The dual objectives of this thesis were to study the physiological role of GIP in the enteroinsular axis and to chemically characterize a side fraction identified in the purification of GIP. In the physiological studies, the dependency of the insulinotropic action of GIP on the prevailing state of glycaemia was confirmed in dogs using a system of steady state hyperglycaemia. GIP, from both exogenous and endogenous sources, was demonstrated to potentiate insulin release in the presence of moderate hyperglycaemia. Both glucose and fat administered enterally released immunoreactive-GIP (IR-GIP) and potentiated immunoreactive insulin (IRI) release during moderate hyperglycaemia (150 mg% above basal). Intravenous administration of GIP at 2.0 μg/kq.h was also capable of eliciting insulinotropic action during moderate hyperglycaemia. A mixture of ten amino acids was demonstrated to potentiate insulin release with mild hyperglycaemia (40 mg% above fasting) regardless of routes (intravenous, intraduodenal and oral) of administration. However, the release of IR-GIP was not demonstrated following the administration of the amino acid mixture. Arginine and alanine infused individually did not potentiate insulin release in the presence of mild hyperglycaemia. Intraduodenal hydrochloric acid infusion was also demonstrated not to release IR-GIP in the presence of mild hyperglycaemia. The interactions of GIP with tricarboxylic acid cycle intermediates and pyruvate were studied in euglycaemic conditions in dogs. Intravenous administration of individual metabolites (a-ketoglutaric acid, succinate and pyruvate) on an equimolar basis were shown not to be insulinotropic in the presence or the absence of concurrent GIP infusion (0.4 μg/kg.h). The presence of a minor peptide component in the stage III GIP was initially identified by thin layer chromatography. Confirmation of the presence of this minor component was obtained from polyacrylamide-urea gel. These techniques were unsuitable for preparative separation, as were conventional gel filtration and ion exchange separation. Further purification of GIP on high pressure liquid chromatography indicated the presence of a 5% minor peptide component which was eventually shown to contain two less amino acid residues (tyrosine and alanine) than GIP. The amino acid sequence of GIP III indicated the presence of a peptide component with an amino acid sequence different from GIP in that the first two amino acid residues of the N-terminal portion of the molecule (tyrosine and alanine) were missing. The lack of inhibitory activity to pentagastrin-stimulated acid secretion by synthetic GIP led to a reinvestigation of the amino, acid sequence of the molecule. The work in collaboration with Jornvall (Sweden) indicated an error, in that the original sequence included a second glutamine in position 30. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

The interaction of the intestinal insulinotropic hormone GIP (Glucose-dependent Insulinotropic Polypeptide or Gastric Inhibitory Polypeptide) with the stimulus-secretion coupling mechanism of glucose-induced insulin secretion was investigated using the isolated, perfused, rat pancreas technique. The action of GIP in potentiating insulin secretion which had been initiated by a number of metabolic compounds other than glucose (D-glycer-aldehyde, 2-ketoisocaproate, L-leucine + L-glutamine) and the concentration-dependent nature of this action led to the formulation of the hypothesis that the insulinotropic effect of GIP required prior oxidation of these insulin-stimulating metabolites. It was therefore likely that the mechanism whereby GIP potentiated glucose-induced insulin secretion required an interaction located at a level involving effects secondary to glucose degradation in the B-cell. This effect may have required an intact N-terminus on the GIP molecule since a GIP₃-₄₂ homologue, which lacked the N-terminal Tyrosine-Alanine, possessed greatly diminished insulinotropic activity. Additional biological actions of GIP were suggested by the stimulation by the hormone of pancreatic somatostatin release and of the release of lipoprotein lipase-like activity from isolated rat adipocytes. A technique using reversed phase high pressure liquid chromatography (HPLC) was developed allowing the purification and fodination-state analysis of a pure bioactive ¹²⁵I-GIP molecule. Preliminary investigations of receptor binding activity of this purified ¹²⁵I-GIP to isolated rat adipocytes were also performed. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

The existence of a stimulatory intestinal phase of gastric acid secretion has been suspected for some time, and recently the importance of this phase has been recognized. The intestinal phase is of particular interest in relation to the profound gastric hypersecretion associated with portacaval anastomosis. The results of many studies in dogs, and recently in man, have demonstrated conclusively that shunt-related gastric hyper-secretion is due to unmasking of the intestinal phase by hepatic bypass of a humoral stimulant that is normally inactivated by the liver. Definitive experiments have shown that this humoral agent is a hormone that arises in the jejunum. Elaboration of the hormone is triggered both by the entry of food into the jejunum and by a brief period of jejunal distension with a balloon.

The effects of cholecystokinin octapeptide (CCK-8), and its antagonist proglumide, upon conditioned behavior in the rat was studied. First, the effects of CCK-8 and proglumide upon passive avoidance behavior was investigated. Rats were trained to avoid a darkened chamber by presenting electrical footshock (two seconds of intensity levels) inside the chamber. Directly following the footshock, injections of CCK-8 or proglumide were given, with avoidance behavior measured 24 hours following the injection. CCK-8 was found to produce reductions in the passive avoidance latency at doses ranging from 30 ug/Kg to 500 ug/Kg. This effect was found to be dependent upon the current intensity used during conditioning. The CCK-8 effect was found when the current was at 0.25 mA, but at no other current setting tested. Proglumide (5 mg/Kg) was found to block the CCK-8 effect upon passive avoidance behavior. A lower dose of proglumide (2 mg/Kg) was found to produce reductions in the passive avoidance latency. These results suggest that CCK-8 may play a role in passive avoidance conditioning in rats. The effects of CCK-8 upon an appetitively conditioned behavior were then investigated. Rats were trained to locate and drink from a drinking tube that contained a 10 percent sucrose solution. Following 30 seconds exposure to the solution, injections of CCK-8 were given, with the latency to begin drinking from the tube measured 24 hours later. CCK-8 was found to produce increases in the latency to begin drinking, at doses of 20 ug/Kg and 100 ug/Kg. CCK-8 also produced a reduction in the amount of sucrose solution consumed during the test period. When CCK-8 was given following exposure to regular tap water, no increase in drinking latency or reduction in consumption was found. These results suggest that CCK-8 can act as an aversive stimulus and is capable of producing conditioned taste aversions. The results of this dissertation project demonstrate that CCK-8 can influence the acquisition of conditioned behavior in the rat when the octapeptide is paired with the presentation of an unconditioned stimulus (shock or sucrose).

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Gel filtration coupled with radioimmunoassay of fractions has demonstrated the existence of an 8000 dalton immunoreactive form of GIP (glucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide), which may be a precursor in the biosynthetic pathway. A monoclonal antibody to GIP has been shown to have highly suitable characteristics for affinity purification of different species of IR-GIP. An enzyme-linked immunosorbent assay (ELISA) was developed for GIP, employing the monoclonal antibody and was used for screening fractions for peptides with the same antigenic determinant i.e. IR-foras of GIP. Classical strategy used in peptide purification may result in loss of related peptides if they are sensitive to the pH or temperature conditions used. Tissue from hog duodenal and jejunal mucosa was boiled and extracted into acetic acid. Peptides were then adsorbed to alginic acid, eluted with 200 mM HC1, precipitated with NaCl and desalted on Sephadex G-25. The desalted material was adjusted to pH 7.0 with 200mM ammonia and extracted with methanol. The methanol insoluble fraction demonstrated the highest content of IR-GIP₈₀₀₀⋅ The overall acidic charge on the larger IR-GIP oUUU moiety suggested the possibility that it might not be adsorbed to alginic acid. The monoclonal antibody to porcine GIP₅₀₀₀ was coupled to cyanogen bromide activated Sepharose -4B. The peptide fraction which was not adsorbed to alginic acid was applied to the column and the fraction which bound to the ligand was eluted with 100 mM HC1. The immunoreactive material was rotary evaporated to dryness and further purified to a monocomponent by HPLC. A µBondapak C₁₈ column and a linear gradient of acetonitrile in water containing 0.1% TFA was used for HPLC. Amino acid analyses revealed the following composition: Asx (6), Thr (2), Ser (3), Glx (3), Pro (3), Gly (4), Ala (8), Val (5), Met (1), He (0), Leu (7), Tyr (1), Phe (3), His (4), Lys (5), Arg (3), Trp (+). The N-terminal residue was identified as valine using the dansylation method. Cleavage of the molecule with trypsin and separation of the tryptic peptides on HPLC showed 2 peptides with elution times similar to tryptic peptides of GIP. Application of monocomponent IR-GIP designated IR-LGIP C, and GIP to the HPLC system confirmed the two peptides to be separate entities. Biological activity was assessed in the isolated perfused rat pancreas, a model used for measurement of the insulin releasing effect of GIP. IR-LGIP C did not demonstrate insulinotropic activity. It is unlikely that this polypeptide is a proform of GIP. It shares common immunoreactivity but lacks the necessary common core of amino acid residues. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Interdigestive myoelectric activity in rat intestine has been recorded and characterized. The interdigestive pattern of activity can be disrupted by oral glucose and high doses of the duodenal ulcerogen cysteamine. Intravenous glucose had no effect on the interdigestive myoelectric pattern, nor did high doses of porcine motilin. Attempts were made to develop a hybridoma cell line secreting antibodies that would recognize rat Intestinal immunoreactive motilin (IR-M). The murine myeloma cell line NS1 was fused with murine B-cells primed against porcine motilin. One hundred of the monoclonal cell lines produced secreted monoclonal antibodies that recognized porcine motilin. Attempts to identify a cell line secreting antibodies with the ability to stain rat intestinal tissue, however, produced only negative results. Rat intestinal IR-M has been characterized with respect to immunocytochemistry (ICC), radioimmunoassay (RIA), and chromatographic properties. The biological activity of partially purified rat intestinal IR-M has also been evaluated utilizing a rabbit isolated duodenal muscle strip preparation. Five different antisera and one monoclonal antibody directed against natural porcine motilin were utilized in an effort to detect IR-M containing cells in rat intestinal tissues. A variety of techniques were employed including tissue fixation with either Bouins, paraformaldehyde, or benzoquinone. In addition a variety of staining methods including, fluorescein conjugated second antibody, peroxidase-antiperoxidase or peroxidase conjugated second antibody techniques were used. All methods using these antibodies failed to detect IR-H in the rat small intestine. Porcine motilin was able to displace ¹²⁵I-motilin from antisera 13-3, 72X and M03. These antisera were utilized in a motilin RIA to evaluate acid extracts of rat intestinal tissue for IR-M. Only antisera 13-3 and 72X were capable of detecting IR-M in gut extracts, and these antisera gave different distributions of IR-M In the proximal small bowel. Rat intestinal tissue was extracted into 2% trifluoroacetic acid and the soluble fraction clarified by centrifugation. This acid extracted material was precipitated with sodium chloride then dissolved in methanol at pH 6.0. Methanol soluble material was precipitated with ether and the ether precipitate then dissolved in water and chromatographed on Sep-Pak C₁₈ cartridges (Waters). Sep-Pak cartridges were eluted with 50% acetonitrile: 0.1% TFA. The 50% eluate was then fractionated further using cation exchange, gel filtration and reverse phase high pressure liquid chromatography (HPLC). Rat intestinal IR-M peaks from cation exchange chromatography on SP-Sephadex-C25 (Pharmacia) were concentrated and examined for contractility in a rabbit duodenal muscle strip preparation. Purification after SP-Sephadex-C25 was approximately 20 fold. Desensitization of rabbit duodenum to porcine motilin could be demonstrated by pre-treatment with motilin. Contractile activity of partially purified rat intestinal IR-M was not inhibited by pretreatment with motilin. Chromatography on Bio-Gel P-10 (Biorad) eluted with 0.2M acetic HPLC, using a linear gradient of water/acetonitrile (10-45% acetonitrile in 30 min), rat intestinal IR-M did not co-elute with natural porcine motilin. In conclusion, the molecular weight of rat intestinal IR-M appeared to be similar to porci ne motilin as these two substances demonstrated co-elution on gel permeation chromatography. The lack of co-elution with porcine motilin on HPLC indicates that other molecular characteristics of rat intestinal IR-M, such as hydrophobicity, are not similar to porcine motilin. Furthermore, partially purified rat intestinal IR-M did induce a contractile response in rabbit duodenal muscle strips but porcine motilin did not desensitize this preparation to the contractile activity of rat intestinal extracts. This suggests that the contractile activity of these two compounds is induced via different receptor mechanisms. It is concluded that the immunoreactive motilin found in rat intestinal extracts does not resemble natural porcine motilin in structure or biological activity. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Several feeding-related factors can affect taste sensitivity or preferences and therefore may be part of a homeostatic regulatory mechanism. Cholecystokinin (CCK), a hormone which reduces food intake in several species, has also been postulated to interact with the orosensory characteristics of food. To test this hypothesis, the effects of CCK-8 and food deprivation on the short-term intakes of water, sucrose solutions (0.05 to 1.0 M), and saline solutions (0.05 and 0.15 M) were determined. In most cases, CCK (2 μg/kg) reduced sucrose intake when measured either as the amount consumed or the number of licks in a short period (nine minutes). Additionally, CCK reduced the intake of 0.15 M NaCl in satiated rats and water intake in both hungry and satiated rats. Rats usually consumed more sucrose when hungry than when satiated or fed ad libitum; CCK-induced suppression of intake, however, was generally greater in the satiated or ad libitum conditions than in the hungry condition. There was no systematic effect of sucrose concentration on the amount of CCK-induced suppression of intake, which suggests that CCK regulates rather than interferes with ingestion. To determine whether the CCK-induced suppression is due to a change in the peripheral taste signal, the integrated chorda tympani responses to sucrose and NaCl tastes were recorded in rats anesthetized with either urethane, Innovar-Vet, or a combination of urethane and alpha-chloralose. The only significant effect of CCK was the slight increase in the initial response to 0.3 M sucrose after the infusion of a total of 10 μg of CCK-8 into rats anesthetized with Innovar-Vet. In general, therefore, the effect of CCK on sucrose intake does not appear to be due to a peripheral taste change; an analysis of single taste fibers, however, would be more conclusive. An examination of the effects of CCK on central gustatory and reward areas might yet provide a mechanism for the CCK effect on taste-motivated ingestion.