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Studies on the physiological release and insulinotropic action of gastric inhibitory polypeptide Schubert, Harold Edward 1974

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STUDIES ON THE PHYSIOLOGICAL RELEASE AND INSULINOTROPIC ACTION OF GASTRIC INHIBITORY POLYPEPTIDE By Harold Edward Schubert B.Sc, Univ e r s i t y of B r i t i s h Columbia, 1973 A Thesis Submitted i n P a r t i a l F u l l f i l l m e n t of the Requirements for the Degree of Master of Science In the Department o f Phys iology We accept t h i s thesis as conforming to the required standard Supervisor The University of B r i t i s h Columbia September, 1974 In presenting th is thes is in pa r t i a l fu l f i lment o f the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l ica t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT The e f f e c t of highly p u r i f i e d g a s t r i c i n h i b i t o r y polypeptide (GIP) on immunoreactive i n s u l i n (IRI) secretion in the conscious fasted dog was investigated. S i g n i f i c a n t increased i n IRI release were observed with intravenous administration of three d i f f e r e n t doses of GIP. These were accompanied by depression i n f a s t i n g serum glucose l e v e l s . Preliminary studies were undertaken to determine whether t h i s i n s u l i n o t r o p i c action of GIP could be a t t r i b u t e d to a p a r t i c u l a r segment of the GIP molecule. GIP fragments produced by cleavage with cyanogen bromide (CNBr) and trypsin (TPCK) showed no s i g n i f i c a n t stimulation of IRI release. The p o s s i b i l i t y that GIP might i t s e l f enhance glucose uptake or potentiate insulin-induced glucose uptake was studied using the r a t hemidiaphragm preparation. No such e f f e c t was observed. The release of IR-GIP by o r a l glucose and f a t was investigated and t h i s release Was shown to occur i n a dose-related manner. Fat was found to be the most potent secretagoge for IR-GIP reported to date but no s i g n i f i c a n t change i n serum IRI l e v e l s was observed following o r a l f a t . In l i g h t of t h i s and other recent work, the possible involvement of GIP i n carbohydrate metabolism i s discussed. i TABLE OF CONTENTS PAGE ABSTRACT i LIST OF TABLES i v LIST OF FIGURES V ACKNOWLEDGEMENTS v i i INTRODUCTION 1 MATERIALS AND METHODS 8 Animal and Tissue Preparations 8 1. Chronic Dogs 8 2. Rat Hemidiaphragms 8 Polypeptide Degradations 9 1. Non-enzymatic Cleavage with Cyanogen Bromide 9 2. Enzymatic Cleavage with Trypsin 10 Ion Exchange Chromatography 10 High Voltage Electrophoresis 12 1. Equipment and Reagents 12 2. Standards and Markers 13 3. Procedure for Q u a l i t a t i v e Electrophoresis 14 4. Detection of Peptides . 16 Assay Methods 17 1. Assay of Glucose i n Sera and KRB Solutions 17 2. Radioimmunoassay of Gastric I n h i b i t o r y Polypeptide 17 3. Radioimmunoassay of In s u l i n 17 Sources of Hormone, Sugar and Fat Preparations 18 i i PAGE Analysis of Data 18 RESULTS 20 E f f e c t of GIP Infusions on Serum Levels of IRI and Glucose i n the Fasted Dog 20 E f f e c t of Infusion of GIP Fragments on Serum Levels of IRI and Glucose i n the Fasted Dog. . 29 E f f e c t of GIP Alone and i n Combination with I n s u l i n on Glucose Uptake by the Rat Hemi-diaphragm 37 Release of IR-GIP by Oral Glucose i n the Fasted Dog 39 Release of IR-GIP by Oral Fat, i n the Fasted Dog, and i t s E f f e c t on Serum IRI Levels . . . 44 DISCUSSION . 49 BIBLIOGRAPHY 60 i i i LIST OF TABLES Table Page I. E f f e c t of Infusion of 0.9% Saline on Serum Concentrations of IRI and Glucose i n the Fasted Dog 21 I I . E f f e c t of Infusion of 0.25 jjg/kg GIP on Serum Concentrations of IRI-GIP, IRI and Glucose i n the Fasted Dog 22 I I I . E f f e c t of Infusion of 0.50 u.g/kg GIP on Serum Concentrations of IR-GIP, IRI and Glucose i n the Fasted Dog 23 IV. E f f e c t of Infusion of 1.5 u,g/kg GIP on Serum Concentrations of IR-GIP, IRI and Glucose i n the Fasted Dog 24 V. E f f e c t of Infusion of CNBr-GIP Fraction I I I (C-terminal residues 15-43) at 5.0 }\g/kg on Serum Concentrations of IR-GIP in the Fasted Dog 34 VT. E f f e c t of 100 ml Oral Water on Serum Concen-trat i o n s of IR-GIP, IRI and Glucose i n the Fasted Dog 42 VII. E f f e c t of 100 ml Oral Lipomul on Serum Concentrations of IRI and Glucose i n the Fasted Dog 47 LIST OF FIGURES Figure Page 1. Standards and markers used on high voltage electrophoresis at pH 6.5 15 2. Changes i n serum immunoreactive i n s u l i n (IRI) concentrations produced by 5 min intravenous infusion of 0.25, 0.50 and 1.5 AgAg of GIP . . . . 25" 3. Changes i n serum glucose concentrations produced by 5 min intravenous infusions of 0.25, 0.50 and 1.5 u.g/kg GIP 27 4. Pattern of changes i n serum concentrations of IR-GIP, IRI and glucose when GIP was infused at 1.5 u.g/kg/5 min 28 5. Changes i n serum IRI concentrations pro-duced by 5 min intravenous infusions of the peptides r e s u l t i n g from CNBr treatment of GIP - 30 6. E l u t i o n p r o f i l e of 2.0 mg of CNBr-treated GIP on a 0.5 x 7.0 cm column of carbo-xymethylcellulose (CM 11) . 31 7. High voltage electrophoresis at pH 6.5 of GIP and the peptides obtained by CNBr cleavage of GIP, a f t e r separation on CM 11 32 8. High voltage electrophoresis at pH 6.5 of GIP and a t r y p t i c digest of GIP 35 Figure Page 9. Complete sequence of g a s t r i c i n h i b i t o r y polypeptide and the major fragments which have been assayed for i n s u l i n o t r o p i c a c t i v i t y 36 10. E f f e c t s of i n s u l i n , GIP and GIP + i n s u l i n on glucose uptake by the r a t hemidiaphragm preparation 38 11. Serum glucose concentrations following o r a l glucose at 0.25, 0.50, 1.0 and 2.0 g/kg i n the dog 41 12. Serum IR-GIP concentrations following o r a l glucose at 0.25, 0.50, 1.0 and 2.0 g/kg i n the dog 41 13. V a r i a t i o n of peak serum IR-GIP response with o r a l glucose load i n the dog . . . . . . . 43 14. Serum IR-GIP concentrations following 25 ml, 50 ml and 100 ml o r a l Lipomul i n the dog. . 45 15. Changes i n serum IRI concentrations following 100 ml oralWaterand following 100 ml o r a l Lipomul i n the dog . „ 48 v i ACKNOWLEDGEMENTS I would f i r s t l i k e to thank Dr. J . C. Brown for his constant enthusiasm, encouragement and guidance i n th i s research. The generous assistance and companionship of Dr. R. A. Pederson and Miss J . Dryburgh was much appreciated. I wish to acknowledge, with thanks, Mr. K. Henze, for the preparation and photography of the fi g u r e s . I am g r a t e f u l for the help of Miss P. Hunter i n preparing the rough d r a f t of t h i s t h e s i s . The Medical Research Council of Canada i s ack-nowledged for f i n a n c i a l support i n t h i s work. v i i 1 INTRODUCTION Earl y i n t h i s century, Moore et a l . (1906) suggested that the duodenum produced a hormone which stimulated the i n t e r n a l secretion of the pancreas. In 1929, Zunz and La Barre suggested that crude s e c r e t i n contained an endocrine pancreas - stimulating factor, which they termed " i n c r e t i n " , as well as the exocrine pancreas - stimulating factor, "excretin". Since then the search for t h i s i n s u l i n o t r o p i c factor, " i n c r e t i n " , has been the subject of much in v e s t i g a -t i o n . Recent development of radioimmunological methods for accurate determination of serum i n s u l i n l e v e l s has a c c e l e r -ated t h i s research. In 1965, Mclntyre established that, i n man, the intraduodenal administration of glucose produced a s i g n i f i c a n t l y greater r i s e i n plasma i n s u l i n l e v e l s than the intravenous administration of a comparable glucose load. This greater i n s u l i n response occurred even though lower serum glucose l e v e l s were achieved following o r a l glucose. This evidence provided substantiation of the old claims of Moore and Zunz et a l . . The difference i n the i n s u l i n response to o r a l ingestion and intravenous infusion of glucose i s such that i t has been estimated that h a l f of the i n s u l i n secreted a f t e r an o r a l glucose load i s released by g a s t r o i n t e s t i n a l hormones (Perley and Kipnis, 1967)„ 2 These studies led to i n v e s t i g a t i o n of the i n s u l i n -releasing potencies of a l l known g a s t r o i n t e s t i n a l hormones and proposals of several new i n s u l i n o t r o p i c gut peptides. For some established hormones c o n f l i c t i n g evidence has been reported whereas, for several new peptides, further p u r i f i c a t i o n and characterization are required before assessment of i n s u l i n o t r o p i c a c t i v i t y can be undertaken. Dupre et a l . reported that administration of crude se c r e t i n increased the concentration of i n s u l i n i n peripheral plasma (Dupre and Beck, 1966) and increased the rate o f disappearance of intravenous glucose (Dupre, 1965). Insulinotropic action of highly p u r i f i e d s e c r e t i n has been reported i n man (Chisholm, 1969; Deckert, 1968) and i t has been shown to act s y n e r g i s t i c a l l y with glucose in stimulating i n s u l i n release (Dupre et a l . , 1969). The r e s u l t s of these in vivo experiments are contradicted by r e s u l t s of more recent i n v i t r o studies using the r a t (Schatz et a l . , 1974) and rabbit (Turner, 1969) i n which se c r e t i n produced no enhancement of i n s u l i n s e cretion. I t has been suggested that the i n s u l i n o t r o p i c action of secretin requires the presence of functioning exocrine pancreas tissue (Schatz et a l . , 1974). Therefore, t h i s action of s e c r e t i n may be a non-specific side e f f e c t of increased metabolism i n exocrine pancreas t i s s u e . I t has also been reported that s e c r e t i n enhances the rate of glucose disappearance from the blood independent of any action i t may have on the endocrine pancreas (Chisholm, 3 1969; L i c k l e y et a l . , 1970). There are c o n f l i c t i n g reports as to whether or not serum s e c r e t i n l e v e l s are elevated a f t e r o r a l glucose (Chisholm e t a l . , 1969; Buchanan et a l . , 1973; Boden et a l . , 1974). These c o n f l i c t s may be due to poor r e l i a b i l i t y of the radioimmunoassay for s e c r e t i n . The release of sec r e t i n by glucose i s not consistent with the evidence that intraduodenal glucose administration does not stimulate exocrine pancreatic secretion (Sum and Preshaw, 1967). The evidence accumulated to date leaves the status of s e c r e t i n as a ph y s i o l o g i c a l stimulant o f i n s u l i n release uncertain. The involvement of g a s t r i n i n the release of i n s u l i n i s a l s o in dispute due to c o n f l i c t i n g reports. Unger et a l . (1967) reported that pure porcine g a s t r i n II showed 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 dog whereas J a r r e t t and Cohen (1967) found no such a c t i v i t y with synthetic human ga s t r i n I, porcine g a s t r i n II or pentagastrin. I t was shown that, i n man, serum g a s t r i n l e v e l s rose o n l y s l i g h t l y a f t e r an o r a l glucose load (Rehfeld et a l . , 1973). An infusion of g a s t r i n , mimicking the small p h y s i o l o g i c a l response to or a l glucose, did not elevate serum i n s u l i n l e v e l s but i t did s l i g h t l y potentiate the i n s u l i n response to intravenous i n j e c t i o n of 25g. glucose (Rehfeld et a l . , 1973). I t i s not c e r t a i n , however, that these infusion studies simulate natural conditions since serum g a s t r i n i s known to be comprised of several molecular forms. 4 An impure extract of canine jejunal mucosa, containing glucagon-like immunoreactivity, has been shown to stimulate i n s u l i n release i n dogs (Unger et a l . , 1968) and l e v e l s of t h i s material were reported to r i s e a f t e r heavy o r a l g l u -cose loading in man (Samols et a l . , 1965). In contrast, Marco et a l . (1970) reported that t h i s glucagon-like substance was not released when monosaccharides were admin-ist e r e d intraduodenally. D e f i n i t e r e s o l u t i o n of t h i s c o n f l i c t w i l l require further 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 of the glucagon-like substance. Turner et a l . (1973), using a polypeptide f r a c t i o n from porcine duodenal-jejunal mucosa, " i n s u l i n releasing polypeptide (IRP)", have shown potentiation of the i n s u l i n response to an intravenous glucose load in the r a t with improvement i n glucose tolerance. Very large doses of IRP were required to produce t h i s e f f e c t . This poly-peptide f r a c t i o n has recently been found to contain small quantities o f g a s t r i c i n h i b i t o r y polypeptide (GIP) (personal communication to J.C. Brown), a known potentiator of i n s u l i n release (Dupre e t a l . , 1973). Whether the potentiation of i n s u l i n release produced by IRP was due to i t s GIP content cannot be ascertained u n t i l data on the p u r i f i c a t i o n and biochemical characterization of IRP i s made a v a i l a b l e . In early studies with CCK-PZ preparations, stimula-t i o n of i n s u l i n release was observed (Dupre et a l . , 1969j Unger et a l . , 1967). These preparations were r e l a t i v e l y 5 crude and subsequent studies with "pure" CCK-PZ have shown i t to be without i n s u l i n o t r o p i c action i n the r a t (Rabino-v i t c h and Dupre, 1972). The fact that GIP has been shown to be present i n crude CCK-PZ preparations, to the extent of 10-15% by weight (Kuzio et a l . , 1974), suggests that i t may have been the active i n s u l i n o t r o p i c agent i n these preparations. The study reported in t h i s thesis deals with the i n s u l i n o t r o p i c action and p h y s i o l o g i c a l release of g a s t r i c i n h i b i t o r y polypeptide (GIP). Gastric i n h i b i t o r y polypeptide, i s o l a t e d i n 1969 from a side f r a c t i o n produced i n the p u r i f i c a t i o n of porcine CCK-PZ, was found to be capable of i n h i b i t i n g g a s t r i n -stimulated acid secretion i n e x t r i n s i c a l l y denervated pouches of the fundus of the stomach, i n the dog. (Brown et a l . , 1969; Brown, Mutt and Pederson, 1970). F i n a l p u r i f i c a t i o n and s t r u c t u r a l analysis of GIP showed i t to be a straight-chain 43-residue polypeptide having areas of homology with porcine s e c r e t i n and glucagon (Brown and Dryburgh, 1971). Phy s i o l o g i c a l studies i n the dog showed that GIP would i n h i b i t acid secretion stimulated by pentagastrin, i n s u l i n hypoglycemia and histamine. The a b i l i t y of the polypeptide to i n h i b i t histamine- and i n s u l i n hypoglycemia-stimulated acid secretion showed i t to possess g a s t r i c i n h i b i t o r y properties d i f f e r e n t from those of s e c r e t i n and CCK-PZ (Pederson and Brown, 1972) . GIP was also 6 shown to i n h i b i t pepsin secretion and g a s t r i c motor a c t i v i t y (Pederson, 1971) and to stimulate jejunal and i l e a l secretion i n the dog (Barbezat and Grossman, 1971) . I t was without e f f e c t on the exocrine pancreas and g a l l bladder (Pederson, 1971). A s p e c i f i c and s e n s i t i v e radioimmunoassay for GIP has been developed using guinea pig antisera r a i s e d to porcine GIP (Kuzio et a l . , 1974). The same antisera allowed l o c a l i z a t i o n of d i s t i n c t GIP c e l l s i n duodenal and jejunal mucosa of dog and man, using the immuno-fluorescent antibody technique (Polak, 1973). The r a d i o -immunoassay has been used to investigate mechanisms for the release of GIP i n man. Mean f a s t i n g l e v e l s of GIP have been reported a t approximately 200pg/ml serum. Cataland et a l . (1974) reported that o r a l glucose provided a potent stimulus for release of GIP and ingestion of fat has a l s o been shown to release GIP (Brown, Dryburgh, Pederson, 1974) whereas p r o t e i n and alcohol were without e f f e c t . Kuzio et a l . (1974) showed that, following a mixed meal i n fasted human subjects, serum GIP l e v e l s exceeded 1.0 ng/ml a f t e r 45 minutes i n a l l subjects tested. This GIP response was biphasic and, i n l i g h t of l a t e r studies, i t was suggested that the e a r l y GIP peak was due to glucose while the second peak was due to the fat content of the meal. Dupre et a l . (1973) found that infusion of GIP i n man, at a rate of 1.0 u.g/min. for 30 minutes, potentiated 7 i n s u l i n release and improved the tolerance of an i n t r a -venous glucose load. No e f f e c t was obtained when GIP was infused alone at the same rate. The peak serum immunoreactive GIP l e v e l s reached were no higher than 1.0 ng/ml which i s well within the range that can be obtained by ph y s i o l o g i c a l stimulation of GIP release. The experiments reported i n t h i s thesis were under-taken i n order t o : -1) e s t a b l i s h a model of i n s u l i n release by GIP, i n the dog, and to use t h i s model to determine whether the i n s u l i n o t r o p i c a c t i v i t y of GIP can be l o c a l i z e d to a p a r t i c u l a r s i t e within the molecule. 2) e s t a b l i s h whether GIP i t s e l f possesses any insulin-? l i k e a c t i v i t y . 3) characterize the serum GIP responses to o r a l glucose and f a t . 8 MATERIALS AND METHODS Animal and Tissue Preparations 1 Chronic Dogs Four conscious dogs of ei t h e r sex, weighing 18-23 kg. were used i n t h i s study. Dogs were fasted 18 hours p r i o r to use and were restrained by a harness within a metal stand during experiments. Blood was c o l l e c t e d using an indwelling catheter i n a foreleg vein and serum was a l i -quoted for measurement of serum glucose, immunoreactive i n s u l i n (IRI) and immunoreactive GIP (IR-GIP). GIP and GIP fragments were administered i n ph y s i o l o g i c a l saline using a second indwelling catheter connected to a harvard infusion pump. Phys i o l o g i c a l s a l i n e was infused i n control experiments. Glucose and f a t solutions were administered o r a l l y to dogs using a glass syringe f i t t e d to 30cm of 0.2cm (I.D.) polyethylene tubing. The end of the tube was held inside the cheek near the pos t e r i o r molars. Liquid deposited here induced dogs to swallow. 2 Rat Hemidiaphragms Rat hemidiaphragms were obtained from 120-150g. Wistar r a t s of ei t h e r sex which had been fasted at l e a s t 24 hours. Hemidiaphragms were incubated i n 2 ml. of Krebs-Ringer-bicarbonate-glucose (KRBG) so l u t i o n i n a Dubnoff Metabolic Incubating Shaker (Precision S c i e n t i f i c , 66722) at 37 C. Insulin, GIP, or i n s u l i n and GIP were added to the incubation media i n experimental f l a s k s . 9 Glucose concentrations in the incubation media were deter-mined upon addition of the prepared hemidiaphragms and af t e r an incubation period o f 90 minutes. The media were gassed with 95% 02/5% CO2 for 5 minutes p r i o r to addition of hemidiaphragms, afte r which flasks were stoppered for the duration of the incubation period. Following the method of Vallance - Owen and Hurlock, rat s were k i l l e d by a blow on the head, the abdomen was opened and the diaphragm excised. The diaphragm was placed i n iced KRB where i t was trimmed and divided into two approximately equal halves. The c e n t r a l tendon portion was discarded. Polypeptide Degradations 1 Non-enzymatic Cleavage with Cyanogen Bromide Cyanogen bromide (CNBr) cleavage of the peptide at the methionyl residue (Gross and witkop, 1962) was performed at 22°C i n a small sealed l y o p h i l i z i n g flask wrapped i n aluminum f o i l to exclude a l l l i g h t . The peptide was dissolved i n 70% (v/v) formic a c i d to give a concentra-t i o n of 10 mg/ml. Cyanogen bromide was also dissolved i n 70% formic acid to a concentration of 10 mg/ml. These solutions were mixed to obtain a peptide:CNBr r a t i o of 10:1 (v/v). A f t e r allowing the reac t i o n to proceed for s i x hours the reaction mixture was d i l u t e d 20:1 with d i s t i l l e d water and then l y o p h i l i z e d . 10 Cyanogen bromide was obtained from Eastman Organic Chemicals and was stored at -20°C u n t i l immediately p r i o r to use. 2 Enzymatic Cleavage with Trypsin Trypti c digestion of peptide was performed i n 1.0% NH4HCO3 soluti o n at 22°C. The enzyme was taken up i n 1.0% NH4HCO3 to give a concentration of 2.0 mg/ml and the peptide was dissolved i n 1.0% NH4HCO3 to a concentration of 1.0 mg/ml. The peptide s o l u t i o n was made up i n a small t e s t tube and an amount of the enzyme so l u t i o n was added to i t every 2 hr for 6 hr. The i n i t i a l enzyme : substrate r a t i o was 1:50 (w/w). After 6 hr the reaction mixture was l y o p h i l i z e d , then redissolved i n 0.5 ml d i s t i l l e d water and held i n a b o i l i n g water bath for 6 min to denature the enzyme. The solu t i o n was then centrifuged and the supernatant was decanted and r e l y o p h i l i z e d . Trypsin 'TPCK' (toluenesulphonylamidophenylethyl-* chloromethylketone treated) was obtained from Worthington Biochemical Corp., Freehold, N.J.. Products of t r y p t i c d igestion were subjected to high voltage electrophoresis together for confirmation of complete dig e s t i o n . Ion Exchange Chromatography A cation exchanger based on c e l l u l o s e , CM 11 (What-man, England), was used. The CM 11 was suspended i n 11 0.5 M ammonia soluti o n and the fines were removed. The exchanger was washed with water and then with 0.5 M ammonia so l u t i o n and 0.5 M HC1 r e s p e c t i v e l y . A f i n a l wash was then given with ammonium bicarbonate a t the ion i c strength which was to be used as the s t a r t i n g condition for chromatography. The CM 11 i n buffer was s t i r r e d to form a s l u r r y which was then poured into a chromatography column which contained a small volume of buffer. The s l u r r y was allowed to s e t t l e under g r a v i t y . A r e s e r v o i r of buffer was connected to the top of the column and allowed to flow through the column by g r a v i t y . This flow of buffer packed the column and additions of s l u r r y were made to obtain a column of the desired height. F i l t e r paper (3 mm) was cut to s i z e and placed on the top of the c e l l u l o s e i n order to s t a b i l i z e the c e l l u l o s e -l i q u i d i n t e r f a c e . Buffer s o l u t i o n was allowed to run through the column for several hours p r i o r to use. Immediately before use of the column, buffer s o l u -t i o n was removed from the top of the column to the l e v e l of the c e l l u l o s e . An amount of the mixture to be separated dissolved i n as small a volume of buffer s o l u t i o n as possible was applied to the top of the column and allowed to sink into the c e l l u l o s e . Buffer was then replaced on top of the c e l l u l o s e and the top of the column reconnected to the buffer r e s e r v o i r . Development of the column was performed by stepwise increases i n buffer pH. F i n a l e l u t i o n of adsorbed material was ensured by passage of 12 0.2M NH4HCO3 buffer at pH 8.0 through the column. Suitable fractions of the effluent volume were c o l l e c t e d on a f r a c t i o n c o l l e c t o r , (LKB u l t r o r a c , LKB productur, Sweden). E l u t i o n of peptides was monitored by measuring the absorbance, at 280 nm, of each f r a c t i o n , (precision spectrophotometer, Bausch & Lomb, Rochester, N.Y., 3-26-50). High Voltage Electrophoresis 1 Equipment and Reagents High voltage electrophoresis was performed on Whatman 3mm paper using the v e r t i c a l s t r i p method of Michl (1951, 1959) as modified by Ryle et a l . (1955). The apparatus consisted of glass chromatographic tanks, 22.5 i n . wide by 22.5 i n . deep (Chromotanks, Shandon, Eng.). These were f i t t e d with glass buffer troughs at the top, glass supporting rods to prevent siphoning of buffer by the paper and Perspex l i d s supporting glass cooling c o i l s . Platinum electrodes,; mounted i n glass holders, were placed i n each upper buffer trough (cathode) and at the bottom of the tanks, which were f i l l e d with buffer to a depth of 5 cm. The electrodes were connected, v i a connecting blocks mounted ext e r n a l l y on the tanks, to a Canadian Research I n s t i t u t e model EPSK-200 D.C. power supply (Don M i l l s , Ontario). Cold water was run through the cooling c o i l s to maintain temperature e q u i l i b r a t i o n i n the tanks at a l l times and 13 to cool the solvents during runs. The tanks were located i n a fume hood equipped with Perspex doors, allowing observation of runs. The buffer system used was described by Ambler (1963) and i s as follows: pH 6.5 Buffer - R e d i s t i l l e d p y r i d i n e : g l a c i a l a c e t i c a c i d : deionised water, (100:3:879 by volume). Coolant - Toluene: r e d i s t i l l e d pyridine, (92: 8 by volume). Varsol was obtained from Imperial O i l Co. 2 Standards and Markers A mixture of taurine, serine and aspartic acid was used for standard amino acids on high voltage runs at pH 6.5. Standards were applied as a 1.0 u.1 spot, on each side of the sample area, on the o r i g i n l i n e . The standards were useful both as a check on the detection reagent and for c a l c u l a t i o n of r e l a t i v e 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 sample peptides. Colour markers were also applied, as 1.0 )\1 spots, on a l l runs i n order to follow the progress of runs and to check for uniformity of running conditions on both sides of electrophoretograms. The markers used were methyl green ( C . l . no. 42590, Fisher c e r t i f i e d b i o l o g i c a l stain) and £-dinitrophenyl l y s i n e (Cal Biochem, Los Angeles). 14 A t y p i c a l run at pH 6.5 i s i l l u s t r a t e d in Figure 1, showing positions of standard amino acids and colour markers. High voltage runs at pH 6.5 were terminated when the methyl green markers had run to the l e v e l of the bottom of the upper buffer trough above which point they would no longer be running v e r t i c a l l y . This point was approximately 15 cm from the o r i g i n . The neutral l i n e was indicated by the centres of the £-dinitrophenyl ly s i n e spots. C-DNP-LYS i s neutral at pH 6.5 so that any movement of t h i s amino ac i d away from the o r i g i n i s due to electroendosmosis. Electrophoretic m o b i l i t i e s of peptides were calculated r e l a t i v e to the movement of aspartic acid from the neutral l i n e : i . e . the distance from the neutral l i n e to the front of the sample peptide spot was divided by the distance from the neutral l i n e to the front of the aspartic a c i d spot. 3 Procedure for Q u a l i t a t i v e Electrophoresis Sheets of Whatman 3mm paper, 57 cm long by 46 cm, were cut to the desired width and the o r i g i n l i n e s and positions for spotting markers, standards and samples, were marked on the sheets with p e n c i l . The o r i g i n was positioned 25 cm up from the anode. The markers, standards and samples were spotted on the paper and dried using cool a i r from a h a i r dryer. The paper was then wetted with the appropriate buffer, care being taken that each side of the o r i g i n l i n e was . 15 0"^ i > I v / \ / \ L Y S -O- - - O T f l U 0 f W Figure 1. Standards and Markers used on high voltage electrophoresis at pH 6.5. 1. Markers - M.G., methyl green;£-DNP Lys, £-dinitrophenyl l y s i n e . 2. Amino acid standards - Tau, taurine; Ser, serine; Asp, as p a r t i c a c i d . 16 wetted uniformly to avoid movement of spots by f l u i d drag. During spotting and wetting of the paper, the o r i g i n l i n e was elevated above the glass working surface by glass rods spaced equally on eit h e r side of i t . The uppermost end of the paper was fixed between a p a i r of glass rods i n order to hold t h i s end i n the upper buffer trough. After p o s i t i o n i n g the paper i n the tank and the passage of current at 4KV for the required time, the paper was hung to dry in a gentle a i r current i n a fume hood. When thoroughly dry, the paper could be treated with detection reagent to locate peptide spots. 4 Detection of Peptides Peptide spots on paper were detected using the cadmium-ninhydrine reagent of Heilman et a l . (1957) . The reagent was prepared by mixing 100 ml of a 0.5% (w/v) ninhydrine (Triketo Hydrinine Hydrate, Mann Research Labs., Inc.) sol u t i o n i n acetone with 15 ml of a stock solution of cadmium acetate. This stock s o l u t i o n was prepared from 5 g cadmium acetate, 250 ml g l a c i a l a c e t i c acid and 500 ml d i s t i l l e d water. The Cd-ninhydrin reagent was s e n s i t i v e to approx-imately 1-5 nanomoles/cm of peptide. Papers were dipped i n t h i s reagent only when thoroughly dry and then were l e f t i n a fume hood for colour development and drying. 17 Assay Methods 1 Assay of Glucose i n sera and KRB Solutions Glucose concentrations were measured in duplicate using an automatic Beckman Glucose analyzer, (Beckman Instruments International S.A., Geneva, Switzerland). 2 Radioimmunoassay of Gastric Inhibitory Polypeptide Concentrations of immunoreactive g a s t r i c i n h i b i t o r y polypeptide (IR-GIP) i n sera were determined i n duplicate using the assay method described by Kuzio et a l . (1974). This assay detected serum IR-GIP concentrations within the range of 0.1 to 6.0 ng/ml. Fasting serum IR-GIP level s i n dogs were found i n most instances to be below 0.1 ng/ml. For t h i s reason the mean fa s t i n g serum IR-GIP concentration has been considered as Non-detectable (N.D.). I t must be kept i n mind that N.D. does not imply zero but le s s than 0.1 ng/ml. IR-GIP concentrations above the range detected by the RIA are recorded as >6.0 ng/ml. In cases where experimental serum IR-GIP concentrations are compared with control l e v e l s for tests of s i g n i f i c a n c e , non-detectable values are considered as 0.10 ng/ml. 3 Radioimmunoassay of I n s u l i n Immunoreactive i n s u l i n (IRI) concentrations i n sera and KRB were determined i n duplicate using the Phadebas (R) I n s u l i n Test (Pharmacia AB, Uppsala, Sweden) and the Amersham/Searle Ins u l i n Immunoassay K i t (Amersham/Searle Corporation: An A c t i v i t y of G.D. Searle & Co. and the Radiochemical Centre, Des Plaines, I l l i n o i s ) . 18 Sources of Hormone, Sugar and Fat Preparations I n s u l i n (zinc i n s u l i n ) was obtained from Connaught Laboratories, Toronto. Gastric i n h i b i t o r y polypeptide was p u r i f i e d according to the method described by Brown et a l . (1971 & 1970). Glucose was obtained as a 50% s t e r i l e s o l u t i o n i n d i s t i l l e d water from Abbott Laboratories, Chicago, 111.. Lipomul (The Upjohn Co. of Canada, Don M i l l s , Ontario) was used as a palatable f a t emulsion i n experiments where o r a l f a t was administered to dogs. Each 15 ml con-tains 10 gm Corn O i l , d-Alpha Tocopheryl Acetate, Butylated Hydroxyanisole, Polysorbate 80, Glyceride Phosphates, sodium saccharin and a r t i f i c i a l c i t r u s v a n i l l a f l a v o r . Preservatives contained are sodium henzoate, 0.05%, benzoic a c i d , 0.05%, and sor b i c a c i d 0.07%. Analysis of Data In a l l experiments involving dogs, serum l e v e l s of glucose and IRI showed considerable v a r i a b i l i t y between dogs and between experiments i n each dog, a f t e r 18 hr fa s t i n g . Therefore, i n some cases, changes i n serum IRI and glucose concentration were used for comparison of experimental r e s u l t s . This required c o l l e c t i o n of at l e a s t 2 blood samples, 15 minutes apart, p r i o r to each experiment. For each time period o f each experiment, change i n serum glucose or IRI concentration was calculated as the difference from the mean fa s t i n g concentration. 19 For each type of experiment the changes observed at a p a r t i c u l a r time period were compared to the changes observed at the same time period i n control experiments for tests of s i g n i f i c a n c e . In each case where data i s treated i n t h i s manner, raw data i s tabulated whereas mean changes i n serum glucose and IRI l e v e l s are shown g r a p h i c a l l y . 20 RESULTS E f f e c t of GIP Infusions on Serum Levels of IRI and Glucose i n the Fasted Dog GIP was infused intravenously at 3 doses (0.25, 0.50 and 1.5 u.g/kg body weight) in 5.2 ml 0.9% saline using a 10 ml glass syringe and a Harvard infusion pump at speed 4 (1.03 ml/min). Two preinfusion blood samples were c o l l e c t e d , 15 min apart. Blood samples were c o l l e c t e d at 3, 4, 5, 7, 10, 15, 20, 30 and 45 min af t e r beginning GIP infusions. Immediate graded increases i n serum IRI concentrations were observed a f t e r beginning GIP infusions. Determinations of serum con-centrations of glucose, IRI and IR-GIP, with GIP infusions of 0.25, 0.50 and 1.50 u.g/kg, are shown i n Tables I I , I I I , and IV, re s p e c t i v e l y . Serum concentrations of IRI and glucose during control experiments, i n which 0.9% saline was infused, are shown i n Table I. A l l r e s u l t s are expressed as mean i S.E.. Serum IR-GIP concentrations during control experiments were, for the most part, non-detectable (i.e.<0.10 ng/ml). When 0.25 u.g/kg and 0.50 Mg/kg GIP were infused, the mean peak serum IR-GIP l e v e l s , reached at the 5 min period, were 2.57 ± 0.14 ng/ml and 5.03 - 0.97 ng/ml r e s p e c t i v e l y . The peak serum IR-GIP concentration obtained with infusion of 1.5 u;g/kg was above the detectable range of the GIP radioimmunoassay but, by sample d i l u t i o n , i t was estimated to be approximately 15 ng/ml. TABLE I E f f e c t of Infusion of 0.9% Saline on Serum Concentrations of IRI and Glucose i n the Fasted Dog, Dog An Sc Ro V i An Re Exp't No„ a b c d e f 0.9% sa l i n e i n f . Time (min) -15 0 3 4 5 7 10 15 20 25 30 45 Serum IRI Cone . (uU/ml) 10 12 10 7 8 10 10 9 13 7 9 9 20 10 10 9 17 18 18 21 16 18 24 N.S.* 18 18 15 12 15 14 14 8 7 10 23 15 18 16 N.S. 6 18 25 12 10 18 15 3 17 9 15 6 8 9 10 9 6 8 12 11 12 16> 15 16 17 19 16 20 21 16 20 16 N.S. X 15.2 14.3 11.4 9.8 14.3 15.5 13.8 12.5 13.0 13.7 14.3 13.3 . 1.9 1.2 1.8 1.7 1.9 2.3 1.8 2.7 1.9 2.0 3.7 1.7 Serum Glucose Cone. (mg%) An a Sc b Ro c V i d An e Re f 83 87 95 93 98 93 86 84 86 86 80 91 97 97 97 90 98 96 81 85 83 83 83 N.S. 94 91 93 92 89 89 95 92 91 96 98 99 75 82 N.S. 81 80 83 80 78 81 81 75 85 74 80 83 83 86 84 84 79 82 87 84 83 79 82 87 87 91 87 90 93 88 92 93 N.S. X 83.7 86.5 91 o0 87.7 90.3 88.7 86.0 85.2 85. 2 87.5 85.5 89.5 . 4.0 2.7 2.6 2.0 2.9 2.1 2.3 2.6 1. 6 2.3 3.5 3.6 * No Sample TABLE II E f f e c t of Infusion of 0.25 ug/kg GIP on Serum Concentrations of IR-GIP, IRI and Glucose i n the Fasted Dog. """""""" " ~~ GIP Infusion Exp't j [ Time (min)  D o9" No. -15 0 3 4 5 7 10 15 20 25 30 45* Ro l a Sc l b Sc l c Ro Id Serum IR-GIP Cone, (ng/ml) is.E, X N.S. 0.21 2.40 2 .30 2.50 2.05 1.60 1.45 0.98 0.72 0.56 0.84 N.D.* N.D. 1.45 2 .10 3 .00 2.50 1.55 N.S. 0.78 1.10 0.76 0.53 N.D. 0.34 1.95 2 .25 2.40 2.15 1.90 1.20 1.00 0.82 0.73 0.70 0.33 N.D. 2.45 2 .35 2.40 2.40 2.45 1.65 1.60 1.15 0.92 0.83 2.06 2 .25 2.58 2.28 1.88 1.43 1.09 0.95 0.74 0.73 0.23 0 .05 0.14 0.11 0.21 0.13 0.18 0.11 0.07 0.07 Serum IRI Cone. (uU/ml) Ro l a 10 12 N.S. 23 17 14 8 9 9 13 11 13 Sc l b N.S. 11 12 11 10 13 11 N.S. 9 10 13 6 Sc l c 15 12 25 23 27 14 10 13 14 12 17 15 Ro Id 14 15 18 19 28 18 16 16 15 10 25 17 X 13.0 12.5 18.3 19 .0 20.5 14.8 11.3 12.7 11.8 11.3 16.5 12.8 ±S.E. 1.5 0.9 3.8 2.8 4.3 1.1 1.7 2.0 1.6 0.7 3.1 2.4 Serum Glucose i Cone. (mg%) Ro l a 95 97 96 97 94 97 96 95 93 94 95 98 Sc l b N.S. 93 91 95 94 97 95 92 92 92 96 91 Sc l c 87 90 95 97 92 92 91 87 92 92 92 95 Ro Id 99 99 102 99 94 97 91 94 95 99 99 100 + * 93.7 94.8 96.Q 97 .0 93.5 95.8 93.3 92.0 93.0 94.3 95.5 96.0 -S.E. 3.5 2.0 2.3 0 .8 0.5 1.2 1.3 1.8 0.7 1.7 1.4 2.0 * Non-detectable fc No Sample TABLE III E f f e c t of Infusion of 0.50 ug/kg GIP on Serum Concentrations of IR-GIP, IRI and Glucose i n the Fasted Dog. — GIP Infusion Dog Sc Be Ro Ro Exp't No. 2a 2b 2d 2f X ±S.E. Time (min) -15 0 3 4 5 7 10 15 20 25 30 45 Serum IR-GIP Cone, (ng/ml) 0.19 N.D.* 2.75 4.00 6.00 6.00 2.70 1.65 1.45 1.25 1.15 0.70 N.D. N.D. N.S.t N.S. N.S. 6.00 6.00 2.90 2.90 3.00 1.75 1.45 N.D. N.D. 3.40 4.90 3.10 3.00 2.40 2.40 2.10 1.25 1.15 0.88 0.20 0.27 3.30 3.80 6.00 4.10 3.60 2.60 1.75 N.S. 1.40 1.05 3.15 4.23 5.03 4.78 3.68 2.39 2.05 1.83 1.36 1.02 0.20 0.34 0.97 0.74 0.82 0.27 0.31 0.58 0.14 0.16 Serum IRI Cone. (uU/ml) Sc 2a N.S. 6 28 28 31 18 12 7 11 10 11 4 Be 2b 10 8 15 18 16 12 N.S. 7 7 11 6 12 Sc 2c 13 8 16 22 23 16 13 15 11 8 14 12 Ro 2d 10 12 25 42 33 40 27 11 12 10 14 8 Ro 2e N.S. 6 19 46 38 30 21 16 16 14 15 12 Ro 2f 13 5 20 24 22 36 27 5 5 N.S. 6 20 , X 11.5 7.5 20.5 30.0 27.2 25.3 20.0 10.2 10.3 10.6 11.0 11.3 -S.E. 0.9 1.0 2.1 4.6 3.3 4.7 3.3 1.9 1.6 1.0 1.7 2.2 Sc Be Sc Ro Ro Ro 2a 2b 2c 2d 2e 2f Serum Glucose Cone. (mg%) -S.E, 94 100 97 97 95 . 100 95 90 97 99 100 101 87 92 82 89 81 84 N.S. 86 85 N.S. 87 89 99 100 101 96 92 96 92 99 92 88 93 91 91 91 98 105 101 99 94 92 92 91 97 101 93 94 93 79 86 83 83 83 93 95 94 96 92 93 97 98 98 100 87 90 82 N.S. 94 94 92.7 95.0 94. 7 94. 0 92. 2 93.7 90.2 90.0 89.8 93.3 94.2 95.3 1.6 1.6 2. 7 3. 7 3. 1 3.3 2.3 2.2 2.7 2.4 1.8 2.0 *Non-detectable fcNo Sample TABLE IV E f f e c t of Infusion of 1.5 ug/kg GIP on Serum Concentrations of IR-GIP. IRI and Glucose i n the Fasted Dog. ~ — — c-Dog Ro Be Be Sc Sc Exp't No. 3a 3g 3h 3d 3e X ±S.E. GIP Infusion Time (min) -15 0 3 4 5 7 10 15 20 25 30 45 Serum IR-GIP Cone, (ng/ml) N.D.* N.D. >6.00 >6.00 >6 .00 >6.00 >6.00 4.20 4.00 3.70 3.50 2.60 N.D. N.D. it II H H H 4.90 4.50 3.60 3.40 2.50 0.17 N.D. II II II H H 6.00 5.00 4.30 3.20 1.50 0.25 0.30 II ti H •! II 5.00 5.00 4.50 3.60 2.50 N.D0 N.D. H II II II II 6.00 5.00 4.70 4.00 2.90 >6.00 >6.00 >6 .00 >6.00 >6.00 5.22 4.70 4.16 3.54 2.40 0.35 0.20 0.22 0.13 0.24 Serum IRI Cone. (u.U/ml) Ro 3a N.S.t 27 75 84 98 64 32 27 22 21 17 19 Ro 3b 12 5 84 50 54 25 22 6 5 5 5 5 Ro 3c N.S. 18 46 58 54 54 26 13 13 8 9 10 Sc 3d 12 20 24 38 50 53 18 13 11 11 10 8 Sc 3e 12 3 31 47 N.S. N.S. 29 11 5 11 11 9 Sc 3f 17 23 25 44 47 53 26 9 20 19 14 19 X 13.3 ±S.E. 1.2 16. 0 47.5 53.5 60.6 49.8 25. 5 13.2 12. 7 12. 5 11.0 11.7 4. 0 10.7 6.7 9.4 6.5 2. 0 3.0 3. 0 2. 6 1.7 2.4 Serum Glucose Cone. (mg%) Ro 3a 93 95 93 92 87 89 79 76 75 77 86 87 Ro 3b 105 104 107 106 103 100 91 89 87 93 92 101 Ro 3c 94 98 100 99 96 96 94 83 81 91 105 106 Sc 3d 94 96 95 95 100 90 91 76 80 82 86 91 Sc 3e 88 104 99 95 N.S. N.S0 92 79 79 78 93 92 Sc 3f 89 78 89 86 76 82 85 81 89 92 91 104 X 93.8 95. 8 97. 2 95. 5 92.4 91.4 88.7 80.7 81.8 85 .5 92. 2 96.8 ±S.E 0 2.5 3. 9 2. 6 2. 7 4.9 3.1 2.3 2.0 2.1 3 .0 2. 8 3 .2 *Non-detectable No Sample 25 I.V. G.I.P O 5 K> 15 20 25 45 Time In Minutes 2. Changes i n serum immunoreactive i n s u l i n (IRI) concentrations produced by 5 min. intravenous infusions of 0.25, 0.5 and 1.5 }xg/kg of GIP. Physiological saline was infused i n control experiments. 26 Figure 2 shows the changes i n serum IRI concentra-tions obtained with the 3 doses of GIP infused. IRI peaks were monophasic and maximum l e v e l s occurred 4 -5 min af t e r i n i t i a t i o n of GIP infusions. The increments i n IRI over control values, 5 min a f t e r the s t a r t of GIP infusions, were 8.2 ± 3 . 6 uU/ml with 0.25 u.g GIP per kg, 18.5 ± 1 . 8 uU/ml with 0.50 »g/kg and 50 ± 13 uU/ml with 1.50 wg/kg. The mean of paired differences for incremental IRI, i n the presence or absence of GIP, was s i g n i f i c a n t at the 5 min period for a l l 3 doses of GIP infused (0.25 wg/kg, P <0.01; 0.5 Mg/kg, P •< 0.0005? 1.5 jug/kg, P < 0.0005) . Figure 3 shows the depressions i n serum glucose concentration observed following GIP inf u s i o n s . In these experiments, the maximum depression i n serum glucose as compared to control l e v e l s (observed 15 min aft e r the s t a r t of GIP infusions) was 2.0 ± 0.9 mg% with 0.25 ng GIP per kg, 3.0 ± 2.4 mg% with 0.50 u.g/kg and 12.3 ± 1.8 mg% with 1.5 Mg/kg. The mean of paired differences for incremental serum glucose l e v e l s , i n the presence or absence of GIP, was s i g n i f i c a n t only with a GIP dose of 1.5 u.g/kg (P <0.0025). Figure 4 i l l u s t r a t e s the pattern of serum IRI and glucose responses to infusion of GIP at a dose of 1.5 Mg/kg. 27 F i g . 3. Changes in serum glucose concentrations produced by 5 min. intravenous infusions of 0.25, 0.5 and 1.5 ug/kg of GIP. Physiological s a l i n e was infused i n c o n t r o l experiments . 2 8 o o n C E ~" 3 16 12 8 H GIP 1.5 Lug/kg / \ / \ / \ o — o Controls - Saline Infused • • GIP Infused-1.5/jg/kg Estimated / + 10 o o £ w a> (0 - - 10 A Time in Minutes F i g . 4 . Pattern of changes i n serum concentrations o f IR-GIP, IRI and glucose when GIP was infused at 1.5 u.g/kg/5 min. 29 E f f e c t of Infusion of GIP Fragments on Serum Levels of  IRI and Glucose in the Fasted Dog. Fragments obtained by cyanogen bromide cleavage. Two mg of GIP were subjected to chemical cleavage with CNBr. The r e s u l t i n g peptide mixture was assayed for in s u l i n o t r o p i c a c t i v i t y by infusion into one fasted dog at 1.5 u,g/kg over 5 minutes. S l i g h t stimulation of i n s u l i n release was observed (Figure 5) although serum glucose l e v e l s showed no change from control l e v e l s . Separation of the peptide mixture was achieved by chromatography on a 0.5 x 7.0 cm column of carbo-xymethylcellulose (CM 11). Fractions of 0.9ml were co l l e c t e d at a flow rate of 0.6ml/min and the absorbance of each was measured at 280 nm. Figure 6 shows separation of the reaction mixture into 3 peaks using 0.01 M and 0.2 M NH4HCO3 buf f e r s . The pH of the buffers was adjusted by gassing with CO2. Two peaks were eluted with 0.01 M NH4HCO3, one at pH 7.0, the other at pH 7.8. A t h i r d peak was eluted with 0.2 M NH4HCO3 at pH 7.8. Fractions under the peaks were pooled and l y o p h i l i z e d . Tubes 1-4 constituted f r a c t i o n I; tubes 12-18, f r a c t i o n II and tubes 23-28, f r a c t i o n I I I . High voltage e l e c t r o -phoresis of the 3 fractions at pH 6.5 and 4KV showed each f r a c t i o n to be a single ninhydrin-positive peptide (Figure 7). Electrophoretic m o b i l i t i e s at pH 6.5 r e l a t i v e to an aspartic acid marker were -0.219, 0.117 and 0.271 for fractions I, II and III r e s p e c t i v e l y . 30 E \ 3 •2. o c o o 3 (A or H E 3 w <o CO + I 0 H o—o Controls n = 6 • - • CNBr-G.IPn=l CNBr-G.I.R n= 2 unseparated (1.5 / jg /kg) Fr.I uncleaved (1 .5 / jg /kg) ~ i 1 1 r-10 20 30 Time in Minutes 40 F i g . 5 . Changes i n serum IRI concentrations produced by 5 min. intravenous infusions of the peptides r e s u l t i n g from CNBr treatment of GIP (1.5 ng/kg) before and a f t e r separation of the peptides on CM 1 1 . P h y s i o l o g i c a l saline was infused in control experiments. 31 0.2MNH 4 HC0 3 0.01 M N H 4 H C O 3 pH 7.8 O.OIMNH.HGOL 4 3 pH 7.8 pH 7.0 f w Sample Number F i g . 6. El u t i o n p r o f i l e of 2.0 mg of CNBr-treated GIP on a 0.5 x 7.0 cm column of carboxymethyl-c e l l u l o s e (CM 11). Sample siz e was 0.9 ml co l l e c t e d at a flow rate of 0.6 ml/min. The f i r s t peak i s composed of N-terminal residues 1-14, the second peak, uncleaved GIP and the t h i r d peak C-terminal residues 15-43. 32 F i g . 7 . High voltage electrophoresis at pH 6 . 5 of GIP and the peptides obtained by CNBr cleavage of GIP, a f t e r separation on CM 11. A. M.G.,£-DNP Lys. B.Tau, Ser, Asp. C. GIP D. CNBr-GIP, Fr. I I I . E. CNBr-GIP, Fr. I I . F. CNBr-GIP, Fr. I. 33 Untreated GIP, run on the same electrophoretogram, showed an electrophoretic mobility of 0.107. The following assignment of peptide composition was made: Fraction I - N-terminal residues 1-14; f r a c t i o n II -uncleaved GIP; f r a c t i o n III - C-terminal residues 15-43. Amino aci d analyses were not performed. These CNBr peptides have been described by Brown and Dryburgh (1971). In s i x experiments fractions I and I I I were assayed i n dogs, using the procedure described, at doses equal to 5 times the molar equivalent of 1.5 pg/kg untreated GIP. No stimulation of i n s u l i n release or depression of serum glucose l e v e l s was observed. When f r a c t i o n I was infused serum IR-GIP showed no change from control l e v e l s . When fr a c t i o n III (GIP C-terminal, residues 15-43) was infused high l e v e l s of serum IR-GIP were detected (Table V). Thus the GIP fragment c o n s i s t i n g of residues 15-43 appeared to cross-react to a high degree with antiserum raised to whole GIP. Figure 5 shows the change i n serum IRI when f r a c t i o n II (uncleaved GIP) was infused i n 2 dogs at 1.5 ucj/kg over 5 minutes. A maximum increment i n IRI of 18.5 - 0.5 uU/ml was obtained at the 7 minute period. Serum glucose showed no change from control l e v e l s . T r y p t i c digest of GIP. One mg of GIP was subjected to enzymatic cleavage with t r y p s i n . The digestion was shown to be complete by high voltage electrophoresis at TABLE V E f f e c t of Infusion of CNBr-GIP Fraction III (C-terminal residues 15-43) at 5.0 uo/ka on Serum Concentrations of IR-GIP in the Fasted Dog. — U 3 a — Dog No. Ro 4a Ro 4b Sc 4c Fraction III Infusion Exp't j I Time (min) X ±S.E. -15 0 3 4 5 7 10 15 20 25 30 45 N.D.* N.D. >6.00>6.00>6.00>6 .00 >6.00 4.40 3.20 3.40 3.20 1.75 0.14 0.20 II i t II H it 4.70 3.30 3.20 2.50 1.65 N.D. 0.24 H II II II i i 4.90 3.60 3.00 2.30 1.35 >6.00>6.00>6.00>6 .00 >6.00 4.67 3.37 3.20 2.67 1.58 0.14 0.12 0.12 0.27 0.12 *Non-detectable 35 F i g . 8. High voltage electrophoresis at pH 6.5 of GIP and a t r y p t i c digest of GIP. A. M.G., €-DNP Lys. B. Tau, Ser, Asp. C. GIP. D. Tryptic digest of GIP. A ^ Tyr U . 0 1 . Oly Thr M I I . S.r A.p Tyr S T U . A l * K.t A.p Ly . . . . A r , Cln Cln A.p Ph. V . ! A.n Trp L . U L . u A l * Cln C n Ly. Cly L y . L y . * . r tap Trp Ly . . . . A .n „ . rhr « , » Tyr A l . Clu Oly Thr Ph. 1 1 * S*r A.p Tyr tor 1 1 . A l . M*t L * " L " " j ^ W O W U F F I I VAL ASM T*P LEU LEU ALA CLN GUI LII CLY Lit LTB » R ASP TRP LTI Mil ASH | L I m « L I Tyr Ala Clu aiy Thr Ph. 1 1 . 8 * r A.p Tyr S.r I I . A l . mt A.p Ly . i Cln Cln Lys S*r Aap Trp Ly . H I . A M II* Thr r.\n M l . A.n II* Thr Cln U) F i g . 9 0 Complete sequence of g a s t r i c i n h i b i t o r y polypeptide and the major fragments which have been assayed for in s u l i n o t r o p i c a c t i v i t y i n the dog. A. Complete sequence of GIP. B. CNBr fragments. C 0 Major t r y p t i c fragments. (Brown and Dryburgh, 1971). The fragment i n block l e t t e r s showed cross r e a c t i v i t y with antiserum raised to whole GIP. 37 pH 6.5 and 4 KV (Figure 8). No ninhydrin-positive peptide spot i n the digest was comparable to that of untreated GIP run on the same electrophoretogram. The peptides obtained by t r y p t i c digestion of GIP have been described by Brown and Dryburgh (1971) . In two dogs, the unsep-arated t r y p t i c digest showed no stimulation of i n s u l i n release or depression of serum glucose l e v e l s even when infused at 7.5 u.g/kg over 5 minutes. Serum IR-GIP showed no change from control l e v e l s . Figure 9 shows the complete sequence of GIP, the fragments obtained by CNBr cleavage and the major products of t r y p t i c digestion of GIP. E f f e c t of GIP Alone and i n Combination with I n s u l i n on  Glucose Uptake by the Rat Hemidiaphragm Figure 10 shows the e f f e c t of i n s u l i n , GIP and i n s u l i n plus GIP on glucose uptake by the r a t hemidia-phragm preparation. In s i x paired experiments, the glucose uptake by r a t hemidiaphragms i n control flasks containing KRBG sol u t i o n was 127.7 ± 9 . 1 mg% per gm of tissue weight. Flasks containing 12.5 ng/ml GIP showed no s i g n i f i c a n t d ifference i n glucose uptake compared with controls (129.0 ± 17.0 mg% per gm t i s s u e , P>0.10). The addition of 2 x 10 U/ml i n s u l i n to the i n -cubation medium yielded a mean glucose uptake value of 218.3 ± 17.8 mg% per gm tissue weight. When i n s u l i n (2 x 10~ 3 U/ml) and GIP (12.5 u.g/ml) were both added to 38 400-1 300 H E o \ E 200 4 ID J£ o a. a) co o o o I O O H Control G.I. P. Insulin Insulin Insulin (12.5/ig/ml) (2Ox l0" s U/ml ) (20xl0" 5 U/ml) (2j0xr0"ZU/ml) and G.I.P. (12.5 jug/ml ) 10. E f f e c t s of i n s u l i n (2 x 10~ z and 2 x 10" 3 U/ml), GIP (12.5 ug/ml) and GIP + i n s u l i n (12.5 ug/ml and 2 x 10~ 3 U/ml) on integrated glucose uptake by the r a t hemidiaphragm preparation. 39 the incubation media, glucose uptake was not s i g n i f i -cantly d i f f e r e n t from that induced by i n s u l i n alone (236.8 ± 24.9 mg% per gm tissue weight, P>0.10). Increasing the i n s u l i n concentration by a factor of 10 (2 x 10~ 2 U/ml) resulted i n a glucose uptake of 332.8 i 32.8 mg% per gm of hemidiaphragm t i s s u e . Release of IR-GIP by Oral Glucose i n the Fasted Dog In 8 experiments i n each of 4 dogs, glucose was administered o r a l l y at 4 doses; 0.25, 0.50, 1.0 and 2.0 gm per kg body weight. For a l l experiments, the stock glucose solution (50% i n d i s t i l l e d water) was d i l u t e d with d i s t i l l e d water to give a 20% s o l u t i o n . Thus glucose concentration i n the ingested sol u t i o n remained constant for a l l experiments whereas volume vari e d . In control experiments, 100 ml d i s t i l l e d water was admin-is t e r e d . Three fas t i n g blood samples were c o l l e c t e d , 15 minutes apart. Glucose or water was then administered af t e r which blood samples were c o l l e c t e d at 5, 10, 15, 30, 45, 60, 75, 90, 105, 120, and 135 minutes. Sera were aliquoted for measurement of glucose and IR-GIP concentrations. Serum glucose l e v e l s observed i n these experiments are shown in Figure 11 and Figure 12 shows the pattern of IR-GIP release observed with each o r a l glucose load. Serum glucose and IR-GIP concentrations, measured for control experiments, are shown in Table VI. F i g . 11. Serum glucose concentrations following o r a l glucose at 0.25, 0.50, 1.0 and 2.0 g/kg i n the dog. F i g . 12. Serum IR-GIP concentrations following o r a l glucose at 0.25, 0.50, 1.5 and 2.0 g/kg, in the dog. 41 Oral Glucose Load • — • 0.25 g/kg o o 0.50 •• &—& 1.0 » A A 2.0 150-1 l 1 1 1 1 1 1 1 1 1 0 30 60 90 120 Time in Minutes Oral Glucose Load • — • 0.25 g /kg n = 5 o-o 0.50 " n = 8 A—A 1.0 " n = 9 2.0 " n = 7 L S I Time in Minutes TABLE VT E f f e c t of 100 ml Oral H 20 on Serum Concentrations of IR-GIP, IRI and Glucose i n the Fasted Dog. Oral H ?0 ^ Time (min)  -30 -15 0 15 30 45 60 75 90 105 120 135 150 165 180 Serum IR-GIP Cone, (ng/ml) N.S . 0.20 N.D.* 0.20 N.D. N.D. 0.20 0.23 N.D. N.D. N.D. N.D. N.D. N.S. 0.23 0.25 N.D. N.D. 0.30 N.D. 0.20 0.25 0.13 0.20 N.D. 0.30 0.18 0.40 N.D. 0.25 N.D. N.D. 0.15 N.D. 0.20 0.25 N.D0 N.D. 0.15 N.D. 0.22 0.13 N.D. 0.20 N.D. Re 5a N.S. 14 15 Sc 5c 12 15 16 Re 5d 9 7 9 Re 5e 8 8 6 X 9.7 11.0 11.5 is.E. 1.2 2.0 2.4 Serum IRI Cone. (MU/ml) 16 19 13 16 14 16 14 18 14 16 18 19 16 16 8 12 7 15 15 11 N.S. 10 13 9 13 15 10 10 13.0 14. 5 11.3 15.5 15.8 13. 3 13.3 2.4 1. 6 2.0 1.0 1.1 1. 6 1.8 14 16 15 12 13 15 14 15 17 18 9 12 14 9 7 15 16 9 7 13 13.3 14.5 13.3 11.3 12.8 1.4 1.0 1.4 2.2 2.3 Serum Glucose Cone. (mg%) Re 5a NoSo 74 75 74 77 73 70 75 77 83 77 74 78 N.S. 83 Ro 5b 87 94 94 97 98 100 91 91 99 97 93 98 98 99 99 Sc 5c 88 91 89 90 90 88 84 87 87 86 83 85 86 88 91 Re 5d 56 62 71 77 76 77 86 85 86 N.S. 91 90 95 93 97 Re 5e 86 92 87 92 85 88 92 92 83 89 92 96 91 91 95 X 79.3 82.6 83.2 86 .0 85.2 85.2 84.6 86.0 86.4 88.8 87 .2 88. 6 89 .6 92.8 93 ±S.E. 7.8 6.3 4.4 4 .5 4.1 4.7 3.9 3.0 3.6 3.0 3 .1 4. 3 3 .5 2.3 2 *Non-detectable ^No Sample 43 F i g . 13. Va r i a t i o n of peak serum IR-GIP response with o r a l glucose load i n the dog. 44 It i s apparent from Figure 12 that, with a glucose load of 0.25 g/kg, serum IR-GIP l e v e l s were not s i g n i f i -cantly elevated above control l e v e l s . Mean peak serum GIP l e v e l s achievgd were 0.770 ± 0.227 ng/ml with 0.50 g glucose/kg, 1.08 ± 0.20 ng/ml with 1.0 g/kg and 1.44 ± 0.22 ng/ml with 2.0 g/kg. At the 15 min. period, the IR-GIP response to 0.50 gm glucose/kg was s i g n i f i c a n t l y greater than the response to 0.25 g/kg (P<0.021). At the 45 min. period, the IR-GIP response to 1.0 gm glucose/kg was s i g n i f i c a n t l y greater than the response to 0.50 g/kg (p <0.007). And at'the 75 min. period, the IR-GIP response to 2.0 gm glucose/kg was s i g n i f i -cantly greater than the response to 1.0 g/kg (P<0.01). (Mann-Whitney Test) Figure 13 i l l u s t r a t e s the v a r i a t i o n of peak serum IR-GIP response with the 4 o r a l glucose loads used. Release of IR-GIP by Oral Fat, in the Fasted Dog, and  Its E f f e c t on Serum IRI Levels. In 16 experiments, Lipomul was administered o r a l l y to dogs at 3 doses; 25 ml, 50 ml and 100 ml. In control experiments, 100 ml d i s t i l l e d water was administered. Three f a s t i n g blood samples were c o l l e c t e d , 15 minutes apart. Lipomul or water was administered a f t e r which blood samples were c o l l e c t e d at 15 minute i n t e r v a l s for 180 minutes. Sera were aliquoted for measurement of glucose, IRI and IR-GIP concentrations. Levels of serum 45 F i g . 14. Serum IR-GIP concentrations following 25 ml, 50 ml and 100 ml o r a l Lipomul, i n the dog. 46 glucose and IRI, a f t e r 100 ml o r a l Lipomul, are recorded i n Table VII. Data from control experiments i s recorded i n Table VI. Figure 14 shows the pattern of IR-GIP release with each dose of o r a l Lipomul. Mean peak serum IR-GIP l e v e l s achieved with each o r a l lipomul dose were; 1.25 i 0.45 ng/ml at the 45 min. period with 25 ml Lipomul, 1.91 i 0.30 ng/ml at the 105 min. period with 50 ml and 2.99 i 0.43 ng/ml at the 135 min. period with 100 ml. At these peak periods, the IR-GIP response to 25 ml Lipomul, was s i g n i f i c a n t l y elevated over control l e v e l s (P< 0.029), the IR-GIP response to 50 ml Lipomul was s i g n i f i c a n t l y greater than the response to 25 ml (P< 0.021) and the IR-GIP response to 100 ml Lipomul was s i g n i f i c a n t l y greater than the response to 50 ml (P< 0.0009) (Mann-Whitney T e s t ) . The changes in serum IRI concentration following 100 ml o r a l Lipomul and following 100 ml o r a l H 20 are plotted i n Figure 15. No s i g n i f i c a n t e levation of. serum IRI l e v e l s was observed following 100 ml o r a l Lipomul although mean serum IR-GIP l e v e l s exceeded 2.0 ng/ml from the 60 min. period to the 180 min. period. TABLE VTI E f f e c t of 100 ml Oral Lipomul on Serum Concentrations of IRI and Glucose i n the Fasted Dog Dog Re Ro V i Sc Ro V i Re Ro V i Sc Ro V i Exp't No. 6a 6b 6c 6D 6e 6f 6a 6b 6c 6d 6e 6f + X -S.E. Oral Lipomul Time (min) :s.E. -30 -15 0 15 30 45 60 75 90 105 120 135 150 165 180 Serum IRI Cone. (uU/ml) N.S.* 10 18 12 15 11 11 16 17 9 13 N.S. N.S. N.S. N.S. 19 18 18 17 24 14 19 19 21 17 19 20 19 22 25 21 22 23 21 22 19 23 25 26 28 25 28 28 25 25 22 19 24 24 24 22 22 17 21 27 22 20 23 23 24 21 19 17 17 21 19 14 19 20 20 17 19 19 18 19 13 13 13 10 11 13 16 N.S. 16 17 19 13 17 14 19 19.2 16.8 18.8 16.8 19.5 16 .3 17.5 19.2 20.2 19.7 19.2 20.0 21.2 20.4 22.4 1.6 1.8 1.7 2.2 2.2 1 .7 1.9 1.6 1.4 2.9 1.7 2.4 1.9 1.9 1.4 Serum Glucose Cone. (mg%) 76 83 84 86 82 81 83 87 79 78 84 81 N.S. N.S. N.S. 70 77 80 84 91 94 87 89 100 96 94 95 100 98 97 75 75 73 77 79 85 73 79 76 80 91 94 73 79 85 80 77 83 82 82 72 82 81 84 91 93 87 88 93 90 72 80 80 83 86 88 87 79 81 86 88 84 82 87 84 72 68 70 72 75 80 69 N.S. 71 73 78 81 76 80 85 74.2 76.7 78.3 80.7 82.5 83 .3 80.2 83.0 81.8 84.0 88.0 87.0 83 .8 87.4 88.2 1.5 2.1 2.3 2.1 2.3 3 .1 3.1 2.1 4.1 3.5 2.5 2.5 4.8 3.7 2.4 *No Sample 48 F i g . 15. Changes i n serum IRI concentrations following 100 ml o r a l H 20 and following 100 ml oral Lipomul i n the dog. 49 DISCUSSION I t has been well established that o r a l or intraduo-denal administration of glucose r e s u l t s i n a considerably greater r i s e i n serum IR-insulin l e v e l s than intravenous administration of glucose at the same rate (Mclntrye e t a l . , 1965). Attendant with t h i s greater r i s e i n serum IRI i s a faster rate of glucose disappearance from the blood. These phenomena have been at t r i b u t e d to an i n s u l i n -otropic factor present i n the duodenum and jejunum which is released into the blood by the presence of glucose i n the gut lumen. Most of the known g a s t r o i n t e s t i n a l hormones have been investigated i n an attempt to i d e n t i f y t h i s i n s u l i n o t r o p i c agent. None of the established g a s t r o i n t e s t i n a l hormones were found to be su i t a b l e candidates for t h i s agent (Dupre et a l . , 1969). Several impure extracts of duodenal and jejunal mucosa have been proposed as i n s u l i n o t r o p i c agents (Turner et a l . , 1973; Marco et a l . , 1970) but v e r i f i c a t i o n of these claims awaits further p u r i f i c a t i o n of these extracts. Highly p u r i f i e d g a s t r i c i n h i b i t o r y polypeptide was recently implicated as an i n s u l i n o t r o p i c agent by the work of Dupre et a l . (1973). GIP was shown to potentiate IRI release and improve tolerance of an intravenous glucose load, i n man, at a dose of only 1.0 ucr/min for 30 min. Peak serum IR-GIP l e v e l s obtained in these experiments were assayed at l e s s than 1.0 ng/ml. Dupre 50 et a l . could show no elevation in serum IRI l e v e l s when GIP was infused without glucose, at a rate of 1.0 u.g/min for 30 min. The studies reported i n t h i s thesis show that exogenous porcine GIP w i l l release IRI i n the conscious fasted dog. Five min infusions of 0.25, 0.50 and 1.5 u.g GIP per kg dog weight produced increments i n serum IRI of 8.2 ± 3.6 uU/ml, 18.5 ± 1.8 uU/ml and 50 ± 13 uU/ml, res p e c t i v e l y (Figure 2). Therefore, at these doses, GIP i s a potent stimulus for IRI release. Peak serum IR-GIP l e v e l s reached in these experiments were 2.57 ± 0.14 ng/ml with a GIP dose of 0.25 wg/kg, 5.03 ± 0.97 ng/ml with 0.50 «g/kg and approximately 15 ng/ml with 1.5 Mg/kg. Although these l e v e l s of IR-GIP exceed the ph y s i o l o g i c a l l e v e l s previously reported i n the l i t e r a t u r e , present studies have shown that the IR-GIP l e v e l s obtained a f t e r 0.25 u.g/kg/5 min GIP, infused intravenously, are we l l within p h y s i o l o g i c a l range. Comparison of these infusion experiments with those of Dupre suggests that, at low serum concentrations, GIP may potentiate the IRI response to glucose whereas, at higher l e v e l s , i t may stimulate i n s u l i n release independently. Although the higher l e v e l s of exogenous IR-GIP measured i n these experiments may be above the p h y s i o l o g i c a l range, the re s u l t s obtained e s t a b l i s h that the IRI response to GIP i s dose-related. The depressions i n serum glucose l e v e l s observed following GIP infusions (Figure 3) 51 i n d i c a t e t h a t the IRI r e l e a s e d by GIP i s b i o l o g i c a l l y a c t i v e i n promoting g l u c o s e uptake. P r e l i m i n a r y i n v e s t i g a t i o n s to determine whether the i n s u l i n o t r o p i c a c t i o n o f GIP can be a t t r i b u t e d to a p a r t i c u l a r segment o f the molecule, gave n e g a t i v e r e s u l t s . When GIP was t r e a t e d w i t h CNBr, p r o d u c i n g cleavage a t the methionine r e s i d u e , o n l y the GIP remaining uncleaved a f t e r treatment r e t a i n e d i n s u l i n - r e l e a s i n g a c t i v i t y ( F igure 5 ) . Fragments o f GIP o b t a i n e d by d i g e s t i o n w i t h t r y p s i n had no e f f e c t on serum IRI l e v e l s when i n f u s e d i n t o f a s t e d dogs a t h i g h doses. F i g u r e 9 summarizes the major fragments o f GIP which were assayed f o r i n s u l i n o t r o p i c a c t i v i t y . I t i s not unusual f o r g a s t r o i n t e s t i n a l hormones t o r e t a i n t h e i r b i o l o g i c a l a c t i v i t y i n a s m a l l f r a c t i o n o f the m olecule. Such i s the case f o r g a s t r i n (Tracy and Gregory, 1964) and c h o l e c y s t o k i n i n ( O n d e t t i e t a l . , 1970). The f a c t t h a t a fragment o f the GIP molecule w i t h i n s u l i n - r e l e a s i n g a c t i v i t y was not found i n t h i s study does not p r e c l u d e the e x i s t a n c e of one. I t i s p o s s i b l e t h a t the e n t i r e molecule i s r e q u i r e d to m a i n t a i n a c o n f i g u r a t i o n n e c e s s a r y f o r p r e s e n t a t i o n o f an a c t i v e s i t e to r e c e p t o r s i t e s . I t i s a l s o p o s s i b l e t h a t i n s u l i n o t r o p i c a c t i v i t y i s c o n t a i n e d i n a segment of the GIP molecule which cannot be i s o l a t e d by c o n v e n t i o n a l chemical or enzymatic cleavages. 52 Although unsuccessful i n locating a fragment of the GIP molecule with i n s u l i n - r e l e a s i n g a c t i v i t y , the fragment studies allowed preliminary l o c a l i z a t i o n of a highly antigenic f r a c t i o n of the molecule. The C-terminal fragment of GIP, obtained by cyanogen bromide cleavage, consisting of residues 15 - 43, showed a high degree of cross-r e a c t i v i t y with antiserum raised to porcine GIP (Table V). Observation of depressions i n serum glucose con-centration following GIP infusions suggested the p o s s i -b i l i t y that GIP i t s e l f might possess some i n s u l i n - l i k e a c t i v i t y . Chisholm et a l . (1969) and L i c k l e y et a l . (1970) found that s e c r e t i n could enhance the rate of glucose disappearance from the blood independent of any action i t had on the pancreas. Using the r a t hemi-diaphragm preparation, GIP was found to have no e f f e c t on glucose uptake, compared to controls, at a concentration of 12.5 ng/ml (Figure 10). GIP was also without s i g n i f i -cant e f f e c t on the glucose uptake stimulated by 2 x 10, U/ml i n s u l i n . One c r i t e r i o n that must be s a t i s f i e d by a candidate for a g a s t r o i n t e s t i n a l i n s u l i n o t r o p i c agent i s that i t must be released into the blood following ingestion of substances which normally stimulate i n s u l i n release, p a r t i c u l a r l y glucose. Experiments reported in t h i s study show that IR-GIP i s released into the blood i n substantial amounts following ingestion of glucose in dogs (Figure 12). The lack of a s i g n i f i c a n t IR-GIP 53 response to 0.25 g glucose per kg suggests that eit h e r the number of GIP c e l l s exposed to glucose (volume eff e c t ) and/or the time of t h i s exposure was c r i t i c a l . The much greater IR-GIP response to 0.50 g glucose per kg could not be explained by volume e f f e c t alone and thus indicates that IR-GIP release from GIP c e l l s also varies with the time of exposure to glucose. In t h i s case, release of IR-GIP would have an inverse r e l a t i o n -ship with the i n t e s t i n a l glucose absorptive capacity. The patterns of serum glucose and IR-GIP concentrations with glucose loads of 1.0 g/kg and 2.0 g/kg appear to confirm t h i s suggestion. With these glucose loads, serum glucose concentrations l e v e l o f f at the 30 min period (Figure 11), possibly due to saturation of the glucose absorptive mechanism, whereas serum IR-GIP le v e l s continue to increase. With o r a l glucose loads of 0.50, 1.0 and 2.0 g/kg peak serum IR-GIP leve l s achieved were 0.770 ± 0.227 ng/ml, 1.08 ± 0.20 ng/ml and 1.44 ± 0 . 2 2 ng/ml, respec-t i v e l y . Peak serum IR-GIP l e v e l s obtained with 1.0 and 2.0 g glucose per kg exceed the l e v e l s of exogenous IR-GIP reported by Dupre et a l . (1973) to be e f f e c t i v e i n potentiating the i n s u l i n response to intravenous glucose and improving glucose tolerance. The body of evidence accumulated to date suggests that GIP i s a strong candidate for an i n s u l i n o t r o p i c agent. The polypeptide, GIP, has been shown to be 54 present i n mucosal c e l l s of the duodenum and jejunum of dog and man (Polak et a l . , 1973). In the present study using dogs, IR-GIP has been shown to be released into the blood, i n a dose-related manner, by the presence of glucose i n the gut lumen. Serum l e v e l s o f exogenous IR-GIP, below those which can be achieved by physio-l o g i c a l means, were shown by Dupre to be e f f e c t i v e i n potentiating the IRI response to I.V. glucose i n man. In addition, at higher doses, exogenous GIP i s a potent stimulus for IRI release in dogs, independent of glucose. I t i s concluded from t h i s evidence that endogenous IR-GIP, released by glucose i n the small bowel, f a c i l i -tates IRI release from the pancreas eit h e r by potentiating the IRI response to serum glucose or by independent stimulation of IRI release. This conclusion, however, requires the assumption that endogenously released IR-GIP i s i d e n t i c a l to exogenous porcine GIP i n i t s i n s u l i n - r e l e a s i n g properties. In order to t e s t t h i s assumption i t was desirable to stimulate release of endogenous IR-GIP with a secretagogue which d i d not elevate serum glucose l e v e l s . Thus the e f f e c t s of elevated endogenous IR-GIP on IRI release could be studied without attendant IRI release by glucose. Pat was previously found to be a potent stimulant of IR-GIP release (Brown, Dryburgh and Pederson, 1974). In the present study, f a t was shown to be the most potent secretagogue for IR-GIP release found to date. With o r a l Lipomul, at doses of 25 ml, 50 ml and 100 ml, 55 peak serum IR-GIP l e v e l s obtained were 1.25 ± 0.45 ng/ml, 1.91 ± 0.3 0 ng/ml and 2.99 ± 0.43 ng/ml, respectively (Figure 13). The i n i t i a l rates of IR-GIP release i n these experiments were comparable to those observed following o r a l glucose. The IR-GIP response to o r a l f a t , however, became much greater i n magnitude and was more prolonged. This greater response may be pr i m a r i l y due to the slow passage of f a t from the stomach and the time required for absorption of f a t by the i n t e s t i n e . No s i g n i f i c a n t changes i n IRI secretion were observed i n these experiments (Figure 14). This ^finding might indicate that IR-GIP released by o r a l glucose or fat requires elevated serum glucose l e v e l s for i n s u l i n o t r o p i c a c t i v i t y . This suggestion, however, i s contradicted by.evidence obtained with the GIP infusion studies. When GIP was infused into dogs, at a dose of 0.25 ug/kg, peak serum IR-GIP l e v e l s achieved were 2.57 ± 0.14 ng/ml and serum IRI l e v e l s were elevated by 8.2 ± 3.6 uU/ml (Figure 2). Following 100 ml o r a l Lipomul, serum IR-GIP l e v e l s reached a peak of 2.99 - 0.43 ng/ml but no s i g n i f i c a n t change in serum IRI leve l s was observed. These apparently contradictory findings could have several explanations. F i r s t , the IRI release obtained with GIP infusions may not represent a physio-l o g i c a l response due to the pattern of serum IR-GIP l e v e l s . The r i s e in serum IR-GIP l e v e l s obtained 5 6 when GIP was infused was much more rapid than was observed following o r a l glucose or f a t . Rate of change i n serum IR-GIP leve l s may influence the IRI response i n that the GIP molecule may be quickly modified in the blood. Some modifications could destroy i n s u l i n o t r o p i c a c t i v i t y while not a f f e c t i n g c r o s s - r e a c t i v i t y with antiserum raised to the complete GIP molecule. Results of the fragment studies allow for t h i s p o s s i b i l i t y . This suggestion would not eliminate the p o s s i b i l i t y that IR-GIP released following o r a l glucose potentiates the IRI response to elevated serum glucose l e v e l s . Secondly, the c o n f l i c t i n g evidence could be explained by hypothesizing the existance of two immunologically i d e n t i c a l but chemically d i s t i n c t forms of GIP, one released by glucose, the other by f a t . I t i s possible that the GIP released by glucose possesses a chemical e n t i t y which confers on i t i n s u l i n o t r o p i c a c t i v i t y . Or the GIP released by fat may possess a blocking group which prevents i n s u l i n o t r o p i c a c t i v i t y . I f t h i s be the case then the porcine GIP used i n infusion studies would be of the type released by glucose. The existance of two forms of GIP would not make i t unique in r e l a t i o n to other peptide hormones. Gastrin has been shown to be present i n the blood and in tissue extracts in several forms (Yalow, 1974) . Gastrins I and II d i f f e r only by the presence of a sulphate group on g a s t r i n II (Bentley et a l . , 1966) but t h i s small 57 difference confers on g a s t r i n II the a b i l i t y to stimulate g a l l bladder contraction. Gastrin I and g a s t r i n II are equally capable of stimulating g a s t r i c acid secretion. In addition, peptide hormones present i n tissue extracts are often d i f f e r e n t from t h e i r counterparts found i n the blood. This i s true to some extent for g a s t r i n (Yalow, 1974), parathyroid hormone (Berson and Yalow, 1968) and i n s u l i n (Kitabchi, 1970)„ These differences are often not detected by immunological assay methods. F i n a l l y , GIP may show variations between species which are not detected by the antisera used i n the current routine assay. Such differences could r e s u l t i n d i f f e r -ences i n i n s u l i n o t r o p i c a c t i v i t y . To date GIP has only been p u r i f i e d and characterized from hog duodenal and jejunal mucosa. In summary, i t i s appropriate to consider the major actions of GIP and how these may r e l a t e to i t s possible ph y s i o l o g i c a l r o l e s . Exogenous porcine GIP was found to be a potent i n h i b i t o r of pentagastrin-, histamine- and i n s u l i n hypoglycemia-stimulated acid secretion i n e x t r i n s i c a l l y denervated pouches of the fundus of the stomach, i n dogs (Pederson, 1971). GIP was also shown to i n h i b i t pepsin secretion and g a s t r i c motor a c t i v i t y i n the dog. Investigations into mechanisms of release of IR-GIP have shown i t to be released into the blood i n substantial amounts by the presence of glucose or fat i n the gut 58 lumen. Recent experiments i n t h i s laboratory (P. Moccia) have confirmed that release of endogenous IR-GIP by duodenal i n s t i l l a t i o n of glucose or fat i s followed by a high degree of i n h i b i t i o n of g a s t r i c acid secretion stimulated by pentagastrin or histamine. This body of evidence strongly indicates a physio-l o g i c a l r o l e for GIP as an enterogastrone. The i n h i b i t o r y actions of GIP would seem appropriate for normal digestive function. When fats are being digested, g a s t r i c acid and pepsin are not required whereas considerable time i s required for handling of f a t by the small bowel. Si m i l a r l y , when starch i s being digested, low g a s t r i c pH i s not desirable, since s a l i v a r y amylases function optimally between pH 4 - 11, and pepsin i s not required. The possible involvement of GIP i n release of i n s u l i n i s less e a s i l y r a t i o n a l i z e d . I t would seem appropriate that GIP f a c i l i t a t e i n s u l i n release when glucose i s digested and the evidence accumulated strongly indicates this p o s s i b i l i t y . However, IR-GIP i s released i n greater quantities by fat but i n s u l i n release in t h i s case i s not observed nor i s i t appropri-ate. Serum l e v e l s of exogenous GIP les s than those which can be obtained with ingestion of f a t are e f f e c t i v e i n stimulating i n s u l i n release. 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