n e , l y s i n e and t h r e o n i n e which were b e l i e v e d not to cause CCK-PZ r e l e a s e d i d e l i c i t an e l e v a t i o n of plasma IR-GIP and IRI. In dogs (Yovos et a l . (1982)), intravenous i n f u s i o n of the same mixture of ten amino a c i d s and intravenous i n f u s i o n of p o r c i n e GIP had shown g r e a t e r i n s u l i n response than intravenous amino a c i d s alone (41±2 vs 29±6 MU/ml). However, an e a r l i e r r e p o rt (Ohneda et a l . (1968)) using the same amino a c i d mixture i n dogs had demonstrated no d i f f e r e n c e i n i n s u l i n and glucagon response between the intravenous and i n t r a d u o d e n a l routes of a d m i n i s t r a t i o n . Endogenous r e l e a s e of IR-GIP has been demonstrated f o l l o w i n g the intraduodenal i n f u s i o n of f a t i n dogs (Brown et a l . (1975)) and i n man (Falko et a l . (1975)) 21 and the long c h a i n f a t t y a c i d s have been shown to be more potent than medium chain f a t t y a c i d s ( O ' D o r i s i o et a l . (1976)). Fat i n g e s t i o n has been shown to e l e v a t e plasma IR-GIP f o r up to 5 hours before l e v e l s returned to the f a s t i n g s t a t e (Pederson, Schubert and Brown (1975)). When these experiments were performed i n a c o n t r o l l e d euglycaemic s i t u a t i o n , plasma glucose and IRI d i d not change s i g n i f i c a n t l y . However, in the presence of an i n t r a v e n o u s i n f u s i o n of g lucose, f a t i n g e s t i o n induced a g r e a t e r increment of IRI r e l e a s e than intravenous i n f u s i o n of glucose alone. Glucose t o l e r a n c e was improved a f t e r o r a l f a t . T h i s suggested that the endogenous r e l e a s e of IR-GIP by f a t i n g e s t i o n had a p o t e n t i a t i n g e f f e c t on IRI r e l e a s e i n the presence of a hyperglycaemic s t a t e . Endogenously r e l e a s e d IR-GIP by e i t h e r glucose or f a t e x h i b i t e d at l e a s t three immunoreactive components a f t e r column chromatography. The f i r s t component was e l u t e d i n the v o i d volume. The second had a molecular weight between 7500 and 8000 d a l t o n s , and the t h i r d had a molecular weight of 5000 d a l t o n . The f i r s t component was b e l i e v e d to be GIP b i n d i n g to a l a r g e r p r o t e i n . The second and the t h i r d component have a l s o been i d e n t i f i e d in e x t r a c t s of hog duodenal mucosa. However, the p h y s i o l o g i c a l r e l a t i o n s h i p or d i f f e r e n c e s between the two 22 components i s not p r e s e n t l y known (Brown, Dryburgh et a l . (1975)). Intraduodenal p e r f u s i o n of h y d r o c h l o r i c a c i d i n a n a e s t h e t i z e d r a t s has been demonstrated to s t i m u l a t e the endogenous r e l e a s e of IR-GIP i n a dose dependent manner (Ebert et a l . (1979)). Serum IR-GIP was e l e v a t e d to a l e v e l which had an i n s u l i n o t r o p i c e f f e c t , i n the presence of hyperglycaemia. The e a r l y phase of i n s u l i n r e l e a s e , produced by duodenal r e l e a s e of IR-GIP by h y d r o c h l o r i c a c i d was a b o l i s h e d by concurrent i n f u s i o n of GIP a n t i s e r a from guinea p i g . Intraduodenal i n f u s i o n of h y d r o c h l o r i c a c i d i n man over a 10 minutes p e r i o d e l e v a t e d serum IR-GIP l e v e l s i n a dose dependent manner (Ebert et a l . (1979)). The peak of IR-GIP response l a s t e d f o r about 30 minutes a f t e r the c e s s a t i o n of intr a d u o d e n a l i n f u s i o n of 100 ml of 0.1 N HC1. I n j e c t i o n of 1.0 CU(Crick, Harper and Raper u n i t s ) / k g of s e c r e t i n e l i c i t e d a pu l s e IRI r e l e a s e with no e l e v a t i o n of IR-GIP. T h i s o b s e r v a t i o n prompted the authors to conclude that the endogenous IR-GIP r e l e a s e d by duodenal p e r f u s i o n with a c i d i n f l u e n c e d the r e l e a s e of IRI and was independent of the s e c r e t i n l e v e l . These r e s u l t s appeared to support the o b s e r v a t i o n that p a t i e n t s with duodenal u l c e r have a s i g n i f i c a n t l y 23 higher f a s t i n g l e v e l of GIP (Arnold et a l . (1978)), and r e l e a s e s i g n i f i c a n t l y more IR-GIP f o l l o w i n g the i n g e s t i o n of a mixed meal. However, in dogs, int r a d u o d e n a l i n f u s i o n of h y d r o c h l o r i c a c i d d i d not produce an i n c r e a s e i n serum IR-GIP (Brown, Dryburgh et a l . (1975)). C r e u t z f e l d t et a l . (1976) p o s t u l a t e d that the r e g u l a t i o n of the r e l e a s e of GIP depended upon the glucose a b s o r p t i v e c a p a c i t y of the small bowel. The GIP response to a standard t e s t meal in p a t i e n t s with untreated c o e l i a c d i s e a s e was compared with a c o n t r o l group. The r e s u l t s i n d i c a t e d a decrease in r e l e a s e of IR-GIP and IRI i n the p a t i e n t s with untreated c o e l i a c d i s e a s e . The h i s t o c h e m i s t r y i n d i c a t e d 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 number of GIP producing c e l l s between the two groups. Furthermore, in p a t i e n t s with s t e a t o r r h e a of p a n c r e a t i c o r i g i n , the IR-GIP response to a standard t e s t meal was i n c r e a s e d f o l l o w i n g the a d d i t i o n of p a n c r e a t i n suggesting that the g r e a t e r a b s o r p t i o n of n u t r i e n t gave r i s e to a l a r g e r GIP r e l e a s e . C r e u t z f e l d t (1979) f u r t h e r propose a feedback i n h i b i t i o n of GIP r e l e a s e by i n s u l i n . The IR-GIP response i n normal s u b j e c t s r e c e i v i n g o r a l f a t c o n c u r r e n t l y with intravenous i n f u s i o n of glucose was lower than in s u b j e c t s r e c e i v i n g 24 o r a l f a t alone ( C l e a t o r and Gourlay (1975)). However, Andersen, E l a h i , Brown, Tobin et a l . (1978) showed that r e l e a s e of IR-GIP by o r a l glucose was not suppressed i n the presence of a square wave of i n s u l i n a e m i a . The l a t t e r had been produced using the euglycaemic clamp. The e n t e r o i n s u l a r a x i s i s a complex system of r e g u l a t o r y mechanisms f o r i n s u l i n s e c r e t i o n i n v o l v i n g the g a s t r o i n t e s t i n a l t r a c t . These r e g u l a t o r y mechanisms probably i n v o l v e the i n t e r a c t i o n s of n u t r i e n t s with the p a r a c r i n e , endocrine and nervous system. The importance of the i n d i v i d u a l r e g u l a t o r y mechanisms can only be a s c e r t a i n e d i n s i t u a t i o n s i n which the other mechanisms can be c o n t r o l l e d . Techniques such as the glucose clamp pro v i d e an o p p o r t u n i t y to study the i n s u i i n o t r o p i c a c t i o n of duodenal secretogogues or hormones du r i n g steady s t a t e plasma glucose c o n d i t i o n s . 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 , a glucose-dependent i n s u i i n o t r o p i c p o l y p e p t i d e , has f u l f i l l e d most of the c r i t e r i a f o r the candidacy of i n c r e t i n . Although the response of GIP to o r a l glucose has been w e l l e s t a b l i s h e d , the r e l e a s e by other n u t r i e n t s and a c i d have not been demonstrated c o n c l u s i v e l y . The present i n v e s t i g a t i o n was undertaken to f u r t h e r d e l i n e a t e the r o l e of GIP i n the r e l e a s e of 25 i n s u l i n to d i f f e r e n t s t i m u l i i n an i_n v i v o canine model and to examine the p u r i t y and the primary s t r u c t u r e of GIP. 26 METHODS I. PHYSIOLOGICAL STUDIES Experiments have been performed i n random bred dogs, weighing 30-50 kg, to study both the a c i d i n h i b i t o r y and i n s u l i n o t r o p i c e f f e c t of 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). A l l animals were f a s t e d 18 h p r i o r to each experiment and were s t u d i e d i n the unanaesthetized s t a t e . Dogs were t r a i n e d to stand on a s p e c i a l l y designed p l a t f o r m and the weight of the dog was supported by l e g harnesses. Intravenous ( i . v . ) i n f u s i o n s were c a r r i e d out v i a the i n s e r t i o n of c a t h e t e r s (Arbrook, Peterborough) i n t o the b a s i l i c v e i n of the f o r e limb. The c a t h e t e r was connected to a d r i p b o t t l e c o n t a i n i n g 0.9% NaCl to prevent blood c l o t t i n g d u r i n g the pre- and p o s t - i n f u s i o n per i o d s . Blood samples were obtained from a c a t h e t e r (Bard, Sunderland, England) i n s e r t e d r e t r o g r a d e l y i n t o the saphenous v e i n of the dogs' hind limb and d r i p b o t t l e s with 0.9% NaCl were connected to the c a t h e t e r between blood sample c o l l e c t i o n s . Blood was c o l l e c t e d with a sy r i n g e (Luer-lok t i p , Becton, M i s s i s s a u g a ) , the f i r s t 1.0 ml of blood c o l l e c t e d was d i s c a r d e d to prevent 27 d i l u t i o n with s a l i n e . The subsequent 3 ml of blood was t r a n s f e r r e d to a 12x75 mm tube c o n t a i n i n g 3.3 mg of EDTA ( E t h y l e n e d i a m i n o t e t r a a c e t a t e , F i s h e r ) . A f t e r the blood was thoroughly mixed with EDTA , a 200 y l a l i q u o t was t r a n s f e r r e d to a h e p a r i n i z e d microfuge tube (Beckman, F u l l e r t o n , C a l i f o r n i a ) and c e n t r i f u g e d f o r 15 sec using a Beckman 152 Microfuge at 10,000 rpm. A 10 M1 a l i q u o t of plasma from the supernatant was used for the de t e r m i n a t i o n of glucose by the glucose oxidase method (Beckman Glucose A n a l y z e r , F u l l e r t o n , C a l i f o r n i a ) . A l l plasma glucose d e t e r m i n a t i o n s were performed i n d u p l i c a t e . The remaining 2.8 ml of blood, mixed with EDTA, were c e n t r i f u g e d i n a desk top c e n t r i f u g e at 4C f o r 10 min at 2,500 rpm. The plasma obtained was a l i q u o t e d , fro z e n and s t o r e d at -20C p r i o r to a s s a y i n g f o r i n s u l i n and GIP . A. G a s t r i c A c i d I n h i b i t o r y S t u d i e s  1. S u r g i c a l Procedures Anaesthesia was induced with an i . v . i n j e c t i o n of 10-16 ml (dependent upon the a c t u a l weight of the animal) of 5% sodium p e n t o t h a l (Abbott, Montreal) and maintained with 0.5% Fluothane (Ayerst, Montreal) throughout the 28 e n t i r e procedure. A Heidenhain pouch of approximately 50 ml i n volume was c r e a t e d from the g r e a t e r c u r v a t u r e of the stomach. Blood supply to the pouch was v i a the g a s t r o e p i p l o i c blood v e s s e l s . The Heidenhain pouch was p o s i t i o n e d c l o s e to the abdominal w a l l and opened to the e x t e r i o r v i a a metal cannula ( F i g . 1). A Thomas cannula was implanted i n the most dependent part of the stomach and was normally kept c l o s e d by a t e f l o n screw cap ( F i g . 1). A Mann-Bollman f i s t u l a was prepared by i s o l a t i o n of a 25 crn length of ileum, the d i s t a l end of which was then anastomosed to the duodenum, about 2-3 cm below the p y l o r u s and the proximal end e x t e r i o r i z e d . The c o n t i n u i t y of the g a s t r o i n t e s t i n a l t r a c t was renewed by an end-end anastomosis of the two s e c t i o n e d ends of the ileum ( F i g . 1). 2. C o l l e c t i o n and A n a l y s i s of G a s t r i c A c i d G a s t r i c s e c r e t i o n was s t i m u l a t e d by the intravenous i n f u s i o n of 2.0 Mg/kg.h of p e n t a g a s t r i n (Ayerst, M o n t r e a l ) . The f l u i d from the Heidenhain pouch was c o l l e c t e d f o r a n a l y s i s , w h i l s t that produced from the g a s t r i c remnant was d r a i n e d i n t o a p o l y e t h y l e n e - b o t t l e v i a the open Thomas cannula and d i s c a r d e d . The c o l l e c t i o n 29 F i g . 1 The c h r o n i c canine p r e p a r a t i o n employed i n the s t u d i e s of g a s t r i c a c i d i n h i b i t i o n and i n s u l i n r e l e a s e . The s u r g e r i e s r e s u l t e d i n a Heidenhain pouch, Thomas cannula to the stomach and a Mann Bollman f i s t u l a f o r a c c e s s i b i l i t y to the duodenum. Heidenhain Pouch Thomas / Cannula Mann Bollman Fistula 31 of s e c r e t i o n from the Heidenhain pouch was made at 15 min i n t e r v a l s . At the beginning of a 15 min p e r i o d , a 10 ml volume of d i s t i l l e d water was in t r o d u c e d i n t o the pouch and allowed to remain there f o r the d u r a t i o n of the p e r i o d . At the end of the p e r i o d , f l u i d from the pouch was d r a i n e d and the pouch was washed with an a d d i t i o n a l 10 ml of d i s t i l l e d water which was then pooled with the o r i g i n a l sample. The t o t a l volume of the mixture was recorded. A c i d i h the g a s t r i c sample was determined by t i t r a t i n g 5 ml a l i q u o t s of the sample with 0.01M HCl to pH 7.0 employing a Metrohm H e r i s a u Automatic T i t r a t o r set-up. The a c i d p r o d u c t i o n was expressed as MEq/15 min. A comparison of p e n t a g a s t r i n c o n t r o l experiments with or without GIP i n f u s i o n s i s shown on F i g . 2. The values f o r a c i d s e c r e t i o n were expressed as the r a t i o of the mean of four p l a t e a u p e r i o d s . The percent of a c i d i n h i b i t i o n was d e f i n e d as the d i f f e r e n c e between the mean of the p l a t e a u p e r i o d s and the two pe r i o d s showing the maximum amount of i n h i b i t i o n d u r i n g the t e s t . B. Glucose Homeostatic Stud i e s  1. Glucose Clamp A l l dogs showed no s i g n s of carbohydrate i n t o l e r a n c e 32 F i g . 2 The g a s t r i c a c i d i n h i b i t o r y e f f e c t of GIP on 2.0 nq/kq.h of 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 . P e n t a g a s t r i n c o n t r o K A A) (n=9) and i n the presence of 1.0 Mg/kg.h of GIP (o o) (n=7). A l l data were expressed as mean±standard e r r o r of the mean (SEM). 15 Min. Periods 34 when o r a l glucose t o l e r a n c e t e s t s were performed ( F i g . 3). The experimental procedures f o r hyperglycaemic clamping i n these s t u d i e s was s i m i l a r to that d e s c r i b e d by other workers ( I n s e l et a l . (1975), Defronzo, Tobin et a l . (1979), Sherwin et a l . (1974)), i n that a square wave of hyperglycaemia was produced by intravenous i n f u s i o n over 12 min of a g r a d u a l l y d e c r e a s i n g priming i n f u s i o n of 20% dextrose (Abbott, Montreal) in water, to r a i s e the plasma glucose r a p i d l y to the d e s i r e d l e v e l . The plasma glucose l e v e l was maintained at the predetermined value f o r the d u r a t i o n of the experiment, by a s e r v o - c o n t r o l l e d negative feedback formula which determined the rate of glucose to be i n f u s e d using a v a r i a b l e - s p e e d i n f u s i o n pump (Harvard Apparatus Co. M i l l s Mass). Plasma glucose l e v e l s were determined every 15 min f o r 1 h p r i o r to the s t a r t of the hyerglycaemic clamp to e s t a b l i s h the ba s a l glucose c o n c e n t r a t i o n . For the f i r s t 10 min of the hyperglycaemic clamp, the blood samples were taken every 2 min and then every 5 min u n t i l the end of the experiment. The hyperglycaemic clamp was maintained i n separate experiments at 40, 100 and 150 mg/dl above the ba s a l plasma glucose l e v e l (G+40, G+100 and G+150). Glucose i n f u s i o n s were c a r r i e d out f o r 150-165 min and m e t a b o l i t e s , secretogogues and hormones to be t e s t e d were 35 F i g . 3 O r a l glucose t o l e r a n c e t e s t . Plasma IRI, glucose and IR-GIP l e v e l s f o l l o w i n g an o r a l glucose l o a d of 1.0 g/kg administered as 25% dextrose i n d i s t i l l e d water over a 5 min p e r i o d at time 60 min (n=7). A l l data are expressed as mean±SEM. 36 Oral glucose a. 25-0-| 1—• 1 r i i 0 60 120 180 240 300 Mm. Oral glucose (1 2500 n 0-( 1 1 1 1 1 0 60 120 180 210 300 Min. 37 given 60 min a f t e r the establishment of the hyperglycaemic clamp. a) I n t e r a c t i o n of Intravenous Amino A c i d s Mixture with  Hyperglycaemia Intravenous amino a c i d s were ad m i n i s t e r e d as a s o l u t i o n c o n t a i n i n g 30 g of 10 amino a c i d s (Thomas, M a z z a f e r r i et a l . (1976), Thomas, Si n a r et a l . (1978)), a d j u s t e d to 300 mosmol with NaCl and to pH 7.4 with HC1. The t o t a l volume of the i n f u s a t e was 1.0 1 and the c o n c e n t r a t i o n and the source of each amino a c i d are co n t a i n e d i n Table I. The d u r a t i o n of the i . v . i n f u s i o n of the 30 g of amino a c i d mixture was e i t h e r 20 or 40 min. b) I n t e r a c t i o n of E n t e r a l l y Administered N u t r i e n t s and  Secretogogues with Hyperglycaemia I n f u s a t e s were a d m i n i s t e r e d over 40 min p e r i o d s i n t o the duodenum v i a the Mann-Bollman f i s t u l a , u s i ng a small diameter f l e x i b l e tube a t t a c h e d to a r a t e a d j u s t a b l e p e r i s t a l i c pump. Substances t e s t e d i n c l u d e d : 50 ml of 0.1 M HC1, 1.0 1 of 30 g mixture of the 10 amino a c i d s and 500-900 ml of glucose s o l u t i o n s . The o s m o l a r i t y of a l l 38 Table I The composition and the commercial sources of the i n d i v i d u a l amino a c i d i n the 30 g mixture. The t o t a l volume of the mixture was 1 1, pH 7.4 t i t r a t e d with HCl and 300 mosmol ad j u s t e d with NaCl. Amino a c i d Cone . T o t a l weight Source (mM) (grams) L - h i s t i d i n e 21 .5 3.36 Eastman L-methionine 23.6 3.52 Eastman L - i s o l e u c i n e 16.7 2.19 Eastman L-threonine 19-1- 2.28 Eastman L-p h e n y l a l a n i n e 18.7 3.09 Eastman L-tryptophan 4.9 1 .00 F i s h e r L -leuc ine 36.0 4.72 Eastman L - a r g i n i n e 9.5 1 .66 Eastman L - l y s i n e 31 .0 5.66 Eastman L - v a l i n e 30.8 3.61 Eastman 40 the intraduodenal i n f u s a t e s was ad j u s t e d to 300 mosmol and the pH to 7.4 with NaHC0 3 or HC1, except i n the case of 0.1 M HC1. Experiments were performed which were i d e n t i c a l i n format to the clamp experiments except that blood glucose was not e l e v a t e d . c) I n t e r a c t i o n of Exogenous GIP with Hyperglycaemia GIP was d i s s o l v e d i n 0.9% NaCl and i n f u s e d over a 60 min p e r i o d at a dose of 1.0 or 2.0 Mg/kg.h. 2. I n t e r a c t i o n of GIP with Intermediates of T r i c a r b o x y l i c  A c i d Cycle and Pyruvate The glucose m e t a b o l i t e s t e s t e d were i n f u s e d at a dose of 3.33 mM/kg.h. D e t a i l s of the pH, o s m o l a r i t y and the source of the in t e r m e d i a t e s t e s t e d are presented i n Table I I . Blood samples were taken at i n t e r v a l s of 15 min f o r 75 min p r i o r to the i . v . i n f u s i o n of the inter m e d i a t e s t e s t e d , 5 min d u r i n g the 30 min p e r i o d of i n f u s i o n and 15 min du r i n g the 45 min p o s t - i n f u s i o n p e r i o d . GIP was i n f u s e d i n t r a v e n o u s l y i n separate 41 Table II The c o n c e n t r a t i o n , pH, o s m o l a r i t y and the commercial source of each s o l u t i o n of glucose intermediates used i n the i n s u i i n o t r o p i c s t u d i e s . ' a -k e t o g l u t . ' denoted a - k e t o g l u t a r a t e . 42 M e t a b o l i t e Cone. pH Osmol. Cone. Source ( g / i ) (mosmdl/1) (mmol/kg.h) Glucose 175 4.4 1010 3.33 Abbott Sodium 107 7.7 1728 3.33 Eastman pyruvate Sodium 157 8.5 1496 3.33 Sigma succ i n a t e o - k e t o g l u t . 142 7.4 2244 3.33 Eastman 43 experiments 30 min a f t e r the s t a r t of the experiment (at doses of 0.4, 1.0 and 2.0 Mg/kg.h). C o n t r o l experiments were performed as above, but without i n f u s i o n of GIP . C. Radioimmunoassay  1 . GIP The radioimmunoassay f o r GIP , developed by Kuzio et a l . (1974) and d e s c r i b e d i n d e t a i l by Brown and Dryburgh (1979), .was employed i n s t u d i e s to determine the c o n c e n t r a t i o n of the immunoreactive GIP (IR-GIP) i n canine serum samples and i n f r a c t i o n s produced during p u r i f i c a t i o n procedures. a) P r e p a r a t i o n of B u f f e r , L a b e l l e d GIP and A n t i s e r a The assay d i l u e n t b u f f e r was 0.04 M phosphate (made up from Na 2HPO u and NaH 2P0 4 pH 6.5 with 5% c h a r c o a l e x t r a c t e d human plasma and 0.75% T r a s y l o l (10,000 KlU/ml). The i o d i n a t e d GIP . ( 1 2 5 1 - G I P ) was prepared using the chloramine-T technique (Greenwood and Hunter (1963)). The i o d i n a t i o n procedure c o n s i s t e d of adding 1.0 mCi of c a r r i e r f r e e sodium , 2 5 - I ( s p e c i f i c a c t i v i t y of 100 mCi/ml) to 5 Mg of GIP d i s s o l v e d i n 100 M1 of 0.4 M phosphate b u f f e r , pH 7.5. T h i s was f o l l o w e d by mixing achieved through g e n t l e e x p u l s i o n of a i r from a 1.0 ml t u b e r c u l i n s y r i n g e a t t a c h e d to an a p p r o p r i a t e 44 m i c r o p i p e t t e . The i o d i n a t i o n r e a c t i o n was i n i t i a t e d by the a d d i t i o n of 40 Mg of chloramine-T i n 10 M1 of 0.4 M phosphate b u f f e r and the r e a c t i o n was allowed to proceed for 15 sec before being terminated by the a d d i t i o n of 252 Mg of sodium metabisulphate i n 20 M1 of 0.4 M phosphate, pH 7.5. The 1 2 5 I - G I P was p u r i f i e d from the r e a c t i o n mixture on a column of Sephadex G-25 f i n e (0.9x27.5 cm). The column was e q u i l i b r a t e d and developed with 0.2 M a c e t i c a c i d c o n t a i n i n g 2% T r a s y l o l and 0.5% bovine serum albumin. The f r a c t i o n s i z e c o l l e c t e d was 300 M1 and a l i q u o t s of 10 M1 were counted f o r 0.1 min and the radiochromatogram was p l o t t e d . The f r a c t i o n s c o n t a i n i n g 1 2 5 I - G I P were pooled f o l l o w i n g d e t e r m i n a t i o n of a b i l i t y to adsorbed to c h a r c o a l . The guinea p i g a n t i s e r a (GP-22 and GP-24) obtained f o l l o w i n g immunization of guinea p i g s with GIP conjugated to bovine serum albumin by the c a r b o d i i m i d e method (Goodfriend et a l . (1964)), were used i n the assay. The a n t i s e r a have demonstrated no c r o s s r e a c t i v i t y with s e c r e t i n , glucagon, CCK-PZ, g a s t r i n , m o t i l i n , i n s u l i n and 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 ) , when a c o n c e n t r a t i o n up to 10 ng per ml was i n t r o d u c e d i n t o the assay system. 45 b) Assay Procedures The assay was c a r r i e d out i n 12x75 mm s i l i c o n i z e d g l a s s t e s t tubes. The r e a c t i o n mixture c o n s i s t e d o f : 1) 100 M1 of d i l u t e d a n t i s e r a , f i n a l t i t r e of 1:10,000. 2) 100 /xi of 1 2 5 I - G I P with a count of 5,000 cpm. 3) 100 M1 of GIP standard or samples to be t e s t e d . 4) assay d i l u e n t b u f f e r making the f i n a l volume to 1.0 ml. The mixture was incubated at 4C f o r 48 h. . A f t e r i n c u b a t i o n , the bound and f r e e components were separated by the a d d i t i o n of 200 M1 a l i q u o t s of dextran coated c h a r c o a l s o l u t i o n (1.25% c h a r c o a l , 0.25% dextran, 5% c h a r c o a l e x t r a c t e d plasma i n 0.04 M phosphate pH 6.5). The tubes were vortexed and then c e n t r i f u g e d at 2,800 rpm for 30 min. The samples were c a r e f u l l y decanted and the f r e e 1 2 5 I - G I P component, adsorbed to the c h a r c o a l was counted f o r 2 min i n an automated 7-counter ( S e a r l e , 1285). 46 2. I n s u l i n a) P r e p a r a t i o n of B u f f e r , L a b e l l e d I n s u l i n , A n t i s e r a and  Standards The l a b e l l e d i n s u l i n ( 1 2 5 I - I n s u l i n ) was prepared using the Chloramine-T technique (Greenwood and Hunter (1963)) and a l l r e a c t a n t s were d i s s o l v e d i n 0.2 M phosphate b u f f e r pH 7.5. The i o d i n a t i o n procedure c o n s i s t e d of adding 1.0 mCi of c a r r i e r f r e e 1 2 5 i to 5 Mg p o r c i n e i n s u l i n (NOVO, Denmark) d i s s o l v e d i n 10 M1 of phosphate b u f f e r , f o l l o w e d by the a d d i t i o n of 100 Mg of chloramine-T in 25 M1 f o r 10 sec and then 240 Mg of sodium metabisulphate i n 100 M1 0.2 M phosphate, pH 7.5. The s e p a r a t i o n of 1 2 5 I - I n s u l i n from the i o d i n a t e d r e a c t i o n mixture was accomplished by a d s o r p t i o n of 1 2 5 I -I n s u l i n to 10 mg of M i c r o f i n e S i l i c a QUSO G32 ( P h i l a d e l p h i a Quartz, Pennsylvania) washing three times to remove f r e e 1 2 5 I . The f i r s t wash was with 1.8 ml of 0.4 M phosphate b u f f e r pH 7.5; t h i s was f o l l o w e d by v i g o r o u s v o r t e x i n g f o r 30 sec and c e n t r i f u g a t i o n at 3,000 rpm f o r 15 min. The supernatant was decanted and the p e l l e t was washed twice with 3 ml of d i s t i l l e d water fol l o w e d each time by v o r t e x i n g and c e n t r i f u g a t i o n as d e s c r i b e d above. The d e s o r p t i o n of the 1 2 5 I - I n s u l i n from 47 the QUSO was c a r r i e d out with 5 ml of conc e n t r a t e d HCl:ethanol:H 20 (3:150:50{vol:vol}) s o l u t i o n , then v i g o r o u s l y vortexed f o r 30 sec and c e n t r i f u g e d as b e f o r e . The supernatant was s t o r e d at -20C. Guinea p i g i n s u l i n a n t i s e r a GP-7 ( g i f t of Dr. K.D. Buchanan, B e l f a s t ) was used i n the assay and standard ( S e a r l e I n s u l i n K i t , Amersham, O a k v i l l e , O n t ario) was s e r i a l l y d i l u t e d from 160 MU/ml to 1.25 MU/ml using the d i l u e n t b u f f e r . The d i l u e n t b u f f e r , 0.04 M phosphate pH 7.5 with 5% c h a r c o a l e x t r a c t e d plasma, was used f o r a l l assay d i l u t i o n s . b) Assay Procedures The assay was c a r r i e d out i n 12x75 mm non-s i l i c o n i z e d g l a s s or p l a s t i c t e s t tubes. The r e a c t i o n mixture c o n s i s t e d o f : 1) 100 M1 of d i l u t e d a n t i s e r a at a f i n a l d i l u t i o n of 1 : 400,000. 2) 100 M1 of i n s u l i n standard or sample to be t e s t e d . 3) D i l u e n t b u f f e r to a f i n a l volume of 900 M1. The mixture was incubated f o r 24 h at 4C. One hundred m i c r o l i t r e s of 1 2 5 I - I n s u l i n (1 Ox 10 3 cpm) was 48 added and i n c u b a t i o n was allowed to continue f o r a f u r t h e r 24 or 48 h. The f r e e 1 2 5 I - I n s u l i n was separated by the a d d i t i o n of a 200 jul a l i q u o t of dextran coated c h a r c o a l , (0.5% dextran i n 0.04 M phosphate pH 7.5) v o r t e x i n g , 10 min i n c u b a t i o n at 4C and c e n t r i f u g a t i o n at 2,800 rpm f o r 20 min. Free 1 2 5 I - I n s u l i n was measured i n the same manner as 1 2 5 I - G I P . I I . PEPTIDE PURIFICATION AND CHARACTERIZATION STUDIES  A. P u r i f i c a t i o n Methodology I. Column Chromatography a) Ion Exchange Chromatography The c a t i o n exchangers, carboxymethyl c e l l u l o s e (Whatman, CM11, London) and carboxymethyl-Sephadex 25 (CM-Sephadex 25), were used i n the p u r i f i c a t i o n of GIP. The c a t i o n exchangers were i n i t i a l l y p r e c y c l e d with a l t e r n a t e washes of 0.5 M NaOH, d i s t i l l e d water, 0.5 M HC1 and d i s t i l l e d water. The a c t i v a t e d c a t i o n exchangers were poured i n t o g l a s s columns with f i n a l bed dimensions of 0.9x25 cm and 1.5x19 cm with flow r a t e s of 30 ml/h and 120 ml/h f o r CM-Sephadex 25 and C M - C e l l u l o s e I I , r e s p e c t i v e l y . A p i e c e of f i l t e r paper (Whatman 3 MM, England) was p l a c e d on top of each g e l bed p r i o r to e q u i l i b r a t i o n of the column. 49 S t a r t i n g m a t e r i a l f o r the p u r i f i c a t i o n of GIP (EG I ) , weighing approximately 50 mg, was d i s s o l v e d i n 5 ml of 0.01 M N H 4 H C O 3 and the pH a d j u s t e d to 7.05 with 0.01 M NH 3 and a p p l i e d to the CM-Cellulose 11 column and e l u t e d i n i t i a l l y with 400 ml of 0.01 M of NH f tHC0 3, pH 7.8, f o l l o w e d by 80 ml of 0.2 M N H „ H C0 3 . The f r a c t i o n s i z e c o l l e c t e d was 5 ml and the absorbance was measured at 280 nm at a path width of 1.0 cm. E i g h t m i l l i g r a m s of GIP, (EG I I I ) , were a p p l i e d to the CM-Sephadex 25 column and e l u t e d with 200 ml of 0.01 M NH„HC0 3 pH 7.8. The f r a c t i o n volume was 1.5 ml and the absorbance was measured at 280 nm. The c o l l e c t e d f r a c t i o n s were pooled a c c o r d i n g to the absorbance p r o f i l e f o l l o w e d by l y o p h i l i z a t i o n . b) Gel F i l t r a t i o n Chromatography Gel f i l t r a t i o n techniques, based on the molecular e x c l u s i o n p r i n c i p l e , were employed in the f i n a l stage of p u r i f i c a t i o n of GIP from EG II to EG I I I . Sephadex G-25 f i n e (Sephadex, Pharmacia, Sweden) was a c t i v a t e d by s w e l l i n g i n water, f o l l o w e d by m u l t i p l e washings i n the e l u t i n g b u f f e r (0.2 M a c e t i c a c i d , A r i s t a r , BDH, England) 50 at 22C f o r 24 h. F i n e s were removed by the r e p e t i t i v e d e c a n t a t i o n of the supernatant a f t e r g e n t l e s t i r r i n g . The prepared Sephadex beads were poured i n t o a g l a s s column with f i n a l bed dimensions of 0.6x120 cm and a flow r a t e of 18 ml/h. The column was e q u i l i b r a t e d by running 3-4 bed volumes of 0.2 M a c e t i c a c i d p r i o r to each run and the column was s t o r e d i n 0.02 M sodium azid e in 0.2 M a c e t i c a c i d . P a r t i a l l y p u r i f i e d GIP (EG I I ) , weighing 10-15 mg, d i s s o l v e d i n 500 p.1 of 0.2 M a c e t i c a c i d , was a p p l i e d to the column and then e l u t e d with 60 ml of 0.2 M a c e t i c a c i d . The f r a c t i o n s i z e c o l l e c t e d was 1.5 ml and'the absorbance measured at 280 nm. The c o l l e c t e d f r a c t i o n s were pooled and l y o p h i l i z e d a c c o r d i n g to the absorbance p r o f i l e o b t a i n e d . 2. High Pressure L i q u i d Chromatography High pressure l i q u i d chromatography (HPLC) was used in the f u r t h e r p u r i f i c a t i o n of GIP. S e v e r a l b u f f e r systems were t e s t e d f o r the optimal s e p a r a t i o n and recovery of GIP. The f i r s t b u f f e r system was composed of 40 mM ammonium a c e t a t e , pH 4.0 (BDH, Toronto) and 51 a c e t o n i t r i l e (Burdick and Jackson Lab, Michigan) at a r a t i o of 70:30 ( v / v ) . The second system c o n s i s t e d of 40 mM ammonium a c e t a t e , pH 4.0 and 99% ethanol ( F i s h e r , New Jerse y ) at a r a t i o of 60:40 ( v / v ) . The t h i r d system c o n s i s t e d of 250 mM phosphoric a c i d ( F i s h e r , New J e r s e y ) , pH a d j u s t e d to 2.5 with t r i e t h y l a m i n e (Eastman, New York), and a c e t o n i t r i l e i n a volume r a t i o of 71:29 r e s p e c t i v e l y . The f o u r t h b u f f e r system c o n s i s t e d of t r i f l u o r o a c e t i c a c i d ( P i e r c e , I l l i n o i s ) , a c e t o n i t r i l e and d i s t i l l e d water at a v a r y i n g volume r a t i o . A l l b u f f e r s were degassed and passed through a 0.22 Mm m i l l i p o r e f i l t e r ( M i l l i p o r e , Massachusetts) p r i o r to d e l i v e r y to the column. GIP weighing 20 Mg was d i s s o l v e d i n 20 M1 of b u f f e r and t r a n s f e r r e d to the HPLC i n j e c t o r model U6K (Waters, Massachusetts) using a 25 M1 sy r i n g e (Hamilton, Nevada). A s o l v e n t d e l i v e r y system M-45 (Waters, Massachusetts) was used to pump b u f f e r at a flow rat e of 1.0 ml/min to the column (reverse phase, C18, Waters, Massachusetts) at pres s u r e s depending on the sol v e n t system used. The absorbance of the elu e n t from the column was measured with a v a r i a b l e wavelength d e t e c t o r (Waters, model 450, Massachusetts). The absorbance was r e g i s t e r e d on a c h a r t 52 recorder ( R e c o r d a l l S e r i e s 5000, F i s h e r , New J e r s e y ) at a paper speed of 0.25 cm/min. The eluent was then c o l l e c t e d , pooled a c c o r d i n g to the absorbance p r o f i l e and l y o p h i l i z e d . B. C h a r a c t e r i z a t i o n of Peptides  1. Gel E l e c t r o p h o r e s i s  a) P o l y a c r y l a m i d e Gel In the p r e p a r a t i o n ' of a l l the p o l y a c r y l a m i d e g e l s used i n t h i s study, the p o l y m e r i z a t i o n of acrylamide monomer (Eastman, New York) and N-N'-methylene-b i s a c r y l a m i d e (Eastman, New York) was i n i t i a t e d by f r e e r a d i c a l s generated by ammonium persul p h a t e ( F i s h e r , New J e r s e y ) , and the r e a c t i o n was subsequently c a t a l y z e d by tetramethylenediamine (TEMED, Eastman, New Y o r k ) . The s l a b g e l apparatus c o n s i s t e d of two p i e c e s of custom made g l a s s p l a t e s ( H o l t , Vancouver) a r e c t a n g u l a r p l a t e with a dimension of 20x16.5 cm, the second p l a t e was e s s e n t i a l l y r e c t a n g u l a r shape (17.5x16.5 cm) but with two square notches (1.5x1.5 cm) p r o t r u d i n g lengthwise out of the angle on each si d e of the p l a t e . The two g l a s s p l a t e s were h e l d together by two s t r i p s of greased (high vacuum grease, Don Corning, Michigan) 15x20 cm of t e f l o n 53 s t r i p s running lengthwise on the edge of each p l a t e and two binder c l i p s ( M e r i t , Quebec) were p l a c e d on each s i d e of the p l a t e assembly to maintain a t i g h t s e a l . The p l a t e assembly was p l a c e d v e r t i c a l l y i n a g e l forming stand; the bottom was s e a l e d with p a r a f i l m and r e s t e d on a p i e c e of foam (25x4x2 cm). The square notched ends c o n s t i t u t e d the top of the p l a t e assembly. Tension was maintained throughout the p l a t e assembly by the attachment of two s p r i n g - l o a d e d metal hooks to the two edges of the top of the p l a t e assembly, thereby ensuring a t i g h t s e a l between the p l a t e bottoms and the p a r a f i l m . The s l a b p o l y a c r y l a m i d e g e l was prepared by c a r e f u l l y t r a n s f e r r i n g the degassed g e l s o l u t i o n to the p l a t e assembly in the g e l forming stand. A p o l y e t h y l e n e tube att a c h e d to a s y r i n g e was used. A g e l s u r f a c e former was then p l a c e d i n the top end of the p l a t e assembly, and the g e l was l e f t at room temperature for at l e a s t 24 h to complete p o l y m e r i z a t i o n . The g e l s u r f a c e former was removed and the g e l s l o t s were washed 2-3 times with the e l e c t r o l y t e b u f f e r before sample a p p l i c a t i o n . The p l a t e assembly c o n t a i n i n g the polymerized g e l was p l a c e d v e r t i c a l l y between the upper and lower b u f f e r r e s e r v o i r s of the e l e c t r o p h o r e t i c apparatus (Raven, 54 England). Vacuum grease was a p p l i e d to the c o n t a c t s u r f a c e between the upper r e s e r v o i r and the top of the g e l p l a t e assembly in order to maintain a t i g h t s e a l , t h i s s e a l was f u r t h e r r e i n f o r c e d by two binder c l i p s compressing the g l a s s p l a t e s a g a i n s t the upper b u f f e r r e s e r v o i r . The bottom of the p l a t e assembly was immersed in the lower b u f f e r r e s e r v o i r . E l e c t r o l y t e b u f f e r was p l a c e d i n both r e s e r v o i r s i n c o n t a c t with the g e l s u r f a c e s . Samples d i s s o l v e d i n the a p p r o p r i a t e b u f f e r were a p p l i e d to each sample s l o t v i a a p i e c e of p o l y e t h y l e n e tube (PE-50, Intramedic, New J e r s e y ) at t a c h e d to a s y r i n g e . Samples were e j e c t e d g e n t l y onto the g e l s u r f a c e beneath the e l e c t r o l y t e b u f f e r . A d i r e c t c u r r e n t was a p p l i e d to the p o l y a c r y l a m i d e g e l from a power supply (Heathkit, O n t a r i o ) v i a two platinum w i r e s , one i n each r e s e r v o i r . The e l e c t r o p h o r e s i s was c a r r i e d out at 22C, and the d u r a t i o n depended on the g e l system used. Upon completion of the e l e c t r o p h o r e s i s , the p o l y a c r y l a m i d e g e l was removed from the p l a t e assembly and s u b j e c t e d to s t a i n i n g and (or) e x t r a c t i o n procedures. 55 i ) Johns Polyacrylamide Gel Johns procedure (1967) was used f o r the p r e p a r a t i o n of the s l a b p o lyacrylamide g e l . A 15 ml volume of acrylamide s o l u t i o n (40% acrylamide monomer {w/v} and 0.6% N-N'-methylene-bisacrylamide {w/v}) was mixed with 15 ml of 0.6% ammonium persul p h a t e {w/v} and 6 ml of TEMED s o l u t i o n (0.5% TEMED i n 4.6 M a c e t i c a c i d {v/v}). The g e l s o l u t i o n was then degassed under vacuum f o r 4 min and t r a n s f e r r e d i n t o the s l a b g e l p l a t e assembly f o r p o l y m e r i z a t i o n . The e l e c t r o l y t e b u f f e r used was 0.01 M a c e t i c a c i d . Samples weighing 10-30 ng, were d i s s o l v e d i n sucrose s o l u t i o n (1.0 M sucrose i n 0.002 M a c e t i c a c i d ) to give a f i n a l c o n c e n t r a t i o n of 1.0 uq/nl. These were a p p l i e d to each sample s l o t . E l e c t r o p h o r e t i c procedures c o n s i s t e d of an i n i t i a l 8 h p e r i o d of p r e - e l e c t r o p h o r e s i s , at an amperage of 30 mA and constant v o l t a g e of 300 V, followed by 8-10 h of e l e c t r o p h o r e s i s at an amperage of 15 mA and constant v o l t a g e of 250 V. i i ) Panyim C h a l k l e y Gel Polyacrylamide s l a b g e l c o n t a i n i n g urea was prepared 56 a c c o r d i n g to the procedures d e s c r i b e d by Panyim and Chalkey (1969). A volume of 10 ml of acrylamide s o l u t i o n c o n s i s t i n g of 60% acrylamide monomer (w/v) and 0.4% of N-N'-methylene-bisacrylamide (w/v) i n water was mixed with 5.0 ml TEMED s o l u t i o n (4% TEMED i n 43.2% g l a c i a l a c e t i c a c i d ) and 25 ml of 0.2% ammonium per s u l p h a t e (w/v) i n 10.0 M urea. The g e l s o l u t i o n was degassed under vacuum fo r 4 min and t r a n s f e r r e d i n t o the s l a b g e l p l a t e assembly f o r p o l y m e r i z a t i o n . The e l e c t r o p h o r e t i c b u f f e r used was 0.9 M a c e t i c a c i d i n d i s t i l l e d water. Samples weighing 10-30 ug were d i s s o l v e d i n 10-30 M1 of 0.9 N a c e t i c a c i d with 15% sucrose, which were a p p l i e d to the sample s l o t s i n the g e l . The e l e c t r o p h o r e t i c procedure c o n s i s t e d of an i n i t i a l 10 h p e r i o d of pre-e l e c t r o p h o r e s i s at an amperage of 30 mA and constant v o l t a g e of 120 V and a f u r t h e r 8-10 h Of e l e c t r o p h o r e s i s at an amperage of 15 mA and a constant v o l t a g e of 250 V. i i i ) D i s c o n t i n o u s Sodium Dodecyl-Sulphate Gel Di s c o n t i n o u s sodium d o d e c y l - s u l p h a t e (SDS) (Biorad, C a l i f o r n i a ) p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s , a technique f o r s e p a r a t i o n of macromolecules on the b a s i s 57 of molecular weight, was m o d i f i e d by Laemmli (1970) from the SDS g e l developed by Weber and Osborn (1969, 1975). The components of the 20% acrylamide g e l were as f o l l o w s : 20 ml of acrylamide s o l u t i o n (30% acrylamide monomer and 0.8% N-N'-methylene-bisacrylamide), 7.5 ml of 1.5 M T r i s c h l o r i d e s o l u t i o n pH 8.8, 300 ul of 10% SDS, 10 M1 of TEMED and 2.15 ml of d i s t i l l e d water. The g e l s o l u t i o n was degassed under vacuum f o r 4 min and 150 jul of 10% ammonium persulphate was added to the s o l u t i o n which was then t r a n s f e r r e d to the s l a b g e l assembly. Isobutanol ( F i s h e r , New Jersey) was c a r e f u l l y l a y e r e d on top of the ge l s o l u t i o n to ensure an even g e l s u r f a c e d u r i n g the 3 h p o l y m e r i z a t i o n and removed a f t e r completion of p o l y m e r i z a t i o n . M a t e r i a l s f o r 5% acrylamide s t a c k i n g g e l were as f o l l o w s : 2.5 ml of acrylamide s o l u t i o n (30% acrylamide monomer and 0.8% N-N'-methylene-bisacrylamide i n water), 1.88 ml of 0.5 M of T r i s HC1 s o l u t i o n pH 6.8, 150 y l of 10% SDS (Biorad, C a l i f o r n i a ) , 7.5 ul of TEMED and 10.32 ml of d i s t i l l e d water. The g e l s o l u t i o n was degassed f o r 4 min, 150 M1 of 10% ammonium persul p h a t e was then added before o v e r l a y i n g on the polymerized s e p a r a t i n g g e l . P e p t i d e s , weighing 10-30 ixq, were d i s s o l v e d i n 10-30 58 Ml of sample b u f f e r (0.0625 M T r i s HC1 pH 6.8, 2% SDS, 10% g l y c e r o l , 5% /3-mercaptoet.hanol and 0.001% bromophenol blue i n water) and a p p l i e d to the sample s l o t s . E l e c t r o p h o r e s i s was performed with an e l e c t r o d e b u f f e r (0.025 M T r i s c h l o r i d e , 0.192 M g l y c i n e and 0.1% SDS and pK a d j u s t e d to 6.8) and c a r r i e d out f o r 8 h at an amperage of 25 mA and constant v o l t a g e of 160 V. iv) Sodium Dodecyl-Sulphate Urea Gel Sodium dodecyl sulphate (SDS) urea p o l y c r y l a m i d e g e l was designed f o r the s e p a r a t i o n of o l i g o p e p t i d e s (Lagunoff and P r i t z l (1976), Swank and Munkers (1971)) on the b a s i s of d i f f e r e n c e s i n molecular weights. P r e p a r a t i o n of the g e l i n v o l v e d the mixing of 10.32 ml of acrylamide s o l u t i o n (38.7% acrylamide monomer {w/v} and 3.8% N-N-rnethylene b i s a c r y l a m i d e {w/v} i n water), 40 ml of 1% SDS i n 1.0 M T r i s phosphate b u f f e r pH 5.0, 19.2 g urea (Biorad, C a l i f o r n i a ) , 15 mg of ammonium per s u l p h a t e and d i s t i l l e d water made up to a f i n a l volume of 40 ml (A h l r o t h and Mutt (1970)). The pH was a d j u s t e d to 6.8 with 2.0 M T r i s base before the a d d i t i o n of 15 M1 of TEMED. The s o l u t i o n was degassed f o r l min before pouring i n t o pyrex tubing 5 mm i n diameter and 120 mm i n l e n g t h . 59 The g e l s u r f a c e s were o v e r l a i d with water d u r i n g the 3 h r e q u i r e d f o r p o l y m e r i z a t i o n , and removed when p o l y m e r i z a t i o n was completed. L y o p h i l i z e d p e p t i d e s , weighing 20-50 uq, were d i s s o l v e d i n 40 M1 of sample b u f f e r (0.01 M H 3 P O „ , 1% SDS, 1% bromophenol blue, 8.0 M urea, pH a d j u s t e d to 6.8 with T r i s base and 1% j3-mercaptoethanol f r e s h l y added). A volume of 10 ul of 70% sucrose was added to the sample s o l u t i o n f o l l o w e d by a 10 min i n c u b a t i o n at 60C. The sample s o l u t i o n were allowed to c o o l to room temperature before a p p l y i n g to the sample s l o t s . E l e c t r o l y t e b u f f e r (0.1 M H 3 P O A , 0.1% SDS, pH a d j u s t e d to 6.8 with T r i s base) was employed duri n g the e l e c t r o p h o r e s i s which was c a r r i e d out at 1.6 mA/gel f o r 15-20 h. A f t e r e l e c t r o p h o r e s i s , the g e l s were removed from the tubings and subjected to s t a i n i n g or v a r i o u s e x t r a c t i o n procedures. b) S t a i n i n g Procedures i ) Coomassie Blue with T r i c h l o r o a c e t i c A c i d P r e c i p i t a t i o n S t a i n i n g was c a r r i e d out f o l l o w i n g p r e c i p i t a t i o n of the p e p t i d e s by a g i t a t i o n i n 50% t r i c h l o r o a c e t i c a c i d 60 (TCA) f o r 30 min i n the g e l matrix. The g e l was then t r a n s f e r r e d to a s t a i n i n g s o l u t i o n c o n t a i n i n g 0.05% Coomassie blue R-250 (B i o r a d , C a l i f o r n i a ) i n 12.5% TCA f o r 1 h, followed by d e s t a i n i n g i n 10% TCA ov e r n i g h t (Chrambach et a l . (1967)). i i ) Coomassie Blue with Methanol and A c e t i c A c i d  Prec i p i t a t i o n A f t e r the completion of the e l e c t r o p h o r e s i s , the s l a b g e l was s t a i n e d i n 0.25% Coomassie blue R-250 i n methanol:acetic acidrwater (5:1:5 {vol}) f o r 2 h. The g e l was d e s t a i n e d in methanol:acetic acid:water (2:1:5 {vol}) o v e r n i g h t with a g i t a t i o n . 2. Thin Layer Chromatography Thin l a y e r chromatography (TLC), a technique based on p a r t i t i o n chromatography, was employed i n the s e p a r a t i o n of p e p t i d e s . P e p t i d e s , weighing 20-60 uq, were d i s s o l v e d i n 20-30 M1 of water and c a r e f u l l y a p p l i e d as spots of 3 mm diameter using 10 M1 d i s p o s a b l e p i p e t t e s ( M i c r o p i p e t t e , DADE, F l o r i d a ) onto a s i l i c a TLC p l a t e ( S i l i c a r , TLC-7GF, M a l l i n c k r o d t ) . The p l a t e s were a i r 61 d r i e d then p l a c e d i n a se a l e d 22x22x9 cm chromatographic tank (Dessago, H e i d e l b e r g , West Germany) and developed i n a s o l v e n t mixture of n-butanol ( F i s h e r , New J e r s e y ) , a c e t i c a c i d ( A r i s t a r , England), p y r i d i n e ( F i s h e r , New Jersey) and d i s t i l l e d water i n a r a t i o of 5:1:3.4:4 volume f o r 8 h at 22C. When the sol v e n t f r o n t had reached to w i t h i n 1.0 cm of the top of the p l a t e , i t was p l a c e d in a fume hood f o r d r y i n g . D e t e c t i o n of the peptide was accomplished by spray i n g the TLC p l a t e with 0.5% n i n h y d r i n ( P i e r c e , I l l i n o i s ) i n acetone with 3 ml of 0. 67% cadium a c e t a t e i n a 34% g l a c i a l a c e t i c a c i d s o l u t i o n ( v / v ) . A 4-5 h d r y i n g p e r i o d was r e q u i r e d before the completion of the c o l o u r development. The d i s t a n c e t r a v e l l e d by the peptide was measured and expressed as a r a t i o to the d i s t a n c e t r a v e l l e d by the so l v e n t f r o n t (Rf v a l u e ) . C. Amino A c i d Composition and Sequence A n a l y s i s 1. Enzymatic degradation P e p t i d e s , weighing 1-2 mg, were d i s s o l v e d i n 250-500 ul of d i s t i l l e d water i n an a c i d washed Pyrex t e s t tube (12x75 mm). An equal volume of 2% NH 4HC0 3 (Mc/B, Ohio) was added to make a f i n a l peptide c o n c e n t r a t i o n of 2 62 mg/ml. The enzymes, d i s s o l v e d i n 1.0% NH<,HC03 were added i n 20 M1 a l i q u o t s . Incubation was c a r r i e d out at 22C. Upon the completion of the i n c u b a t i o n p e r i o d , the s o l u t i o n was l y o p h i l i z e d . The peptide mixture was r e d i s s o l v e d i n 500 M1 of d i s t i l l e d water and the enzymes denatured by h e a t i n g f o r 6 min i n a b o i l i n g water bath. The sample was then r e l y o p h i 1 i z e d f o r f u r t h e r i n v e s t i g a t i o n . a) T r y p s i n and Chymotrypsin An a l i q u o t of 20 y l of 2.0 mg Of t r y p s i n (TPCK t r e a t e d Worthington, O n t a r i o ) or chymotrypsin (Worthington, O n t a r i o ) i n 1.0% NH„HC0 3 was added to a s o l u t i o n c o n t a i n i n g 2 mg of p e p t i d e at time 0, 2 h, 4 h, and the r e a c t i o n was allowed to proceed f o r an a d d i t i o n a l 2 h at 22C before l y o p h i 1 i z a t i o n . The enzyme was i n a c t i v a t e d by b o i l i n g and the mixture c e n t r i f u g e d . The supernatant was l y o p h i l i z e d and the p e p t i d e s produced by the enzymatic degradation were separated e i t h e r on high v o l t a g e e l e c t r o p h o r e s i s or by column chromatography. 63 2. Chemical Cleavage and M o d i f i c a t i o n Two m i l l i g r a m s of pept i d e d i s s o l v e d i n 200 M1 of 70% formic a c i d ( F i s h e r , New Jer s e y ) were p l a c e d i n a 250 ml f l a s k . Two m i l l i l i t r e s of a s o l u t i o n c o n t a i n i n g 10 mg/ml of cyanogen bromide (Eastman, Rochester) i n 70% formic a c i d were added to the peptide s o l u t i o n to give a f i n a l peptide:cyanogen bromide (CNBr) molar r a t i o of 1:470. The r e a c t i o n was allowed to proceed f o r 6 h at 22C (Gross and Witkop (1961), Gross and Witkop (1962)). A f t e r , the completion of the i n c u b a t i o n p e r i o d , 40 ml of d i s t i l l e d water was added to the r e a c t i o n mixture which was then l y o p h i l i z e d . The s e p a r a t i o n of CNBR t r e a t e d peptide fragments was accomplished on CM-11. a) O x i d a t i o n i ) Hydrogen Peroxide Peptide, weighing 400 /xg, was p l a c e d i n an a c i d washed Pyrex 12x75 mm t e s t tube and d i s s o l v e d i n 10% H 2 0 2 s o l u t i o n i n 0.05 M a c e t i c a c i d to give a f i n a l p e p t i d e c o n c e n t r a t i o n of 1.0 mg/ml (Mutt (1964)). The r e a c t i o n was allowed to proceed f o r 45 min at 22C. Twenty volumes of water were added to the mixture which was then 64 l y o p h i l i z e d . i i ) Performic A c i d Performic a c i d was prepared by the a d d i t i o n of 1.0 ml of 30% H 2 0 2 to 9 ml of 90% formic a c i d ( F i s h e r , New Jersey) at 22C f o r 1 h. Peptide, weighing 500 Mg, vras d i s s o l v e d i n 1.0 ml of per f o r m i c a c i d s o l u t i o n p r e v i o u s l y cooled to 0C and the r e a c t i o n was allowed to continue f o r 4 h at 0C (Moore (1963)). Twenty volumes of water were added to the peptide s o l u t i o n on completion of the experiment, followed by l y o p h i l i z a t i o n . 3. I s o l a t i o n of Peptide Fragments  a) Paper Chromatography Enzyme t r e a t e d p e p t i d e fragments were separated on descending paper chromatography as d e s c r i b e d by Waley and Watson (1954). The products of peptide cleavage, weighing 100-200 Mg, d i s s o l v e d i n 10-20 M1 of water were a p p l i e d to 50 MM paper (Whatman, England) f o r a n a l y t i c a l chromatographic s e p a r a t i o n . The peptide samples were spotted 10 cm from the top of the paper, and 3 cm apart from each other, a f t e r the spots were a i r d r i e d , the 65 paper was t r a n s f e r r e d to a g l a s s chromatographic tank (America O p t i c a l Co., Richmond, C a l i f o r n i a ) 31x31x62 cm equipped with a s t a i n l e s s s t e e l rack, and the tank was l i n e d with f i l t e r paper s a t u r a t e d with s o l v e n t p r i o r to the run. The top of the paper was p l a c e d i n a g l a s s trough and h e l d i n p l a c e by a g l a s s rod. A volume of 200-300 ml of s o l v e n t , n - b u t a n o l : a c e t i c a c i d : p y r i d i n e : H 2 0 (5:1:3.3:4 {v/v}), was c a r e f u l l y i n t r o d u c e d i n t o the g l a s s trough and the paper was developed i n a s e a l e d g l a s s tank at 22C f o r 12-14 h. The s o l v e n t f r o n t was marked and the paper was d r i e d i n the fume hood. D e t e c t i o n of p e p t i d e s i n v o l v e d the s p r a y i n g the n i n h y d r i n s o l u t i o n as d e s c r i b e d e a r l i e r and the d i s t a n c e t r a v e l l e d by the peptide fragments was expressed as a r a t i o of the s o l v e n t f r o n t ( R f ) . Preparatory paper chromatography was prepared s i m i l a r to the a n a l y t i c a l procedures except with some m o d i f i c a t i o n s . Mixtures of p e p t i d e s to be separated, weighing 1-2 mg, were d i s s o l v e d i n 500 p.1 of water and a p p l i e d to the chromatographic paper as a narrow band. A f t e r completion of the run the area of the paper c o n t a i n i n g the peptide fragments was i d e n t i f i e d by c u t t i n g guide s t r i p s from each edge of the band 66 c o n t a i n i n g the samples and s t a i n i n g these W i t h I .-linhvdrin. The p o s i t i o n s of the p e p t i d e s to be e l u t e d were marked and a p p r o p r i a t e s t r i p s of paper were removed. E l u t i o n of the p e p t i d e fragments from the s t r i p s of paper was performed in a covered b u f f e r trough system using 0.2 M a c e t i c a c i d . One end of the paper s t r i p was tapered and the t i p was r e s t e d a g a i n s t the w a l l of a t e s t tube; w h i l s t the other end of the paper s t r i p was p l a c e d between two g l a s s s l i d e s i n the b u f f e r r e s e r v o i r . B u f f e r was poured i n t o the trough, and allowed to migrate because of the c a p i l l a r y a c t i o n produced by the two g l a s s s l i d e s . A f t e r the accumulation of 500 ul of s o l u t i o n , the t e s t tube was removed and the e l u t e d peptide recovered by l y o p h i l i z a t i o n . The e n t i r e e l u t i o n procedure was performed under a Perpex cover and the completeness of e l u t i o n of the peptide was confirmed by the negative r e a c t i o n of the e l u t e d paper s t r i p with n i n h y d r i n . b) High Vol t a g e E l e c t r o p h o r e s i s High v o l t a g e e l e c t r o p h o r e s i s as d e s c r i b e d by Ryle et a l . (1955) was employed f o r the a n a l y t i c a l as w e l l as the p r e p a r a t o r y s e p a r a t i o n of p e p t i d e fragments. The 67 apparatus c o n s i s t e d of a g l a s s chromatographic tank 58x58 cm deep and 24 cm i n width (Chromotank, Shandon, England) in a fume hood. The tank was f i t t e d with a g l a s s b u f f e r trough s i t t i n g on metal supports at the top and a metal rod was p l a c e d i n f r o n t of the g l a s s trough f o r s u p p o r t i n g the paper chromatogram d u r i n g the run. The temperature of the tank was r e g u l a t e d by a continous flow of c o l d water through a c o o l i n g c o i l mounted at the rear of the tank. The power supply was a model PSK-200DC (Canadian Research I n s t i t u t e , Don M i l l s , O n t a r i o ) . Platinum e l e c t r o d e s were used f o r both the cathode ( i n the b u f f e r t r o u g h ) , and the anode (placed i n the b u f f e r at the bottom of the t a n k ) . The b u f f e r was composed of 879 ml of d e i o n i z e d water and 100 ml of p y r i d i n e ( F i s h e r , New J e r s e y ) with pH a d j u s t e d to 6.5 with g l a c i a l a c e t i c a c i d . The c o o l a n t was 92% toluene (Caledon, Ontario) and 8% p y r i d i n e ( v o l / v o l ) (Yamamihro (1964)). The upper r e s e r v o i r and the bottom of the tank were f i l l e d with b u f f e r to a depth of 5 cm. The remaining space i n the tank was f i l l e d with c o o l a n t to l e v e l 5 cm higher than that a t t a i n e d by the paper. The a n a l y t i c a l s e p a r a t i o n of p e p t i d e s was accomplished on a sheet of Whatman 3MM paper (57x46 cm). 68 Peptides to be separated weighing 30-40 Mg, were d i s s o l v e d i n 20-30 M1 of water and a p p l i e d as spots 25 cm from the anode (bottom) and 32 cm from the cathode ( t o p ) . The spots were 2.5 cm a p a r t . An i n d i c a t o r dye mixture c o n s i s t i n g of methylene green (C.I. NO. 42590, F i s h e r , New, Jersey) and e - d i n i t r o p h e n y l l y s i n e (Calbiochem, Los An g e l e s ) , and 10 nmol of an amino a c i d standard mixture which c o n s i s t e d of a s p a r t i c a c i d , s e r i n e , and t a u r i n e (Calbiochem, Los An g e l e s ) , were spotted 2.5 cm and 5 cm r e s p e c t i v e l y from the border of the paper on each s i d e . A f t e r a l l the a p p l i e d spots were d r i e d with c o o l a i r , the paper was p l a c e d between two g l a s s rods., 1 .5 cm from the top of the paper (cathode) and the g l a s s rods were h e l d together by a p o l y v i n y l washer on each s i d e . The paper was wetted with b u f f e r . The e f f e c t of s o l v e n t drag on the a p p l i e d sample was prevented by the simultaneous wetting with b u f f e r on each s i d e of the a p p l i c a t i o n spots. The paper was then t r a n s f e r r e d to a chromatogrphic tank. The top of the paper ( f i t t e d with two g l a s s rods) was p l a c e d i n s i d e the b u f f e r trough and the paper was allowed to hang down over the metal rod. The e l e c t r o p h o r e s i s was c a r r i e d out at a constant v o l t a g e of 4 kV and was terminated a f t e r the i n d i c a t o r 69 dye had reached 3 cm from the metal rod. The chromatogram was d r i e d i n the fume hood before spraying with n i n h y d r i n as d e s c r i b e d in s e c t i o n I I , B2. The d i s t a n c e migrated by each n i n h y d r i n p o s i t i v e sample spot was measured from the p o s i t i o n of the n e u t r a l amino a c i d standards ( t a u r i n e and s e r i n e ) to the p o s i t i o n of the sample spots thus n e u t r a l i z i n g the e f f e c t due to e l e c t r o e n d o s m o s i s . The degree of s e p a r a t i o n was expressed as the r a t i o of d i s t a n c e migrated by the p e p t i d e spot to the d i s t a n c e migrated by the a s p a r t i c a c i d ( e l e c t r o p h o r e t i c m o b i l i t y ) . The procedure f o r p r e p a r a t o r y s e p a r a t i o n of the p e p t i d e mixtures was the same as the a n a l y t i c a l procedure except with two m o d i f i c a t i o n s . Peptide was a p p l i e d i n a narrow band, and guide s t r i p s were cut from each edge adjacent to the sample zone and s t a i n e d with n i n h y d r i n to l o c a t e the p o s i t i o n of the separated p e p t i d e fragments. The procedures f o r the e l u t i o n of peptides from the paper were as d e s c r i b e d e a r l i e r . 4. Amino A c i d A n a l y s i s Determination of the amino a c i d composition of the 70 p e p t i d e s was c a r r i e d out using the Dionex MSB/SS amino a c i d a n a l y z e r k i t (Sunnyvale, C a l i f o r n i a ) . The p u r i f i e d p e p t i d e , c o n t a i n i n g 30-40 nmol was t r a n s f e r r e d to an a c i d washed 15x70 mm tube (Kimax, F i s h e r , New J e r s e y ) , t h i s was f o l l o w e d by the a d d i t i o n of 200 ul of 6.0 M HC1. The h y d r o l y s e s of p e p t i d e s were c a r r i e d out i_n vacuo at 1 10C f o r 22 h. Upon completion of the h y d r o l y s e s , the tubes were allowed to c o o l to room temperature and, c e n t r i f u g e d at 2,500 rpm i n a desk top c e n t r i f u g e . The tubes were subsequently opened and the h y d r o l y s a t e s d r i e d i n a d e s i c c a t o r under continuous vacuum in the presence of NaOH p e l l e t s . The d r i e d h y d r o l y z e d p e p t i d e s were d i s s o l v e d i n sample b u f f e r , pH 2.0 with a sodium ion c o n c e n t r a t i o n of 0.20 M (Femto-Buffer, system 2, Durrum, C a l i f o r n i a ) to a f i n a l c o n c e n t r a t i o n of approximately 5 nmol/20 ul f o r a p p l i c a t i o n to the a n a l y z e r . The amino a c i d s were separated on a s i n g l e column system, u t i l i z i n g a c a t i o n exchange r e s i n DC-5A (Durrum, C a l i f o r n i a ) made up from p o l y s t y r e n e c r o s s l i n k e d with d i v i n y l b e n z e n e . The r e s i n bed was t i g h t l y packed i n t o the column to ensure good r e s o l u t i o n and yet p r o v i d e an adequate flow r a t e and p r e s s u r e . E l u t i n g b u f f e r was pumped through the column at a constant flow r a t e of 12 7 1 ml/h (pressures u s u a l l y 1400-1600 p . s . i . ) . Three d i f f e r e n t b u f f e r s were used to e l u t e amino a c i d s from the column. The i n f o r m a t i o n concerning the sequence, t i m i n g and d u r a t i o n of each b u f f e r and the temperature of the column was s t o r e d i n a programmer (CP-3, Dionex, C a l i f o r n i a ) , which allowed automation throughout the e n t i r e a n a l y s i s (Table I I I ) . N i n h y d r i n (NIN-Solve,. P i e r c e , I l l i n o i s ) with 1.7 ml of t i t a n o u s c h l o r i d e s o l u t i o n ( P i e r c e , I l l i n o i s ) added was used f o r the d e t e c t i o n of the separated amino a c i d s . The n i n h y d r i n flow r a t e was maintained at 12 ml/h (400-500 p . s . i . ) which gave a t o t a l flow r a t e ( b u f f e r and n i n h y d r i n ) of 24 ml/h i n the system. The r e a c t i o n mixture was heated i n a b o i l i n g water bath f o r c o l o u r development and then absorbance was read at 590 nm i n a spectrophotometer (Model 56, Glenco, Texas). Peak areas were recorded and measured using a c h a r t recorder and i n t e g r a t o r (3380A I n t e g r a t o r , Hewlett Packer, C a l i f o r n i a ) . The i d e n t i f i c a t i o n and q u a n t i f i c a t i o n of amino a c i d s i n the sample were based on comparison with 5 nmol a l i q u o t s of an amino a c i d standard mixture ( P i e r c e , I l l i n o i s ) . 72 Table I I I The composition of b u f f e r and the program used i n Dionex MSB/SS amino a c i d a n a l y z e r with DC-5A exchange r e s i n . The column was t i g h t l y packed to a height of 13 cm. B u f f e r Sodium Cone. mol/1 pH Temp °C A A 0.200 0.200 3.25 3.25 45 45 B 0.200 4.25 45 B 0.200 4.25 65 C 1 .000 7.4 65 Regen. A 0.200 3.25 65 45 Duration Purpose (min) 12. 0 E q u i 1 i b r a t ion 23. 5 E l u t i o n of Asp to V a l 8. 0 E l u t i o n of Met to Leu. 9. 5 E l u t i o n of Tyr to Phe. 42. 0 E l u t i o n of His to Arg. 12. 0 Regeneration 7. 0 E q u i l i b r a t i o n 74 5. N-Terminus Determinations The N-terminal amino a c i d r e s i d u e s of. the separated p e p t i d e s were determined using the d a n s y l a t i o n (DNS) procedure (Gray (1967)). A l i q u o t s of pep t i d e s of 1-5 nmol were p l a c e d i n an a c i d washed 4.0x30 mm tube to which 10 ul of 0.1 M NaHC0 3 was added. The tube was c e n t r i f u g e d at 2,500 rpm and then l y o p h i l i z e d . T h i s was followed by the a d d i t i o n of 10.. M1 of d i s t i l l e d water and 5 ul of 2.5 mg/ml of DNS-chloride (Sigma, S t . L o u i s ) , a f t e r which the tube was covered with p a r a f i l m and incubated at 37C f o r 1 h. The da n s y l a t e d peptide was hydr o l y z e d at 110C f o r 18 h f o l l o w i n g a d d i t i o n of 20 M1 of 6.0 M HC1. The tube was opened and the h y d r o l y s a t e d r i e d under vacuum i n the presence of NaOH p e l l e t s . The d r i e d h y d r o l y s a t e was d i s s o l v e d in 2.5 M1 of 50% p y r i d i n e p r i o r to i d e n t i f i c a t i o n by TLC. I d e n t i f i c a t i o n of the d a n s y l a t e d amino a c i d r e s i d u e s was c a r r i e d out on a 5x5 cm TLC polyamide p l a t e (Chen, Chin T r a d i n g Co. Taiwan). A volume of 0.50 M1 of the h y d r o l y s a t e was a p p l i e d as a f i n e spot (placed 1.0 cm from the two s i d e s forming a r i g h t angular edge) on each s u r f a c e of the p l a t e such that the two spots were i n 75 c o rresponding p o s i t i o n s . On one s i d e of the p l a t e , 5 M1 of 5 . 0 rnnol of d a n s y l a t e d amino a c i d standard (Sigma, S t . Lou i s ) c o n t a i n i n g a r g i n i n e , g l y c i n e , glutamic a c i d , i s o l e u c i n e , p h e n y l a l a n i n e , p r o l i n e and s e r i n e (5.0 nmol) d i s s o l v e d i n acetone was a p p l i e d i n the same l o c a t i o n as the sample. The p l a t e was d r i e d before c a r r y i n g out ascending chromatography i n a se a l e d 5.0 cm diameter g l a s s j a r with a s o l v e n t l e v e l of 0.50 cm. The chromatographic run was terminated when the solvent f r o n t had reached 0.5 cm from the top of the p l a t e ; the p l a t e was subsequently d r i e d i n a stream of c o o l a i r . The sol v e n t system used was as f o l l o w s : DIMENSION SOLVENT COMPOSITION 1 1 H 2O:90% formic a c i d (200:3 {v/v}) 2 2 b e n z e n e : g l a c i a l a c e t i c a c i d (9:1 {v/v}) 2 3 h e x a n e : b u t a n o l : g l a c i a l a c e t i c a c i d (3:3:1 {v/v}) 2 4 0.10 M ammonia:ethanol (9:1 {v/v}) 76 A l l p l a t e s (5x5 cm) were developed i n s o l v e n t 1 i n the f i r s t dimension and d r i e d before developing i n so l v e n t 2 i n the second dimension. The d a n s y l a t e d amino a c i d s were i d e n t i f i e d by viewing the p l a t e s under an u l t r a v i o l e t l i g h t source. The s e p a r a t i o n of the da n s y l a t e d amino a c i d p a i r s : DNS-serine and DNS-thre o n i n e , DNS-aspartic a c i d and DNS-glutaminic a c i d , and DNS-glycine and DNS-alanine was accomplished by deve l o p i n g the polyamide p l a t e s i n s o l v e n t 3 f o l l o w i n g s o l v e n t 2 i n the second dimension. Solvent 4 was used to d i s t i n g u i s h DNS-arginine, e-DNS-lysine and a-DNS-h i s t i d i n e f o l l o w i n g s o l v e n t 2 i n the second dimension. C o n f i r m a t i o n of the i d e n t i t i e s of the d a n s y l a t e d amino a c i d r e s i d u e s was accomplished by comparing the r e l a t i v e p o s i t i o n of the sample with the standards on the p l a t e . ANALYSIS OF DATA A l l the r e s u l t s were analyzed using unpaired t - t e s t . 77 RESULTS I PHYSIOLOGICAL STUDIES A. GLUCOSE CLAMP EXPERIMENTS Steady s t a t e hyperglycaemia experiments were performed i n dogs. The degree of s t a b i l i t y of the steady s t a t e glucose clamp was expressed by the c o v a r i a n c e . T h i s was d e f i n e d to be the standard d e v i a t i o n of the mean plasma glucose c o n c e n t r a t i o n d i v i d e d by the mean plasma glucose c o n c e n t r a t i o n . The accuracy of the plasma l e v e l a t t a i n e d d u r i n g the experiment was expressed as the mean plasma glucose d i v i d e d by the d e s i r e d glucose c o n c e n t r a t i o n . Both of these parameters were c a l c u l a t e d from the glucose c o n c e n t r a t i o n 20 min a f t e r s t a r t i n g the intravenous glucose i n f u s i o n u n t i l the end of the experiment. The covar i a n c e of a l l the experiments performed was below 10% and the accuracy was ±3% from the d e s i r e d l e v e l (Table I V ) . The summary of the mean glucose i n f u s i o n r a t e s and mean plasma IRI of a l l the glucose experiments were shown on Table V. 78 Table IV Summary of the e v a l u a t i o n s of the glucose clamp experiments performed i n t h i s study. 'G+x' denoted 'x mg/dl' of plasma glucose maintained above the f a s t i n g l e v e l . 'I.D.' denoted i n t r a d u o d e n a l i n f u s i o n . 'I.V.' denoted intravenous i n f u s i o n . 'Fast.' denoted f a s t i n g . 'Test' denoted the l e v e l of plasma glucose a t t a i n e d . 'Stab.' denoted the s t a b i l i t y of the clamp. 'Accur.' denoted the accuracy of the clamp. A l l r e s u l t s were expressed as mean±SD (standard d e v i a t i o n ) . 79 Exper iments Mean plasma G l u . Clamp E v a l u a t . (mg/dl) ( %) F a s t . Test Stab Accur C o n t r o l G+40 1 02±5 40 2 1 00 G+100 97±6 1 00 5 100 G+150 100±5 1 49 4 100 GIP i n f u s i o n G+40+1 .0 Mg/kg.h 97±8 40 2 100 G+100+1.0 Mg/kg.h 1 01 ±9 1 03 3 101 G+150+1.0 Mg/kg.h 1 0 1 ±6 1 48 4 99 G+150+2.0Mg/kg.h 99±4 151 3 1 00 O r a l Lipomul G+40 103±2 37 3 98 G+1 50 1 08±4 1 49 4 100 o r a l / I . D . glucose G+40+1.D. 95±2 46 5 1 04 G+100+1.D. 93±5 1 06 5 1 03 G+40+oral 1 04±5 38 3 99 G+100+Oral 1 0 1 ± 3 97 6 98 Amino a c i d Mix. G+40+1.V. (20 min) 103±8 39 4 99 G+40+1.V. (40 min) 97±4 38 5 99 G+40+oral 1 04±6 38 3 99 G+40+1.D. 98±3 38 3 . 99 O r a l FreAmine G+40 1 06±1 38 3 101 O r a l amino a c i d G+40+arginine 99±1 36 7 97 G+40+alanine 1 02±4 38 3 99 I.D. HC1 G+40 99±4 39 2 99 80 Table V Summary of the mean glucose i n f u s i o n r a t e s and mean plasma IRI d u r i n g glucose clamp. ' t 2 0 . 6 0 ' denoted time 20 to 60 min, the p e r i o d before the i n f u s i o n of t e s t substances. ' t 6 5 . 1 5 0 ' denoted time 65 to 150 min, the p e r i o d d u r i n g and a f t e r the i n f u s i o n of t e s t substances. 'G+x' denoted 'x mg/dl' of plasma glucose maintained above the f a s t i n g l e v e l . 'I.D.' denoted intraduodenal i n f u s i o n . A l l r e s u l t s were expressed as mean±SD (standard d e v i a t i o n ) . 81 Expsriments C o n t r o l G+40 G+100 G+1 50 GIP i n f u s i o n G+40+1.0 Mg/kg. h G+100+1.0 Mg/kg.h G+150+1.0 Mg/kg.h G+150+2.0 Mg/kg.h O r a l Lipomul G+40 G+1 50 Oral/I.D. Glue. G+40+1,D. G+100+1.D. G+40+oral G+100+oral Amino a c i d Mix. G+40+1.V.(20 min) G+40+1.V.(40 min) G+40+oral G+40+1.D. Or a l FreAmine G+40 O r a l amino a c i d G+40+arginine G+40+alanine I.D. HC1 G+40 Mean G l u . I n f . (mg/kg.min) f-20-60 t 6 5 . 1 5 o Mean plasma IRI (MU/ml) t 2 0 • 60 t 6 5 - 1 5 O 5.2±0.7 12.9±2.6 14.5±5.5 5.9±0.7 17.013.5 22.5±1.0 46±7 132±16 227±24 40±7 183±27 283126 6.211 .6 13.912.4 15.715.5 18.311 .4 9.110.5 17.211.1 23.512.2 22.212.7 7617 1 74120 201119 156125 75 + 9 178117 225126 261164 7.611 17.9+3 13.911 25.5+3 2311 0 225136 2915 337141 8.2+0.9 10.7+1 .8 7.511 .8 12.711 .8 3.4+1 .8 13.412.3 4.511 .6 13.212.8 61110 9611 7 7211 3 207128 6311 4 195142 86+1 3 318151 6.310.8 4.710.3 8.6+1.4 6.710.9 10.2+3.5 10.513.5 12.0+1.0 12.812.2 113122 85120 99124 51110 260127 354194 215195 249163 5.711.0 10.811.9 3819 61112 6.111.6 6.611 .2 12.012.1 11.0+1.7 2914 3519 4211 1 4811 0 6.9+1.0 7.310.8 3415 2814 82 1. Exogenously Administered GIP on IRI Release i n a State  of Euglycaemia and Hyperglycaemia a) Euglycaemia 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 was i n t r a v e n o u s l y a d m i n i s t e r e d at doses of 1.0 and 2.0 Mg/kg.h (n=3) f o r a 1 h d u r a t i o n . No s i g n i f i c a n t change i n the l e v e l s of plasma glucose and plasma IRI was observed between the p r e - i n f u s i o n p e r i o d and the i n f u s i o n p e r i o d f o r both dosages of GIP ( F i g . 4 and 5). The plasma IR-GIP l e v e l s (expressed as meantSEM) du r i n g the i n f u s i o n of 1.0 Mg/kg.h of GIP in c r e a s e d from a bas a l value of 215±21 pg/ml to a peak of 1067±240 pg/ml 60 min a f t e r the beginning of the i n f u s i o n . The l e v e l s r e t u r n e d to bas a l 15 min a f t e r the c e s s a t i o n of the i n f u s i o n . When 2.0 Mg/kg.h of GIP was admin i s t e r e d i n t r a v e n o u s l y the IR-GIP became, e l e v a t e d from a b a s a l value of 125±100 pg/ml to a peak of 2080±562 pg/ml 50 min a f t e r the beginning of the i n f u s i o n , which then returned to the p r e i n f u s i o n s t a t e 15 min a f t e r c e s s a t i o n of the i n f u s i o n . b) Steady State Hyperglycaemia Maintained at G+40 When steady s t a t e hyperglycaemia was maintained at G+40 (n=7), plasma IRI l e v e l s (expressed as meantSEM) 83 F i g . 4 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 i n f u s i o n s t u d i e s i n euglycaemic s t a t e . Plasma glucose, IRI and IR-GIP l e v e l s f o l l o w i n g i . v . i n f u s i o n of 1.0 Mg/kg.h of GIP (n=3) at time 0 min over a 60 min p e r i o d i n euglycaemic s t a t e . A l l r e s u l t s were expressed, as meaniSEM. Plasma glucose (mg/dl) Plasma IR - GIP (pg/ml) Plasma IRI (/JU/ml) 00 85 F i g . 5 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 i n f u s i o n s t u d i e s i n euglycaemic s t a t e . Plasma glucose, IRI and IR-GIP l e v e l s f o l l o w i n g i . v . i n f u s i o n of 2.0 Mg/kg.h of GIP (n=3) at time 0 min over a 60 min p e r i o d i n euglycaemic s t a t e . A l l r e s u l t s were expressed as meantSEM. 86 j. 2.0 /jg/kg.h GIP •o N § 5 0 -« CO O O O E CO o QL £ 0- T -30 0 +30 —r~ 90 mm 2.0 /jg/kg.h GIP 3000 i — r -30 0 *30 2.0 / j g / k g . h GIP | 100-cr »—* o E (0 o OL 50-o - t -- 3 0 i r— 0 +30 9 0 mm mm. 87 i n i t i a l l y became e l e v a t e d from the b a s a l s t a t e of 15±2 MU/ml to 47±7 yU/ml durin g t 2 0 . 6 o (20 min to 60 min), and then decreased to 40±7 yuU/ml d u r i n g t 6 5 . 1 5 0 ( 65 min to 150 min) ( F i g . 6). T h i s suggested that 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 the mean plasma IRI d u r i n g t 2 o - 6 o and t 6 5 . i 5 0 (p>0.05). When GIP at a dose of 1.0 ixg/kg.h was i n f u s e d commencing at time 60 min f o r 1 h d u r a t i o n i n 5 dogs, there was a l s o no s i g n i f i c a n t d i f f e r e n c e between the mean plasma IRI l e v e l s d u r i n g t 2 0 . 6 o and t 6 5 - i 5 o ( F i g . 7 ) . The mean glucose i n f u s i o n r a t e f o r the maintenance of the steady s t a t e hyperglycaemia alone (5.2±0.7 mg/kg.min durin g t 2 0 - 6 o and 5.9±0.7 mg/kg.min du r i n g t E 5 . 1 5 0 ) , was a l s o unchanged and 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 r a t e of glucose i n f u s i o n was observed before and d u r i n g the GIP i n f u s i o n of 1.0 jig/kg.h. Serum IR-GIP remained unchanged d u r i n g the intravenous glucose i n f u s i o n , unless exogenous GIP was a d m i n i s t e r e d i n t r a v e n o u s l y . c) Steady State Hyperglycaemia Maintained at G+100 Steady s t a t e hyperglycaemia maintained at G+100 (n = 5) r e s u l t e d in a mean plasma IRI s e c r e t i o n of 132±16 88 F i g . 6 Hyperglycaemic clamp maintained at G+40. Plasma glucose, IRI and glucose i n f u s i o n r a t e d u r i n g G+40 (n=7). A l l r e s u l t s were expressed as meaniSEM. A Plasma glucose (mg/d l ) Glucose infusion (mg/kg. min.) { + ro - _ ro cn I O Cn o Cn O , ° ° & ° CD-1 O 90 F i g . 7 E f f e c t of 1.0 Mg/kg.h GIP d u r i n g hyperglycaemia maintained at G+40. Plasma g l u c o s e , IRI and glucose i n f u s i o n r a t e d u r i n g G+40 with i . v . i n f u s i o n of 1.0 Mg/kg.h of GIP at time 60 min over a 60 min p e r i o d (n--5). A l l r e s u l t s were expressed as meaniSEM. Glucose infusion (mg/kg.min.) 92 MU/ml du r i n g t 2 0 . 6 o which i n c r e a s e d to 183±27 MU/ml during t 6 5 . 1 5 0 ( F i g . 8). The mean plasma IRI i n c r e a s e d from 174±20 MU/ml durin g t 2 0 . 6 0 to 178±17 MU/ml du r i n g t 6 5 - i 5 o when 1.0 Mg/kg.h GIP was i n f u s e d (n=5) ( F i g . 9). The d i f f e r e n c e s between the mean plasma IRI l e v e l s ( during the p e r i o d t 6 5 . 1 5 0 ) i n the two se t s of experiments were not s t a t i s t i c a l l y d i f f e r e n t (p>0.05). In summary, the mean IRI r e l e a s e 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 hyperglycaemia with ' or without exogenous po r c i n e GIP i n f u s i o n at t h i s plasma glucose l e v e l . The mean r a t e of glucose i n f u s i o n to maintain the steady s t a t e hyperglycaemia was 12.9±2.6 mg/kg.min du r i n g t - 2 0 - 6 0 which i n c r e a s e d to 17. 0±3.5 mg/kg.min durin g t 6 5 . , 5 0 i n the c o n t r o l experiments and when GIP was admini s t e r e d the mean r a t e was 13.9±2.4 mg/kg.min d u r i n g t 2 0 . 6 0 and 17.2±1.1 mg/kg.min durin g t 6 5 . 1 5 0 . The amount of glucose u t i l i z a t i o n 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 se t s of experiments (p>0.05). d) Steady State Hyperglycaemia Maintained at G+150 Maintenance of steady s t a t e hyperglycaemia at G+150 produced a mean plasma IRI c o n c e n t r a t i o n of 227±24 MU/ml 93 F i g . 8 Hyperglycaemic clamp maintained at G+100. Plasma glucose, IRI and glucose i n f u s i o n r a t e d u r i n g G+100 (n=5). A l l r e s u l t s were expressed as meantSEM. Glucose infusion (mg/kg. min ) A plasma glucose (mg/dl) CO -* o 95 F i g . 9 E f f e c t of 1.0 Mg/kg.h GIP durin g hyperglycaemia maintained at G+100. Plasma glucose, IRI and glucose i n f u s i o n r a t e d u r i n g G+100 with i . v . i n f u s i o n of 1.0 Mg/kg.h of GIP at time 60 min over a 60 min p e r i o d (n=5). A l l r e s u l t s were expressed as meantSEM. 97 du r i n g t 2 o - 6 o and 283±26 MU/ml durin g t e 5 . 1 5 o (p>0.lO) (n=5) ( F i g . 10). When 1.0 Mg/kg.h of GIP was i n f u s e d t h e IRI l e v e l s were found to be 201±19 MU/ml at t 2 0 . e o and 225±26 MU/ml at 6 5 . 1 5 o (n=5) ( F i g . 11). I n c r e a s i n g t h e c o n c e n t r a t i o n of GIP i n f u s e d to 2.0 Mg/kg.h i n 5 dogs r e s u l t e d i n plasma IRI l e v e l s of 156±25 MU/ml durin g t 2 o - e o and 261±64 MU/ml du r i n g t 6 5 . 1 5 0 ( F i g . 12). There was no s t a t i s t i c a l d i f f e r e n c e i n t h e mean plasma I R I l e v e l s between p r e - i n f u s i o n and i n f u s i o n p e r i o d s of 1.0 Mg/kg.h GIP. However, there was a s i g n i f i c a n t i n c r e a s e (p>0.l0) i n I R I s e c r e t i o n between the two p e r i o d s when 2.0 Mg/kg.h of GIP was i n f u s e d and there was a l s o a suggestion of a l a t e e l e v a t i o n of plasma IRI between time 120 min and the end of the experiment. The mean r a t e of glucose i n f u s i o n i n the c o n t r o l experiments was 14.5±5.5 mg/kg.min d u r i n g t 2 0 . 6 o and 22.5±1.0 mg/kg.min du r i n g t 6 5 . 1 5 0 ; and when 1.0 Mg/kg.h GIP was given the mean i n f u s i o n r a t e s were 15.7 ± 5.5 mg/kg.min durin g t 2 0 . 6 o and 23.5±2.2 mg/kg.min durin g t 6 5 . , 50 . When GIP i n f u s i o n was i n c r e a s e d to 2.0 Mg/kg.h the mean glucose i n f u s i o n r a t e s were 18.3±1.4 mg/kg.min du r i n g t 2 0 . 6 o and 22.2±2.7 mg/kg.min durin g t 65 . 1 5 0 . 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 glucose i n f u s i o n r a t e s was 98 F i g . 10 Hyperglycaemic clamp maintained at G+150. Plasma glucose, IRI and glucose i n f u s i o n r a t e d u r i n g G+150 (n=5). A l l r e s u l t s were expressed as mean±SEM. 1 00 F i g . 11 E f f e c t of 1.0 Mg/kg.h GIP duri n g hyperglycaemia maintained a t G+150. Plasma g l u c o s e , IRI and glucose i n f u s i o n r a t e d u r i n g G+150 with i . v . i n f u s i o n of 1.0 Mg/kg.h of GIP at time 60 min over a 60 min p e r i o d (n=5). A l l r e s u l t s were expressed as meantSEM. Glucose infusion (mg/kg.min.) A plasma glucose (mg/dl) 1 02 F i g . 12 E f f e c t of 2.0 MgAg.h GIP du r i n g hyperglycaemia maintained at G+150. Plasma glucose, IRI and glucose i n f u s i o n r a t e d u r i n g G+150 with i . v . i n f u s i o n of 2.0 Mg/kg.h of GIP at time 60 min over a 60 min p e r i o d (n=5). A l l r e s u l t s were expressed as mean±SEM. Glucose infusion ( mg/kg. min ) 1 04 found amongst the three d i f f e r e n t groups of experiments. 2. I n s u i i n o t r o p i c A c t i o n of I n t r a d u o d e n a l l y and O r a l l y  Administered Glucose During Hyperglycaemia A glucose load of 0.9 g/kg was a d m i n i s t e r e d o r a l l y over a p e r i o d of 5 min and compared to the same dose given i n t r a d u o d e n a l l y but over a 40 min p e r i o d . The experiments were performed i n the absence and presence of c o n t r o l l e d hyperglycaemia of 40 mg/dl and 100 mg/dl above b a s a l . a) Euglycaemia The plasma glucose l e v e l s f o l l o w i n g o r a l glucose a d m i n i s t r a t i o n were e l e v a t e d from a f a s t i n g value of 106±2 mg/dl to a peak of 150±10 mg/dl at time 115 min (n=5). T h i s e l e v a t i o n of plasma glucose was s u s t a i n e d and decreased g r a d u a l l y to 130±7 mg/dl at time 180 min before r e t u r n i n g to the f a s t i n g l e v e l at time 200 min ( F i g . 13). Intraduodenal glucose a d m i n i s t e r e d to 6 dogs e l e v a t e d the plasma glucose from a f a s t i n g value of 102±3 mg/dl to a peak of 171±17 mg/dl at time 110 min. T h i s e l e v a t i o n of glucose was of short d u r a t i o n and decreased to 116±19 mg/dl at time 145 min before r e t u r n i n g to the f a s t i n g l e v e l at 180 min ( F i g . 14). 1 05 F i g . 13 O r a l glucose l o a d . Plasma glucose, IRI and IR-GIP l e v e l s d u r i n g o r a l glucose of 0.9 g/kg i n 20% dextrose s o l u t i o n and i n f u s e d at time 60 min over a 5 min p e r i o d (n=5). A l l r e s u l t s were expressed as meantSEM. 106 F i g . 14 Intraduodenal glucose l o a d . Plasma glucose, IRI and IR-GIP l e v e l s d u r i n g i n t r a d u o d e n a l glucose of 0.9 g/kg i n 5% dextrose s o l u t i o n pH 7.4, 300 mosmol and 37C i n f u s e d at time 60 min over a 40 min p e r i o d (n=6). A l l r e s u l t s were expressed as mean±SEM. 108 I. d. glucose I.d. glucose 300.0 E X 5 2000 o e o 0. I 0 0 0 I.d. glucose 150 -. E \ ID —- 100 -i—< cr M O 5 0 -E CO Pla i 0 o i — 6 0 120 i 1 180 2 4 0 Min. 109 The IRI response to o r a l glucose showed an e l e v a t i o n from a b a s a l l e v e l of 13±4 MU/ml to a peak of 64±15 MU/ml at time 95 min. T h i s e l e v a t i o n decreased g r a d u a l l y to 33±5 MU/ml at time 170 min before r e t u r n i n g to the pre-s t i m u l a t i o n l e v e l at time 190 min. Intraduodenal glucose a d m i n i s t r a t i o n e l e v a t e d the IRI from a mean bas a l l e v e l of 13±2 MU/ml to a peak of 96±17 MU/ml at time 85 min. The r a t e of i n c r e a s e was much g r e a t e r than when given v i a the o r a l route. The plasma IRI then decreased to 40±10 MU/ml at time 130 min before r e t u r n i n g to b a s a l l e v e l s at t i nie. 1 4 0 m i n. The IR-GIP response to glucose a d m i n i s t e r e d o r a l l y showed an i n c r e a s e from b a s a l l e v e l s of 309±110 pg/ml to a peak of 3840±702 pg/ml at time 120 min. The plasma IR-GIP decreased to 2460±541 pg/ml at time 180 min reaching <1000 pg/ml at time 200 min. Intraduodenal a d m i n i s t r a t i o n of glucose e l i c i t e d a r i s e of IR-GIP from the f a s t i n g s t a t e of 3431152 pg/ml to a peak of 2496±652 pg/ml at time 110 min and the rate of in c r e a s e was s l i g h t l y g r e a t e r than when glucose was admi n i s t e r e d o r a l l y . Plasma IR-GIP l e v e l s decreased to 1555±626 at time 180 min reaching <1000 pg/ml at time 190 min. 1 10 b) Hyperglycaemia Maintained at G+40 In the presence of a steady s t a t e hyperglycaemia maintained at G+40, the mean IRI f o l l o w i n g the o r a l glucose load was 72±13 MU/ml d u r i n g t 2 0 . 6 o and 86± 1 3 MU/ml durin g t 65 . 15 0 (n = 6) ( F i g . 15). A f t e r the intraduodenal a d m i n i s t r a t i o n of the glucose load i n 6 dogs the l e v e l s were 61±10 MU/ml du r i n g t 2 o - 6 o and 63±14 MU/ml durin g t 6 5 . 1 5 0 ( F i g . 16). The i n c r e a s e of mean IRI between 1 2 o - 6 o and t 6 5 . i 5 0 was not s t a t i s t i c a l l y s i g n i f i c a n t , nor was t h i s mean value d i f f e r e n t from that obtained i n the experiments i n which a steady s t a t e hyperglycaemia alone was produced. There was a l s o no s i g n i f i c a n t d i f f e r e n c e i n mean IRI between the intraduodenal and o r a l route of glucose a d m i n i s t r a t i o n ( F i g . 15 and 16). The glucose i n f u s i o n r a t e s were d i f f e r e n t when glucose was admi n i s t e r e d v i a the intr a d u o d e n a l and o r a l routes a f t e r establishment of a G+40 clamp ( F i g . 15 and 16). The r a t e of glucose i n f u s i o n had to be decreased from 8.2 mg/kg.min at time 65 min, to 1.6 mg/kg.min at time 80 min, to maintain steady s t a t e glycaemia f o l l o w i n g intraduodenal glucose and then at a mean of 2.3 mg/kg.min F i g . 15 O r a l glucose d u r i n g hyperglycaemia maintained at G+40. Plasma glucose, IRI and glucose i n f u s i o n r a t e du r i n g G+40 with o r a l glucose of 0.9 g/kg i n 20% dextrose s o l u t i o n and i n f u s e d at time 60 min over a 5 min p e r i o d (n=6). A l l r e s u l t s were expressed as mean±SEM. 112 Oral glucose Oral glucose F i g . 16 Intraduodenal i n f u s e d glucose d u r i n g hyperglycaemia maintained at G+40. Plasma glucose, IRI and glucose i n f u s i o n r a t e d u r i n g G+40 with intraduodenal glucose of 0.9 g/kg in 5% dextrose s o l u t i o n pH 7.4, 300 mosmol and 37C i n f u s e d at time 60 min over a 40 min p e r i o d (n=9). A l l r e s u l t s were expressed as meaniSEM. 114 I.d. glucose I.d. glucose 1 1 5 between time 85 min to time 130 min before having to be in c r e a s e d to 6.3±2.9 mg/kg.min at time 150 min. I t was necessary to decrease the intravenous glucose i n f u s i o n g r a d u a l l y from 9.2±1.9 mg/kg.min at time 65 min to 3.7±1.9 mg/kg.min at time 90 min f o l l o w i n g the o r a l glucose l o a d . The glucose i n f u s i o n r a t e was maintained at a mean of 3.8±0.7 mg/kg.min from time 95 min to time 150 min and no in c r e a s e i n the r a t e of glucose i n f u s i o n was necessary towards the end of the experiments. The p a t t e r n of IR-GIP r e l e a s e f o l l o w i n g i n t r a d u o d e n a l glucose a d m i n i s t r a t i o n showed a r i s e from 720±223 pg/ml at f a s t i n g to a peak of 2850±644 pg/ml at time 110 min. IR-GIP l e v e l s began to d e c l i n e at time 140 min and reached 1735±319 pg/ml at time 160 min ( F i g . 15 and 16). T h i s p a t t e r n was c o n s i s t e n t with the IR-GIP response i n the absence of steady s t a t e hyperglycaemia. IR-GIP r e l e a s e to o r a l glucose i n c r e a s e d from a mean f a s t i n g l e v e l of 943±547 pg/ml to a peak of 2253±318 pg/ml at time 120 min. The IR-GIP remained e l e v a t e d u n t i l the end of the experiment. The peak IR-GIP was lower than when o r a l glucose was ad m i n i s t e r e d i n the absence of a steady s t a t e hyperglycaemia. However the p a t t e r n of prolonged e l e v a t i o n of IR-GIP f o l l o w i n g o r a l glucose 1 1 6 a d m i n i s t r a t i o n was present i n experiments both with or without steady s t a t e hyperglycaemia. c) Hyperglycaemia Maintained at G+100 A steady s t a t e hyperglycaemia was maintained at G+100 and 0.9 g/kg of glucose was ad m i n i s t e r e d e i t h e r o r a l l y or i n t r a d u o d e n a l l y i n 4 experiments each at time 60 min ( F i g . 17 and 18). The mean plasma IRI was 96±17 MU/ml at 1 2 o , 6 o and 195+.42 MU/ml at t 65 . 1 5 0 f o l l o w i n g i n t r a d u o d e n a l glucose a d m i n i s t r a t i o n (p>0.05). Mean IRI l e v e l s of 207±28 MU/ml at t 2 0 . 6 o and 318±51 MU/ml at t 6 5 . 15 0 were observed f o l l o w i n g o r a l glucose a d m i n i s t r a t i o n (p>0.05). The high mean IRI at t 2 o - 6 o was due to the h y p e r s e c r e t i o n of i n s u l i n by one animal. A f t e r the s t a r t of the in t r a d u o d e n a l a d m i n i s t r a t i o n of glucose i t was necessary to r a p i d l y decrease the intravenous i n f u s i o n of glucose from 14.9±1.7 mg/kg.min at time 65 min to 8.9±1.8 mg/kg.min at time 95 min. At time 120 min the glucose i n f u s i o n was i n c r e a s e d to 15.8±0.5 mg/kg.min. The mean r a t e of intravenous glucose i n f u s i o n r e q u i r e d to maintain the predetermined hyperglycaemia was 10.7+1.8 mg/kg.min d u r i n g t 2 0 . 6o and 1 3 . 4 ± 2 . 3 mg/kg.min during t 6 5 . 1 5 0 f o l l o w i n g intraduodenal 1 17 F i g . 17 O r a l glucose d u r i n g hyperglycaemia maintained at G+100. Plasma glucose, IRI and glucose i n f u s i o n r a t e d u r i n g G+100 with o r a l g l u c o s e of 0.9 g/kg i n 20% dextrose s o l u t i o n and i n f u s e d at time 60 min over a 5 min p e r i o d (n=4). A l l r e s u l t s were expressed as mean±SEM. Oral glucose 1 19 F i g . 18 Intraduodenal glucose i n f u s i o n d u r i n g hyperglycaemia maintained at G+100. Plasma glucose, IRI and glucose i n f u s i o n r a t e d u r i n g G+100 with intraduodenal glucose of 0.9 g/kg i n 5% dextrose s o l u t i o n pH 7.4, 300 mosmol and 37C i n f u s e d at time 60 min over a 40 min p e r i o d (n=4). A l l r e s u l t s were expressed as mean±SEM. 120 121 glucose a d m i n i s t r a t i o n . Square wave hyperglycaemia was maintained, f o l l o w i n g o r a l glucose a d m i n i s t r a t i o n , by the intravenous i n f u s i o n of glucose, at 12.7±1.8 mg/kg.min d u r i n g t 2 o - 6 o and 13.2±2.8 mg/kg.min d u r i n g t 6 5 . 1 5 0 . F o l l o w i n g the i n g e s t i o n of the o r a l glucose load a decrease i n intravenous glucose a d m i n i s t r a t i o n from 16.0±2.4 mg/kg.min at time 65 min to 9.7±1.7 mg/kg.min at time 115 min was necessary. At time 150 min the i n f u s i o n r a t e was i n c r e a s e d to 11.9±2.3 mg/kg.min. 3. I n s u l i n o t r o p i c A c t i o n of O r a l , Intraduodenal and  Intravenous A d m i n i s t r a t i o n of Amino A c i d Mixture A d m i n i s t r a t i o n of 30 g of a mixture of ten amino a c i d s (composition shown in Table I) was c a r r i e d out at time 60 min in the presence and the absence of steady s t a t e hyperglycaemia. The amino a c i d load was a d m i n i s t e r e d o r a l l y over a p e r i o d of 20 min, i n t r a d u o d e n a l l y over a p e r i o d of 40 min and over 20 min or 40 min f o r the intravenous route. 1 22 a) Euglycaemia  i ) Plasma Glucose O r a l a d m i n i s t r a t i o n of the amino a c i d mixture e l i c i t e d an i n i t i a l i n c r e a s e i n plasma glucose from 102±3 mg/dl at time 60 min to 106±3 mg/dl at time 85 min (n=6) ( F i g . 19). T h i s was f o l l o w e d by a gradual decrease of plasma glucose to 98±3 mg/di at time 115 min before r e t u r n i n g to the f a s t i n g l e v e l at time 125 min. While the in t r a d u o d e n a l route of a d m i n i s t r a t i o n i n 6 dogs e l i c i t e d no i n i t i a l hyperglycaemia, the plasma glucose began to decrease from 100±3 mg/dl at time 75 min to 93±2 mg/dl at time 130 min before g r a d u a l l y r e t u r n i n g to the f a s t i n g l e v e l at time 170 min ( F i g . 20). The intravenous route of a d m i n i s t r a t i o n over a 40 min p e r i o d e l e v a t e d plasma glucose from 94±3 mg/dl at time 60 rnin to 121 ±9 mg/dl at time 70 min, d e c r e a s i n g to 108±14 mg/dl at time 85 min (n=3) ( F i g . 21). When the i n f u s i o n was c a r r i e d out over a 20 min p e r i o d i n 3 dogs the plasma glucose was e l e v a t e d from 95±8 mg/dl at time 60 min to 110±6 mg/dl at time 70 min before g r a d u a l l y d e c r e a s i n g to 81±6 mg/dl at time 120 min. Plasma glucose had returned to 99±6 mg/dl a f t e r 150 min ( F i g . 21). 123 F i g . 19 I n s u l i n o t r o p i c a c t i o n of o r a l l y a d m i n i s t e r e d amino a c i d mixture. Plasma glucose, IRI and IR-GIP f o l l o w i n g 30 g of amino a c i d mixture i n 1 1 ad m i n i s t e r e d at time 60 min f o r a .period of 20 min (n=6). A l l r e s u l t s were expressed as mean±SEM. 124 Oral amino Acids I25-N Ol 100 I <u 75 </) O O 0 1 50 D E w r l 2 5 — i 1 i I 60 120 180 240 Min. Oral amino acids (1 Oral amino acids ( i 100-H-1 E \ 50 CO ^ 0 1 1 180 2 4 0 Min. o 6 0 n 1 1 120 180 240 Min. 125 F i g . 20 I n s u i i n o t r o p i c a c t i o n of i n t r a d u o d e n a l l y a d m i n i s t e r e d amino a c i d mixture. Plasma glucose, IRI and IR-GIP f o l l o w i n g 30 g of amino a c i d mixture i n 1 1 ad m i n i s t e r e d at time 60 min i n t r a d u o d e n a l l y over a p e r i o d of 40 min (n=6). A l l r e s u l t s were expressed as meantSEM. 126 I.d. amino acids I25 n ~ lOO-i •D N o> E 75 o. 50H o E CO I 25 H 60 120 ~l 1 180 240 Min. I. d. amino acids a . O I CC — *—t E \ c a . (0 ^ o Q. lOOO-i 5 0 0 H — I — 6 0 I 1 1 120 180 2 4 0 Min. I.d. amino acids 100- i • - i Min. 1 27 F i g . 21 I n s u i i n o t r o p i c a c t i o n of i n t r a v e n o u s l y a d m i n i s t e r e d amino a c i d mixture. Plasma glucose and IRI f o l l o w i n g 30 g of amino a c i d mixture i n 1 1 administered at time 60 min over a) a 20 min (n=3) or b) a 40 min p e r i o d (n=3). A l l r e s u l t s were expressed as mean±SEM. 128 ( b ) 1 29 i i ) Plasma IRI The plasma IRI f o l l o w i n g o r a l a d m i n i s t r a t i o n of the amino a c i d mixture was e l e v a t e d from the b a s a l l e v e l of 14±3 MU/ml at time 60 min to a peak value of 43±7 MU/ml at time 85 min ( F i g . 19). The l e v e l s then g r a d u a l l y decreased to 26±8 MU/ml at time 180 min'. Intraduodenal amino a c i d a d m i n i s t r a t i o n e l i c i t e d an e l e v a t i o n of IRI from 17±2 MU/ml at time 60 min to 68±6 MU/ml at time 105 min ( F i g . 20). The l e v e l then decreased to 23±7 MU/ml at time 140 min. The intravenous i n f u s i o n of the amino a c i d mixture over a 40 min p e r i o d e l e v a t e d IRI from the bas a l l e v e l of 15±5 MU/ml at time 60 min to a peak of 166±95 MU/ml at time 95 min, fo l l o w e d by a gradual d e c l i n e to 80±36 MU/ml e l e v a t e d from a b a s a l value of 15±5 MU/ml at time 60 min to 150±92 MU/ml at time 90 min fol l o w e d by a d e c l i n e to 20±5 MU/ml at time 190 min ( F i g . 21). i i i ) Plasma IR-GIP Plasma IR-GIP was r e l e a s e d by the a d m i n i s t r a t i o n of the amino a c i d mixture by both the o r a l and intraduodenal routes ( F i g . 19 and 20). IR-GIP was e l e v a t e d from a b a s a l of 181±56 pg/ml at time 60 min to a peak of 493±121 pg/ml at time 90 min f o r the o r a l a d m i n i s t r a t i o n , and 128±30 130 pg/ml at time 60 min to 382±158 pg/ml at time 90 min. The e l e v a t i o n of IR-GIP was short l i v e d and retu r n e d to the ba s a l l e v e l s w i t h i n 20 min f o r both i n t r a d u o d e n a l and o r a l routes of a d m i n i s t r a t i o n . b) Hyperglycaemia Maintained at G+40 i ) Plasma IRI Intravenous, o r a l and intraduodenal a d m i n i s t r a t i o n of 30 g of the amino a c i d mixture was c a r r i e d out i n the presence of hyperglycaemia maintained at G+40. There was a s t a t i s t i c a l l y s i g n i f i c a n t (p<0.05) e l e v a t i o n of IRI when compared with steady s t a t e hyperglycaemia alone ( F i g . 22, 23, 24 and 25). Intravenous a d m i n i s t r a t i o n of the amino a c i d load over a 40 min p e r i o d e l e v a t e d the mean IRI from 85±20 MU/ml d u r i n g t 2 0 - 6 o to 354±94 MU/ml d u r i n g t 6 5 . 1 5 0 (n=3), whereas intravenous i n f u s i o n over a 20 min p e r i o d e l e v a t e d the mean IRI from 113±22 MU/ml d u r i n g t 2 0 - s o to 260±27 MU/ml d u r i n g t 6 5 . 1 5 0 (n=3). Intraduodenal i n f u s i o n of the amino a c i d s gave a comparable mean plasma IRI response to the o r a l route, i n c r e a s i n g from 51±10 MU/ml during t 2 0 . 6 0 to 249±63 MU/ml at t 6 5 . 1 5 o (n=6). The weakest response was from o r a l a d m i n i s t r a t i o n , f o l l o w i n g 131 F i g . 22 I n s u l i n o t r o p i c a c t i o n of intravenous amino a c i d mixture at G+40. Plasma glucose, IRI and glucose i n f u s i o n r a t e of 30 g of amino a c i d mixture i n 1 1 at time 60 min over a 20 min p e r i o d (n=3). A l l r e s u l t s were expressed as mean±SEM. 132 1 33 F i g . 23 I n s u l i n o t r o p i c a c t i o n of intravenous amino a c i d mixture d u r i n g hyperglycaemia maintained at G+40. Plasma glucose, IRI and glucose i n f u s i o n r a t e of 30 g of amino a c i d mixture i n 1 1 at time 60 min over 40 min p e r i o d (n=3). A l l r e s u l t s were expressed as mean±SEM. 1 35 F i g . 24 I n s u i i n o t r o p i c a c t i o n of intraduodenal i n f u s i o n of amino a c i d mixture d u r i n g hyperglycaemia maintained at G+40. Plasma glucose, IRI and glucose i n f u s i o n r a t e of G+40 with 30 g amino a c i d mixture i n 1 1 37C at time 60 min over a 40 min p e r i o d (n=6). A l l r e s u l t s were expressed as mean±SEM. 136 I.d. amino acids 1 37 F i g . 25 I n s u l i n o t r o p i c a c t i o n of o r a l amino a c i d mixture d u r i n g hyperglycaemia maintained at G+40. Plasma gl u c o s e , IRI and glucose i n f u s i o n r a t e of G+40 with 30 g of amino a c i d mixture i n 1 1 at time 60 min over a 20 min p e r i o d (n=6). A l l r e s u l t s were expressed as mean±SEM. 138 W o o O \ w E o — a. 50n + 25H -25--60 Oral amino acids ~r 0 i— 60 — l 1 120 180 Min. Oral amino acids -60 Q. i—i O i or — H-l E E CL CO — o 0_ 1000 1 500 H o - t --60 I 1 1 •460 120 180 Min. Oral amino acids n ~r~ 0 400 300H e ZD it: M 2 00 100 Oral amino acids -60 0 + 60 -1 1 120 180 Min. • 60 I 1 120... 180 Mm. 139 which the mean plasma IRI was e l e v a t e d from 99±24 MU/ml dur i n g t 2 0 . s o to 215±95 MU/ml du r i n g t 6 5 . 1 5 0 (n=6). i i ) Glucose I n f u s i o n The amount of glucose r e q u i r e d to maintain steady s t a t e hyperglycaemia was s i g n i f i c a n t l y g r e a t e r i n experiments i n which amino a c i d s were i n f u s e d (p>0.05), r e g a r d l e s s of the route by which they were adm i n i s t e r e d , than d u r i n g the steady s t a t e hyperglycaemia alone ( F i g . 22, 23, 24 and 25). Intravenous amino a c i d i n f u s i o n over a 40 min p e r i o d r e q u i r e d that the r a t e of glucose i n f u s i o n had to be i n c r e a s e d from a mean of 4.7±0.3 mg/kg.min durin g t 2 0 . 6 o to 10.5±3.5 mg/kg.min d u r i n g t 6 5 . 1 5 o ( F i g . 23), whereas when the intravenous i n f u s i o n was over a 20 min p e r i o d , the glucose i n f u s i o n r a t e had to be i n c r e a s e d from 6.3±0.8 mg/kg.min du r i n g t 2 o - 6 o to 10.2±3.5 mg/kg.min durin g t 6 5 . 1 5 0 ( F i g . 22). Intraduodenal i n f u s i o n of amino a c i d s n e c e s s i t a t e d that s i m i l a r changes in the r a t e of i n f u s i o n of glucose had to be undertaken, from 6.7±0.9 mg/kg.min d u r i n g t 2 0 . e o to 12.8±2.2 mg/kg.min durin g t 6 5 . 1 5 0 ( F i g . 24). The lowest mean glucose i n f u s i o n r a t e was r e q u i r e d i n the experiments i n 1 40 which the amino a c i d s were ad m i n i s t e r e d o r a l l y ( F i g . 25). The i n f u s i o n rate had to be i n c r e a s e d from 8.6±1.4 mg/kg.min duri n g t 20 . 6 0 to 12.0±1.0 MU/ml du r i n g t 6 5 - 1 5 0 ' i i i ) Plasma IR-GIP There was no change i n serum IR-GIP f o l l o w i n g both o r a l and intr a d u o d e n a l a d m i n i s t r a t i o n of the amino a c i d mixture. 4. O r a l A d m i n i s t r a t i o n of a Commerical P r e p a r a t i o n of  Amino A c i d Mixture A commerically a v a i l a b l e p r e p a r a t i o n of amino a c i d s (Freamine, McGraw Lab, M i s s i s s a u g a , O n t a r i o ) , weighing 8.5 g i n 305 ml (pH a d j u s t e d to 7.0 with NaHC0 3 and 300 mosmol with NaCl) was ad m i n i s t e r e d o r a l l y at 60 min a f t e r the establishment of a steady s t a t e hyperglycaemia of G+40 (n=3). F i v e minutes were allowed f o r a d m i n i s t r a t i o n . The p r e p a r a t i o n e l i c i t e d a s i g n i f i c a n t i n c r e a s e (p>0.05) in IRI when compared to steady s t a t e hyperglycaemia alone ( F i g . 26). The mean IRI i n c r e a s e d from 38±9 MU/ml at t 2 0 . 6 0 to 61±12 MU/ml at t 6 5 . 1 S 0 . During steady s t a t e hyperglycaemia maintained at G+40 the corresponding values were 46±7 MU/ml at t 2 0 . 6 0 and 40±7 MU/ml at 141 F i g . 26 I n s u l i n o t r o p i c a c t i o n of a commercially a v a i l a b l e amino a c i d mixture during hyperglycaemia maintained at G+40. Plasma glucose, IRI, IR-GIP and glucose i n f u s i o n r a t e of G+40 with 8.5 g of Freamine amino a c i d mixture i n 1 1 at time 60 min over a 5 min p e r i o d (n=3). A l l r e s u l t s were expressed as mean±SEM. Oral fre-amine Oral fre—amine £ IOOO n o i 1 43 t 6 5 - 1 5 0 • The glucose i n f u s i o n had to be r a p i d l y i n c r e a s e d a f t e r the o r a l a d m i n i s t r a t i o n of Freamine and i t was nececessary to keep i t at a high l e v e l u n t i l the end of the experiment. The r a t e was i n c r e a s e d from 5.7±1.0 mg/kg.min durin g t 2 0 . to to 10.8±1.9 mg/kg.min du r i n g t 6 5 . 15 0 . The corresponding r a t e s of glucose i n f u s i o n were 5.2±0.7 mg/kg.min du r i n g t 2 0 . 6 0 and 5.9±0.7 mg/kg.min dur i n g t 65 . , 50 , when steady s t a t e hyperglycaemia alone was induced. A s l i g h t e l e v a t i o n of plasma IR-GIP was de t e c t e d f o l l o w i n g o r a l a d m i n i s t r a t i o n of the amino a c i d s . The l e v e l rose from the bas a l of 230±90 pg/ml at time 60 min to 500±180 pg/ml at time 90 min and returned to the ba s a l l e v e l at time 100 min. 5. I n s u i i n o t r o p i c A c t i o n of O r a l l y Administered A l a n i n e  at G+40 O r a l a d m i n i s t r a t i o n of 5 g of a l a n i n e d i s s o l v e d i n 150 ml of 0.9% NaCl d u r i n g a steady s t a t e hyperglycaemia at G+40 produced no s i g n i f i c a n t e l e v a t i o n of plasma IRI (n=6) ( F i g . 27). The mean IRI was 35±9 MU/ml during t 2 0 . 6 0 and 48±10 MU/ml du r i n g t 6 5 . i 5 o , f o l l o w i n g the 1 44 F i g . 27 I n s u l i n o t r o p i c a c t i o n of o r a l a l a n i n e d u r i n g hyperglycaemia maintained at G+40. Plasma glucose, IRI, IR-GIP and glucose i n f u s i o n r a t e of G+40 with 5 g of a l a n i n e d i s s o l v e d i n 150 ml of 0.9% NaCl at time 60 min over a 5 min p e r i o d (n=6). A l l r e s u l t s were expressed as mean±SEM. 145 Oral alanine tt Oral alanine lOOO-i o a: e e 5 CO o a. 500 -60 0 +60 120 Min. 1 46 a l a n i n e . The glucose i n f u s i o n r a t e had to be s i g n i f i c a n t l y i n c r e a s e d f o l l o w i n g the o r a l a d m i n i s t r a t i o n of a l a n i n e , from a mean of 6.2±1.6 mg/kg.min durin g t 2 0 - 6 o to 12.0±2.1 mg/kg.min durin g t 6 5 . 1 5 0 . During steady s t a t e hyperglycaemia alone the r a t e s of glucose i n f u s i o n were 5.2±0.7 mg/kg.min during t 2 0 . 6 0 and 5.9±0.7 mg/kg.min dur i n g t 6 5 - i so • No s i g n i f i c a n t i n c r e a s e i n plasma IR-GIP c o u l d be observed f o l l o w i n g the o r a l a d m i n i s t r a t i o n of a l a n i n e . 6. I n s u i i n o t r o p i c A c t i o n of O r a l l y Administered A r g i n i n e  at G+40 O r a l l y a d m i n i s t e r e d a r g i n i n e (5 g) d i s s o l v e d i n 150 ml of 0.9% NaCl produced no s i g n i f i c a n t i n c r e a s e i n IRI s e c r e t i o n i n the presence of steady s t a t e hyperglycaemia maintained at G+40 (n=6). The mean IRI was i n c r e a s e d from 29±4 MU/ml du r i n g t 2 0 _ 6 o to 42±11 MU/ml durin g t 6 5 . 1 5 0 ( F i g . 28). The glucose i n f u s i o n r a t e had to be i n c r e a s e d from 6.111.6 mg/kg.min du r i n g t 2 0 . 6 o to 12.0±2.1 mg/kg.min dur i n g t 6 5 . 1 5 o , f o l l o w i n g the o r a l a d m i n i s t r a t i o n of 1 47 F i g . 28 I n s u l i n o t r o p i c a c t i o n of o r a l a r g i n i n e d u r i n g hyperglycaemia maintained at G+40. Plasma glucose, IRI, IR-GIP and glucose i n f u s i o n r a t e of G+40 with 5 g of a r g i n i n e d i s s o l v e d i n 150 ml of 0.9% NaCl at time 60 min over a 5 min p e r i o d (n=6). A l l r e s u l t s were expressed as mean±SEM. 148 Oral arginine 1 49 a r g i n i n e to maintain the hyperglycaemia. During steady s t a t e hyperglycaemia alone, the corresponding f i g u r e s f o r glucose i n f u s i o n r a t e s were 5.2±0.7 mg/kg.min durin g t 2 0 . 6 0 and 5.9±0.7 mg/kg.min durin g t 65 . 1 5 0 . No s i g n i f i c a n t e l e v a t i o n of plasma IR-GIP was observed f o l l o w i n g the o r a l a d m i n i s t r a t i o n of 5 g of a r g i n i n e . 7. I n s u l i n o t r o p i c E f f e c t of. O r a l l y Administered. Lipomul  During G+40 A dose of 50 ml of Lipomul was ad m i n i s t e r e d o r a l l y , at time 60 min a f t e r e s t a b l i s h i n g a steady s t a t e hyperglycaemia at e i t h e r 40 mg/dl or 150 mg/dl above b a s a l . F i v e minutes were allowed f o r i n g e s t i o n . a) Plasma IRI When o r a l Lipomul was admin i s t e r e d d u r i n g G+40 (n=6), no s i g n i f i c a n t i n c r e a s e i n IRI c o u l d be observed (p>0.05). The mean IRI was 23±10 MU/ml du r i n g t 2 0 - 6 o and 29±5 MU/ml durin g t £ 5 . 1 5 0 . These values were comparable with those obtained when steady s t a t e hyperglycaemia alone was s t u d i e d ( F i g . 29). In the presence of steady s t a t e hyperglycaemia 1 50 F i g . 29 E f f e c t of o r a l Lipomul on IRI s e c r e t i o n d u r i n g hyperglycaemia maintained at G+40. Plasma glucose, IRI and glucose i n f u s i o n r a t e of G+40 with 50 ml of Lipomul at time 60 min over a 5 min p e r i o d (n=6). A l l r e s u l t s were expressed as meantSEM. 151 Oral lipomul 2500 -, oH 1 1 1 1 -60 0 +60 I20 I80 Min. 1 52 maintained at G+150 i n 5 dogs, i n g e s t i o n of Lipomul induced an in c r e a s e i n IRI r e l e a s e from 225±36 MU/ml during t 2 0 - 6 o to 337±41 juU/ml during t 65 . 1 5 0 (p>0.05), whereas the corresponding values f o r hyperglycaemia alone were 227±24 MU/ml durin g t 2 0 - 6 o and 283±26 MU/ml du r i n g t 6 5 . i 5 o (p>0.1u) ( F i g . 30). b) Glucose I n f u s i o n The mean glucose i n f u s i o n r a t e r e q u i r e d to maintain the hyperglycaemia at 40 mg/dl above ba s a l was 5.2±0.7 mg/kg.min durin g t 2 0 . 6 0 and 5.9±0.7 mg/kg.min d u r i n g t 6 5 . 1 5 0 . In the s e r i e s when Lipomul was ingested at time 60 min, the mean glucose i n f u s i o n r a t e was 7.611.6 mg/kg.min durin g t 2 0 - 6 o and 13.9+1.6 mg/kg.min du r i n g t S 5 . i so ( F i g . 29). The r a t e of glucose i n f u s i o n had to be s i g n i f i c a n t l y i n c r e a s e d f o l l o w i n g the o r a l a d m i n i s t r a t i o n of Lipomul duri n g hyperglycaemia maintained at G+150 (p>0.05) ( F i g . 30). The rate then f e l l to pre-Lipomul l e v e l s a f t e r 60 min. The mean glucose i n f u s i o n r a t e was 17.9±3.2 mg/kg.min durin g t 2 0 . 6 o and 25.5±3.0 mg/kg.min du r i n g t 6 5 . | 5 0 . During hyperglycaemia alone, 150 mg% above b a s a l , the glucose i n f u s i o n r a t e s were 14.5±5.5 mg/kg.min 153 F i g . 30 E f f e c t of o r a l Lipomul on IRI s e c r e t i o n during hyperglycaemia maintained at G+150. Plasma glucose, IRI and glucose i n f u s i o n r a t e of G+150 with 50 ml of Lipomul at time 60 min over a 5 min p e r i o d (n=5). A l l r e s u l t s were expressed as meantSEM. 154 1 55 d u r i n g t 2 o - 6 o and 22.5±1.0 mg/kg.min durin g t 6 5 . 1 5 o c) Plasma IR-GIP O r a l a d m i n i s t r a t i o n of Lipomul d u r i n g G+40 e l e v a t e d plasma IR-GIP from a mean b a s a l l e v e l of 160±113 pg/ml at time 60 min to 1720±686 pg/ml at time 135 min. The e l e v a t e d l e v e l of plasma IR-GIP p e r s i s t e d f o r the d u r a t i o n of the experiment ( F i g . 29). The plasma IR-GIP response to o r a l Lipomul i n the presence of hyperglycaemia maintained at G+150 was not measured because of c o n s e r v a t i o n of GIP antiserum f o r other experiments. 8. I n s u i i n o t r o p i c E f f e c t of Intraduodenal A d m i n i s t r a t i o n  of H y d r o c h l o r i c A c i d IRI and IR-GIP responses to the intraduodenal i n f u s i o n of 50 ml of 0.1 M HC1 over 40 min were s t u d i e d i n the presence and absence of a steady s t a t e hyperglycaemia maintained at G+40. Plasma glucose c o n c e n t r a t i o n s d i d not change f o l l o w i n g the intr a d u o d e n a l a d m i n i s t r a t i o n of HC1 i n the euglycaemic s t a t e (n=4) ( F i g . 31). No demonstrable in c r e a s e i n plasma IRI was observed a f t e r the 1 56 F i g . 31 E f f e c t of h y d r o c h l o r i c a c i d on IRI s e c r e t i o n in the euglycaemic s t a t e . Plasma glucose, IRI and IR-GIP l e v e l s of i n t r a d u o d e n a l i n f u s i o n of 50 ml of 0.1 M HC1 at time 60 min over a 40 min p e r i o d (n=4). A l l r e s u l t s were expressed as meam+SEM. 1 58 a d m i n i s t r a t i o n of HC1. The mean IRI responses to steady s t a t e hyperglycaemia maintained at G+40 were 34±5 MU/ml du r i n g t 2 0 - 6 o and 28±4 MU/ml du r i n g t 6 5 . i 5 o ( F i g . 32). These values were not s i g n i f i c a n t l y d i f f e r e n t from the mean IRI responses to steady s t a t e hyperglycaemia a l o n e . No i n c r e a s e i n glucose i n f u s i o n r a t e was necessary to maintain the steady s t a t e hyperglycaemia when HC1 was int r o d u c e d i n t o the duodenum at time 60 min.. The mean i n f u s i o n r a t e s were 6. 9 ± 1 . 0- mg/kg.min du r i n g t 2 0 . 6 o and 7.3±0.8 mg/kg.min during t 6 5 . 1 5 0 . The values d u r i n g t 6 5 . , 5 0 were comparable to the mean glucose i n f u s i o n r a t e s with hyperglycaemia alone (40 mg/dl above b a s a l ) . No e l e v a t i o n of plasma IR-GIP was observed f o l l o w i n g the i n t r a d u o d e n a l i n f u s i o n of h y d r o c h l o r i c a c i d a l o n e . B. I n t e r a c t i o n of some Intermediates of T r i c a r b o x y l i c  A c i d C y c l e with GIP Six dogs were used f o r the study of the i n s u i i n o t r o p i c a c t i o n of i n t r a v e n o u s l y a d m i n i s t e r e d i n t e r m e d i a t e s of the t r i c a r b o x y l i c a c i d c y c l e i n the absence or presence of 0.6 Mg/kg.h p o r c i n e GIP ( d i s s o l v e d i n 0.9% NaCl) i n t r a v e n o u s l y . GIP was i n f u s e d f o r 2 h f o l l o w i n g a 30 min basal p e r i o d . A l l m e t a b o l i t e s were 159 F i g . 32 E f f e c t of h y d r o c h l o r i c a c i d on i n s u l i n s e c r e t i o n d u r i n g hyperglycaemia maintained at G+40. Plasma glucose, IRI and glucose i n f u s i o n r a t e of G+40 with i n t r a d u o d e n a l i n f u s i o n of 50 ml of 0.1 M HC1 at time 60 min over a 40 min p e r i o d (n=6). A l l r e s u l t s were expressed as meantSEM. Plasma IRI co-1 o 161 i n f u s e d at 33.3 mmol/kg.h at pH 7.4 f o r 30 min. Intravenous a d m i n i s t r a t i o n of s u c c i n i c a c i d , a-k e t o g l u t a r a t e and pyruvate d i d not e l e v a t e plasma IR-GIP l e v e l s ( F i g . 33). The f a s t i n g IR-GIP ranged from 125 to 600 pg/ml. S u c c i n i c a c i d , pyruvate and a - k e t o g l u t a r a t e i n f u s i o n d i d not a l t e r the plasma glucose c o n c e n t r a t i o n or IRI from the f a s t i n g s t a t e ( F i g . 34 and 35), e i t h e r i n the absence or the presence of an intravenous i n f u s i o n of 0.6 Mg/kg.h of GIP. 1 62 F i g . 33 E f f e c t of i . v . i n f u s i o n of i n t e r m e d i a t e s of t r i c a r b o x y l i c a c i d c y c l e on plasma IR-GIP. Tested i n t e r m e d i a t e s i n c l u d e d s u c c i n i c a c i d (• •) (n=4), a-k e t o g l u t a r a t e (A A) (n=4), pyruvate (x x) (n=4) and glucose (o o) (n=4) at 33.3 mmol/kg.h, pH 7.4 at 45 min f o r a 30 min p e r i o d . 164 F i g . 34 E f f e c t of i n f u s i o n of in t e r m e d i a t e s of t r i c a r b o x y l i c a c i d c y c l e on plasma g l u c o s e . a) 0.6 Mg/kg.h of GIP i n f u s i o n (n=3) at time 0 min f o r 2 h. b) 33.3 mmol/kg.h of s u c c i n i c a c i d alone (o o) or with GIP (• •) (n=3). c) 33.3 mmol/kg.h of a - k e t o g l u t a r i c a c i d alone (o o) or with GIP (• •) (n=2). d) 33.3 mmol/kg.h of pyruvate alone (o o) or with GIP (• •) (n=3). A l l r e s u l t s were expressed as meantSEM. Plasma glucose (mg/dl) o 3 Plasma glucose (mg/dl) P lasmo glucose ( m g / d l ) 3 Plasma glucose ( m g / d l ) 166 F i g . 35 E f f e c t of i n f u s i o n of int e r m e d i a t e s of t r i c a r b o x y l i c a c i d c y c l e on plasma IRI. a) 0.6 Mg/kg.h of GIP i n f u s i o n (n=3) from time 0 to 120 min. b) 33.3 mmol/kg.h s u c c i n i c a c i d alone (o o) or with GIP (• •) (n=3). c) 33.3 mmol/kg.h of a - k e t o g l u t a r i c a c i d alone (o o) or with GIP (• •) (n=2). d) 33.3 mmol/kg.h pyruvate alone (o o) or with GIP (• •) (n=3). A l l r e s u l t s were expressed as meantSEM. 167 GIP infusion 50 cr o E ft IA-T^^^^A 0--30 + 30 60 9 0 Min. 1 2 0 00 GIP infusion Succinic acid infusion ( b ) GIP infusion (X Ketoglutaric acid GIP infusion Pyruvate infusion ^ 50 r 90 ... 120 Mm. -30 0 1-30 —I— 60 90 Min. -1 120 ( c ) 168 I I . Peptide P u r i f i c a t i o n And C h a r a c t e r i z a t i o n S t u d i e s  A . P u r i f i c a t i o n of GIP 1. F r a c t i o n a t i o n of EG Stage I on CM-Cellulose Column S t a r t i n g m a t e r i a l EG stage I weighing 50 mg was d i s s o l v e d i n 0.01 M N H 4 H C O 3 at a c o n c e n t r a t i o n of 10 mg/ml and the pH a d j u s t e d to 7.05 with 0.01 M N H 3 . The s o l u t i o n was a p p l i e d to a 1.5x19 cm of CM-Cellulose 11 column and developed i n i t i a l l y with 0.01 M NHi,HC03 pH 7.8 fol l o w e d by 0.2 M of NHflHC03 pH 8.1 at a flow rat e of 120 ml/h (Brown, Mutt and Pederson (1970)). The eluent was c o l l e c t e d as f r a c t i o n s of 5.0 ml and the absorbance was measured at 280 nm through a 1.0 cm l i g h t path. The absorbance column p r o f i l e ( F i g . 36) i n d i c a t e d three d i s t i n c t i v e peaks and the f r a c t i o n s comprising each peak were pooled s e p a r a t e l y and designated as EG II ( A ) , EG II (B) and EG II (C) r e s p e c t i v e l y . 2. F r a c t i o n a t i o n of EG Stage II on Sephadex G-25 EG stage II Fr (B) weighing 15 mg d i s s o l v e d in 1 ml of 0.20 M a c e t i c a c i d was a p p l i e d to a 0.6x120 cm Sephadex G-25 column and developed with 0.20 M a c e t i c a c i d a flow ra t e of 18 ml/h (Brown, Mutt and Pederson 1 69 F i g . 36 Absorbance p r o f i l e of EG I on CM C e l l u l o s e 11. S t a r t i n g m a t e r i a l EG stage I weighing 50 mg d i s s o l v e d i n 5 ml of 0.01 M of NH aHC0 3 and pH a d j u s t e d to 7.05 with 0.01 M NH 3. Column was developed f i r s t with 0.01 M NH„HC0 3 pH 7.8 fo l l o w e d by 0.2 M NH aHC0 3 pH 8.1 at flow r a t e 120 ml/h. The f r a c t i o n s i z e was 5.0 ml and absorbance was measured at 280 nm through a 1.0 cm l i g h t path. 170 171 (1970)). The f r a c t i o n s i z e was 1.5 ml and the absorbance was measured at 280 nm through a 1.0 cm l i g h t path. The absorbance column p r o f i l e ( F i g . 37) i n d i c a t e d a shoulder before the peak, and the f r a c t i o n s comprising the shoulder and the peak were pooled s e p a r a t e l y and d e s i g n a t e d as EG III (A) and EG III (B) (GIP) r e s p e c t i v e l y . 3. F r a c t i o n a t i o n of GIP on CM-Sephadex C25 Column GIP weighing 8.1 mg was d i s s o l v e d i n 1.0 ml of 0.01 M NH„HC0 3 and the pH a d j u s t e d to 7.05 with C0 2 or 0.01 M NH 3. The s o l u t i o n was a p p l i e d to a 0.9x25 cm CM-Sephadex C25 column and developed with 0.01 M NH„HC0 3 _ pH 7.8 at a flow r a t e of 30 ml/h (Brown, F r o s t et a l . (1980)). The eluent f r a c t i o n s of 1.5 ml were c o l l e c t e d and the absorbance was read at 280 nm through a 1.0 cm l i g h t path. The absorbance p r o f i l e i n d i c a t e d a s i n g l e non-symmetrical peak. The peak was d i v i d e d i n t o four f r a c t i o n s and each f r a c t i o n was s e p a r a t e l y l y o p h i l i z e d ( F i g . 38). a) P h y s i o l o g i c a l A c t i o n s of the F r a c t i o n s of GIP Post CM- Sephadex C25 I n h i b i t i o n of 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 1 72 F i g . 37 Absorbance p r o f i l e of EG II on Sephadex G-25. EG stage II weighing 15 mg d i s s o l v e d in 1.0 ml of 0.2 M a c e t i c a c i d was a p p l i e d to the column. The column was developed with 0.2 M a c e t i c a c i d at a flow rate of 180 ml/h. The f r a c t i o n s i z e was 1.5 ml and absorbance was measured at 280 nm through a 1.0 cm l i g h t path. 173 o Fraction Number 174 F i g . 38 Absorbance p r o f i l e of GIP on CM-Sephadex C25. 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 weighing 8.1 mg, d i s s o l v e d in 1.0 ml of NH aHC0 3 pH a d j u s t e d to 7.05 with C0 2, was a p p l i e d to the column. The column was developed with 0.01 M NH,HC03 pH 7.8 at a flow rate of 30 ml/h. The f r a c t i o n s i z e was 1.5 ml and absorbance was measured at 280 nm through a 1.0 cm l i g h t path. 175 W//////,OQ\y\ N H 4 H C 0 , p H 7 S % M 0.18 -i F r a c t i o n N u m b e r 176 in the dog by the four f r a c t i o n s ranged from 31±4% to 40±17% when ad m i n i s t e r e d at a dose of 1.0 M g/kg.h. 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 a c i d i n h i b i t o r y a c t i v i t y amongst the four f r a c t i o n s t e s t e d . Blood samples taken at time 0, 15, and 45 rnin d u r i n g the i n f u s i o n p e r i o d of the f r a c t i o n s a l l showed r i s e s i n IR-GIP with peaks o c c u r r i n g at 45 min. However, the peak value of IR-GIP ranged from 500 pg/ml f o r Fr D to 2900 pg/ml f o r F r . E . 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 o t r o p i c a c t i v i t y i n the i s o l a t e d r a t pancreas system of the f r a c t i o n s was observed (Brown, F r o s t et a l . (1980)). b) Peptide A n a l y s i s of the F r a c t i o n s of EG III Post CM- Sephadex C25 Column The four f r a c t i o n s were s u b j e c t e d to v a r i o u s analyses i n c l u d i n g p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (Johns (1967)), N-terminal amino a c i d r e sidue d e t e r m i n a t i o n (Gray (1967)), amino a c i d composition and t r y p t i c d i g e s t i o n and i d e n t i f i c a t i o n . The four f r a c t i o n s t e s t e d showed no d i f f e r e n c e s on p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s or N-terminal a n a l y s i s . The N-terminal residue was t y r o s i n e . When the f r a c t i o n s underwent 1 77 t r y p t i c d i g e s t i o n f o l l o w e d by high v o l t a g e e l e c t r o p h o r e s i s ( F i g . 39), the s t a i n e d peptide fragments on the chromatogram r e v e a l e d the presence of peptide Tr 1 in f r a c t i o n D, w h i l s t a l l other f r a c t i o n s showed a t y p i c a l GIP t r y p t i c d i g e s t p a t t e r n . The amino a c i d composition s t u d i e s of the f r a c t i o n s showed no s i g n i f i c a n t d i f f e r e n c e s at the s e n s i t i v i t y range s t u d i e d . In the immunoreactivity s t u d i e s , the percentage of IR-GIP i n f r a c t i o n B, C, D and E were found to be 45, 83 ,85 and 80% r e s p e c t i v e l y . 4. P u r i f i c a t i o n of GIP on High Pressure L i q u i d  Chromatography S e v e r a l b u f f e r systems were employed i n high pressure l i q u i d chromatography f o r analyses of GIP. The f i r s t s o l v e n t system was of 70% (v/v) of 40 mM ammonium a c e t a t e , pH 4.0 with 30% (v/v) a c e t o n i t r i l e . The absorbance p r o f i l e f o r GIP at 225 nm ( F i g . 40) i n d i c a t e d three d i s t i n c t i v e peaks with C showing the highest absorbance followed i n order by A and B. The next solvent system c o n s i s t e d of 60% 40 mM ammonium a c e t a t e and 40% et h a n o l . The absorbance p r o f i l e of GIP at 225 nm ( F i g . 41) i n d i c a t e d two merging peaks with a minor peak 178 F i g . 39 T r y p t i c fragments of f r a c t i o n s of EG III post CM Sephadex C-25 column on high v o l t a g e e l e c t r o p h o r e s i s . (1) and (9) are markers. (2) and (8) are standards (10 nmol of a s p a r t i c a c i d , s e r i n e and t a u r i n e ) . (3) i s t r y p s i n . (4) i s 90 ug of f r a c t i o n B. (5) i s 88 ug of f r a c t i o n C. (6) i s 97 Mg of f r a c t i o n D. (7) i s 89 Mg of f r a c t i o n E. 'Tr' denoted the t r y p t i c d i g e s t e d fragment number. 179 • t # • • • # T r 7 # T r 6 § T r 5 9 T r 4 § • • T r 3 # T r 2 T r 1 7 8 9 \ \ 180 F i g . 40 High pressure l i q u i d chromatography p u r i f i c a t i o n of GIP in so l v e n t system of 70% of 40 mM ammonium ac e t a t e pH 4.0 and 30% a c e t o n i t r i l e . Absorbance was c a r r i e d out 225 nm. a) 20 M1 of s o l v e n t . b) 5 Mg of GIP. c) 10 Mg of GIP. d) 20 Mg of GIP. The arrow i n d i c a t e d i n j e c t i o n of sample. 181 182 F i g . 41 High pressure l i q u i d chromatography p u r i f i c a t i o n of GIP i n 60% of 40 mM ammonium ace t a t e pH 4.0 and 40% of a b s o l u t e e t h a n o l . Absorbance was c a r r i e d out at 225 nm. a) 20 M1 of s o l v e n t . b) 5 Mg of GIP. c) 10 Mg of GIP. d) 40 Mg of GIP. The arrow i n d i c a t e d i n j e c t i o n of sample. 184 l e a d i n g . The t h i r d s o l v e n t system c o n s i s t e d of 71% of 0.25 M phosphoric a c i d , pH 2.5 a d j u s t e d with t r i e t h y l a m i n e and 29% a c e t o n i t r i l e . The absorbance p r o f i l e of GIP at 205 nm. ( F i g . 42) i n d i c a t e d two separated peaks with the minor peak e l u t i n g f i r s t from the column. The recovery of the p e p t i d e s from the column i n v o l v e d f i r s t l y o p h i l i z i n g the e l u e n t f o l l o w e d by the removal of phosphate s a l t on Sephadex G-25 column. The percentage of recovery of p e p t i d e s a f t e r the d e s a l t i n g procedure was very low i n comparison to t r i f l u o r o a c e t i c a c i d and a c e t o n i t r i l e s o l v e n t system. F i n a l l y , the f o u r t h s o l v e n t system c o n s i s t e d of 0.1% t r i f l u o r o a c e t i c a c i d with v a r i o u s r a t i o s of H 20 to a c e t o n i t r i l e . A system using these s o l v e n t s p r o v i d e d the best s e p a r a t i o n and recovery of p e p t i d e from the column. The absorbance p r o f i l e at 205 nm i n d i c a t e d GIP to c o n t a i n two peaks with the minor peak e l u t i n g f i r s t from the column ( F i g . 43). The optimal c o n d i t i o n s f o r s e p a r a t i o n were determined to be 68% H 20 and 32% a c e t o n i t r i l e i n 0.1% t r i f l u o r o a c e t i c a c i d . 185 F i g . 42 High pressure l i q u i d chromatography p u r i f i c a t i o n of GIP i n 29% a c e t o n i t r i l e , 71% of 250 mM phosphoric a c i d pH a d j u s t e d to 2.5 with t r i e t h y l a m i n e . Abosrbance was c a r r i e d out at 205 nm. a) 10 M1 of s o l v e n t . b) 5 Mg of GIP. c) 10 Mg of GIP. The arrow i n d i c a t e d i n j e c t i o n of sample. 187 F i g . 43 High pressure l i q u i d chromatography p u r i f i c a t i o n of GIP i n 0.1% t r i f l u o r o a c e t i c a c i d , 68% H 20 and 32% a c e t o n i t r i l e . Absorbance was c a r r i e d out at 205 nm. a) 20 ixl of s o l v e n t . b) 5 jug of GIP. c) 10 uq of GIP. The arrow i n d i c a t e d i n j e c t i o n of sample. 188 189 B. PEPTIDE ANALYSIS 1 . Polyacrylamide Gel E l e c t r o p h o r e s i s a) Johns Method A comparison of GIP with i t s s i d e f r a c t i o n s on po l y a c r y l a m i d e g e l (Johns (1967)) was c a r r i e d out. O c c a s i o n a l analyses had shown that GIP ( I I I ) p r e p a r a t i o n s c o n t a i n e d two components of v a r y i n g c o n c e n t r a t i o n s , i n t e n s i t y , with the minor component m i g r a t i n g f a s t e r than the major component, f o l l o w i n g d i f f u s i o n s t a i n i n g ( F i g . 44). A l l the s i d e f r a c t i o n s c o n t a i n e d m u l t i p l e bands. The i n t e n s i t y of the minor band v a r i e d a l s o with d i f f e r e n t batchs of the GIP ( I I I ) p r e p a r a t i o n used. The e x i s t e n c e of the minor band was e q u i v o c a l , i f the e l e c t r o p h o r e s i s was c a r r i e d out i n short tube g e l s (0.6x7.0 cm) and s t a i n e d and d e s t a i n e d e l e c t r o p h o r e t i c a l l y . b) Panyim C h a l k l e y Method In t h i s p o l y a c r y l a m i d e g e l system which c o n t a i n e d urea (10), the dual band p a t t e r n of GIP was u n e q u i v o c a l l y observed ( F i g . 45). The minor component migrated f a s t e r , r e l a t i v e to the major component, i n t h i s g e l system. 1 90 F i g . 44 Comparison of GIP with i t s s i d e f r a c t i o n on po l y a c r y l a m i d e g e l . 1. 10 Mg EG 11-111 F r . A 2. 40 Mg EG I-I I F r . A 3. 28 Mg GIP 4. 61 Mg EG I-I I F r . C 5. 40 Mg EG 11-111 F r . A 6. 40 Mg EG I-I I F r . A 7. 34 Mg EG I-II F r . C 8. 35 Mg GIP 9. 47 Mg GIP 1 92 F i g . 45 Polyacrylamide-urea g e l e l e c t r o p h o r e s i s of o x i d i z e d GIP. 1. 50 Mg of performic a c i d t r e a t e d GIP and 30 Mg of GIP. 2. 50 Mg of performic a c i d t r e a t e d GIP. 3. 30 Mg of GIP. 4. 40 Mg of H 2 0 2 t r e a t e d GIP and 30 Mg of GIP. 4. 40 Mg of H 2 0 2 t r e a t e d GIP. 5. 40 Mg of GIP. 6. 20 Mg of bovine serum albumin. 193 » 1 2 3 4 5 6 +• 1 94 The p o s s i b i l i t y that the dual band p a t t e r n observed was due to the p r o d u c t i o n of o x i d i z e d and reduced GIP was i n v e s t i g a t e d . GIP. p r e p a r a t i o n s o x i d i z e d with e i t h e r H 2 0 2 or p e r f o r m i c a c i d were analyzed on t h i s p o l y a c r y l a m i d e g e l system. The r e s u l t s i n d i c a t e d that the presence of dual band p a t t e r n and the r e l a t i v e i n t e n s i t y and m o b i l i t y between the major and the minor components remain unchanged when compared to the u n o x i d i z e d GIP ( F i g . 45). 2. Amino A c i d Composition Amino a c i d analyses were c a r r i e d out on GIP (EG I I I ) and the two components obtained from GIP (EG I I I ) on HPLC in the a c e t o n i t r i l e and t r i f l u o r o a c e t i c a c i d s o l v e n t system. The analyses i n d i c a t e d the amino a c i d composition of the major peak of GIP (EG I I I ) post-HPLC to be s i m i l a r to GIP (EG I I I ) , whereas the the amino a c i d composition of the minor peak of GIP post-HPLC had one l e s s t y r o s i n e and a l a n i n e r e s i d u e s . C. Amino A c i d Sequence A n a l y s i s  1. Cyanogen Bromide Cleavage Two m i l l i g r a m s of GIP t r e a t e d with cyanogen bromide were d i s s o l v e d in 1.0 ml of 0.01 M NH«HC0 3, pH a d j u s t e d to 6.4 with C0 2 and a p p l i e d to a 0.5x7.0 cm column of CM-1 95 11. The peptide fragments were e l u t e d with 0.01 M NH„HC0 3 , pH 7.0, 0.01 M NH„HC0 3 , pH 7.8 and 0.2 M NH„HC0 3, in that order. The f r a c t i o n s i z e c o l l e c t e d was 0.9 ml and the absorbance was measured at 280 nm at a path width of 1.0 cm. The absorbance p r o f i l e i n d i c a t e d three peaks ( F i g . 46). The f r a c t i o n s c o n t a i n e d i n each peak were pooled and then l y o p h i l i z e d . Amino a c i d analyses r e v e a l e d that the composition of the peptide i n peak I was s i m i l a r to that of G I P T . ^ (Brown and Dryburgh (1971)). In a d d i t i o n , peak I c o n t a i n e d homoserine which was an end product of the r e a c t i o n of methionine with cyanogen bromide (Brown and Dryburgh (1971)). In a s i n g l e experiment performed, the composition of peak II was comparable to GIP except with lower content i n a s p a r t i c a c i d , i s o l e u c i n e and glutamic a c i d . Peak III was shown to have s i m i l a r composition as G I P i 5 . « 2 sequence ( C - t e r m i n a l ) . D a n s y l a t i o n of the N-terminal amino a c i d r e s i d u e s of peak I, II and III r e v e a l e d t y r o s i n e , t y r o s i n e and a s p a r t i c a c i d (or asparagine) r e s p e c t i v e l y . T h i s was a c o n f i r m a t i o n of the e a r l i e r p u b l i s h e d r e s u l t (Brown (1971)). 196 F i g . 46 Absorbance p r o f i l e of cyanogen bromide t r e a t e d GIP on CM-11 column. Two m i l l i g r a m s of cyanogen bromide t r e a t e d GIP d i s s o l v e d i n 1.0 ml of 0.01 M NHftHC03 pH adj u s t e d to 6.4 with C0 2 and a p p l i e d to a CM-11 column. The column was developed i n i t i a l l y with 0.01 M NHftHC03 pH 7.0, followed by 0.01 M NH„HC0 3 pK 7.8 and f i n a l l y 0.2 M NH„HC0 3. The f r a c t i o n s i z e c o l l e c t e d was 0.9 ml and absorbance was c a r r i e d out at 280 nm. 197 Fraction Number 1 98 Thin l a y e r chromatography s t u d i e s c a r r i e d out i n n-butanol, a c e t i c a c i d , p y r i d i n e and H 20 i n a volume r a t i o of 5:1:3.4:4 re v e a l e d that peak I c o n t a i n e d two components with Rf values of 0.80 f o r the major component and 0.77 f o r the minor. Peak II was s i m i l a r i n Rf value (0.66) with GIP. Peak III c o n t a i n e d one component with Rf of 0.52. Polyacrylamide g e l e l e c t r o p h o r e s i s (Johns (1967)) performed i n 0.6x7.0 cm tube g e l i n d i c a t e d the m o b i l i t y of Peak II to be i d e n t i c a l to GIP, and Peak III to be slower than GIP. Whereas Peak I c o u l d not be d e t e c t e d using t h i s system. 2. Trypti'c D i g e s t i o n Two m i l l i g r a m s of GIP t r e a t e d with t r y p s i n were analyzed on high v o l t a g e paper e l e c t r o p h o r e s i s at pH 6.5. The r e s u l t i n d i c a t e d the presence of s i x to seven n i n h y d r i n p o s i t i v e components ( F i g . 39), t h i s was in agreement with the e a r l i e r s t u d i e s (Brown (1971), Brown and Dryburgh (1971)). The fragments were desi g n a t e d as Tr 1 to Tr 7 s t a r t i n g from the anode. Each peptide was e l u t e d from the paper. Tr 3 was run on a paper chromatography system as o u t l i n e d by Waley and Watson 1 99 (1954) and two components were obtained and designated as Tr 3a and Tr 3b. The separated t r y p t i c fragments were analyzed on TLC in a n-butanol, a c e t i c a c i d , p y r i d i n e and H 20 system (volume r a t i o of 5:1:3.4:4 r e s p e c t i v e l y ) . Most of the t r y p t i c components showed a s i n g l e n i n h y d r i n p o s i t i v e component (Table VI) except Tr 2 which i n d i c a t e d two components of Rf values of 0.65 and 0.59. D. Thin Layer Chromatography Analyses of GIP on t h i n l a y e r chromatography i n d i c a t e d two n i n h y d r i n p o s i t i v e components with a Rf value of 0.66 f o r the major component and 0.62 f o r the minor component. In c o n t r a s t , f r a c t i o n C of GIP p u r i f i e d on HPLC r e v e a l e d a s i n g l e n i n h y d r i n p o s i t i v e component with a Rf value of 0.66 which corresponded to the major component of GIP. I I I . S t r u c t u r e and A c i d I n h i b i t o r y A c t i v i t y  A. Chemical M o d i f i c a t i o n of GIP The tryptophan r e s i d u e s i n GIP have been m o d i f i e d by e i t h e r d i m e t h y l s u l p h o x i d e (Savige and Fontana (1977)) or a - n i t r o p h e n y l s u l p h e n y l c h l o r i d e (Glazer et a l . (1975)). 200 Table VI E l e c t r o p h o r e t i c m o b i l i t y and Rf values of GIP t r y p t i c p e p t i d e s . E l e c t r o p h o r e t i c m o b i l i t y from high v o l t a g e e l e c t r o p h o r e s i s was expressed as r e l a t i v e to a s p a r t i c a c i d standard. Rf values on t h i n l a y e r chromatography of GIP t r y p t i c fragments were expressed as r a t i o of the d i s t a n c e t r a v e l l e d to the s o l v e n t f r o n t . 201 T r y p t i c Frag. E l e c t r o p h . M o b i l . Rf value on E h r l i c h from High Voltage Thin Layered Reaction E l e c t r o p h . Chromatog. Tr t 0.71 Tr 2 0.39 0.59, 0.63 Tr 3a 0.03 0.55 + + + Tr 3b 0.03 0.49 +++ Tr 4 -0.35 0.55 + + + Tr 5 -0.54 0.63 Tr 6 -0.83 0.46 Tr 7 -0.94 0.40 202 The extent of the m o d i f i c a t i o n was confirmed by a s h i f t i n peak u.v. absorbance from 280 nm to 250 nm. The r e l a t i v e a c i d i n h i b i t o r y a c t i v i t y (Table VII) of a-n i t r o p h e n y l s u l p h e n y l c h l o r i d e t r e a t e d GIP (53±7%) was comparable with the GIP c o n t r o l (51±5%) at the same dose, whereas the d i m e t h y l s u l p h o x i d e t r e a t e d (22±5%) was l e s s than the GIP c o n t r o l . The treatment of GIP by dimethylsulphoxide a l s o o x i d i z e d the methionine r e s i d u e to methionine sulphoxide. Reduction of methionine sulphoxide to methionine by i n d o l y l - . 3 - p r o p i o n i c a c i d was c a r r i e d out f o l l o w i n g d i m e t h y l s u l p h o x i d e treatment which y i e l d e d an a c i d i n h i b i t o r y a c t i v i t y of 21% i n a s i n g l e experiment. The l o s s of a c i d i n h i b i t o r y a c t i v i t y by d i m e t h y l s u l p h o x i d e was not r e v e r s e d by i n d o l y l - 3 -p r o p i o n i c a c i d . However the degree of completion of r e d u c t i o n of methionine sulphoxide was not determined. The d i f f e r e n c e i n the a c i d i n h i b i t o r y a c t i v i t y between the two treatments of tryptophan r e s i d u e s c o u l d s t i l l be due to o x i d a t i o n of methionine r e s i d u e s by dimethylsulphoxide which d i d not occur with o~ n i t r o p h e n y l s u l p h e n y l c h l o r i d e (Savige and Fontana (1977), G l a z e r et a l . (1975)). When the methionine r e s i d u e i n GIP was o x i d i z e d by 203 Table VII G a s t r i c a c i d i n h i b i t o r y a c t i v i t y of m o d i f i e d p o r c i n e GIP, s i d e f r a c t i o n s of GIP and s y n t h e t i c GIP to 2.0 Mg/kg.h of 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 . Most of the s t u d i e s were performed as s i n g l e experiments unless otherwise i n d i c a t e d . 204 Poreine GIP Substance i n f u s e d GIP GIP GIP Dosage (Mg/kg.h) 1 .0 2.0 4.0 % Inhib. 51 ±5 61 87 DMSO o x i d i z e d GIP 1.0 DMSO and I PA 1.0 t r e a t e d GIP Hydrogen peroxide 1.0 Mo d i f i e d t r e a t e d GIP GIP Performic a c i d 1.0 t r e a t e d GIP a- n i t r o p h e n y l s u l p h e n y l 1.0 t r e a t e d GIP Iod i n a t e d GIP 1.0 22±5 21 33 20±1 53±1 28 EG I-I I F r . A Side EG 11-111 F r . A f r a c t i o n Desamido III F r . B of GIP Desamido III F r . A 4.0 1 .0 4.0 1 .0 18 24±4 57±4 1 0±1 0 Synthet i c GIP Yan. NY-TM VII 92 Yan. NY-TM VII 26-DE 1-GH430H Yan. NY-TM VII 10-F32-2 Yan. NY-TM VII 26-DE 3-3 Yan. NY-TM IX 54-F3 H43 OH Wuensch 1-38 Wuensch prep. A 1 .0 1 .0 2.0 1 .0 1 .0 5.0 2.0 1 7 6 0±3 6 8 24 8 205 H 2 0 2 and performic a c i d , the a c i d i n h i b i t o r y a c t i v i t y of the m o d i f i e d GIP was reduced from the c o n t r o l value of 51±5% to 33% and 20±1% r e s p e c t i v e l y (Table V I I ) . When the t y r o s i n e r e s i d u e was m o d i f i e d by i o d i n a t i o n using the chloramine-T method (Greenwood and Hunter (1953)), the a c i d i n h i b i t o r y a c t i v i t y of the mod i f i e d GIP was reduced from 51±5% to 28%. However, chloramine-T a l s o o x i d i z e d the methionine r e s i d u e (Schechter et a l . (1975)). B. S y n t h e t i c GIP  1. Peptide A n a l y s i s S y n t h e t i c p r e p a r a t i o n s of GIP produced by the l a b o r a t o r i e s of Wuensch (Germany) and Yanaihara (Japan) were compared with p o r c i n e GIP on poly a c r y l a m i d e g e l e l e c t r o p h o r e s i s , and by paper e l e c t r o p h o r e s i s and amino a c i d compositions of p e p t i d e s produced by enzymatic d e g r a d a t i o n . In p o l y a c r y l a m i d e g e l s t u d i e s (Johns (1967)), the e l e c t r o p h o r e t i c m o b i l i t y of a l l the Yanaihara s y n t h e t i c p e p t i d e s examined was d i f f e r e n t from p o r c i n e GIP, i n a d d i t i o n m u l t i p l e bands were observed i n most of the s y n t h e t i c p r e p a r a t i o n s which was i n d i c a t i v e of h e t e r o g e n e i t y . 2 0 6 When comparisons were made of the t r y p t i c p e ptides of s y n t h e t i c p r e p a r a t i o n s by Yanaihara with p o r c i n e GIP on h i g h v o l t a g e paper e l e c t r o p h o r e s i s , the e l e c t r o p h o r e t i c m o b i l i t i e s of some of the fragments of the s y n t h e t i c p r e p a r a t i o n s corresponded to p o r c i n e GIP, but a l l s y n t h e t i c p r e p a r a t i o n s contained a d d i t i o n a l fragments which were not present i n p o r c i n e GIP. The Rf values of some of the pe p t i d e fragments ob t a i n e d by the t r y p t i c d i g e s t i o n of the s y n t h e t i c G I P 1 . 3 8 (Wuensch) corresponded to those of p o r c i n e GIP. However there were more pept i d e fragments i n the s y n t h e t i c GIP than p o r c i n e GIP. ( F i g . 39 and 47). A s y n t h e t i c fragment, GIP,.!, (Wuensch) was t r e a t e d with cyanogen bromide. The cleavage product was f u r t h e r d i g e s t e d with chymotrypsin, without i n i t i a l p u r i f i c a t i o n . The fragments were separated on high v o l t a g e paper e l e c t r o p h o r e s i s and i t was observed that two out of the three n i n h y d r i n p o s i t i v e spots had i d e n t i c a l Rf values and amino a c i d compositions with those of chymotrypsin t r e a t e d p o r c i n e G I P , . ^ ( F i g . 48). 207 F i g . 47 High v o l t a g e e l e c t r o p h o r e s i s of t r y p t i c fragments of Wuensch s y n t h e t i c GIP. (1) and (6) are markers. (2) and (5) are standards (10 nM of a s p a r t i c a c i d , s e r i n e and t a u r i n e ) . (3) i s s y n t h e t i c Wuensch GIP (1-38). (4) i s t r y p s i n . # 1 2 3 4 5 6 1 + 209 F i g . 48 Comparison of amino a c i d composition of chymotrypsin t r e a t e d N-terminal fragment of Wuensch s y n t h e t i c GIP (1-13) with p o r c i n e GIP. The complete sequence of GIP (1-13) i s shown. The N-terminal amino a c i d of each fragment i s shown o u t s i d e the p a r e n t h e s i s . 1 1 0 Tyr-Ala-Glu-Gly-Thr-Phe-I le-Ser Asp Tyr-Ser I le-Ala A A I Chymotrypsin Frag, Wiinsch Synth, peptide Natural peptide 1 Tyr(Ala,Glu,Gly,Thr,Phe) Tyr(Ala,GIU,Gly,Thr,Phe) 2 (lle.Thr.Ala.Ser) |le(ser,Asp,T yr) 3 Ser(lle,A1a) (ser.lle.Ala) 21 1 2. P h y s i o l o g i c a l A c t i v i t y The g a s t r i c a c i d i n h i b i t o r y a c t i v i t y of a l l the s y n t h e t i c p e p t i d e s s t u d i e d (Yanaihara and Wuensch) ranged from 0 to 30% ( t a b l e V I I ) . At equal doses on a weight b a s i s , a l l the s y n t h e t i c p r e p a r a t i o n s had lower a c t i v i t y than p o r c i n e GIP ( F i g . 49 and 50). C. Side F r a c t i o n of EG I I I F r . A F o r t y m i l l i g r a m s of EG I I I (A), a s i d e f r a c t i o n o b t ained d u r i n g the p u r i f i c a t i o n of GIP on Sephadex G-25, were d i s s o l v e d i n 5.0 ml of 0.01 M NH„HC0 3 pH a d j u s t e d to 7.05 with 0.01 M NH 3 and a p p l i e d to a 1.5x19 cm CM-11 column. The p e p t i d e s were e l u t e d from the column using 0.01 M NH„HC0 3 pH 7.8 at a flow r a t e of 120 ml/h. F r a c t i o n s of 3.0 ml volume were c o l l e c t e d and the absorbance measured at 280 nm through a 1.0 cm l i g h t path. A f t e r p l o t t i n g the absorbance p r o f i l e , the f r a c t i o n s under the main peak were pooled, l y o p h i l i z e d and designated as Desamido I I . Further p u r i f i c a t i o n of t h i s s i d e f r a c t i o n i n v o l v e d g e l f i l t r a t i o n on columns of e i t h e r Sephadex G-25 or G-50 f i n e . Eleven m i l l i g r a m s of Desamido II d i s s o l v e d in 1.0 212 F i g . 49 G a s t r i c a c i d i n h i b i t o r y a c t i v i t y of 2.0 Mg/kg.h of s y n t h e t i c GIP (Yanaihara) and p o r c i n e GIP t o 2.0 Mg/kg.h of p e n t a g a s t r i n - s t i m u l a l t e d a c i d s e c r e t i o n (• • ) . Tested p r e p a r a t i o n s i n c l u d e d NY TM IX 54 No. 2 F3 H-430H (o o ) , NY TM IV 54-1-43-OH DE 2-2 LH 60 E3 (A A) and p o r c i n e GIP (A A ) . A l l s t u d i e s were s i n g l e experiment. 2.0/jq/kg.h Synthetic GIP or Natural GIP 1 2.0 /jg/kg.h Pentagastrin 1 r 1 T 1 -45-30 0 3 0 6 0 9 0 105 Time in 15 mia interval after acid plateau 214 F i g . 50 G a s t r i c a c i d i n h i b i t o r y a c t i v i t y of 2.0 Mg/kg.h of s y n t h e t i c GIP (Wuensch) and p o r c i n e GIP to 2.0 Mg/kg.h of 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 (• • ) . Tested p r e p a r a t i o n s i n c l u d e d sample A (o o ) , sample B (A A) and p o r c i n e GIP (A A ) . A l l s t u d i e s were s i n g l e experiment. 215 3 Q. O - g o a £ E X o 110-IOOH 9 0 -8 0 -7 0 -6 0 ^ 5 0 -4 0 -3 0 -2 0 -10-0 -20/jg/kg.h Synthetic GIP| or Natural GIP 2 .0 / jg /kg .h Pentagastrin 1 1 - 4 5 - 3 0 0 3 0 6 0 9 0 105 Time in 15 min. interval after acid plateau 216 ml of 0.2 M a c e t i c a c i d was a p p l i e d to a 0.6x120 cm column and was e l u t e d with 0.2 M a c e t i c a c i d at a flow rate of 12 ml/h. The f r a c t i o n s i z e c o l l e c t e d was 2.0 ml and absorbance was measured at 280 nm through a 1.0 cm l i g h t path. A f t e r the absorbance p r o f i l e was p l o t t e d ( F i g . 51 and 52), f r a c t i o n s comprising the main peak were d i v i d e d , pooled and d e s i g n a t e d as Desamido III f r a c t i o n A and B. 1. Peptide A n a l y s i s The IR-GIP content in f r a c t i o n s c o l l e c t e d from Sephadex G-25 or G-50 columns d i d not correspond to the peak p r o t e i n absorbance r e a d i n g s . A p l a t e a u was achieved d u r i n g the d e c l i n e of p r o t e i n absorbance p r o f i l e ( F i g . 51 and 52). The r e s u l t s i n d i c a t e d the presence of e i t h e r a r e s i d u a l amount of GIP or other peptide m a t e r i a l which possessed IR-GIP a c t i v i t y . The p o l y a c r y l a m i d e g e l e l e c t r o p h o r e i s (Johns (1967)) s t u d i e s showed that Desamido II was heterogeneous with two major bands, one of which corresponded to GIP. The other moved slower. Desamido III (A) gave e s s e n t i a l l y the same p a t t e r n as Desamido II but with an i n c r e a s e i n the r e l a t i v e amount of the non-GIP p e p t i d e . Desamido III (B) 217 F i g . 51 Absorbance p r o f i l e and IR-GIP content of Desamido II on Sephadex G-25 column. Eleven m i l l i g r a m s of Desamido II d i s s o l v e d i n 1.0 ml of 0.2 M a c e t i c a c i d was a p p l i e d to the column and e l u t e d with 0.2 M a c e t i c a c i d at a flow rate of 12 ml/h. The f r a c t i o n s i z e c o l l e c t e d was 2.0 ml and the absorbance was measured at 280 nm through a 1.0 cm l i g h t path. The content of IR-GIP was expressed as ug/ml. 218 Fraction Number 219 F i g . 52 Absorbance p r o f i l e and IR-GIP content of Desamido II on Sephadex G-50 column. Eleven m i l l i g r a m s of Desamido II d i s s o l v e d i n 1.0 ml of 0.2 M a c e t i c a c i d was a p p l i e d to the column and e l u t e d with 0.2 M a c e t i c a c i d at a flow r a t e of 12 ml/h. The f r a c t i o n s i z e c o l l e c t e d was 2.0 ml and the absorbance was measured at 280 nm through a 1.0 cm light, path. The content of IR-GIP was expressed as Mg/ml. 2 2 0 * ft it * * * Fraction Number 221 gave a s i n g l e band with an e l e c t r o p h o r e t i c m o b i l i t y i d e n t i c a l to that of GIP. S e v e r a l N-terminal amino a c i d r e s i d u e s were det e c t e d i n Desamido III (A) ( t y r o s i n e , glutamic a c i d {or glutamine}, s e r i n e , g l y c i n e , a l a n i n e , p r o l i n e , i s o l e u c i n e and a r g i n i n e ) which f u r t h e r i n d i c a t e d h e t e r o g e n e i t y . The p a t t e r n of t r y p t i c d i g e s t e d p e p t i d e s of Desamido III (A) on high v o l t a g e paper e l e c t r o p h o r e s i s i n d i c a t e d the presence of fragments other than those expected from GIP. Most of the e x t r a fragments were b a s i c . 2. P h y s i o l o g i c a l S t u d i e s The g a s t r i c a c i d i n h i b i t o r y a c t i v i t y to 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 was found to be s i g n i f i c a n t l y lower in EG I-I I (A), EG 11-111 (A) and Desamido II than GIP at equal dose. Whereas 4.0 jug/kg.h of Desamido III (A) demonstrated an a c i d i n h i b i t o r y a c i t i v i t y of 57±4% compared to 87% f o r the same dose of GIP. 222 DISCUSSION Numerous p o l y p e p t i d e s have been i s o l a t 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 but only a l i m i t e d number have been e s t a b l i s h e d as g a s t r o i n t e s t i n a l hormones. S e v e r a l c r i t e r i a have to be f u l f i l l e d i n order f o r a p o l y p e p t i d e to be c o n s i d e r e d as a g a s t r o i n t e s t i n a l hormone. F i r s t l y , the p o l y p e p t i d e must be i s o l a t e d i n i t s pure form; secondly, i t s p h y s i o l o g i c a l a c t i o n through the endogenous p h y s i o l o g i c a l r e l e a s e and exogenous a d m i n i s t r a t i o n of the p o l y p e p t i d e must be demonstrated. T h i r d l y , d e t e c t i o n of the p o l y p e p t i d e i n blood f o l l o w i n g a d m i n i s t r a t i o n of the p h y s i o l o g i c a l stimulus must be shown. 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) i s a p o l y p e p t i d e which c o u l d be c o n s i d e r e d to be a g a s t r o i n t e s t i n a l hormone. The p o l y p e p t i d e was i s o l a t e d i n i t i a l l y from a s i d e f r a c t i o n d e r i v e d from the p u r i f i c a t i o n of CCK-PZ. The bioassay used to monitor p u r i f i c a t i o n was f o r i n h i b i t o r y a c t i v i t y to 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 i n p a r a s y m p a t h e t i c a l l y denervated g a s t r i c pouches of dogs (Brown, Mutt and Pederson (1970)). The p u r i f i c a t i o n procedures used were p r i n c i p a l l y ion exchange and g e l f i l t r a t i o n chromatography. Assessment of p u r i t y was based upon the demonstration of a s i n g l e absorbance peak from 223 the f i n a l stage of chromatography, a s i n g l e p r o t e i n band on p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s , s t a i n e d and d e s t a i n e d c o u n t e r - e l e c t r o p h o r e t i c a l l y and amino a c i d a n a l y s e s . P h y s i o l o g i c a l a c t i v i t y was assessed by measurement of a c i d i n h i b i t i o n i n g a s t r i c pouch dogs and d e t e r m i n a t i o n of i n s u i i n o t r o p i c a c t i o n i n the i s o l a t e d p e r f u s e d r a t pancreas. The amino a c i d sequence of GIP was determined by f i r s t c l e a v i n g the peptide by chemical and enzymatic degradation, i s o l a t i o n of the fragments then sequencing of the p e p t i d e s (Brown (1971), Brown and Dryburgh (1971)) using manual techniques. A radioimmunoassay was developed to enable the d e t e c t i o n , of immunoreactive GIP (IR-GIP) (Kuzio et a l . (1974)) which e v e n t u a l l y allowed the measurement of IR-GIP r e l e a s e in response to secretogogues and other s t i m u l i (Brown, Dryburgh et a l . (1975)). T h i s study was undertaken to i n v e s t i g a t e the p u r i t y of GIP and to c l a r i f y the r e l a t i o n s h i p of GIP with secretogogues i n the 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 . The apparent d i v e r s e nature of the p h y s i o l o g i c a l a c t i v i t i e s a s c r i b e d to GIP ( a c i d i n h i b i t o r y and i n s u i i n o t r o p i c ) , o c c a s i o n a l o b s e r v a t i o n of dual p a t t e r n of p u r i f i e d GIP, and the f a i l u r e of s y n t h e t i c products to 224 have b i o l o g i c a l a c t i v i t y a l l were suggestive that the b i o l o g i c a l a c t i v i t y may be present i n two separate e n t i t i e s . The p u r i t y of GIP EG III (Brown, Mutt and Pederson (1970)) was a p p r a i s e d by 3 experimental techniques: a n a l y s i s of d i f f e r e n t f r a c t i o n s of GIP from CM Sephadex C25 column chromatography, the a n a l y s i s of GIP on p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and the m o b i l i t y of GIP on t h i n l a y e r chromatography (TLC). A n a l y s i s of GIP was c a r r i e d out on CM Sephadex C25, e l u t i n g with 0.01M NH 4HC0 3 pH 7.8. The absorbance p r o f i l e demonstrated a s i n g l e peak ( F i g . 38). The peak was d i v i d e d i n t o four f r a c t i o n s : B, C, D and E (Brown, F r o s t et a l . (1980)). A l l four f r a c t i o n s demonstrated no d i f f e r e n c e i n p h y s i o l o g i c a l a c t i v i t y i n respect to a c i d i n h i b i t o r y a c t i v i t y and i n s u i i n o t r o p i c a c t i o n i n i s o l a t e d rat pancreas. N-terminal analyses showed i d e n t i c a l r e s i d u e s f o r a l l four f r a c t i o n s and the amino a c i d compositions were s i m i l a r amongst the four f r a c t i o n s . However, high v o l t a g e paper e l e c t r o p h o r e s i s of the t r y p t i c d i g e s t e d f r a c t i o n s demonstrated one f r a c t i o n to contain,an e x t r a a c i d i c d i p e p t i d e ( F i g . 39). 225 Polyacrylamide g e l e l e c t r o p h o r e t i c a n a l y s i s of GIP f o l l o w e d by d i f f u s i o n s t a i n i n g and d e s t a i n i n g procedures i n d i c a t e d two bands. One, a minor component which moved f a s t e r than the major component ( F i g . 44). The nature of the g e l technique used i n d i c a t e d that the minor component c o u l d be more b a s i c or (and) possessed a smaller molecular weight than the major component. The degree of s e p a r a t i o n of the two components was shown to be f u r t h e r enhanced in a p o l y a c r y l a m i d e - u r e a g e l system developed by Panyim and C h a l k l e y (1967). T h i s r e s u l t was i n c o n t r a s t to the s i n g l e component demonstrated when g e l s were s t a i n e d and d e s t a i n e d c o u n t e r - e l e c t r o p h o r e t i c a l l y ( A h l r o t h and Mutt (1970)). The reason f o r the d i f f e r e n t o b s e r v a t i o n s i n the two systems was probably due to r e t a r d a t i o n i n m o b i l i t y of the f a s t moving minor component a f t e r r e a c t i o n with the dye, while the major component was s t i l l moving at the same speed before c o n t a c t i n g the dye. The end r e s u l t would be a merging phenomenon in which the r e s o l u t i o n of the two components was l o s t . Other g e l e l e c t r o p h o r e s i s s t u d i e s such as sodium do d e c y l s u l p h a t e (SDS) p o l y a c r y l a m i d e g e l and urea-SDS p o l y a c r y l a m i d e g e l s were performed, but the degree of r e s o l u t i o n was poor probably because of the low molecular weights of the p o l y p e p t i d e s . 226 The dual components of GIP observed on the pol y a c r y l a m i d e - u r e a g e l e l e c t r o p h o r e s i s was u n l i k e l y to be an a r t i f a c t of o x i d i z e d or reduced product of GIP, si n c e o x i d a t i o n of GIP with e i t h e r H 20 2 or performic a c i d d i d not a l t e r the r e l a t i v e i n t e n s i t y of the two components ( F i g . 45). When GIP was run on TLC p l a t e s , developed i n a b u t a n o l : a c e t i c a c i d : p y r i d i n e system, 2 n i n h y d r i n p o s i t i v e components were o c c a s i o n a l l y observed. The major one had an Rf value of 0.66 and the minor one was s l i g h t l y l e s s . T h i s degree of s e p a r a t i o n was enhanced when the water content i n the developing b u f f e r was in c r e a s e d from 30% to 37.5%, the Rf values of the two components were 0.67 and 0.71, the l a t t e r being the major component. An a n a l y s i s of the peptide products of GIP t r e a t e d with cyanogen bromide was c a r r i e d out on TLC. Cyanogen bromide rea c t e d with the methionine residue of GIP which c l e a v e d the GIP molecule i n t o two fragments: the N-te r m i n a l p e p t i d e ( r e s i d u e 1-14) and C-terminal peptide ( r e s i d u e 15-42). Both of these fragments and the uncleaved GIP were separated on CM-11 column ( F i g . 46). When a p r e p a r a t i o n c o n t a i n i n g the N-terminal peptide of 227 cyanogen bromide t r e a t e d GIP was run on TLC, two n i n h y d r i n p o s i t i v e components were observed with Rf values of 0.65 and 0.69, whereas the C-terminal fragment of cyanogen bromide t r e a t e d GIP demonstrated only a s i n g l e component. A l s o GIP t r y p t i c d i g e s t e d fragments Tr 2 (amino a c i d r e s i d u e s 1-17) demonstrated 2 components with Rf valuers of 0.59 and 0.65 on TLC. These o b s e r v a t i o n s suggested the p o s s i b i l i t y that the two p o l y p e p t i d e components present in GIP might share s i m i l a r C -terminal amino a c i d compositions and sequences, whereas the N-terminal p o r t i o n s of the molecule may d i f f e r . The evidence s u p p o r t i n g the h e t e r o g e n i e t y of GIP p r e p a r a t i o n s l e d 'to attempts to separate the d i f f e r e n t components p r e p a r a t i v e l y . E l u t i o n of p o l y p e p t i d e d i r e c t l y from the unstained g e l u s i n g a c e t i c a c i d or d i s t i l l e d water achieved extremely low r e c o v e r i e s as w e l l as the a d d i t i o n a l problem of i m p u r i t i e s from the p o l y a c r y l a m i d e g e l . The main problem a s s o c i a t e d with recovery of p o l y p e p t i d e from TLC p l a t e s was the presence of l a r g e q u a n t i t i e s of c a l c i u m phosphate. The percentage of p o l y p e p t i d e recovered a f t e r the removal of calcium using d i a l y s i s was too low f o r chemical i n v e s t i g a t i o n s to be pursued. 228 High pressure l i q u i d chromatography with v a r i o u s s o l v e n t systems were t e s t e d f o r the s e p a r a t i o n of p o l y p e p t i d e s from GIP. Solvents such as: ammonium a c e t a t e : a c e t o n i t r i l e , ammonium a c e t a t e : e t h a n o l , and phosphoric a c i d : t r i e t h y l a m i n e : a c e t o n i t r i l e were not s u i t a b l e because of low recovery of p o l y p e p t i d e s . A so l v e n t system of w a t e r : a c e t o n i t r i l e : t r i f l u o r o a c e t i c a c i d was found to be the optimal system f o r the s e p a r a t i o n of the p o l y p e p t i d e s from GIP. Absorbance p r o f i l e s i n d i c a t e d three d i s t i n c t i v e peaks with the two major peaks occupying 75% and 22% of the t o t a l area although t h i s was very v a r i a b l e . The recovery of peptide m a t e r i a l from the column was over 70% as i n d i c a t e d by rechromatography of the e l u a t e d sample. V a r i o u s chemical and p h y s i o l o g i c a l s t u d i e s were undertaken to i n v e s t i g a t e the nature of v a r i o u s components. A f t e r s e p a r a t i o n on HPLC the peaks were designated as I and II with I e l u t i n g f i r s t ( F i g . 43). The p o l y p e p t i d e i n peak II has demonstrated i n s u i i n o t r o p i c a c t i v i t y i n the i s o l a t e d r a t pancreas i n a potency s i m i l a r to GIP. However, the p o l y p e p t i d e i n peak I demonstrated no i n s u i i n o t r o p i c e f f e c t i n the i s o l a t e d r a t pancreas (Brown, Dahl et a l . (1981)). The c o n f i r m a t i o n of the e a r l i e r p u b l i s h e d amino a c i d 229 sequence of GIP (Brown (1971), Brown and Dryburgh (1971)) was c a r r i e d out i n c o l l a b o r a t i o n with J o r n v a l l et a l . (1981). The sequence of the i n t a c t molecule and the cyanogen bromide t r e a t e d GIP fragments i n d i c a t e d the presence of 42 amino a c i d r e s i d u e s ( F i g . 53). T h i s sequence i s d i f f e r e n t from that r e p o r t e d e a r l i e r (Brown (1971), Brown and Dryburgh (1971)) in that one i n s t e a d of two glutamine r e s i d u e s were found i n p o s i t i o n 29 and 30. T h i s new sequence was confirmed by manual d a n s y l a t i o n of the GIP p e p t i d e s d e r i v e d from t r y p t i c d i g e s t i o n and N-c h l o r o s u c c i n i m i d e cleavage at tryptophan r e s i d u e s with subsequent p u r i f i c a t i o n of the fragments on HPLC. The s e q u e n t i a l degradation of the i n t a c t fragment and the cyanogen bromide t r e a t e d GIP a l s o r e v e a l e d the sequence of the minor component. I t was i d e n t i c a l to the sequence of the major component except that r e s i d u e s 1 and 2 were absent. The amount of contaminant v a r i e d between 7-22% r e l a t i v e to the major component. These data f i t w e l l with the TLC s t u d i e s on the N-terminal fragment of cyanogen bromide t r e a t e d GIP in which two n i n h y d r i n p o s i t i v e components of d i f f e r e n t i n t e n s i t y were observed, whereas the C-terminal fragment r e v e a l e d only one n i n h y d r i n r e a c t i v e component. The components were 230 separated by HPLC, and amino a c i d analyses on the separated fragments support that the minor component has the amino a c i d composition of one l e s s t y r o s i n e and a l a n i n e ( F i g . 53). J o r n v a l l et a l . (1981) s p e c u l a t e d that the two p o l y p e p t i d e s i n the GIP p r e p a r a t i o n might have been d e r i v e d from d i f f e r e n t p a r t l y d u p l i c a t e d genes or d i f f e r e n t regions of a l a r g e r gene. Another hypothesis c o u l d be that the two GIP p o l y p e p t i d e s were d e r i v e d from a degradation process by an aminopeptidase, d i p e p t i d a s e , e l a s t a s e or r e l a t e d enzymes in the i n t e s t i n e d u r i n g e x t r a c t i o n . I t i s h i g h l y probable that the sequence of the major component (1-42) might correspond to the l a r g e r peak of GIP on HPLC, and the minor contaminant (3-42) might correspond to the smaller peak. Although t h i s has been supported by amino a c i d a n a l y s e s , the f i n a l c o n f i r m a t i o n r e q u i r e s sequencing of the two HPLC components. The presence of the p o l y p e p t i d e contaminant i n GIP prompted a search f o r t h i s p o l y p e p t i d e i n other s i d e f r a c t i o n s obtained d u r i n g the p u r i f i c a t i o n of GIP on column chromatography. A l l the s i d e f r a c t i o n s were shown to be a heterogenous mixture of p o l y p e p t i d e s on 231 F i g . 53 The amino a c i d sequence of GIP. 232 1 T yr-A!a-Glu-Gly-Thr4>he-I le-Ser-Asp-T y r-Ser-l le-Ala-Met-Asp 2 0 3 0 L y s- l le -Arg-Gln-Gln-Asp-PheVal^sn-Trp4.euM.eu -AUi -Gln-Lys-4 0 Gly-Lys-Lys-Ser-Asp-Trp-Lys-His-AsrvlSe-Thr-GSn 233 p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s ( F i g . 44). However, the s i d e f r a c t i o n d e r i v e d from EG II Fr A on Sephadex G-25 column ( F i g . 51) showed a band of p o l y p e p t i d e m a t e r i a l with an e l e c t r o p h o r e t i c m o b i l i t y corresponding to the contaminant in GIP. CM-11 and Sephadex G-25 column chromatography were employed i n the p u r i f i c a t i o n of t h i s p o l y p e p t i d e , and the eluant from the column was pooled i n t o two f r a c t i o n s . One f r a c t i o n had a s i n g l e band of the same e l e c t r o p h o r e t i c m o b i l i t y as GIP on polyacrylamide g e l s and possessed 60% of the g a s t r i c a c i d i n h i b i t o r y a c t i v i t y of GIP. The other f r a c t i o n c o n t a i n e d a heterogeneous group of p o l y p e p t i d e s i n c l u d i n g the minor component in GIP and possessed no g a s t r i c a c i d i n h i b i t o r y a c t i v i t y . N-terminus determinations confirmed the h e t e r o g e n e i t y of t h i s f r a c t i o n . Further p u r i f i c a t i o n of t h i s minor component of GIP was not completed. A proposed method i n v o l v i n g HPLC c o u l d be c o n s i d e r e d as one of the p u r i f i c a t i o n s t e p s . An HPLC system using a c e t o n i t r i l e r t r i f l u o r o a c e t i c a c i d as s o l v e n t s has been demonstrated to be e f f e c t i v e i n s e p a r a t i n g p o l y p e p t i d e s of s i m i l a r chemical nature to GIP. The i s o l a t i o n of t h i s minor component of GIP c o u l d enable the study of the s t r u c t u r e / f u n c t i o n r e l a t i o n s h i p of the two components. The p o s s i b i l i t y of one component having a g r e a t e r or 234 l e s s e r i n s u i i n o t r o p i c or g a s t r i c i n h i b i t o r y a c t i v i t y than GIP might be i n d i c a t e d . A l s o the s e n s i t i v i t y of the two components to GIP antiserum c o u l d be assessed s e p a r a t e l y . M o d i f i c a t i o n of amino a c i d r e s i d u e s w i t h i n the molecule i s one of the approaches taken in the study of s t r u c t u r e / f u n c t i o n r e l a t i o n s h i p s of any p o l y p e p t i d e hormone. Mutt (1964) had demonstrated that o x i d a t i o n of the methionine residue i n CCK-PZ with H 2 0 2 l e d to the l o s s of b i o l o g i c a l a c t i v i t y which c o u l d be regenerated by subsequent r e d u c t i o n with c y s t e i n e h y d r o c h l o r i d e . M o d i f i c a t i o n of methionine, tryptophan and t y r o s i n e r e s i d u e s i n GIP have been s t u d i e d . E f f e c t s of s t r u c t u r a l m o d i f i c a t i o n of amino a c i d s on the a c i d i n h i b i t o r y a c t i v i t y of GIP have been s t u d i e d , because i t was co n s i d e r e d e a r l i e r that the minor component may be simply produced because of a m o d i f i c a t i o n to an amino a c i d r e s i d u e . The a c i d i n h i b i t o r y a c t i v i t y of GIP was decreased when the methionine residue was o x i d i z e d with e i t h e r hydrogen peroxide or per f o r m i c a c i d . I o d i n a t i o n of the t y r o s i n e r e s i d u e s i n GIP a l s o reduced the a c i d i n h i b i t o r y a c t i v i t y (Table V I I ) . The e f f e c t of m o d i f i c a t i o n of tryptophan r e s i d u e s in GIP r e s u l t e d i n a v a r i a b l e degree of a c i d i n h i b i t o r y a c t i v i t y depending on 235 the agent used. M o d i f i c a t i o n of tryptophan r e s i d u e s i n GIP by a - n i t r o p h e n y l s u l p h e n y l c h l o r i d e d i d not a l t e r g a s t r i c a c i d i n h i b i t o r y a c t i v i t y (Table V I ) . M o d i f i c a t i o n of tryptophan by d i m e t h y l s u l p h o x i d e has been shown a l s o to cause o x i d a t i o n of methionine r e s i d u e s to methionine sulphoxide (Glazer et a l . (1975), Savige and Fontana (1977)) and i n d o l y l - 3 - p r o p i o n i c a c i d c o u l d reduce the methionine sulphoxide. The m o d i f i c a t i o n of tryptophan r e s i d u e s by d i m e t h y l s u l p h o x i d e alone or with i n d o l y l - 3 -p r o p i o n i c a c i d r e s u l t e d i n a decrease i n g a s t r i c a c i d i n h i b i t o r y a c t i v i t y . However, the assessment of the completeness of the r e a c t i o n of the methionine was not c a r r i e d out. i t i s p o s s i b l e t h e r e f o r e , that o x i d i z e d methionine c o u l d s t i l l be present a f t e r i n d o l y l - 3 -p r o p i o n i c a c i d treatment. I n s u l i n o t r o p i c a c t i v i t y i n the i s o l a t e d r a t pancreas was not a f f e c t e d by m o d i f i c a t i o n of tryptophan with a - n i t r o p h e n y l s u l p h e n y l c h l o r i d e , whereas the a c t i v i t y was decreased when the tryptophan r e s i d u e s were m o d i f i e d by d i m e t h y l s u l p h o x i d e . The i n s u l i n o t r o p i c e f f e c t c o u l d be r e s t o r e d when the methionine was reduced by i n d o l y l - 3 - p r o p i o n i c a c i d . These s t u d i e s i n d i c a t e d that o x i d a t i o n of the methionine r e s i d u e and s t r u c t u r a l m o d i f i c a t i o n of the t y r o s i n e s and p o s s i b l y of the tryptophans a l t e r e d the g a s t r i c a c i d i n h i b i t o r y a c t i v i t y 236 of GIP but m o d i f i c a t i o n of tryptophan with a-n i t r o p h e n y l s u l p h e n y l c h l o r i d e has no e f f e c t on the i n s u l i n o t r o p i c a c t i o n . Attempts to s y n t h e s i z e GIP have met with l i t t l e success i n terms of producing a homogeneous substance with the same p h y s i o l o g i c a l a c t i o n s of p o r c i n e GIP. The s y n t h e t i c GIP p r e p a r a t i o n produced by Yanaihara et a l . (1980) demonstrated h e t e r o g e n e i t y in s e v e r a l p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s systems. In the present study, comparison of the t r y p t i c fragments of s y n t h e t i c and n a t u r a l p o r c i n e GIP on high v o l t a g e e l e c t r o p h o r e s i s showed a d d i t i o n a l fragments i n the s y n t h e t i c p r e p a r a t i o n which were not present i n the n a t u r a l compound. S y n t h e t i c GIP(1~38) prepared by Wuensch et a l . (1978) had Rf v a l u e s corresponding to the p o r c i n e GIP except with one a d d i t i o n a l fragment. The s y n t h e t i c GIP with sequence 1-14 prepared by Wuensch was d i f f e r e n t from n a t u r a l GIP sequence 1-14 as suggested by the d i f f e r e n c e s i n Rf v a l u e s f o l l o w i n g chymotryptic d i g e s t i o n . The p h y s i o l o g i c a l a c t i v i t y , i . e . a c i d i n h i b i t o r y a c t i v i t y and i n s u l i n o t r o p i c a c t i o n i n i s o l a t e d r a t pancreas, of a l l the s y n t h e t i c p r e p a r a t i o n s t e s t e d were s i g n i f i c a n t l y lower than n a t u r a l GIP when compared 237 on a weight b a s i s . In view of the recent p u b l i s h e d c o r r e c t e d sequence of p o r c i n e GIP ( J o r n v a l l et a l . (1981)), the e a r l i e r i n c o r r e c t sequence c o u l d be one of the c o n t r i b u t i n g f a c t o r s to the f a i l u r e of the s y n t h e s i s of a GIP molecule with complete p h y s i o l o g i c a l a c t i v i t y . However, s i n c e the a c t i o n of GIP i s very s e n s i t i v e to the m o d i f i c a t i o n of amino a c i d r e s i d u e s , t e c h n i c a l d i f f i c u l t i e s r e s u l t i n g i n the o x i d a t i o n of the methionine residue c o u l d p o t e n t i a l l y a f f e c t the p h y s i o l o g i c a l a c t i o n of the s y n t h e t i c GIP. In a d d i t i o n , a l l the s y n t h e t i c p r e p a r a t i o n s t e s t e d thus f a r on p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s have been shown to be impure as i n d i c a t e d by m u l t i p l e s t a i n e d bands. The ' e n t e r o i n s u l a r a x i s ' (Unger and E i s e n t r a u t (1969)) concept p o s t u l a t e s the e x i s t e n c e of an i n s u i i n o t r o p i c hormone from the g a s t r o i n t e s t i n a l t r a c t which i s r e l e a s e d when in g e s t e d glucose a c t s on the mucosa of the small i n t e s t i n e . T h i s r e s u l t s i n an i n c r e a s e d r e l e a s e of i n s u l i n i n response to glucose adm i n i s t e r e d o r a l l y as compared to the same loa d given i n t r a v e n o u s l y . The i n t e r a c t i o n of GIP with glucose, an amino a c i d mixture and f a t have been i n v e s t i g a t e d . Most in v i v o s t u d i e s of the i n s u i i n o t r o p i c a c t i o n of 238 n u t r i e n t s or hormones measure two v a r i a b l e s : plasma glucose and i n s u l i n . These v a r i a b l e s are i n v o l v e d i n a feedback c o n t r o l system i n which hyperglycaemia induces i n s u l i n r e l e a s e and subsequently r e s u l t s i n a decrease i n plasma g l u c o s e . T h i s i n turn decreases i n s u l i n s e c r e t i o n . On the other hand, most of the ir\ v i t r o s t u d i e s such as the i s o l a t e d i s l e t c e l l p r e p a r a t i o n and the i s o l a t e d r a t perfused pancreas s t u d i e s are not s u b j e c t e d to t h i s feedback mechanism, because glucose i s perfused at a f i x e d l e v e l and IRI becomes the only v a r i a b l e . The experimental approach used i n t h i s study i n v o l v e d the maintenance . of a steady s t a t e plasma glucose c o n c e n t r a t i o n at a predetermined l e v e l , measurement of the v a r i a b l e plasma IRI and the amount of glucose i n f u s e d fo r the maintenance of the steady s t a t e glycaemia. T h i s i s an i n d i r e c t way of measuring metabolic c l e a r a n c e r a t e of glucose. Two general o b s e r v a t i o n s were made with regard to the f l u c t u a t i o n of plasma glucose and i n s u l i n d u r i n g the steady s t a t e hyperglycaemia experiments. The f i r s t o b s e r v a t i o n was that both the f a s t i n g venous plasma glucose and i n s u l i n taken at 15 min i n t e r v a l s over a 1h p e r i o d have shown v a r i a t i o n by as much as 7 mg% and 15 239 MU/ml r e s p e c t i v e l y . T h i s f l u c t u a t i o n of f a s t i n g glucose and IRI was a l s o r e p o r t e d i n monkeys (Goodner et a l . (1977)). They demonstrated a synchronous, s u s t a i n e d o s c i l l a t i o n of i n s u l i n , glucagon and glucose i n the venous blood. In man, I b e r a l l et a l . (1968) had showed a high frequency of o s c i l l a t i o n i n plasma glucose by as much as 10-30 mg%, whereas Nimmo et a l . (1973) were unable to confirm t h i s o b s e r v a t i o n . The simultaneous sampling of the h e p a t i c and p a n c r e a t i c venous blood i n the canine p o s t a b s o r p t i v e s t a t e have shown a p u l s a t i l e r i s e i n glucose i n hep a t i c venous blood. T h i s was foll o w e d w i t h i n 15 min by a corresponding i n c r e a s e i n IRI in the p a n c r e a t i c venous blood (Anderson et a l . (1967)). This o b s e r v a t i o n c o u l d p o s s i b l y e x p l a i n the o s c i l l a t o r y nature of plasma glucose and p a n c r e a t i c hormone i n the systemic venous blood. The second o b s e r v a t i o n was the f l u c t u a t i o n of plasma IRI d u r i n g a steady s t a t e hyperglycaemia maintained by intravenous glucose i n f u s i o n . There was no d e f i n i t e c y c l i c p a t t e r n of the f l u c t u a t i o n of IRI. The amplitude of the o s c i l l a t i o n i n c r e a s e d as the l e v e l of hyperglycaemia i n c r e a s e d . The reason f o r t h i s o s c i l l a t o r y behaviour d u r i n g glucose i n f u s i o n probably i n v o l v e d the 240 i n t e r a c t i o n of a l l the p a n c r e a t i c hormones. T h i s hypothesis was supported by the o b s e r v a t i o n that i n s u l i n , glucagon and somatostatin l e v e l s from the i s o l a t e d canine pancreas d u r i n g exposure to a constant glucose c o n c e n t r a t i o n (Stagner et a l . (1980)) showed an o s c i l l a t o r y p a t t e r n . Furthermore, t h i s i n t r i n s i c o s c i l l a t i o n was u n a f f e c t e d by a d r e n e r g i c blockade. O l e f s k y et a l . (1973) r e p o r t e d d i s s o c i a t i o n of the plasma IRI response and blood glucose d u r i n g glucose i n f u s i o n i n normal a n a e s t h e t i z e d dogs. These o b s e r v a t i o n s suggested that the l e v e l of blood glucose may not be the primary determinant of the i n s u l i n response to glucose d u r i n g the c h r o n i c phase of i n s u l i n s e c r e t i o n . Bowden et a l . (1980) have observed c y c l i c changes of glucose and IRI d u r i n g constant glucose i n f u s i o n i n conscious dogs. Futhermore the source of the glucose i n the c i r c u l a t i o n i s almost completely from the exogenous i n f u s i o n suggesting that endogenous glucose p r o d u c t i o n c o n t r i b u t e d very l i t t l e i f any to the o v e r a l l glucose p i c t u r e . The degree of p o t e n t i a t i o n of i n s u l i n s e c r e t i o n by GIP i n the presence of hyperglycaemia depends not only on the magnitude of the dose of GIP and the p r e v a i l i n g l e v e l of glycaemia, but a l s o on the s p e c i e s s t u d i e d . In the 241 i s o l a t e d p erfused r a t pancreas (Pederson and Brown (1978)), the glucose c o n c e n t r a t i o n t h r e s h o l d f o r the i n s u i i n o t r o p i c a c t i o n of 5.0 ng/ml p o r c i n e GIP c o u l d be shown to be 100 mg/dl (5.5 mM). As the c o n c e n t r a t i o n of glucose s o l u t i o n was i n c r e a s e d , the i n s u i i n o t r o p i c e f f e c t of a given dose of GIP was a l s o i n c r e a s e d . In man, i n f u s i o n of 1.0 Mg/min of p o r c i n e GIP was shown to be capable of improving intravenous glucose t o l e r a n c e and i n c r e a s i n g i n s u l i n s e c r e t i o n over and above glucose alone (Dupre et a l . (1973)). When 0.4 jxg/kg.h of po r c i n e GIP was i n f u s e d i n the presence of steady s t a t e hyperglycaemia maintained at 54 mg/dl above b a s a l , a 20% inc r e a s e i n IRI s e c r e t i o n was demonstrated ( E l a h i et a l . (1979)). However, when steady s t a t e hyperglycaemia was maintained at 145 mg% above b a s a l , the same dose of GIP e l i c i t e d a 700-800% 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 . In dogs the t h r e s h o l d of GIP necessary to e l i c i t i n s u i i n o t r o p i c a c t i o n was shown to be gr e a t e r than i n man and i n the i s o l a t e d p e r f u s e d r a t pancreas. When 2.0 Mg/kg.h of GIP was i n f u s e d f o r 2 hours i n the presence of an intravenous i n f u s i o n of 0.6 g/kg.h glucose f o r 1 h, an improvement of glucose t o l e r a n c e and a 50% inc r e a s e of IRI over the c o n t r o l was observed (Pederson, Schubert and 242 Brown (1975)). In the present study no i n s u l i n o t r o p i c a c t i o n of GIP was observed when 1.0 jug/kg.h of GIP was i n f u s e d i n the presence of steady s t a t e hyperglycaemia maintained at 40, 100 and 150 mg/dl above b a s a l ( F i g . 7, 9 and 11). When 2.0 ng/kg.h of GIP was i n f u s e d d u r i n g hyperglycaemia maintained at 150 mg/dl above b a s a l , a b r i e f surge of IRI was'observed towards the t e r m i n a t i o n of the GIP i n f u s i o n ( F i g . 12). These r e s u l t s suggested that both a higher t h r e s h o l d of i n f u s e d GIP and a g r e a t e r l e v e l of hyperglycaemia were r e q u i r e d to e l i c i t an i n s u l i n o t r o p i c a c t i o n i n dogs i n comparison with man. T h i s apparent higher t h r e s h o l d for exogenous GIP i n dogs than man c o u l d p o s s i b l y be due to d i f f e r e n c e s i n the p r e p a r a t i o n of the GIP i n f u s a t e s . In the human study ( E l a h i et a l . (1979), the GIP i n f u s a t e was prepared with the a d d i t i o n of 1 ml of the s u b j e c t ' s own serum to minimize the n o n - s p e c i f i c a d s o r p t i o n of GIP to glassware and i n f u s i o n apparatus. Whereas the p r e p a r a t i o n of the GIP i n f u s a t e i n the canine study i n v o l v e d the d i s s o l u t i o n of the p o l y p e p t i d e i n 0.9% NaCl with no p r o t e i n added. T h e r e f o r e , the l o s s of GIP due to n o n - s p e c i f i c a d s o r p t i o n might be expected to be h i g h e r . However, the plasma IR-GIP achieved i n both human and canine s t u d i e s were 243 comparable ( F i g . 4 and E l a h i et a l . (1979)). The o b s e r v a t i o n of a gr e a t e r i n s u l i n response to a dose of glucose a d m i n i s t e r e d o r a l l y r a ther than i n t r a v e n o u s l y has been w e l l e s t a b l i s h e d . In t h i s study the IRI and IR-GIP responses to an o r a l l y or i n t r a d u o d e n a l l y a d m i n i s t e r e d glucose l o a d were examined durin g steady s t a t e hyperglycaemia. E l e v a t i o n of plasma IRI and glucose l e v e l s to the o r a l a d m i n i s t r a t i o n of a glucose l o a d were lower but more prolonged than when an e q u i v a l e n t l o a d was in t r o d u c e d i n t r a d u o d e n a l l y ( F i g . 13 and 14). The reason i s probably due to a delay i n g a s t r i c emptying when the o r a l r a t h e r than the intraduodenal route was used. There was no s i g n i f i c a n t i n c r e a s e above ba s a l i n IRI response a f t e r the o r a l and intr a d u o d e n a l glucose l o a d when steady s t a t e hyperglycaemia was maintained at 40 mg/dl above b a s a l ( F i g . 15 and 16). The IR-GIP response observed f o l l o w i n g the o r a l and in t r a d u o d e n a l glucose l o a d were not s i g n i f i c a n t l y d i f f e r e n t whether or not a m i l d steady s t a t e hyperglycaemia e x i s t e d . When hyperglycaemia was maintained at 100 mg/dl above b a s a l , the same intr a d u o d e n a l and o r a l glucose l o a d e l i c i t e d a gre a t e r i n c r e a s e i n IRI response ( F i g . 17 and 18). The 244 plasma IR-GIP l e v e l s obtained f o l l o w i n g the o r a l and int r a d u o d e n a l glucose loads were comparable to the l e v e l s a chieved by exogenous a d m i n i s t r a t i o n of 2.0 jig/kg.h of GIP ( F i g . 5). Exogenous a d m i n i s t r a t i o n of t h i s dose of GIP was capable of e l i c i t i n g an i n s u l i n response i n the presence of moderate hyperglycaemia. These data i n dogs were i n agreement with data obtained i n human s t u d i e s (Andersen et a l . (1978)) i n which i t was shown that a s i g n i f i c a n t e l e v a t i o n of IRI was observed f o l l o w i n g an o r a l glucose l o a d i n the presence of hyperglycaemia maintained at 143 mg/dl above b a s a l . However, i n g e s t i o n of glucose i n the dog r e s u l t e d i n a 30% i n c r e a s e i n IRI over the bas a l l e v e l s . T h i s was c o n s i d e r a b l y smaller than the 300% inc r e a s e which had been observed i n man (Andersen et a l . (1978)). In the human s t u d i e s (Andersen et a l . (1978)), exogenously or endogenously induced h y p e r i n s u l i n a e m i a d i d not suppress endogenously r e l e a s e d IR-GIP by o r a l g l u c o s e . The data i n dogs suggested no suppression by m i l d hyperglycaemia of IR-GIP r e l e a s e induced by o r a l g l u c o s e . However, the measurement of plasma IR-GIP i n hyperglycaemia maintained at 100 mg/dl above ba s a l was not c a r r i e d out because of lack of a n t i s e r a f o r IR-GIP d e t e r m i n a t i o n s . T h e r e f o r e , no c o n f i r m a t i o n of the suppression of IR-GIP response to 245 h y p e r i n s u l i n a e m i a induced by o r a l glucose c o u l d be made. An amino a c i d mixture c o n t a i n i n g ten amino a c i d s (Table I) was used to study the i n s u i i n o t r o p i c a c t i o n of amino a c i d s f o l l o w i n g d i f f e r e n t , routes of a d m i n i s t r a t i o n : o r a l , i n traduodenal and in t r a v e n o u s . The plasma glucose f o l l o w i n g intravenous a d m i n i s t r a t i o n showed a b r i e f e l e v a t i o n before d e c r e a s i n g to a l e v e l s l i g h t l y lower than the f a s t i n g v a l u e . The i n i t i a l i n c r e a s e i n plasma glucose c o u l d be due to glucagon r e l e a s e and the-subsequent decrease i n plasma glucose c o u l d be due to i n s u l i n r e l e a s e . The change i n plasma glucose was l e s s when the o r a l and in t r a d u o d e n a l route of amino a c i d a d m i n i s t r a t i o n was used. The d i f f e r e n c e c o u l d be due to the r a p i d i n c r e a s e i n the plasma l e v e l s of amino a c i d s achieved when given v i a the intravenous route which s t i m u l a t e d the p a n c r e a t i c i s l e t maximally. The present study showed that IRI response to amino a c i d s was gr e a t e r when the intravenous route r a t h e r than the o r a l or intraduodenal route were used. The e l e v a t i o n was t r a n s i e n t as compared to the more s u s t a i n e d p a t t e r n when the o r a l and in t r a d u o d e n a l routes were employed. T h i s r e s u l t was in d i r e c t c o n f l i c t with r e p o r t s i n man (Raptis et a l . (1973), Thomas et a l . (1976), Thomas et 246 a l . (1978)) and i n dogs (Yovos et a l . (1982)) i n which a greater s t i m u l a t i o n of IRI was seen when the intra d u o d e n a l rather than the intravenous route was used. Thomas et a l . (1976) had demonstrated a r i s e i n serum IR-GIP of about 300 pg/ml f o l l o w i n g the intraduodenal but not the intravenous a d m i n i s t r a t i o n of t h i s amino a c i d mixture i n man. The gr e a t e r degree of IRI re l e a s e f o l l o w i n g i n t r a d u o d e n a l rather than intravenous a d m i n i s t r a t i o n (77±9 versus 43±6 MU/ml) was a t t r i b u t e d to the r i s e i n IR-GIP produced d u r i n g duodenal i n f u s i o n . Yovos et a l . (1982) demonstrated that concurrent i n f u s i o n of 1.0 Mg/kg.h of GIP with intravenous i n f u s i o n of the same amino a c i d mixture was capable of i n c r e a s i n g the IRI response above that seen with the mixture alone (41±2 versus 29±6 MU/ml). However, the i n c r e a s e i n i n s u l i n r e l e a s e o c c u r r e d only d u r i n g the f i r s t 30 min of the i n f u s i o n p e r i o d and d i d not a f f e c t the second h a l f hour of the i n f u s i o n . T h i s r e s u l t suggested that the concurrent i . v. i n f u s i o n of GIP with amino a c i d s might p o t e n t i a t e the f i r s t phase of IRI r e l e a s e . A s l i g h t i n c r ease i n IR-GIP r e l e a s e f o l l o w i n g the o r a l and intra d u o d e n a l a d m i n i s t r a t i o n of the amino a c i d mixture has been observed i n these s t u d i e s . Immunoreactive GIP 247 r e l e a s e was much l e s s than that observed f o l l o w i n g o r a l or i n t r a d u o d e n a l a d m i n i s t r a t i o n of glucose or f a t . When given on an equal weight b a s i s . The IR-GIP was not measured duri n g the intravenous route of a d m i n i s t r a t i o n of the amino a c i d mixture because of the n e c e s s i t y to conserve a n t i s e r a f o r GIP. Ohneda et a l . (1968) had demonstrated 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 IRI response to an amino a c i d mixture administered e i t h e r i n t r a v e n o u s l y or i n t r a d u o d e n a l l y i n dogs. T h e r e f o r e , 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 the IR-GIP response to the i n t r a d u o d e n a l a d m i n i s t r a t i o n of the amino a c i d mixture remains to be c l a r i f i e d . A comparison was made of the e f f e c t s of a mixture of 10 amino a c i d s a d m i n i s t e r e d v i a the o r a l , i n traduodenal or intravenous route to dogs i n the presence of a steady s t a t e m i l d hyperglycaemia. The IRI response was i n c r e a s e d 2-3 f o l d over the p r e - i n f u s i o n l e v e l . The exogenous i n f u s i o n of glucose had to be i n c r e a s e d d u r i n g the same p e r i o d of time i n order to maintain the steady s t a t e hyperglycaemia. An i n c r e a s e which r e f l e c t e d a change in the glucose metabolic r a t e . The p a t t e r n of IRI r e l e a s e 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 the amino a c i d mixture was more s u s t a i n e d i n the presence than in the absence of 248 m i l d steady s t a t e hyperglycaemia. T h i s r e s u l t confirmed the e a r l i e r o b s e r v a t i o n i n dogs (Ohneda et a l . (1968)) i n which a constant intravenous glucose i n f u s i o n with concurrent amino a c i d a d m i n i s t r a t i o n was performed. Blood samples from the p a n c r e a t i c v e i n i n d i c a t e d e l e v a t e d IRI r e l e a s e and a decrease i n the l e v e l of glycaemia, w h i l s t the IR-glucagon l e v e l s remained constant throughout. When 8.5 g of a commercially a v a i l a b l e mixture of 15 amino a c i d s was adm i n i s t e r e d o r a l l y i n the presence of m i l d hyperglycaemia, a 25% i n c r e a s e i n IRI o c c u r r e d . T h i s i n d i c a t e d that the composition and the amount of the amino a c i d mixture was important i n determining the degree of p o t e n t i a t i o n . The p o t e n t i a t i n g e f f e c t of an amino a c i d mixture i n the presence of m i l d hyperglycaemia was independent of the route of a d m i n i s t r a t i o n . The mechanism of t h i s p o t e n t i a t i o n e f f e c t c o u l d i n v o l v e the p a n c r e a t i c i s l e t , p e r i p h e r a l t i s s u e s e n s i t i v i t y and c e n t r a l nervous system. In the i s o l a t e d p e r f u s e d rat. pancreas the i n d i v i d u a l amino a c i d a r g i n i n e , and a mixture of amino a c i d s were shown to modulate i n s u l i n and glucagon r e l e a s e i n the presence of glucose (Pederson and Brown (1978)). However, i n the absence of glucose the amino a c i d s e l i c i t e d a poor 249 i n s u l i n response. A r g i n i n e , i n the absence of hyperglycaemia i n c r e a s e d the second phase of IRI r e l e a s e and a l s o induced a b i p h a s i c glucagon r e l e a s e i n the i s o l a t e d perfused r a t pancreas ( G e r i c h et a l . (1974)). In the presence of glucose, a r g i n i n e p o t e n t i a t e d the b i p h a s i c r e l e a s e of i n s u l i n while s t i l l m a i n t a i n i n g the glu c a g o n o t r o p i c a c t i o n . Pederson and Brown (1978) demonstrated that exogenously i n f u s e d GIP was capable of p o t e n t i a t i n g the i n s u i i n o t r o p i c a c t i o n of a r g i n i n e and t h i s e f f e c t was dependent upon the glucose c o n c e n t r a t i o n in the p e r f u s a t e . Exogenously i n f u s e d GIP a l s o p o t e n t i a t e d the glu c a g o n o t r o p i c a c t i o n of a r g i n i n e , but only i n the presence of low glucose c o n c e n t r a t i o n s . Furthermore, Thomas et a l . (1976, 1978) had demonstrated that i n man, an amino a c i d mixture c o n t a i n i n g a r g i n i n e was capable of r e l e a s i n g IR-GIP and e l i c i t e d a gre a t e r IRI response when admini s t e r e d i n t r a d u o d e n a l l y rather than i n t r a v e n o u s l y . The present study demonstrated that an o r a l dose of a r g i n i n e , e l i c i t e d no IR-GIP r e l e a s e and no p o t e n t i a t i o n of IRI when admin i s t e r e d i n the presence of m i l d hyperglycaemia i n dogs. I t should a l s o be noted the amount of a r g i n i n e used was 3 times the amount used by 250 Thomas et a l . (1976, 1978). T h i s suggested that IR-GIP response observed i n man (Thomas et a l . (1976), Thomas et a l . (1978)) f o l l o w i n g i n t r a d u o d e n a l a d m i n i s t r a t i o n of an amino a c i d mixture was u n l i k e l y to be due to a r g i n i n e . A r g i n i n e has not been demonstrated to be a potent i n s u l i n o t r o p i c amino a c i d i n dogs in the absence of hyperglycaemia (Rocha et a l . (1972)). However, a r g i n i n e c o u l d s t i l l have c o n t r i b u t e d to the o v e r a l l i n s u l i n o t r o p i c a c t i o n of the amino a c i d mixture i n the s t u d i e s of Thomas et a l . (1976, 1978). The i n s u l i n o t r o p i c a c t i o n of a r g i n i n e observed i n the i s o l a t e d p e r f u s e d r a t pancreas i n the presence of hyperglycaemia ( G e r i c h et a l . (1974), Pederson and Brown (1978)) was not confirmed in t h i s canine study. The d i f f e r e n c e i n the t h r e s h o l d to a r g i n i n e c o u l d account f o r the lack of i n s u l i n o t r o p i c a c t i o n i n the dogs. A l a n i n e c o u l d exert a dual r o l e i n glucose metabolism: a d i r e c t a c t i o n on gluconeogenesis and an i n d i r e c t a c t i o n through the r e g u l a t i o n of i n s u l i n and glucagon r e l e a s e . A l a n i n e has been shown to be one of the four amino a c i d s which give r i s e to a s i g n i f i c a n t amount of glucose i n the p e r f u s e d r a t l i v e r (Ross, Hems and Krebs (1966)). A l a n i n e e n t e r s the gluconeogenic pathway 251 by c o n v e r t i n g to pyruvate (Rocha et a l . (1972)). Intravenous and o r a l a d m i n i s t r a t i o n of a l a n i n e i n man w i l l e l e v a t e plasma glucagon l e v e l s , whereas o r a l a l a n i n e was shown to e l e v a t e i n s u l i n ( R o s s i n i et a l . (1975)). A l a n i n e was demonstrated to be one of the poorest i n s u i i n o t r o p i c agents amongst the - twenty amino a c i d s being s t u d i e d i n dogs i n a normoglycaemic s i t u a t i o n (Rocha et a l . (1972)). The i n s u i i n o t r o p i c a c t i o n of 5 g of o r a l a l a n i n e (50% of the amount used i n the human experiments by R o s s i n i et a l . (1975) was s t u d i e d i n the presence of m i l d hyperglycaemia in dogs. The r e s u l t s i n d i c a t e d that there was no p o t e n t i a t i n g e f f e c t on IRI r e l e a s e and there ' was no IR-GIP r e l e a s e f o l l o w i n g o r a l a l a n i n e . Two o b s e r v a t i o n s d u r i n g s t u d i e s on the p h y s i o l o g y and pathophysiology of GIP i n man prompted a renewed i n t e r e s t i n the r e l a t i o n s h i p between the 2 major p h y s i o l o g i c a l e f f e c t s of GIP i . e . the i n s u i i n o t r o p i c and the g a s t r i c i n h i b i t o r y a c t i v i t i e s . The f i r s t o b s e r v a t i o n was that p a t i e n t s with a c t i v e duodenal u l c e r s have a higher f a s t i n g IR-GIP and a g r e a t e r IR-GIP response than c o n t r o l s f o l l o w i n g the ingeston of a mixed meal (Arnold et a l . (1978), Cataland et a l . (1977)) or glucose ( L a u r i t s e n and Moody (1978)). The second o b s e r v a t i o n was that an i n t r a d u o d e n a l i n f u s i o n of h y d r o c h l o r i c a c i d or endogenously s e c r e t e d h y d r o c h l o r i c a c i d cause a greater IRI response during intravenous i n f u s i o n of glucose (Fahrenkrug et a l . (1978)). In r a t s , Ebert et a l . (1979) had demonstrated a dose-dependent r e l a t i o n s h i p between the c o n c e n t r a t i o n of h y d r o c h l o r i c a c i d introduced i n t r a d u o d e n a l l y and r e l e a s e of IR-GIP and that t h i s IR-GIP response was capable of p o t e n t i a t i n g IRI r e l e a s e i n the presence of hyperglycaemia. They a l s o r e p o r t e d that the i n s u l i n o t r o p i c a c t i o n of IR-GIP c o u l d be a b o l i s h e d by the concurrent i n f u s i o n of GIP a n t i s e r a . However, t h e i r data a l s o showed a s t a t i s t i c a l l y s i g n i f i c a n t e l e v a t i o n of serum glucose f o l l o w i n g i n t r a d u o d e n a l a d m i n i s t r a t i o n of h y d r o c h l o r i c a c i d at high c o n c e n t r a t i o n . T h i s r a i s e d the p o s s i b i l i t y that other mechanisms were i n v o l v e d i n the r e g u l a t i o n of plasma glucose in r a t s d u r i n g duodenal i n f u s i o n of h y d r o c h l o r i c a c i d . One p o s s i b l e e x p l a n a t i o n of the e l e v a t e d plasma glucose c o u l d be due to a r i s e i n plasma glucagon by the r e l e a s e of IR-GIP. In man i n t r a d u o d e n a l i n f u s i o n of h y d r o c h l o r i c a c i d e l i c i t e d IR-GIP r e l e a s e but d i d not a l t e r the plasma glucose or IRI (Ebert et a l . (1979)). However, in dogs 253 (Brown, Dryburgh et a l . (1975)), i n t r a d u o d e n a l i n f u s i o n of h y d r o c h l o r i c a c i d d i d not r e l e a s e IR-GIP. The present study confirmed the l a t t e r o b s e r v a t i o n i n which no change in IRI, IR-GIP and plasma glucose were d e t e c t e d f o l l o w i n g i n t r a d u o d e n a l h y d r o c h l o r i c a c i d i n f u s i o n . In a d d i t i o n no IRI r e l e a s e was observed f o l l o w i n g i n t r a d u o d e n a l h y d r o c h l o r i c a c i d i n f u s i o n i n the presence of m i l d hyperglycaemia. Thus i t seemed u n l i k e l y that HC1 was i n v o l v e d i n the e n t e r o i n s u l a r a x i s . The IR-GIP response to intr a d u o d e n a l h y d r o c h l o r i c a c i d appeared to be s p e c i e s dependent, and while the i n c r e t i n e f f e c t of IR-GIP endogenously r e l e a s e d by HC1 might be s i g n i f i c a n t i n some human p a t h o l o g i c s t a t e s , 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 i n he a l t h y man has yet to be i d e n t i f i e d . Fat i n g e s t i o n has been demonstrated to r e l e a s e IR-GIP i n dogs (Pederson, Schubert and Brown (1975)) and in man (Falko et a l . (1975)). The p a t t e r n of IR-GIP response f o l l o w i n g i n g e s t i o n of f a t i s d i f f e r e n t from that f o l l o w i n g o r a l glucose with respect to i n i t i a t i o n of response, magnitude and d u r a t i o n ( S i r i n i k , C r ockett et a l . (1974)). The IR-GIP response f o l l o w i n g o r a l f a t reached a p l a t e a u much l a t e r , achieved a higher l e v e l and 254 remained e l e v a t e d f o r a longer p e r i o d of time than f o l l o w i n g o r a l g l u c o s e . The i n s u l i n o t r o p i c a c t i o n of IR-GIP f o l l o w i n g o r a l f a t depended on the degree of hyperglycaemia. In man, o r a l f a t with concurrent intravenous i n f u s i o n of glucose e l i c i t e d a g r e a t e r IRI response than d i d intravenous glucose alone ( C l e a t o r and Gourlay (1975), but no s t a t i s t i c a l d i f f e r e n c e was observed i n plasma glucose between the two groups. In the absence of hyperglycaemia the r i s e of IR-GIP f o l l o w i n g o r a l f a t d i d not a l t e r the plasma l e v e l of IRI, glucose and n o n - e s t e r i f i e d f a t t y a c i d (Falko et a l . (1975)). In the present study, o b s e r v a t i o n s of the i n s u l i n o t r o p i c a c t i o n of endogenously r e l e a s e d IR-GIP f o l l o w i n g i n t r a d u o d e n a l f a t were c a r r i e d out i n the presence of a steady s t a t e hyperglycaemia i n dogs. No i n s u l i n o t r o p i c a c t i o n was observed f o l l o w i n g o r a l f a t duri n g m i l d hyperglycaemia, but a modest inc r e a s e i n IRI was observed d u r i n g G+150. The e l e v a t i o n of IR-GIP was demonstrated i n the presence of m i l d hyperglycaemia, but IR-GIP was not measured i n the moderate hyperglycaemic s t a t e because of a shortage of GIP a n t i s e r a . T h e r e f o r e , no c o n c l u s i o n s c o u l d be made in regard to the e f f e c t of 255 h y p e r i n s u l i n a e m i a and hyperglycaemia on IR-GIP r e l e a s e f o l l o w i n g o r a l f a t . The molecular mechanism of i n s u l i n r e l e a s e has been shown to be a set of s e q u e n t i a l metabolic, i o n i c and m o t i l e events. The two most accepted t h e o r i e s to e x p l a i n the c o u p l i n g of the metabolic and the i o n i c events are the g l u c o r e c e p t o r theory and the s u b s t r a t e - s i t e (or metabolic f u e l ) h y p o t h e s i s . In the g l u c o r e c e p t o r theory ( A s h c r o f t (1980)) molecular glucose was c o n s i d e r e d to act as a s i g n a l f o r i n s u l i n r e l e a s e by i n t e r a c t i n g with g l u c o r e c e p t o r s on the c e l l membrane s u r f a c e , w h i l s t i n the s u b s t r a t e - s i t e theory (Malaisse et a l . (1979)) the s i g n a l f o r i n s u l i n r e l e a s e was' generated by the metabolism of glucose i n the p a n c r e a t i c c e l l i n the form of a m e t a b o l i t e or c o f a c t o r . The support f o r the s u b s t r a t e - s i t e theory over the glucose r e c e p t o r theory came from s e v e r a l experimental o b s e r v a t i o n s (Malaisse et a l . (1979)). F i r s t the r e l a t i v e i n s u i i n o t r o p i c a c t i v i t y of d i f f e r e n t sugars c o r r e l a t e d with t h e i r metabolism i n the i s l e t c e l l s . Secondly i t has been shown p o s s i b l e 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 e x t r a c e l l u l a r glucose by provoking g l y c o g e n o l y s i s and g l y c o l y s i s from endogenous s t o r e s of carbohydrate. 256 The search f o r the p o s t u l a t e d metabolic s i g n a l i n the s u b s t r a t e - s i t e theory showed that p u r i n e n u c l e o t i d e ( J a i n and Logothetopoulus (1978)), pyruvate, l a c t a t e ( J a i n , Asina and Logothetopouls (1978) and D-gl y c e r a l d e h y d e (Schauder, Mcintosh et a l . (1977) were a l l capable of i n c r e a s i n g i n s u l i n r e l e a s e i n the i s o l a t e d r a t i s l e t p r e p a r a t i o n . The r o l e of cAMP has not been c o n c l u s i v e l y determined (Schauder, Mcintosh et a l . (1977), M a l a i s s e et a l . (1979)). The i n t e r a c t i o n of hormones with these m e t a b o l i t e s has not been w e l l s t u d i e d . Schauder, S c h i n d l e r et a l . (1977) had observed a g r e a t e r i n s u l i n response with a - k e t o i s o c a p r o i c a c i d and D-glyceraldehyde i n the presence of GIP or glucagon. Most of the s t u d i e s of the mechanism of i n s u l i n r e l e a s e u t i l i z e d rn v i t r o p r e p a r a t i o n s such as i s o l a t e d i s l e t p r e p a r a t i o n s or i s o l a t e d p e r f u s e d rat pancreas. In these s t u d i e s an _iri v i v o canine p r e p a r a t i o n was used to study the i n t e r a c t i o n of exogenous GIP with intravenous i n f u s i o n of a - k e t o g l u t a r i c a c i d , glucose, pyruvate and s u c c i n i c a c i d on an equimolar b a s i s . The r e s u l t s i n d i c a t e d no IRI r e l e a s e c o u l d be induced f o l l o w i n g the i n f u s i o n of these m e t a b o l i t e s e i t h e r i n the presence or the absence of a concurrent GIP i n f u s i o n . E a r l i e r J a i n et 257 a l . (1978) had r e p o r t e d that an i n c r e a s i n g c o n c e n t r a t i o n of pyruvate f a i l e d to s t i m u l a t e p r o i n s u l i n b i o s y n t h e s i s and i n s u l i n r e l e a s e i n the f r e s h l y i s o l a t e d i s l e t s . However pyruvate d i d enhance IRI i n the presence of f r u c t o s e , l e u c i n e and glucose (Matschinsky et a l . (1975)). In the i s o l a t e d p e r f u s e d r a t pancreas a-k e t o g l u t a r i c a c i d and pyruvate d i d not r e l e a s e IRI (Lenzen (1977)). The ir\ v i v o canine p r e p a r a t i o n provided a model in which the response of the organism as a whole to the m e t a b o l i t e c o u l d be s t u d i e d . However the disadvantage of t h i s iri v i v o study was the i n a b i l i t y to determine the l o c a l c o n c e n t r a t i o n of the m e t a b o l i t e s i n the pancreas. The problems such as d i f f e r e n t r a t e of metabolism of the s u b s t r a t e by t i s s u e s would c e r t a i n l y d i f f e r e n t i a l l y a l t e r the c o n c e n t r a t i o n of m e t a b o l i t e s i n the c i r c u l a t i o n . The present study demonstrated that the i n s u l i n o t r o p i c a c t i o n of GIP was not expressed with the three m e t a b o l i t e s s t u d i e d . 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 has been examined i n r e s p e c t to i t s p h y s i o l o g i c a l a c t i o n s , g a s t r i c a c i d i n h i b i t i o n and the glucose dependent i n s u l i n o t r o p i c a c t i o n , and i n respect to the p u r i t y of the p r e p a r a t i o n . 258 The p h y s i o l o g i c a l r o l e of . GIP i n the 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 i n a stomach with i n t a c t nervous supply remains to be determined. However t h i s study has shown that m o d i f i c a t i o n of c e r t a i n amino a c i d r e s i d u e s i n GIP c o u l d decrease t h i s a c i d i n h i b i t o r y a c t i v i t y . The dependency of the i n s u l i n o t r o p i c a c t i o n of GIP on the s t a t e of glycaemia was confirmed using dogs in a steady s t a t e hyperglycaemic system. GIP 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 moderate hyperglycaemia r e g a r d l e s s of whether the GIP was from exogenous or endogenous sources. Both glucose and f a t a d m i n i s t e r e d e n t e r a l l y were demonstrated to r e l e a s e IR-GIP and p o t e n t i a t e i n s u l i n r e l e a s e at moderate hyperglycaemia. T h i s c o u l d l e a d to the s p e c u l a t i o n that the a d d i t i o n a l i n s u l i n r e l e a s e provoked by GIP c o u l d p r o v i d e a safeguard a g a i n s t a prolonged hyperglycaemia i n the organism. The r o l e of i n s u l i n in feedback i n h i b i t i o n of IR-GIP r e l e a s e was not answered in t h i s study. An amino a c i d mixture a d m i n i s t e r e d e i t h e r o r a l l y , i n t r a d u o d e n a l l y or i n t r a v e n o u s l y was capable of p o t e n t i a t i n g i n s u l i n r e l e a s e i n the presence of m i l d hyperglycaemia although no d i f f e r e n c e i n the degree of h y p e r i n s u l i n a e m i a was observed. The r o l e of GIP , i n the 259 s t u d i e s with the amino a c i d mixture a d m i n i s t e r e d e n t e r a l l y remains to be c l a r i f i e d . Although IR-glucagon was not measured, these amino a c i d s i n the mixture are known to be gl u c a g o n o t r o p i c (Rocha et a l . (1972)), t h e r e f o r e the i n t e r a c t i o n of i n s u l i n , glucagon and p o s s i b l y GIP co u l d be important d u r i n g p o s t p r a n d i a l p r o t e i n i n g e s t i o n . F i n a l l y the i n s u i i n o t r o p i c a c t i o n of GIP f o l l o w i n g intraduodenal a c i d a d m i n i s t r a t i o n was not confirmed i n dogs d u r i n g m i l d hyperglycaemia. Since the study was not c a r r i e d out at moderate hyperglycaemia, t h i s does not r u l e out the p o s s i b i l i t y of other humoral f a c t o r s r e l e a s e d by a c i d and capable of p o t e n t i a t i n g i n s u l i n r e l e a s e d u r i n g moderate hyperglycaemia. The o b s e r v a t i o n of the d i v e r s e p h y s i o l o g i c a l a c t i o n of GIP, g a s t r i c a c i d i n h i b i t i o n and i n s u i i n o t r o p i c a c t i o n , prompted the i n v e s t i g a t i o n i n t o the p o s s i b i l i t y of the presence of other p o l y p e p t i d e components i n the GIP p r e p a r a t i o n which might account f o r some of the p h y s i o l o g i c a l a c t i o n s a t t r i b u t e d to GIP. The bio c h e m i c a l i n v e s t i g a t i o n using p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and t h i n l a y e r chromatography i n d i c a t e d u n e q u i v o c a l l y the presence of two p o l y p e p t i d e components. Furthermore, p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n d i c a t e d the two 260 components were u n l i k e l y to be the r e s u l t of o x i d a t i o n of GIP. S u c c e s s f u l s e p a r a t i o n of d i f f e r e n t p o l y p e p t i d e components from GIP was accomplished by HPLC, in which two d i s t i n c t i v e peaks were observed, a minor component peak I with absorbance about one f i f t h of that of peak I I . The amino a c i d composition of the p o l y p e p t i d e component i n peak I was s i m i l a r to peak II with the exception that peak I had. one l e s s t y r o s i n e and a l a n i n e r e s i d u e . T h i s r e s u l t compared w e l l with the sequence work on GIP p r e p a r a t i o n s by J o r n v a l l et a l . {1981), i n which a minor p o l y p e p t i d e with a sequence s i m i l a r to GIP except that the N-terminal t y r o s i n e and a l a n i n e r e s i d u e s were l a c k i n g . T h i s sequence work on GIP a l s o i n d i c a t e d a d i f f e r e n c e of one glutamine r e s i d u e i n p o s i t i o n 29 of the e a r l i e r p u b l i s h e d sequence of GIP (Brown (1971), Brown and Dryburgh (1971)). T h i s c o u l d e x p l a i n p a r t i a l l y the 9 lack of success i n o b t a i n i n g s y n t h e t i c GIP p r e p a r a t i o n with a s c r i b e d p h y s i o l o g i c a l a c t i v i t y i n a d d i t i o n to other problems such as p u r i t y and amino a c i d m o d i f i c a t i o n s i n the s y n t h e s i s of GIP. The s t a t e of knowledge of the p h y s i o l o g i c a l a c t i v i t y of the major and the minor components i s not complete with respect to a c i d i n h i b i t i o n and i n s u i i n o t r o p i c a c t i o n , However, the p o l y p e p t i d e s separated by HPLC are p r e s e n t l y being 261 accumulated f o r f u r t h e r i n v e s t i g a t i o n s . The r o l e of GIP i n the e n t e r o i n s u l a r a x i s was i n v e s t i g a t e d i n a canine steady s t a t e hyperglycaemic model. Exogenous GIP was demonstrated to p o t e n t i a t e IRI r e l e a s e i n the presence of moderate but not i n m i l d hyperglycaemia. The hyperglycaemic 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 and the dose of GIP which produced i n c r e a s e d i n s u l i n r e l e a s e were found to be higher i n dogs than i n man, even a f t e r t a k i n g i n t o c o n s i d e r a t i o n the d i f f e r e n c e i n GIP p r e p a r a t i o n s used i n the two s t u d i e s . The i n c r e a s e i n IRI r e l e a s e to p a r e n t e r a l l y a dministered secretogues such as glucose and f a t was a l s o smaller i n dogs than man, even though e l e v a t e d IR-GIP l e v e l s were observed. These r e s u l t s suggested that the endogenous r e l e a s e of IR-GIP d u r i n g hyperglycaemia might have an i n s u l i n o t r o p i c e f f e c t s i m i l a r to that seen with exogenous i n f u s i o n of GIP. However, the potency of GIP as an i n s u l i n o t r o p i c agent i s much weaker i n dogs than i n man at a l l l e v e l s of hyperglycaemia. F u r t h e r c o n f i r m a t i o n came from the human s t u d i e s i n which o r a l glucose e l e v a t e d IRI r e l e a s e i n the presence of moderate hyperglycaemia and a l s o e l e v a t e d IR-GIP (Andersen et a l . (1978)). 262 The hypothesis of the e x i s t e n c e of an e n t e r o i n s u l a r a x i s i s an a t t r a c t i v e mechanism by which the gut senses the q u a l i t y and the q u a n t i t y of the i n g e s t e d m e t a b o l i t e s , which then s i g n a l other organs in the organism f o r the ha n d l i n g of the n u t r i e n t s a f t e r a b s o r p t i o n . The r e s u l t s of t h i s study suggested that the r o l e of GIP as a s i g n a l from the gut to the pancreas i n dogs i s much weaker than that suggested in man. The r o l e of f a t induced h y p e r i n s u l i n a e m i a and the e l e v a t i o n of IR-GIP r e l e a s e i n the presence of hyperglycaemia i s not completely understood. T h i s study d i d not demonstrate a r o l e of IR-GIP i n the p o t e n t i a t i o n of IRI r e l e a s e f o l l o w i n g the i n g e s t i o n of amino a c i d mixtures. A l s o no d i f f e r e n c e i n the degree of IRI r e l e a s e was observed f o l l o w i n g the e n t e r a l and p a r e n t e r a l route of a d m i n i s t r a t i o n of amino a c i d mixtures. T h e r e f o r e , an e n t e r o i n s u l a r a x i s s i m i l a r to that which e x i s t s f o r o r a l glucose was not demonstrated f o r o r a l amino a c i d s . T h i s study a l s o confirmed the e a r l i e r p u b l i s h e d r e s u l t s on the r e l a t i o n s h i p of H C 1 with GIP (Brown, Dryburgh et a l . (1975)), i n which no IR-GIP was observed f o l l o w i n g i n t r a d u o d e n a l H C 1 i n f u s i o n and no p o t e n t i a t i o n of IRI r e l e a s e by H C 1 i n the presence of m i l d hyperglycaemia. 2 6 3 However, the p o s s i b i l i t y that some form of p o t e n t i a t i o n c of IRI r e l e a s e at higher l e v e l s of hyperglycaemia might e x i s t to account f o r the o b s e r v a t i o n of h y p e r i n s u l i n a e m i a induced by concurrent intraduodenal i n f u s i o n of HC1 and intravenous i n f u s i o n of glucose in r a t s (Ebert et a l . (1979)). The glucose clamp model provided a steady s t a t e hyperglycaemia which allowed the measurement of the e f f e c t of secretogogues or hormones to be made at a constant plasma glucose c o n c e n t r a t i o n . The amount of glucose i n f u s e d to maintain the clamp i s a r e f l e c t i o n of the glucose c l e a r a n c e r a t e . The major t e c h n i c a l d i f f i c u l t y i n m a i n t a i n i n g the glucose clamp was the problem of glucose o s c i l l a t i o n i n the beginning of the experiment, however, t h i s c o u l d be r e s o l v e d by changing the glucose i n f u s i o n r a t e s by small increments i n the d i r e c t i o n necessary to o b t a i n the d e s i r e d plasma glucose l e v e l . The glucose clamp d i d not completely simulate a p h y s i o l o g i c a l s i t u a t i o n f o l l o w i n g the i n g e s t i o n of a meal, in which the i n i t i a l r i s e of hyperglycaemia and the d u r a t i o n of steady s t a t e hyperglycaemia were d i f f e r e n t . 264 BIBLIOGRAPHY A h l r o t h A and Mutt V (1970) Polyacrylamide g e l e l e c t r o p h o r e s i s of p o l y p e p t i d e s from the i n t e s t i n a l w a l l , with c o u n t e r m i g r a t i o n of dye. A n a l y t i c a l B iochemistry 37:125-128. 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Brown JC, Dahl M, Kwauk S, Mcintosh CHS, Otte SC and Pederson RA (1981) Mechanism of action of GIP. Brain-Gut Axis: The New Frontier. Florence, Italy meeti ng. I i v to c o n t a i n two l e s s amino a c i d r e s i d u e s ( t y r o s i n e and a l a n i n e ) than GIP. The amino a c i d sequence of GIP III i n d i c a t e d the presence of a peptide component with an amino a c i d sequence d i f f e r e n t from GIP i n that the f i r s t two amino a c i d r e s i d u e s of the N-terminal p o r t i o n of the molecule ( t y r o s i n e and a l a n i n e ) were m i s s i n g . The lack of i n h i b i t o r y a c t i v i t y to 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 s y n t h e t i c GIP l e d to a r e i n v e s t i g a t i o n of the amino a c i d sequence of the molecule. The work i n c o l l a b o r a t i o n with J o r n v a l l (Sweden) i n d i c a t e d an e r r o r , in that the o r i g i n a l sequence i n c l u d e d a second glutamine in p o s i t i o n 30. i v to c o n t a i n two l e s s amino a c i d r e s i d u e s ( t y r o s i n e and a l a n i n e ) than GIP. The amino a c i d sequence of GIP III i n d i c a t e d the presence of a pept i d e component with an amino a c i d sequence d i f f e r e n t from GIP i n that the f i r s t two amino a c i d r e s i d u e s of the N-terminal p o r t i o n of the molecule ( t y r o s i n e and a l a n i n e ) were m i s s i n g . The lack of i n h i b i t o r y a c t i v i t y to 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 s y n t h e t i c GIP l e d to a r e i n v e s t i g a t i o n of the amino a c i d sequence of the molecule. The work i n c o l l a b o r a t i o n with J o r n v a l l (Sweden) i n d i c a t e d an e r r o r , in that the o r i g i n a l sequence i n c l u d e d a second glutamine in p o s i t i o n 30.. 

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