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Control of insulin secretion from the perfused rat pancreas : effects of acetylcholine and a somatostatin… Verchere, Cameron Bruce 1987

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CONTROL OF INSULIN SECRETION FROM THE PERFUSED RAT PANCREAS: EFFECTS OF ACETYLCHOLINE AND A SOMATOSTATIN ANALOG, SMS 201-995 By Cameron Bruce Verchere B.Sc, The University of British Columbia, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Physiology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA APRIL, 1987 ©Cameron Bruce Verchere, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Physiology The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date A p r i l 29, 1987 DE-6(3/81) i i ABSTRACT The effect of varying concentrations of glucose or the gastrointestinal hormones, gastric inhibitory polypeptide (GIP) and somatostatin (SS-14), on the in vitro immunoreactive insulin (IRI) response to the parasympathetic neurotransmitter, acetylcholine (ACh) was investigated. The isolated, vascularly perfused rat pancreas was used i n a l l experiments. Acetylcholine (1.0 jtiM) did not stimulate IRI secretion i n the presence of 2.2 mM glucose. However, i n the presence of 4.4, 6.6, or 8.9 mM glucose, ACh (1.0 jiM) potently stimulated IRI secretion (approximately fourfold). At a higher glucose concentration (17.8 mM), the IRI response to ACh was reduced. GIP also potentiated the IRI response to 1.0 uM ACh. This potentiation was most marked in the presence of 1.0 nM GIP, whereas the effect of concomitant infusion of 0.2 nM GIP and 1.0 pK ACh was only slightly greater than additive. SS-14 potently inhibited ACh-stimulated IRI secretion. These results demonstrated the glucose dependency of cholinergically stimulated IRI secretion, and that physiological levels of glucose and GIP increased B-c e l l sensitivity to cholinergic stimulation. It was suggested that the parasympathetic stimulation of IRI secretion associated with food intake could be affected by postprandial increases i n glucose, GIP, and SS-14. The idea that endogenously released somatostatin may have influenced glucose or GIP-stimulated IRI secretion was not supported by the present experiments, since neither glucose (8 .9 mM) nor GIP (2.0 nM) were found to have a significant effect on the release of pancreatic somatostatin-like immunoreactivity (SLI). i i i Both atropine (1.0 juM) and hexamethonlum (100 ;_M) inhibited the IRI response to ACh. This suggested that the parasympathetic stimulation of IRI secretion was mediated not only by muscarinic receptors on the B-cell, but also by nicotinic receptors on intrapancreatic ganglia. Neither atropine nor hexamethonlum had a significant effect on glucose- or GIP-stimulated IRI secretion, indicating that the IRI response to these stimuli was not mediated by cholinergic receptors. Both SS-14 and the synthetic somatostatin analog SMS 201-995 (SMS; Sandostatin®) inhibited IRI secretion stimulated by 8.9 mM glucose, 2.0 nM GIP, or 1;.0 jiM ACh, but not 17.8 mM glucose. The most potent inhibition by both SS-14 and SMS was observed i n the presence of the weakest IRI stimuli (8.9 mM glucose and 1.0 p.H ACh). These results suggested that the inhibitory effects of somatostatin on the B-cell could be overcome by the presence of strong stimuli. In addition, the inhibitory effects of the native hormone and the analog were found to be approximately equipotent (weight basis), indicating that the increased potency of SMS previously observed in vivo was due to i t s longer half-l i f e i n plasma, and not due to a more potent direct effect on the B-c e l l . i v TABLE OF CONTENTS Abstract i i Table of Contents i v List of Figures vi Acknowledgements v i i Dedication v i i i INTRODUCTION 1 GENERAL METHODOLOGY 22 I. Isolated Perfused Pancreas 22 A. Apparatus 22 B. Animals 25 C. Surgical Procedure 25 D. Solutions 30 1. Perfusate 30 2. Drugs and Peptides 31 II. Peptide Quantification by Radioimmunoassay 31 A. Insulin 32 1. Antiserum 32 2. Iodination 32 (a) . Solutions 32 (b) . Procedure 33 3. Assay buffer 34 4. Standards 35 5. Controls 35 6. Assay protocol 36 7. Calculations 36 B. Somatostatin 37 1. Antiserum 37 2. Iodination 38 (a) . Solutions 38 (b) . Procedure 38 3. Assay buffer 39 4. Standards 39 5. Assay protocol 40 6. Calculations 40 III. S t a t i s t i c a l Analysis 40 APPENDIX TO METHODS - CHEMICAL SOURCES 41 RESULTS 42 I. Effect of Glucose on ACh-stimulated IRI Secretion 42 II. Effect of GIP on ACh-stimulated IRI Secretion 50 III. Effect of Atropine and Hexamethonium on Glucose-, GIP-, and ACh-stimulated IRI Secretion 55 IV. Effect of SS-14 and SMS on Glucose-, GIP-, and ACh-stimulated IRI Secretion 64 V. Effect of Glucose and GIP on SLI Secretion 76 DISCUSSION 80 LIST OF REFERENCES 95 vi LIST OF FIGURES FIGURE 1 Amino Acid Sequence of Somatostatin-14 and SMS 201-995 16 2 Apparatus for the Vascular Perfusion of the Rat Pancreas 23 3 Schematic Diagram Showing the Placement of Cannulae and Associated Ligatures i n the Perfused Rat Pancreas 27 4 The Effect of ACh on IRI Secretion i n the Presence of 4.4 mM Glucose 44 5 The Effect of ACh on IRI Secretion i n the Presence of 8.9 mM Glucose 46 6 Summary of the Effects of the Glucose Concentration on the IRI Response to ACh 48 7 The Effect of ACh on IRI Secretion i n the Presence of 8.9 mM Glucose and 1.0 nM GIP 51 8 Summary of the Effects of GIP on the IRI Response to ACh 53 9 The Effect of Atropine on the IRI Response to ACh 57 10 The Effect of Hexamethonlum on the IRI Response to ACh 59 11 The Effect of Atropine on the IRI Response to GIP 61 12 Summary of the Effects of Atropine and Hexamethonlum on the IRI Response to ACh, Glucose, and GIP 63 13 The Effect of SS-14 and SMS on the IRI Response to 17.8 mM Glucose 67 14 The Effect of SS-14 and SMS on the IRI Response to 8.9 mM Glucose 69 15 The Effect of SS-14 and SMS on the IRI Response to GIP 71 16 The Effect of SS-14 and SMS on the IRI Response to ACh 73 17 Summary of the Effects of SS-14 and SMS on the IRI Response to Glucose, GIP, and ACh 75 18 The Effect of Glucose and GIP on SLI Secretion 78 v i i ACKNOWLEDGEMENTS I would f i r s t like to thank Dr. John Brown for his continual support as a teacher, motivator, and friend. I must also thank Dr. Yin Nam Kwok, with whom I worked very closely over the past two years, for his guidance and friendship. I am indebted to Dr. Raymond Pederson, whose continual effort on behalf of myself and other graduate students i n this department i s greatly appreciated. The help of Dr. Chris Mcintosh, particularly i n the use of his computer, the production of figures and his many ideas, i s gratefully acknowledged. As well, I thank Dr. Alison Buchan for her input and support. To a l l of the above, I thank you for making l i f e i n and out of the lab a great experience. I look forward to working with you i n the future. There are a great many other people who must also be acknowledged. Within the Regulatory Peptide Group, I thank a l l of the other students and staff for making work a lot of fun. I must specially thank Ms. Sue Otte, Dr. Mika Sinanan, Mrs. Herminia Sy, and Mrs. Marie Langton for their help. The assistance of Mr. John Sanker, Mr. Joe Tay, Mr. Jeff Russell, Mr. Dave Phelan, Miss Christine Tang, Mr. Patrick Leung and Mrs. Mary Forsythe, i n the production of this thesis as well as numerous other papers and seminars, i s greatly appreciated. Finally, to a l l other graduate students, faculty, and staff within the Department of Physiology: thank you. You have helped make these past two years quite enjoyable. DEDICATION For Mom and Dad and Steph and AJ 1 INTRODUCTION The regulation of insulin secretion has been shown to involve the interactions of ingested nutrients with the autonomic nervous system and endogenously released endocrine and paracrine substances (Ahren et a l , 1986; Berthoud, 1984; Gerich et a l , 1976; Miller, 1981). Berthoud (1984) demonstrated that the insulin response to a meal was mediated mostly by the parasympathetic nervous system, gastrointestinal hormones, and glucose, as well as synergistic interactions between these stimuli. However, the effects of postprandial changes i n the levels of circulating nutrients and hormones on the parasympathetic stimulation of insulin secretion has not been studied. Determination of the direct interactions of various agents i n the control of insulin release has required i n vitro approaches, since the influence of the central nervous system and circulating factors would be present .in vivo. The isolated, perfused pancreas has been used frequently as an i n vitro model for studying the effects of physiological and pharmacological agents on B-cell secretion. It has been demonstrated that the in. situ architecture of the pancreas, including the i n t r i n s i c innervation, microvasculature, and c e l l - c e l l contacts, remained intact i n this preparation (Pipeleers et a l , 1984; Weir et a l , 1986). In addition, the perfused pancreas has been shown to be responsive to physiological levels of the gastrointestinal hormones gastric inhibitory polypeptide (GIP) (Pederson and Brown, 1976) and somatostatin (SS-14) (Albert! et a l , 1973), whereas pharmacological doses of these peptides were required i n isolated i s l e t preparations (Efendic et a l , 1974; Fujimoto, 1981). In the present study, the perfused pancreas was used to investigate the interactions of glucose, GIP, and SS-14 with the parasympathetic neurotransmitter, acetylcholine (ACh), i n the control of insulin secretion. The source of insulin has been identified as the B-cell, one of several endocrine c e l l types found i n the pancreatic i s l e t s . Shortly after the discovery of the i s l e t s (Langerhans, 1869) several investigators suggested that an "internal secretion" of the pancreas, possibly from the i s l e t s , was involved i n the control of glucose metabolism (for historical review see Bliss, 1982). In 1920, Banting and Best used a crude extract of i s l e t tissue from dogs to relieve the hyperglycemia and glycosuria seen i n animals made diabetic by total pancreatectomy. Purer preparations of the active component, insulin, were soon available and used widely i n the treatment of diabetes. However, research into the mechanisms controlling insulin secretion was limited by the absence of sensitive insulin assay techniques until the development of radioimmunoassay (Berson and Yalow, 1959), which allowed the accurate measurement of insulin levels i n plasma and from i n vitro preparations. In addition to the B-cells, the i s l e t s have been shown to contain at least three other endocrine c e l l types: A-cells (glucagon), D-cells (somatostatin), and PP-cells (pancreatic polypeptide) (Orel, 1982). The presence of other peptides i n i s l e t endocrine c e l l s , such as corticotropin releasing factor (CRF) (Petrusz et a l , 1983) and pancreastatin (Tatemoto et a l , 1986), has also recently been suggested. In most species, the various i s l e t c e l l types have been shown to have an ordered arrangement, suggesting the existence of i n t r a - i s l e t control mechanisms (Samols et a l , 1986). In humans and rats, for example, the i s l e t s have been shown to be comprised of a mantle of B-cells surrounded by a periphery of A-, B-, D-, and PP-cells (Orci, 1982). Studies on the control of insulin secretion have therefore been complicated by the fact that these i s l e t peptides may have exerted a local effect on the B-cell. In particular, A- and D-cells have often been found in contact with B-cells i n the periphery of the i s l e t , suggesting that glucagon and somatostatin may have affected insulin secretion via a paracrine mechanism (Orci and Unger, 1975). In addition, a unique i s l e t microcirculation has been shown to exist, via which i s l e t hormones may travel to influence the secretion of other i s l e t cells downstream (Samols et a l , 1986). Immunocytochemical evidence for the release of pancreatic somatostatin into this microcirculation was recently found i n rat i s l e t s (Aponte et a l , 1985). Thus, the possibility of in t r a - i s l e t influences on insulin secretion must be considered i n any investigation which has used intact i s l e t preparations, including isolated i s l e t s and perfused pancreas studies. As insulin has been shown to be an important regulator of carbohydrate and l i p i d metabolism (Unger et a l , 1978), i t follows that the metabolic fuels, and i n particular glucose, would be involved i n the control of insulin secretion. Numerous studies have demonstrated a stimulatory effect of glucose and other nutrients on insulin release (Gerich et a l , 1976; Hedeskov, 1980). In vitro preparations such as the isolated, perfused pancreas (Grodsky et a l , 1963), isolated i s l e t s , and purified B-cells (Pipeleers et a l , 1982) have demonstrated the potent and direct stimulatory effect of glucose on the B-cell. However, recent studies with dispersed i s l e t cells suggested that glucose alone was not sufficient for the stimulation of insulin release, but rather that the presence of exogenous glucagon or contacts with other i s l e t cells was required (Pipeleers et a l , 1982; Pipeleers et a l , 1985). In addition, the threshold for glucose-stimulated insulin release i n vitro (approximately 5 mM) indicated that glucose alone did not regulate fasting levels of insulin (Gerich et a l , 1976). Instead, at subthreshold concentrations, glucose may have served to modulate the action of other insulinotropic agents ( G r i l l and Rundfeldt, 1979). As w i l l be discussed, most insulin secretagogues have been shown to be dependent on the presence of glucose or other nutrients for their insulinotropic action. The insulin response to a rapid (square wave) increase i n glucose concentration has been shown to be multiphasic (Gerich et a l , 1976); an I n i t i a l peak was followed by a second phase of slowly increasing insulin secretion. This biphasic release pattern was also seen i n response to a number of other insulin secretagogues. It has been suggested to be due to a functional segregation of intracellular mediators of insulin secretion or B-cell secretory granules (storage-limited model) or feedback of an inhibitory factor (signal-limited model) (Landahl and Grodsky, 1982; O'Connor et a l , 1980). A third phase of insulin release, namely a slow decrease after prolonged exposure to glucose, has also been described i n perifused i s l e t s and the perfused pancreas (Grodsky et a l , 1986). The mechanism of glucose-induced insulin release was proposed to be mediated by stereospecific glucoreceptors on the B-cell membrane (Matschinsky et a l , 1975). However, recent evidence suggested a mechanism involving products of intracellular glucose metabolism. Insulin release was stimulated from i s l e t s i n the absence of extracellular glucose by inducing catabolism of endogenous glycogen (Malaisse et a l , 1977). In addition, the insulinotropic effect of glucose and other nutrient secretagogues was shown to correlate well with their rate of metabolism i n the B-cell, while non-metabolized sugars did not stimulate insulin secretion. These studies were embodied by Malaisse et al (1979) as the "fuel hypothesis", which proposed that nutrient-Induced insulin release occurred via increased catabolism of the nutrient i n the B-cell. Although the precise Intracellular mechanism coupling nutrient catabolism to insulin secretion has not yet been elucidated, i t has been suggested to involve increases i n intracellular levels of calcium and cAMP stimulated by the products of catabolism (NADH and ATP), followed by the activation of Ca- and cAMP-dependent protein kinases (Sener and Malaisse, 1984). Other nutrients shown to be involved in the regulation of Insulin secretion have included amino acids and fats (Fajans and Floyd, 1972; Gerich et a l , 1976). The amino acids were shown to vary i n their insulinotropic action, with the basic amino acids, arginine and lysine, being the most potent i n man (Fajans and Floyd, 1972). In the perfused rat pancreas, arginine weakly stimulated a monophasic release of insulin in the absence of glucose, whereas a more potent, biphasic release was seen i n the presence of subthreshold levels of glucose (Gerich et a l , 1974). Free fatty acids also demonstrated a glucose-dependent stimulation of insulin release (Balasse and Ooms, 1973). Insulin secretion has also been shown to be under the influence of the autonomic nervous system (Edwards, 1984; Miller, 1981). The rich innervation of the pancreas, f i r s t described by Langerhans i n 1869, has now been recognized as a complex arrangement of both extrinsic and in t r i n s i c nerves, with cholinergic, adrenergic, and peptidergic fibers terminating on i s l e t cells . Insulin release has generally thought to be increased by cholinergic and beta-adrenergic stimuli and inhibited by alpha-adrenergic inputs. It has been demonstrated that the parasympathetic nervous inputs to the i s l e t s originate i n the dorsal motor nucleus and nucleus ambiguus (Luiten et a l , 1984) and descend to the pancreas via the gastric, celiac, and hepatic branches of the vagus nerves (Berthoud et a l , 1983). A role for the parasympathetic nervous system i n the control of i s l e t secretion was suggested by Britton (1925), who demonstrated that electrical stimulation of the vagus nerve caused a decrease i n blood glucose. It was later confirmed by several groups that this effect was due to vagal stimulation of insulin release (Daniel and Henderson, 1967; Frohman et a l , 1967; Kaneto et a l , 1967). Bergman and Miller (1973), using an i n situ, cross-perfused pancreas preparation i n the dog, found that the vagus nerve had a direct stimulatory effect on the B-cell, suggesting that the response was not mediated by a blood-borne factor. This was further supported by studies which showed the presence of cholinergic nerve fibers terminating on the i s l e t s (Amenta et a l , 1983). In addition, i t has been shown that the parasympathetic, postganglionic neurotransmitter ACh had a stimulatory effect on insulin release i n  vitro, from isolated i s l e t s (Sharp et a l , 1974), pancreatic pieces (Malaisse et a l , 1967), and the perfused pancreas of rats (Loubatieres-Mariani et a l , 1973), pigs (Hoist et a l , 1981a) and dogs (Iversen, 1973). Parasympathetic stimulation of insulin release has been shown to be inhibited by prior administration of atropine, suggesting that the response was mediated primarily through B-cell muscarinic receptors. However, the response to ACh seen i n the perfused pancreas could also have had a component mediated by intrapancreatic ganglia, which were intact i n this preparation and have been shown to possess nicotinic, cholinergic receptors (Stagner and Samols, 1986). Chapal and Loubatieres-Mariani (1978) showed that nicotine, at low concentrations, stimulated insulin release from perfused rat pancreas. Also, i n vivo studies i n pigs demonstrated that the insulin response to ACh was unaffected by atropine but abolished by the nicotinic blocker hexamethonium (Hoist et a l , 1981a). Although many pancreatic postganglionic fibers have been shown to be cholinergic (Amenta et a l , 1983), there has been increasing evidence for the role of a number of peptidergic neurotransmitters i n the pancreas. For example, the identification of nerve fibers i n the pancreas of the pig that contained vasoactive intestinal peptide (VIP) (Larsson et a l , 1978), coupled with the insulinotropic effects of VIP seen i n this species (Lindkaer-Jensen et a l , 1978), suggested that the parasympathetic stimulation of insulin secretion was at least partly mediated by VIPergic nerve fibers. Similarly, i n the rat, although a large part of the insulin response to vagal stimulation was shown to be cholinergic, the role of a number of putative peptidergic transmitters, such as gastrin releasing peptide (GRP), VIP, and the opioid peptides could not be discounted (Ahren et a l , 1986). Most studies have shown that vagally or cholinergically stimulated insulin secretion was glucose dependent. In the absence of glucose or at low concentrations (< 3.0 mM), ACh was usually shown to have no effect on insulin release ( G r i l l and Fak, 1985; Honey and Weir, 1980; Loubatieres-Mariani et a l , 1973; Muller et a l , 1986), although one study, i n isolated i s l e t s , demonstrated a biphasic insulin response to ACh i n the presence of 2.4 mM glucose (Sharp et a l , 1974). In a l l studies, ACh-induced insulin release was potentiated by the presence of threshold levels of glucose (> 5.0 mM). The nature of the interaction between glucose and ACh i n the control of insulin secretion has not yet been determined. One study found that glucose-induced insulin release from isolated rat i s l e t s could be inhibited with atropine, suggesting an action of glucose at the muscarinic receptor (Sharp et a l , 1974), although this has not been reported elsewhere i n the literature. G r i l l and Ostenson (1983) showed that the priming effect of glucose was not mediated by changes i n ACh binding to the muscarinic receptor, since 3 binding of the muscarinic antagonist [H]-methylscopolamine to i s l e t s was not affected by changes i n glucose concentration. Instead, the presence of glucose may have provided an appropriate metabolic state of the B-cell, a necessary prerequisite for cholinergic stimulation. Meglasson et a l (1986) demonstrated that ACh enhanced glucose metabolism i n the i s l e t s , suggesting that cholinergic stimulation of insulin secretion was partly mediated by amplification of the glucose stimulus. However, ACh and glucose have previously been shown to have different effects on the electrical activity of i s l e t c e l l s , indicating that ACh did not act on the B-cell solely by increasing a signal generated by the presence of glucose (Cook et a l , 1981). It has been suggested that the physiological role of the parasympathetic nervous system i n the regulation of insulin secretion was probably to enhance meal-related insulin secretion (Ahren et a l , 1986). This idea has been supported by studies which showed that sectioning of the vagus nerves i n rats (Hakanson et a l , 1971) or administration of atropine i n conscious calves (Bloom and Edwards, 1980) had no effect on fasting levels of insulin secretion, but reduced the insulin response to oral or intravenous glucose. Studies on the effect of vagotomy on the insulin response to a meal i n man have so far been inconclusive (Miller, 1981). One study found that vagotomy had no effect on insulin secretion stimulated by either oral or intravenous glucose (Lund et a l , 1975), whereas a second study showed a decreased insulin response to oral glucose (Russell et a l , 1974). A role for the vagus i n regulating the insulin response to a meal has been indirectly supported by numerous studies which have demonstrated the glucose-dependency of vagal or cholinergic stimulation of insulin release. In addition, the cephalic phase of insulin release, which stimulated insulin secretion prior to increases i n circulating nutrients during a meal, has been shown to be mediated by the vagus nerves (Storlien, 1985). The sympathetic nervous system has also been shown to be involved in the regulation of insulin secretion (Ahren et a l , 1986). El e c t r i c a l stimulation of the splanchnic nerves, i n a range thought to mimic the physiological situation (approximately 10 Hz), inhibited insulin secretion i n a number of species, even in the presence of stimulatory levels of glucose (Edwards, 1984). Miller (1975), using the isolated canine pancreas, demonstrated that the inhibition was due to a direct effect on the pancreas mediated by alpha-adrenergic receptors. This was further supported by Johnson et a l (1976), who showed that scorpion toxin, which caused the release of norepinephrine from sympathetic nerve endings, inhibited insulin release from perifused i s l e t s , and that this effect was blocked by the alpha-adrenergic antagonist phenoxybenzamine. The presence of a beta-adrenergic stimulatory effect was f i r s t shown using the beta-agonist isoproterenol (Porte, 1967), and was supported by later studies that demonstrated a stimulatory effect of alpha-antagonists during splanchnic stimulation or norepinephrine administration, presumably by unmasking of the beta-adrenergic receptors (Edwards, 1984). The physiological significance of this apparent dual role of the sympathetic innervation has not yet been established. However, i t has been suggested that tonic sympathetic activity may have played an important role i n modulating insulin secretion under normal conditions, since infusion of phentolamine or propranolol at rest has been shown to induce a rise or f a l l , respectively, i n circulating levels of insulin (Robertson and Porte, 1973). Recent anatomical studies of the sympathetic innervation of the pancreas revealed the presence of intramural, postganglionic sympathetic nerves i n the pancreas (Baetens et a l , 1985; Luiten et a l , 1986). This finding contrasted with the classical view of the preganglionic sympathetic fibers terminating at cholinergic synapses i n the extramural, celiac ganglion. Therefore, i t should be recognized that the observed effects of parasympathomimetics on insulin secretion i n the perfused pancreas or i n vivo, i n which the i n t r i n s i c innervation of the pancreas was intact, could have been mediated partly by stimulation of nicotinic receptors on sympathetic, intrapancreatic ganglia. As well, the postganglionic fibers of these ganglia might be adrenergic or peptidergic (Ahren et a l , 1986). Therefore, the effects of splanchnic nerve stimulation or cholinergic agonists could have been mediated by neuropeptides released from postganglionic, sympathetic nerve fibers. Galanin, which elicit e d sympathomimetic effects i n canine pancreas and has been found i n intrapancreatic nerves, has been suggested as a potential sympathetic, postganglionic, neurotransmitter i n the pancreas (Dunning et a l , 1986). Investigation into the physiological control of insulin release has been further complicated by the fact that many hormones have demonstrated potent effects on B-cell secretion. In particular, an increasing number of gastroenteropancreatic peptides have been shown to influence insulin release. These peptides may have reached the B-cell by one or more different routes: via the circulation (endocrine transmission), via the i n t e r s t i t i a l space (paracrine), or as neurotransmitters. Gastric inhibitory polypeptide (GIP), a 42 amino acid hormone, was isolated and purified from porcine duodeno-jejunal mucosa (Brown et a l , 1969; Brown et a l , 1970). It was originally described as an enterogastrone candidate, due to i t s a b i l i t y to inhibit gastrin-stimulated acid secretion from denervated stomach pouches (Brown and Pederson, 1970). However, the physiological importance of this effect has not yet been determined, because of the weak acid-inhibitory effect of GIP observed i n the innervated stomach (Soon-Shiong et a l , 1979). A more important role has been suggested for GIP i n the "entero-insular axis", a regulatory system proposed by Unger and Eisentraut (1969), wherein nervous and humoral signals, activated during substrate stimulation of the gastrointestinal tract, would have an effect on pancreatic hormone secretion. The existence of an "incretin" - an intestinal factor that stimulated insulin release - was postulated by Mclntyre et al (1965), who showed that intraduodenal glucose produced a greater increase i n plasma insulin than an equivalent amount of glucose administered intravenously. Dupre et a l (1973) demonstrated that purified porcine GIP had a potent, glucose-dependent, insulinotropic action i n humans. This was later confirmed by studies i n the conscious, fasted dog (Pederson et a l , 1975a) and i n the perfused rat pancreas (Pederson and Brown, 1976). Creutzfeldt (1979) has since suggested that, although other gastrointestinal hormones have insulinotropic effects (e.g. secretin), "GIP i s the strongest incretin candidate". A physiological role for GIP i n the enteroinsular axis has been supported by a number of studies. Kuzio et al (1974) demonstrated that the plasma concentration of immunoreactive GIP i n humans i n response to a meal rose to approximately 0.2 nM. This concentration of GIP was later found to be Insulinotropic i n the perfused rat pancreas i n the presence of threshold levels of glucose (8.9 mM) (Brown et a l , 1980). In addition, oral glucose has been shown to cause an increase i n circulating levels of GIP i n man (Thomford et a l , 1974) and i n dog (Pederson et a l , 1975b). In the latter study i t was shown that, using physiological doses of GIP administered intravenously, the insulin response to oral glucose and to intravenous glucose plus GIP were both increased compared to intravenous glucose alone. The threshold glucose concentration for the insulinotropic action of 1.0 nM GIP was shown to be approximately 5.5 mM i n the rat, with the maximum potentiation seen at 16.0 mM (Pederson and Brown, 1976). This was similar to the threshold of approximately 1.4 mM above basal, seen i n humans (Elahi et a l , 1979). The mechanism of interaction between GIP and glucose at the B-cell has not been elucidated. The presence of high affinity binding sites for GIP on the B-cells of a hamster insulinoma has been described (Maletti et a l , 1984), suggesting that GIP acted directly on the B-cell. It has also been shown that GIP-stimulated insulin release, i n the presence of glucose, was coupled to increases i n i s l e t cAMP levels (Szecowa et a l , 1982; Siegel and Creutzfeldt, 1985). However, investigation into possible intracellular mechanisms of GIP-stimulated insulin secretion has been limited to perfused pancreas studies, since isolated i s l e t preparations have been found to be insensitive to physiological levels of GIP (Fujimoto, 1981; Schauder et a l , 1975). Studies i n the perfused pancreas indicated that the presence of metabolic compounds other than glucose (e.g. glyceraldehyde, arginine) were sufficient for GIP stimulation of insulin release (Dahl, 1983; Pederson and Brown, 1978), suggesting that the action of GIP occurred at a site distal to nutrient catabolism. The reason for the insensitivity of isolated rat i s l e t s , incubated in a static system, to physiological levels of GIP has not been ascertained. Brown et a l (1980) proposed several reasons: damage to B-c e l l GIP receptors during collagenase digestion of the i s l e t s ; accumulation of an endogenous inhibitory substance (e.g. somatostatin) in the incubation medium; damage to the i s l e t microvasculature preventing transport of GIP to the B-cell; or, i f GIP has an indirect action on insulin release, removal of a site of action of GIP other than the B-cell. The idea that GIP receptors were damaged during i s l e t isolation has been supported by two recent studies. F i r s t , GIP receptors have not yet been found on i s l e t s isolated by collagenase digestion, whereas they were recently identified on membrane preparations of B-cell tumors (Maletti et a l , 1984). Second, collagenase isolated i s l e t s were recently shown to regain sensitivity to physiological levels of GIP i n short-term culture (Siegel and Creutzfeldt, 1985). It has recently been suggested that the weak insulin response to GIP seen in isolated i s l e t s may be due partly to removal of the parasympathetic inputs to the B-cell. In rat pancreatic lobules, i n the presence of threshold glucose concentrations (11 mM), GIP was found to have no effect on insulin secretion unless the cholinergic agonist carbachol was added to the incubation medium (McCullough et a l , 1985). Further, the stimulatory effect of GIP i n the presence of carbachol was abolished by atropine. However, no study to date has examined the effect of parasympathomimetics on GIP-stimulated insulin secretion from isolated i s l e t s . Previously, i t had been demonstrated that the insulin response to oral glucose but not intravenous glucose was inhibited by atropine (Henderson et a l , 1976), suggesting that the release of the intestinal factor that potentiated insulin secretion was dependent on cholinergic input. McCullough et a l (1985) reasoned that the inhibition of gut-mediated insulin release by atropine, as well as the weak insulin response to GIP seen in the i s l e t s , could be due to a cholinergic dependence of the action of GIP on the B-cell. A cholinergic interaction with GIP had previously been demonstrated with the effects of GIP on gastric somatostatin secretion (Mcintosh et a l , 1981) and pancreatic glucagon secretion (Ahren and Lundquist, 1982). Studies i n the perfused pancreas, however, have not demonstrated a cholinergic dependence of GIP-stimulated insulin secretion (Muller at a l , 1986; Pederson and Brown, 1976), although i n this preparation the innervation of the i s l e t was almost certainly more intact than i n pancreatic lobules. In addition, atropine was shown to have no effect on GIP-stimulated insulin release i n man (Amland et a l , 1985). Thus, the nature of the interaction between GIP and the parasympathetic input to the B-cell has not yet been fu l l y studied. Somatostatin has also been shown to be a physiologic regulator of insulin secretion. Originally isolated from sheep hypothalamic extracts, somatostatin was f i r s t described as a 14 amino acid peptide that Inhibited growth hormone secretion (Brazeau et a l , 1973). Somatostatin has since been shown to have an inhibitory effect on a number of physiological functions, particularly i n the gut (Mcintosh, 1985; Reichlin, 1983), and inhibition of the secretion of a l l of the known gastroenteropancreatic hormones by somatostatin has been demonstrated (Reichlin, 1985). Somatostatin-like immunoreactivity has been found i n endocrine cells i n the pancreatic i s l e t s and throughout the gastrointestinal tract, and i n nerve cells throughout the central nervous system, gut, and various peripheral organs (Polak and Bloom, 1986). Larger molecular forms of somatostatin (e.g. somatostatin-28) have been described (Pradayrol et a l , 1980), and may play a physiological role i n the regulation of insulin secretion (Marco et a l , 1983); however, cyclic somatostatin-14 (SS-14; Figure 1) has generally been used i n the study of the action of the hormone on the B—cell. Alberti et al (1973) f i r s t demonstrated the Inhibitory effect of SS-14 on insulin secretion. Somatostatin-14 was shown to inhibit the insulin response to both intravenous glucose i n man, and to a square wave glucose stimulus of 8.3 mM i n the perfused canine pancreas, suggesting a direct action of the tetradecapeptide on the B-cell Figure 1: AMINO ACID SEQUENCE OF SOMATOSTATIN-14 AND SMS 201-995. 17 SOMATOSTATIN ALA-GLY-CYS-LYS-ASN-PHE-PHE-TRP I l CYS-SER-THR-PHE-THR-LYS SMS 201-995 dPHE-CfS-PHE-dTRP-LYS-THR-CYS-THR (Albert! et a l , 1973). In the canine pancreas, a potent suppression of both phases of glucose-stimulated insulin release by physiological doses of SS-14 (0.61 nM) was seen. A number of studies have since demonstrated that the inhibitory effects of the peptide can be overcome by the presence of higher concentrations of glucose i i i vivo and i n vitro (Efendic et a l , 1978). In the perfused rat pancreas, pharmacological doses of SS-14 (> 30 nM) were required to inhibit the insulin response to 16.5 mM glucose (Curry et a l , 1974). In man, the glucose-insulin dose response curve was shifted to the right i n the presence of SS-14, and high glucose loads overcame the inhibitory action of SS-14 (Efendic et a l , 1976b). From these studies, a competitive inhibition of glucose-induced insulin release by SS-14 was suggested (Efendic et a l , 1978). However, i t has since been shown that the stimulatory action of glucose was probably not mediated by a glucoreceptor on the B-cell membrane (Malaisse et a l , 1979), indicating that competition between glucose and SS-14 for B-cell receptor sites was unlikely. In addition, SS-14 has been shown to have no effect on glucose metabolism i n the B-cell (Bent-Hansen et a l , 1979), suggesting that i t s inhibitory effect was not mediated by metabolic changes. Pace (1979) proposed that the mechanism of action of SS-14 on the B-cell involved disruption of the transduction of ions across the B-cell membrane which were required for stimulus-secretion coupling. It was suggested that such a mechanism was attractive considering the general inhibitory action of SS-14 on a wide variety of secretory c e l l s . This was supported by experiments that showed that SS-14 increased the permeability of the B-cell membrane to potassium (Pace and Tarvin, 1981), decreased i t s permeability to calcium (Oliver, 1976), and inhibited glucose-induced electrical activity (Pace et a l , 1977) and cAMP accumulation (Bent-Hansen et a l , 1979) i n the i s l e t s . In addition, Curry and Bennett (1976) demonstrated that the inhibition of glucose-induced insulin release by SS-14, i n the perfused rat pancreas, could be reversed by elevating the calcium concentration of the perfusate. Somatostatin-14 has been shown to inhibit the effects of a number of different insulin secretagogues in addition to glucose. Pederson et al (1975c) demonstrated that intravenous administration of the peptide i n conscious dogs inhibited the insulinotropic action of GIP. Similarly, a strong suppression of GIP-stimulated insulin release by SS-14 (6.1 nM) was observed in the perfused rat pancreas (Takemura et a l , 1986). Arginine- and glucagon-stimulated insulin secretion were also inhibited by SS-14 i n both man and perfused rat pancreas (Efendic et a l , 1976a). A role for SS-14 i n the regulation of vagally mediated insulin release was suggested by Bloom et a l (1980), who showed that the insulin response to 2-deoxyglucose i n the conscious, splanchnectomized calf was abolished by intravenous infusion of SS-14. The main source of SS-14 acting on the B-cell i n vivo has not yet been determined. As was previously discussed, the close anatomical relationship between D-cells and B-cells seen i n the i s l e t suggested that pancreatic SS-14 influenced B-cell secretion via a paracrine mechanism, although the physiological evidence for such a mechanism has been equivocal (Samols et a l , 1986). In addition, a true hormonal role for gastrointestinal SS-14 i n modulating postprandial insulin secretion has been implicated i n man (0'Shaughnessy et a l , 1985) and i n dogs (Schusdziarra et a l , 1980). Thus, possible physiological roles of both pancreatic and gastrointestinal SS-14 i n the control of the B-cell have been demonstrated. Brazeau et al (1973) f i r s t suggested that the inhibitory effect of SS-14 on growth hormone secretion had possible c l i n i c a l applications i n the treatment of acromegaly. However, the short h a l f - l i f e of the peptide, coupled with i t s wide array of physiological effects seen at pharmacological doses, limited i t s usefulness as a therapeutic agent. In attempting to make a drug that was longer-lasting, more specific i n i t s effects on growth hormone, and more potent than SS-14, a number of laboratories produced synthetic analogs of the native hormone. Veber et al (1978) had suggested that the tetrapeptide fragment of structure Phe-Trp-Lys-Thr was the essential pharmacophore of somatostatin. Starting from this tetrapeptide sequence, cyclized into a weakly active hexapeptide ring by the addition of cystine at each terminus, Bauer et al (1982) systematically designed a number of analogs and tested them for their influence on the secretion of growth hormone, insulin, and glucagon. One octapeptide analog (Figure 1), named synthetic mini-somatostatin (SMS; SMS 201-995; Sandostatiri^) was found to be at least 20 times more potent jji vivo than the native hormone i n i t s inhibition of growth hormone secretion, and 3 times more potent i n i t s inhibition of insulin secretion (Bauer et a l , 1982). This increased specificity for the inhibition of growth hormone was accompanied by considerably greater sta b i l i t y , with a h a l f - l i f e about 30 times longer than SS-14 i n man, whether administered subcutaneously or intravenously (Kutz et a l , 1986). Cl i n i c a l t r i a l s with SMS have demonstrated i t s potential usefulness i n the treatment of acromegaly (Vance et a l , 1986), gastrointestinal APUDomas (Buchanan et a l , 1986), and i n reducing the postprandial hyperglycemia seen i n insulin-dependent diabetics (Serrano Rios et a l , 1986). In the rat, SMS was 3 times more potent than SS-14 i n inhibiting the insulin response to a glucose bolus, i f both drugs were administered intramuscularly. When injected intravenously, i n the monkey, SMS was 1.3 times more potent than SS-14 i n the inhibition of basal insulin levels (Bauer et a l , 1982). In both experiments, because insulin levels were measured 15 min after administration of SMS or SS-14, the increased potency observed for SMS could have been due to i t s longer h a l f - l i f e i n the circulation, rather than a more potent direct effect of the analog on the B-cell. In vitro assessment of the relative potencies of SMS and SS-14 on the B-cell, using the perfused rat pancreas, has indicated that the analog i s less effective than the native hormone i n inhibiting insulin secretion (Takemura et a l , 1986). In addition, the in a b i l i t y of the analog to inhibit GIP-stimulated insulin release suggested that i t may have acted on the B-cell via a different mechanism than SS-14 (Takemura et a l , 1986). These studies have therefore warranted further comparisons of the inhibitory actions of SMS and SS-14. Thus, the objective of the present experiments was to examine some of the possible interactions between the neurotransmitters, hormones, and nutrients thought to be influencing the B-cell during meal ingestion. Specifically, the effects of glucose, GIP, and SS-14 on ACh-stimulated insulin secretion were studied. 22 GENERAL METHODOLOGY I. Isolated Perfused Rat Pancreas The isolated, vascularly perfused pancreas was used to test the effects of various agents on the secretion of insulin in. vitro. A. Apparatus The apparatus used i n these studies i s shown i n Figure 2. Perfusate was stirred i n flasks that were continuously gassed with a water-saturated 95 % 0^ / 5 % C0 2 mixture to maintain a pH of 7.4. The perfusate was administered at a constant flow rate of either 2 ml/min (Results, sections I-III) or 3 ml/min (Results, sections IV and V) by a peristaltic pump (No. 7553-30; Cole-Parmer Instrument Co., Chicago, 111.). The perfusate was heated to 37.0 °C i n coiled tubi ng whi ch wrapped around a servo-controlled heating block. A temperature probe was used to ensure that the correct temperature was maintained. The perfusate was screened by a fine mesh f i l t e r and air bubbles were removed by a bubble trap before entering the gland. The perfused organ was maintained at a constant temperature of 37.0 - 37.5 °C by the heating block and a 60 watt desk lamp. The entire preparation was covered with plastic wrap to protect the organ and prevent fluctuations i n temperature due to circulating a i r . Venous effluent was collected i n chilled test tubes i n an automated fraction collector. The apparatus allowed the administration of physiological or pharmacological agents at desired concentrations i n two ways. F i r s t , two parallel perfusate circulation systems were employed. Perfusate of 23 Figure 2: APPARATUS FOR THE VASCULAR PERFUSION OF THE RAT PANCREAS. Perfusate was administered from 1.0 1 flasks (a) through the heating c o i l (b), bubble trap, f i l t e r (c), and temperature probe (d) by a peristaltic pump (e). The pancreas (not shown) was heated from below by a heating block (f) and from above by a desk lamp (g). Venous effluent was collected i n chilled tubes i n a fraction collector (not shown). different glucose concentrations from two flasks was circulated independently. While the perfusate from one flask passed through the pancreas, the perfusate from the other flask was recirculated. A change in glucose concentration was then achieved by rapidly interchanging the two systems at the level of the bubble trap, such that perfusate from the second flask passed through the pancreas and the f i r s t flask was then recirculated. Other agents were administered by sidearm infusion from an infusion pump (Model 940; Harvard Apparatus Co. Inc., MiHis, Mass.). These substances were pumped from 10 ml syringes, via polyethylene tubing (PE90; Clay Adams, Parsippany, N.J.), into a rubber bulb at the level of the arterial cannula to achieve the desired f i n a l concentrati on. B. Animals Male Wistar rats (obtained from Animal Care, U.B.C.) weighing 250 - 350 g were used i n a l l experiments. The animals were housed i n metal cages ( 5 - 6 rats/cage) i n a light-controlled room (12 h cycle) with access to laboratory rat food and water. The animals were fasted overnight prior to experimentation (16 - 24 h). C. Surgical Procedure Isolation of the pancreas for vascular perfusion was achieved by a method modified from Penhos et a l (1969). Animals were f i r s t anesthetized with 60 mg/kg sodium pentobarbital (Somnotol®) administered intraperitoneally. The abdomen was opened with a midline incision from the pelvis to the sternum. The abdominal aorta and inferior vena cava were located and the associated connective tissue was cleared around the celiac junction. After careful separation of the two vessels, two loose l i g a t u r e s were placed around the abdominal aorta i n f e r i o r to the superior mesenteric artery (Figure 3 A ) . A t h i r d loose l i g a t u r e was placed around the aorta just below the diaphragm. Next, the righ t and l e f t renal a r t e r i e s and veins were t i e d . A f t e r d i s s e c t i o n of the connective tissue around the spleen, the vasculature between the spleen and pancreas was l i g a t e d and the spleen was removed. The majority of the gut was then i s o l a t e d and removed i n the following order. F i r s t , the descending colon was cut between double l i g a t u r e s . A drainage tube was i n s e r t e d i n t o the duodenum, d i s t a l to the l e v e l of the pancreas and adjacent to the ligament of T r e i t z . The mesenteric arcades were then t i e d , with three l i g a t u r e s , from the drainage tube to the cecum. Next, the colon was l i g a t e d caudal to the cecum. This i s o l a t e d section of gut was then removed leaving only the rectum and a small remnant of duodenum attached to the pancreas. The vascular connections between the stomach and pancreas were then doubly-ligated and cut, taking care not to damage the pancreatic t i s s u e . The pylorus and esophagus, i n c l u d i n g the l e f t g a s t r i c artery, were also sectioned between double l i g a t u r e s . A f t e r d i s s e c t i n g the remaining connective t i s s u e , the stomach was removed. The i n f e r i o r vena cava near the r i g h t adrenal gland was then i s o l a t e d with a simple loose l i g a t u r e . In preparation for cannulation, one loose l i g a t u r e was placed around the p o r t a l vein where i t exits the pancreas, while a second loose l i g a t u r e was placed c l o s e r to the l i v e r which encompassed the p o r t a l vein, b i l e duct, and associated vasculature as i l l u s t r a t e d i n Figure 3B. The aorta was clamped below the superior mesenteric artery to f a c i l i t a t e cannulation of the abdominal aorta without i n t e r r u p t i o n of a r t e r i a l blood flow to the pancreas. The 27 Figure 3: SCHEMATIC DIAGRAM SHOWING THE PLACEMENT OF CANNULAE AND ASSOCIATED LIGATURES IN THE PERFUSED RAT PANCREAS. A. The arterial cannula was secured i n the abdominal aorta by two ligatures and the aorta was ligated just below the diaphragm, so that perfusate was directed to the pancreas via the superior mesenteric artery (S.M.A.) and celiac artery (C.A.). B. The venous cannula was secured i n the portal vein (P.V.) by two ligatures and the inferior vena cava (I.V.C.) was ligated near the right adrenal gland and kidney. abdominal aorta was then cannulated with polyethylene tubing (PE160) to the level of the clamp. The clamp was removed and the cannula was inserted further to a level just inferior to the superior mesenteric artery, where i t was secured with the two ligatures (Figure 3A). The loose aortic ligature near the diaphragm was tied to direct the flow of perfusate through the celiac and superior mesenteric arteries. This maneuver interrupted the blood flow to the pancreas, therefore, the remaining surgical procedure was accomplished i n less than 90 s to minimize the period of anoxia i n the organ. Two m i l l i l i t e r s of heparin (600 U) were introduced via the arterial cannula. After dissecting the connective tissue between the liver and the diaphragm, the animal was sectioned at the level of the diaphragm and the upper half of the animal was discarded. The loose ligatures on the inferior vena cava and on the portal vein nearest the liver were tied. An incision was made in the portal vein adjacent to this ligature, and a cannula (PE160 tubing) was inserted towards the pancreas. This cannula was secured with the remaining portal vein l i gature. The arterial cannula was connected to the perfusion appparatus shown i n Figure 2, and the flow of perfusate was started. Care was taken to avoid the introduction of air bubbles into the system. Perfusion of the pancreas was achieved via the superior mesenteric and celiac arteries. Total venous effluent was collected from the portal vein cannula. Any leak i n the preparation was immediately halted by ligatures or clamps. The preparation was equilibrated for 20 - 30 min before collection of samples to minimize the effects of surgical trauma. During this 30 equilibration period, minute adjustments to the heating unit and perfusion pump were made, i f necessary, to ensure a stable perfusate temperature and flow rate. Samples were collected every minute and stored at -20 °C for radioimmunoassay. Those samples to be assayed for somatostatin were stored as 500 u l aliquots added to 50 ^ 1 aprotinin (Trasylol ). D. Solutions 1. Perfusate The perfusate was a Krebs' solution containing 3 % dextran ( c l i n i c a l grade) and 0.2 % bovine serum albumin (BSA; RIA grade). The Krebs' solution was prepared from a concentrated stock solution of the following composition: 285 ml KCl (154 mM) 243 ml CaCl 2 (102.7 mM) 78 ml MgS04'7H20 (154 mM) 97 ml KH2P04 (154 mM) Prior to experiments, dextran and BSA were dissolved i n saline (154 mM) overnight at 4 °C. On the morning of the experiment, Krebs' concentrate and sodium bicarbonate (6.5 g dissolved i n 500 ml H20) were added i n appropriate volumes to obtain the desired f i n a l concentrations i n the perfusate. The solution was made up to i t s f i n a l volume by the addition of saline and a small amount of concentrated glucose (308 mM). The glucose stock solution was prepared from a commercial 50 % dextrose solution by diluting 55 ml i n 500 ml water and storing at 4 °C. The fi n a l glucose concentration of the perfusate was checked using a Beckman Glucose Analyzer (Beckman Instruments, Inc.; Fullerton, Ca.). Osmolarity of the perfusate was 280 - 285 mOs and was not effected by varying the glucose concentration, as the glucose solution was isotonic. The f i n a l composition of the perfusate was as follows: KCl 4.4 mM CaC_2 2.5 mM MgSOA'7H20 1.2 mM KH2P04 1.5 mM NaHC03 25 mM Dextran 3.0 % BSA 0.2 % NaCl 120 mM Glucose 2.2, 4.4, 6.6, 8.9, or 17.8 mM as desired. 2. Drugs and Peptides A l l drugs and peptides were prepared as concentrated stock solutions on the day of the experiment. Peptides were dissolved i n siliconized test tubes i n 0.01 M acetic acid and 0.2 % BSA. Other agents were dissolved i n d i s t i l l e d water. The concentrations of the stock solutions were calculated after consideration of the perfusion flow rate (2 or 3 ml/min) and the drug infusion rate (0.103 ml/min), so that a f i n a l dilution of 10 pi stock solution i n 1 ml perfusate gave the desired concentration of drug to be delivered to the pancreas. Gastric inhibitory polypeptide was purified by the method of Brown (1969; Brown et a l , 1970), dissolved i n 0.01 M acetic acid and 0.2 % BSA, and stored as 1 pg lyophilized fractions at -20 °C. A l l other agents were obtained commercially from various sources (see Appendix to Methods) and weighed out fresh on the day of the experiment. 32 I I . Peptide Q u a n t i f i c a t i o n by Radioimmunoassay In the present studies, concentrations of radioimmunoassayable i n s u l i n (immunoreactive i n s u l i n ; IRI) and somatostatin (somatostatin-l i k e immunoreactivity; SLI) were determined by s e n s i t i v e 125 radioimmunoassays using s p e c i f i c antisera and I- peptide as l a b e l l e d antigen, as described below. A. Inguljtn, 1. Antiserum Unconjugated porcine i n s u l i n emulsified i n Freund's complete adjuvant had been previously used to r a i s e i n s u l i n antiserum i n guinea pigs. This antiserum (GP01) was o r i g i n a l l y d i l u t e d 1:10 and stored as 0.1 ml l y o p h i l i z e d a l i q u o t s . These were reconstituted i n i n s u l i n assay buffer to achieve a d i l u t i o n of 1:5000 and stored as 1 ml aliquots at -20 °C. On the day of the assay, the antiserum was further d i l u t e d 1:20 i n assay buffer for a f i n a l d i l u t i o n of 1:10^. At t h i s d i l u t i o n , the 125 antiserum consistently bound 45 - 55 % of I- l a b e l l e d porcine i n s u l i n i n the absence of unlabelled i n s u l i n . In a d d i t i o n , i t was s e n s i t i v e to porcine i n s u l i n standards and rat i n s u l i n i n perfusion samples i n the range of 5 - 160 juU/ml. 2. lodjnation 125 I - i n s u l i n was prepared fresh every s i x weeks or as needed, (a) Solutions Unless otherwise ind i c a t e d , a l l solutions were prepared just p r i o r to the i o d i n a t i o n procedure. ( i ) 0.4 M Phosphate buffer, pH 7.5 - 0.4 M Na 2HP0 4 ( a c i d i c ; 11.04 g i n 200 ml H 20) was added slowly to 0.4 M NaH 2P0 4 (basic; 57.58 g i n 1000 ml H 20) to a t t a i n pH 7.5; stored at 4 °C for several weeks. ( i i ) 0.04 M and 0.2 M Phosphate buffer, pH 7.5 - made from appropriate d i l u t i o n of 0.4 M phosphate buffer. ( i i i ) Acid ethanol - 1500 ml 95% ethanol, 500 ml H 20, 30 ml concentrated HCl; stored at 4 °C for several weeks. (i v ) Chloramine T - 4 mg/ml i n 0.2 M phosphate buffer, pH 7.5. (v) Sodium met a b i s u l f i t e - 2.4 mg/ml i n 0.2 M phosphate buffer, pH 7.5. ( v i ) Potassium iodide -10 mg/ml i n 0.2 M phosphate buffer, pH 7.5. ( v i i ) I n s u l i n - 10 - 50 ug of porcine i n s u l i n dissolved i n 10 pi of 0.01 M HCl then brought to a f i n a l concentration of 5 ug/10 p i with 0.2 M phosphate buffer, pH 7.5. (b) Procedure S i l i c o n i z e d glassware was used throughout, and the entire procedure was carried out at room temperature. F i r s t , 10 pi each of 125 i n s u l i n , 0.2 M phosphate buffer, and Na I (1 mCi) were gently mixed i n the reaction v e s s e l . Next, 25 pi of chloramine T was added for 10 s. Sodium metabisulfite (100 pi) was then added and mixed gently for 45 s. F i n a l l y , 50 pi of potassium iodide was added and the reaction was further d i l u t e d with 1.8 ml of 0.04 M phosphate buffer. The I-insulin was then adsorbed onto 10 mg of microfine s i l i c a (QUSO G-32). The mixture was vortexed thoroughly and centrifuged for 30 125 s. The supernatant (containing free I) was discarded, and the pellet was washed twice with 3.0 ml of d i s t i l l e d water and recentrifuged. 125 I-insulin was eluted from the s i l i c a with 3 ml of acid ethanol, added to a s c i n t i l l a t i o n v i a l containing 2 ml acid ethanol and 1.5 ml X 2 3 d i s t i l l e d water, and stored at -20 °C. Incorporation of I by this method was usually 40 - 50 %. This was determined by counting the s i l i c a pellet, and 10 ^1 aliquots of both the vortexed QUSO mixture 125 prior to centrifugation (total counts) and the I-insulin i n acid ethanol by the following formula (C = counts per 0.01 min, corrected for volume): % Incorporation = C, . N + Q, (acid ethanol) (pellet) C ( t o t a l ) 125 For use i n the assay, the dilute I-insulin i n acid ethanol was further diluted i n assay buffer so that a 100 yx\ fraction contained approximately 10000 cpm. 3. Assay buffer Diluent buffer used i n the insulin RIA was 0.04 M phosphate buffer containing 5 % charcoal extracted plasma. Phosphate buffer was diluted from 0.4 M phosphate buffer stock solution, pH 7.5, described earlier. Charcoal extracted human plasma was prepared from outdated blood bank plasma (Red Cross, Vancouver, B.C.) as follows. Erythrocytes were f i r s t separated from the plasma by centrifugation. The supernatant was then filtered through 15 cm sharkskin f i l t e r paper (Schleicher and Schuell, Inc., Keene, N.H.). One percent activated charcoal (Carbon Decolorizing Neutral; Norit) was added to the f i l t r a t e . After continuous stirring for 1 h at 4 °C, the mixture was centrifuged at 10000 rpm for 30 min. The supernatant was fi l t e r e d u n t i l charcoal was no longer visible i n the f i l t r a t e (at least twice). The charcoal extracted plasma was then aliquoted into 10 ml fractions and stored at -20 °C until use. 4. Standards Lyophilized rat insulin (21.3 U/mg; Novo) was dissolved i n diluent buffer to achieve a concentration of 4260 uU/ml and was stored as 1 ml fractions at -20 °C. When required, these fractions were further diluted (1 ml:26.6 ml) in diluent buffer to give a concentration of 160 jiU/ml, aliquoted into 1 or 2 ml fractions, and stored at -20 °C. The 160 pU/ml standards were serially diluted i n assay buffer to produce standard concentrations of 160, 80, 40, 20, 10, 5, and 2.5^uU/ml for use in the assay. 5. Controls Rat insulin controls were obtained from a perfused rat pancreas (Methods, Section I) stimulated with 17.8 mM glucose and 10 mM arginine for 30 min. Total venous effluent collected during this period was pooled and insulin content was determined by RIA. The effluent was then diluted i n an appropriate volume of assay buffer to achieve a concentration of 60 uU/ml, and stored as 1 or 2 ml aliquots at -20 °C. Inter- and intra-assay variation of the control values was 5.2 and 4.2 %, respectively. 6. Assay protocol The contents of the various assay tubes (in microliters) were as follows: Tube Diluent buffer Sample Antiserum I-insulin sample or standard 700 100 100 100 zero binding 800 - 100 100 non-specific binding standard 900 - - 100 sample or control 800 100 100 100 Standards, control, non-specific binding (NSB), and zero-binding tubes were assayed i n tri p l i c a t e ; samples were assayed i n duplicate. Diluent buffer, sample and antiserum were f i r s t mixed i n assay 123 tubes and Incubated for 24 h at 4 °C. I-insulin was then added, and three tubes containing 100 jjl of the label were prepared as total 125 counts. After a further 24 h incubation, the bound and free I-insulin were separated using dextran-coated charcoal. The charcoal was prepared by adding 5.0 g dextran T-70 and 50.0 g activated charcoal to 1.0 1 of 0.04 M phosphate buffer, pH 7.5, and stirred overnight at 4 °C. Two hundred microliters of charcoal was added to each tube. The mixture was vortexed and centrifuged (3000 rpm for 30 min), the supernatant discarded, and the free iodinated hormone adsorbed to the pellet was counted for 2 min i n a gamma spectrometer (Searle Model 1285). 7. Calculations 125 The percent of I-insulin bound (%B) in each tube was calculated by the following formula: 37 % B ( C t o t a l " Csample) " ( C t o t a l " CNSB ) c c t o t a l t o t a l where C = counts per minute. A computer program was used to produce a l o g - l o g i t p l o t of %B v s . standard concentrations (uU/ml) and determine the i n s u l i n concentration of the unknown samples and c o n t r o l s u s i n g the c a l c u l a t e d %B. Concentration of i n s u l i n i n each p e r f u s i o n sample was m u l t i p l i e d by the volume of sample c o l l e c t e d (ml/min) to c o r r e c t f o r changes i n flow r a t e . Where i n s u l i n c o ncentration i s presented as the i n t e g r a t e d output, the f o l l o w i n g formula was used: X T = 0.5*(X t Q + X t l ) * ( t l - tO) + 0.5*(X t l + X t 2 ) * ( t 2 - t l ) + ... + °- 5* ( Xt(n) + X t ( n - 1 ) > where t = time X T = i n t e g r a t e d ouput of i n s u l i n f o r time period tQ to t ^ . X = concentration at time t . B. Somatostatin The assay procedure was performed according to the method of Mcintosh et a l (1978), w i t h the exception of the antibody used. 1. Antiserum A monoclonal antibody r a i s e d to c y c l i c somatostatin-14, SOMA 03 (Buchan et a l , 1985), was used i n the assay. Crude a s c i t e s f l u i d from mice, i n j e c t e d w i t h hybridoma c e l l s that produced SOMA 03, was stored at -20 °C. When re q u i r e d , t h i s was thawed, f i l t e r e d through s t e r i l e f i l t e r s (0.45 ;um; M i l l i p o r e Corp., Bedford, Mass.), d i l u t e d 1:1 i n a s o l u t i o n of 0.9 % s a l i n e , 0.5 % sodium a z i d e , and 0.1 % BSA, and stored as 20 ; i l a l i q u o t s at -70 °C. The antibody was used at a f i n a l d i l u t i o n '38 of 1.4:4.0(10") i n assay buffer, and was shown to be s e n s i t i v e to l e v e l s -18 of somatostatin ranging from 180 - 300 amol (10 ). In addition, SLI i n samples of i s o l a t e d perfused stomach e f f l u e n t , measured by both SOMA 03 ascit e s and conventional rabbit antiserum 26.3.4 (Mcintosh et a l , 1978), were shown to be equivalent (Mcintosh et a l , 1987). 2. Iodinatlon (a) Solutions ( i ) 0.5 M Phosphate buffer, pH 7.5 - 1.0 M KOH was added slowly to 0.5 M NaH^PO^ to pH 7.5. ( i i ) 0.05 M Phosphate buffer - 0.5 M Phosphate buffer, pH 7.5, d i l u t e d 1:10 i n d i s t i l l e d H 2o. ( i i i ) Chloramine T - 2 mg/ml i n 0.05 M phosphate buffer ( i v ) Sodium met a b i s u l f i t e - 5 mg/ml i n 0.05 M phosphate buffer (v) A c e t i c acid / acetone - 100 u l g l a c i a l a c e t i c a c i d , 3.9 ml acetone, 4 ml d i s t i l l e d EL^O. (b) Procedure Synthetic Tyr^-somatostatin (5 jug) was dissolved i n 10 pi d i s t i l l e d water. Ten m i c r o l i t e r s of 0.5 M phosphate buffer (pH 7.5), 10 125 pi (1 mCi) of Na I, and 10 j _ l of chloramine T were added and mixed gently. A f t e r 30 s, the reaction was terminated by the addition of 10 u l of sodium me t a b i s u l f i t e . One m i l l i l i t e r of charcoal extracted plasma and 20 mg of microfine s i l i c a (QUSO G-32) were then added, and the mixture was vortexed and centrifuged f o r 3 min. The supernatant was discarded and the p e l l e t was washed twice with d i s t i l l e d water (1 ml) and c e n t r i f u g e d . I-somatostatin was eluted from the p e l l e t w i t h 1 ml a c e t i c a c i d / acetone, and was d i l u t e d to 500 000 cpm / 10 pi w i t h 0.1 M a c e t i c a c i d c o n t a i n i n g 0.5 % BSA. The d i l u t e l a b e l was stored as 100 pi l y o p h i l i z e d a l i q u o t s at -20 °C. These a l i q u o t s were r e c o n s t i t u t e d on the day of the assay i n 0.002 M ammonium acetate, pH 4.6, and then p u r i f i e d on a CM-cellulose column (CM 52, 0.7 x 8 cm; Whatman L t d . , Maidstone, England) that had been 125 e q u i l i b r a t e d i n the same b u f f e r . The I-somatostatin was el u t e d w i t h 0.2 M ammonium acet a t e , pH 4.6, pumped through the column at a rate of 1 ml/min. Two minute f r a c t i o n s of the elu a t e were c o l l e c t e d and the r a d i o a c t i v i t y was counted. Peak f r a c t i o n s were pooled, n e u t r a l i z e d w i t h 2 M NaOH and d i l u t e d i n assay b u f f e r to 3000 cpm / 10 pi. 3. Assay b u f f e r A stock s o l u t i o n of b u f f e r was prepared by d i s s o l v i n g 4 .9 g sodium b a r b i t a l , 0.32 g sodium a c e t a t e , 2.55 g sodium c h l o r i d e , and 0.10 g me r t h i o l a t e i n 700 ml of d i s t i l l e d water, a d j u s t i n g to pH 7.4 w i t h 1.0 M HCl, and adding d i s t i l l e d water f o r a f i n a l volume of 1.0 1. This was stored at 4 °C and d i l u t e d 1:10 i n d i s t i l l e d water f o r use i n the assay, w i t h the a d d i t i o n of 0.5 % BSA and 500 KlU/ml a p r o t i n i n . 4. Standards Syn t h e t i c c y c l i c somatostatin was d i s s o l v e d i n 0.1 M a c e t i c a c i d c o n t a i n i n g 0.05 % BSA and a l i quoted as 50 pi (10 pg) f r a c t i o n s , l y o p h i l i z e d , and stored at -20 °C. On the day of the assay, a 10 pg a l i q u o t was d i s s o l v e d i n 1.0 ml assay b u f f e r and was s e r i a l l y d i l u t e d to obtai n standards of 250, 125, 62.5, 31.25, 15.625, 7.8, and 3 .9 pg/ml. 5. Assay p r o t o c o l Each assay tube contained 100 pl d i l u e n t b u f f e r , 100 pl standard 125 1 or sample, 100 pl antiserum, and 100 pl I - t y r -somatostatin. A f t e r a 72 h i n c u b a t i o n at 4 °C, bound and f r e e l a b e l l e d somatostatin were separated using dextran-coated c h a r c o a l . The charcoal was prepared by s t i r r i n g 0.25 % dextran T-70, 1.0 % charcoal e x t r a c t e d plasma, and 1.25 % a c t i v a t e d c harcoal i n 0.05 M phosphate b u f f e r pH 7.5, f o r 1 h. Each assay tube received 1 ml of t h i s mixture, and was vortexed and ce n t r i f u g e d at 3000 rpm f o r 30 min. The supernatant was discarded and the d r i e d p e l l e t counted f o r 3 min by a gamma spectrometer ( S e a r l e Model 1285). 6. C a l c u l a t i o n s 125 Per cent of bound I-somatostatin was c a l c u l a t e d as f o r i n s u l i n . Standard curves were prepared on semi-logarithmic graph paper, and the concentration of SLI was read from these curves. I I I . S t a t i B t l c a l A n a l v s i s In a l l experiments, the mean i n t e g r a t e d output of IRI was c a l c u l a t e d f o r the period during which a t e s t substance or a c o n t r o l v e h i c l e was i n f u s e d . A computer program s t a t i s t i c a l l y compared the c o n t r o l and t e s t groups using the Mann-Whitney t e s t . In experiments i n which SLI was measured, the Wilcoxon signed-rank t e s t was used to compare the mean SLI l e v e l s during i n f u s i o n of a t e s t substance to the mean p r e - i n f u s i o n l e v e l s . S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s (p<0.05) are represented by an a s t e r i s k (*) on f i g u r e s . 41 APPENDIX TO METHODS - CHEMICAL SOURCES CHEMICAL GRADEj SOURCE A r i s t a r RIA C l i n i c a l A c e t i c a c i d Acetone A c e t y l c h o l i n e c h l o r i d e Ammonium acetate _ A p r o t i n i n (Trasylol®) Atropine s u l f a t e B a r b i t a l sodium C-IV Bovine serum albumin Calcium c h l o r i d e Carbon d e c o l o r i z i n g n e u t r a l ( N o r i t ; a c t i v a t e d charcoal) Chloramine T CM-cellulose Dextran Dextran T-70 E t h y l m e r c u r i t h i o s a l i c y l i c a c i d sodium s a l t ( m e r t h i o l a t e ) Ethanol (95%) Freund's complete adjuvant Glucose (dextrose) C l i n i c a l Heparin sodium Hexamethonlum bromide I n s u l i n (porcine) I n s u l i n ( r a t ) Magnesium s u l f a t e _ Phentolamine hydrochloride ( R e g i t i n e HCl®) Potassium c h l o r i d e Potassium i o d i d e Potassium phosphate (monobasic) d - l - P r o p r a n o l o l hydrochloride QUSO m i c r o f i n e s i l i c a , G-32 Sodium acetate Sodium azide Sodium bicarbonate Sodium c h l o r i d e Sodium 1 2 5 I o d i d e ( i n NaOH) Sodium m e t a b i s u l f i t e Sodium p e n t o b a r b i t a l (Somnoto Sodium phosphate ( d i b a s i c ) Sodium phosphate (monobasic) Somatostatin-14 ( c y c l i c SMS1201-995 (Sandostatiri 0') Tyr -somatostatin BDH BDH Sigma Baker M i l e s Sigma F i s h e r Sigma F i s h e r F i s h e r F i s h e r Eastman Kodak Whatman Sigma Pharmacia Eastman Kodak Standard Sigma Abbott F i s h e r S i gma Novo Novo F i s h e r CIBA F i s h e r N i c h o l s F i s h e r Sigma P h i l a d e l p h i a Quartz F i s h e r Baker F i s h e r F i s h e r Amersham F i s h e r MTC F i s h e r F i s h e r P eninsula Sandoz Peninsula *Unless otherwise i n d i c a t e d , chemicals were reagent grade. 42 RESULTS I . E f f e c t of Glucose on ACh-stimulated IRI S e c r e t i o n The f i r s t s e r i e s of experiments examined the e f f e c t of varying the glucose concentration of the perfusate on the IRI response to 1.0 pM ACh. I n these experiments, ACh was i n f u s e d i n the presence of 2.2, 4.4, 6.6, 8.9, or 17.8 mM glucose. The data i s presented as the i n t e g r a t e d IRI output (mean ± S.E.M.) during the 10 min p e r i o d 15 - 25 min f o l l o w i n g the i n t r o d u c t i o n of ACh, or during the same 10 min period i n the absence of ACh i n c o n t r o l animals. Figure 4 shows the e f f e c t of i n f u s i o n of 1.0 ^iM a c e t y l c h o l i n e (ACh) on immunoreactive i n s u l i n ( IRI) s e c r e t i o n i n the presence of 4.4 mM glucose. I n the c o n t r o l animals, t h i s concentration of glucose had no e f f e c t on IRI s e c r e t i o n . However, i n t r o d u c t i o n of ACh produced a b i p h a s i c s t i m u l a t i o n of IRI r e l e a s e , c o n s i s t i n g of an i n i t i a l peak followed by a s l o w l y r i s i n g second phase. The i n t e g r a t e d IRI output during i n f u s i o n of 1.0 j^iM ACh (1251 ± 179 /iU/10 min), was approximately 4.5 times greater than the c o n t r o l value, i n the absence of ACh (286 ± 30 ^U/10 min). When the glucose concentration of the perfusate was increased from 4.4 mM to 8.9 mM ( F i g u r e 5 ) , a b i p h a s i c s t i m u l a t i o n of IRI s e c r e t i o n was observed. I n f u s i o n of 1.0 pM ACh, 10 min a f t e r changing the glucose concentration to 8.9 mM, produced a f u r t h e r b i p h a s i c response. The i n t e g r a t e d IRI ouput during ACh i n f u s i o n (6429 ± 964 p\]/l0 min) was approximately 5 times the c o n t r o l l e v e l (1222 ± 213 uU/10 min). The e f f e c t of varying the glucose concentration of the perfusate on 1.0 uM Ach-stimulated IRI release i s summarized i n Figure 6. In the presence of 2.2 mM glucose, ACh had no s i g n i f i c a n t e f f e c t (167 i 8 vs. 182 + 20 pU/10 min). The potent IRI response to ACh observed i n the presence of 4.4 and 8.9 mM glucose (Figures 4 and 5, respectively) was also seen when the perfusate glucose concentration was 6.6 mM, with IRI secretion increased from a con t r o l l e v e l of 417 ± 65 jaU/10 min to 2511 ± 350 juU/10 min during ACh i n f u s i o n . In the presence of 17.8 mM glucose, the e f f e c t of ACh i n f u s i o n was reduced compared to the lower glucose concentrations. Mean integrated IRI output was s i g n i f i c a n t l y increased from a control value of 11227 ± 1873 ;uU/10 min In the absence of ACh to 15184 ± 2512 juU/10 min during Ach administration. 44 Figure 4: THE EFFECT OF ACH ON IRI SECRETION IN THE PRESENCE OF 4.4 mM GLUCOSE. ACh (1.0 JJM) was i n f u s e d during minutes 5-40. Each point represents the mean IRI co n c e n t r a t i o n (;uU/min). E r r o r bars represent the S.E.M.. Closed t r i a n g l e s : 4.4 mM glucose alone, (n=5); open t r i a n g l e s : 4.4 mM glucose + 1.0 pM ACh (n=8). 46 Figure 5: THE EFFECT OF ACH ON IRI SECRETION IN THE PRESENCE OF 8.9 mM GLUCOSE. The glucose concentration was in c r e a s e d from 4.4 to 8.9 mM at minute 5. ACh (1.0 pM) was i n f u s e d during minutes 15-40. Each point represents the mean IRI conc e n t r a t i o n (^uU/min) . E r r o r bars represent the S.E.M.. Closed t r i a n g l e s : 8.9 mM glucose alone, (n=6); open t r i a n g l e s : 8.9 mM glucose + 1.0 juM ACh, (n=6). 48 Figure 6: SUMMARY OF THE EFFECTS OF THE GLUCOSE CONCENTRATION ON THE IRI RESPONSE TO ACH. Pe r f u s i o n s were performed at glucose concentrations ranging from 2.2 - 17.8 mM i n the presence ( c l o s e d bars) or absence (open bars) of 1.0 ^_M ACh. The data represents the mean (+ S.E.M.) i n t e g r a t e d IRI output (mU/10 min) f o r the 10 min period 15-25 min f o l l o w i n g the i n t r o d u c t i o n of ACh. In a l l cases n=6, except 4.4 mM glucose alone (n=5), and 4.4 mM glucose + ACh (n=8). *Denotes s i g n i f i c a n t d i f f e r e n c e (p<0.05) of glucose + ACh compared to ACh alone. 49 2.2 4 .4 6 .6 8.9 17.8 [G lucose] (mM) I I . E f f e c t of GIP on ACh-stimulated IRI S e c r e t i o n The e f f e c t of i n f u s i o n of 1.0 uM ACh on IRI r e l e a s e i n the presence of GIP (0.2 and 1.0 nM) was s t u d i e d . At a glucose concentration of 8.9 mM, which has been shown to be above the threshold 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 (Pederson and Brown, 1978), both 0.2 nM and 1.0 nM GIP produced a b i p h a s i c s t i m u l a t i o n of IRI r e l e a s e . When 1.0 uM Ach was Infused 10 min a f t e r the i n t r o d u c t i o n of GIP, a second b i p h a s i c s t i m u l a t i o n of IRI r e l e a s e was seen. As shown i n Figure 7, a dramatic IRI response to 1.0 j_M ACh i n f u s i o n was observed i n the presence of 8.9 mM glucose and 1.0 nM GIP. The i n t e g r a t e d IRI output during concomitant i n f u s i o n of 1.0 nM GIP and 1.0 juM Ach (27002 ± 4643 juU/10 min) was s i g n i f i c a n t l y greater than the c o n t r o l value obtained w i t h 1.0 nM GIP alone (8432 ± 1295 pU/10 min), and was 1.7 times greater than the sum of the responses to 1.0 nM GIP and 1.0 jiM ACh when they were i n f u s e d s e p a r a t e l y (Figure 8 ) . I n the presence of 0.2 nM GIP, i n f u s i o n of 1.0 JJM ACh produced a s i g n i f i c a n t i n c r e a s e i n the i n t e g r a t e d IRI output from a c o n t r o l value of 3662 ± 648 pU/10 min i n the absence of ACh to 12091 ± 1742 pV/10 min during ACh a d m i n i s t r a t i o n . The observed IRI response to concomitant i n f u s i o n of 0.2 nM GIP and 1.0 ^iM ACh was not s i g n i f i c a n t l y greater than the sum of the i n d i v i d u a l responses to 0.2 nM GIP and 1.0 pit ACh (Fi g u r e 8 ) . 51 Figure 7: THE EFFECT OF ACH ON IRI SECRETION IN THE PRESENCE OF 8.9 mM GLUCOSE AND 1.0 nM GIP. The glucose c o n c e n t r a t i o n was increased from 4.4 to 8.9 mM at minute 5. GIP (1.0 nM) was i n f u s e d during minutes 15-45 and ACh (1.0 juM) during minutes 25-45. Each po i n t represents the mean IRI concentration (jjU/min). E r r o r bars represent the S.E.M.. Closed t r i a n g l e s : 8.9 mM glucose + 1.0 nM GIP, (n=5); open t r i a n g l e s : 8.9 mM glucose + 1.0 nM GIP + 1.0 >uM ACh. 52 5 0 0 0 n 4 0 0 0 £ 3 0 0 0 -J c t r 2 0 0 0 -1 0 0 0 A 0 8.9 m M GIUCOSG 1.0 nM GIP 1 juM A C h 2 0 3 0 4 0 TimQ (min) G I P + A C h GIP 53 Figure 8: SUMMARY OF THE EFFECTS OF GIP ON THE IRI RESPONSE TO ACH. ACh (1.0 ^ M) was i n f u s e d during minutes 25-45 and GIP (0.2 or 1.0 nM) during minutes 15-45, i n the presence of 8.9 mM glucose. The data represents the mean (+ S.E.M.) i n t e g r a t e d IRI output (mU/10 min) f o r the 10 min period 10-20 min f o l l o w i n g the i n t r o d u c t i o n of ACh. Open bars: 8.9 mM glucose + 1.0 uM ACh (n=6); c l o s e d bars: 8.9 mM glucose + 0.2 or 1.0 nM GIP (n=6); hatched bars: 8.9 mM glucose + 0.2 or 1.0 nM GIP + 1.0 ;uM ACh (n=5). Arrows i n d i c a t e the expected e f f e c t s i f the response to GIP and ACh was a d d i t i v e ( i . e . the sum of the i n d i v i d u a l responses to GIP and ACh). The observed IRI response to ACh + GIP was s i g n i f i c a n t l y greater than the a d d i t i v e response i n the presence of 1.0 nM GIP but not 0.2 nM GIP. *Denotes s i g n i f i c a n t d i f f e r e n c e of ACh + GIP compared to GIP alone. In tegra ted IRI Output ( m U / 1 0 min) (lAirf i) MOV Mu S'O) dIO (IAJu 2 " 0 ) dIO + MOV Md l ) MOV (lr \u o"L) dIO (IrNU O' l ) dIO + MOV o i ro o i t —1 t + o T J + > o + O T J • > o co o _l CO CD 3 §: o c o o CO CD 55 I I I . E f f e c t of Atropine and Hexamethonium on Glucose-, GIP-, and ACh- stimulated IRI S e c r e t i o n Since the preceding r e s u l t s suggested the existence of i n t e r a c t i o n s between glucose, GIP, and ACh at the B - c e l l , the f o l l o w i n g experiments were designed to examine i f the IRI responses to these agents were mediated by c h o l i n e r g i c r e c e p t o r s . E i t h e r the muscarinic b l o c k e r , atropine (1.0 jiM), or the n i c o t i n i c , g a n g l i o n i c b l o c k e r , hexamethonium (100 jiM), was i n f u s e d f o r 10 min during the second phase of IRI release s t i m u l a t e d by one of the f o l l o w i n g : Ach (1.0 jiM), glucose (8.9 mM), or GIP (0.2 or 1.0 nM). The IRI response to 1.0 juM ACh (1411 ± 316 pU/10 min) was p o t e n t l y i n h i b i t e d during i n f u s i o n of 1.0 jaM a t r o p i n e to a l e v e l of 607 ± 102 uU/10 min (Figure 9 ) . Hexamethonium (100 JJM; Figure 10) had a s l i g h t l y l e s s potent i n h i b i t o r y e f f e c t on ACh-stimulated IRI s e c r e t i o n (1411 ± 316 vs. 753 ± 156 p.U/10 min). Both atropine (1821 ± 314 vs. 1764 + 227 juU/10 min) and hexamethonium (1628 ± 301 vs. 1764 ± 227 uU/10 min) were found to be without e f f e c t on 8.9 mM glucose-stimulated IRI r e l e a s e . When atropine was i n f u s e d during s t i m u l a t i o n of IRI s e c r e t i o n by 1.0 nM GIP ( i n the presence of 8.9 mM g l u c o s e ) , a s l i g h t , t r a n s i t o r y suppression of IRI r e l e a s e was apparent (Figure 11); however, the mean i n t e g r a t e d IRI ouputs i n the presence (7419 ± 511 uU/10 min) and absence (7843 ± 261 ;jU/10 min) of atropine were not s i g n i f i c a n t l y d i f f e r e n t . S i m i l a r l y , the IRI response to 0.2 nM GIP was not s i g n i f i c a n t l y a f f e c t e d by atropine i n f u s i o n (3117 ± 356 vs. 2750 ± 363 ^U/10 min). Hexamethonium a l s o had no e f f e c t on 0.2 nM GIP-stimulated IRI s e c r e t i o n 56 (3117 + 356 vs. 3274 ± 348 uU/10 min). These r e s u l t s are summarized i n Figure 12. 57 Figure 9: THE EFFECT OF ATROPINE ON THE IRI RESPONSE TO ACH. Atropine (1.0 j_M) was i n f u s e d during minutes 20-30 f o l l o w i n g i n t r o d u c t i o n of 1.0 uM ACh at minute 5, i n the presence of 4.4 mM glucose. Each point represents the mean IRI conc e n t r a t i o n (uU/min). E r r o r bars represent the S.E.M.. Open t r i a n g l e s : 1.0 j_M ACh (n=8); close d t r i a n g l e s : 1.0 ;_M Ach + 1.0 JJM a t r o p i n e , (n=6). I J J M ACh TimQ (min) 59 Figure 10: THE EFFECT OF HEXAMETHONIUM ON THE IRI RESPONSE TO ACH. Hexamethonlum (100 pM) was i n f u s e d during minutes 20-30 f o l l o w i n g i n t r o d u c t i o n of 1.0 ACh at minute 5, i n the presence of 4.4 mM glucose. Each point represents the mean IRI conce n t r a t i o n 0_U/min). E r r o r bars represent the S.E.M.. Open t r i a n g l e s : 1.0 juM ACh (n=8); closed t r i a n g l e s : 1.0 uM ACh + 100 jiM hexamethonlum, (n=6). IJJM ACh TimG (min) 61 Figure 11: THE EFFECT OF ATROPINE ON THE IRI RESPONSE TO GIP. Atropine (1.0 JJM) was i n f u s e d f o r a 10 min period beginning 10 min a f t e r the i n t r o d u c t i o n of GIP (1.0 nM), i n the presence 8.9 mM glucose. Each point represents the mean IRI c o n c e n t r a t i o n (pU/min). E r r o r bars represent the S.E.M.. Closed t r i a n g l e s : 8.9 mM glucose + 1.0 nM GIP (n=5); open t r i a n g l e s : 8.9 mM glucose + 1.0 nM GIP + 1.0 uM atropine (n=5). 62 GIP (1.0 nM) 8.9 mM Glucose + Atropine Time (min) 63 Figure 12: SUMMARY OF THE EFFECTS OF ATROPINE AND HEXAMETHONIUM ON THE IRI RESPONSE TO ACH, GLUCOSE, AND GIP. Atropine (1.0 /iM) or hexamethonium (100 uM) were infused for a 10 min period beginning 10 or 15 min after the introduction of either ACh (1.0 juM), glucose (8.9 mM) or GIP (0.2 or 1.0 nM) + 8.9 mM glucose. The data represents the mean (+ S.E.M.) integrated IRI output (mU/10 min) for the 10 min period during infusion of either atropine (hatched bars), hexamethonium (closed bars), or a control (open bars). *Denotes significant difference (p<0.05) of atropine or hexamethonium compared to controls. 64 65 IV. E f f e c t of SS-14 and SMS on Glucose-, GIP-, and ACh- stlmulated IRI S e c r e t i o n In t h i s s e r i e s of experiments, the i n h i b i t o r y e f f e c t s of SS-14 and SMS on the IRI response to various s t i m u l i were compared. The concentrations of SS-14 (6.1 nM) and SMS (9.4 nM) used i n these experiments were equivalent by weight (10 ng/ml). The peptides were in f u s e d f o r 10 min during s t i m u l a t i o n of IRI r e l e a s e by glucose (8.9 or 17.8 mM), GIP (2.0 nM), or ACh (1.0 juM). As shown i n Figure 13, a potent, b i p h a s i c s t i m u l a t i o n of IRI s e c r e t i o n was observed i n the presence of 17.8 mM glucose. Both SS-14 (6.1 nM) and SMS (9.4 nM) appeared to produce s l i g h t i n h i b i t i o n of t h i s response, although a s t a t i s t i c a l comparison of the i n t e g r a t e d IRI output during i n f u s i o n of SS-14 (13071 ± 1396 uU/10 min) or SMS (13702 ± 964 uU/10 min) to the c o n t r o l value (15147 ± 1452 JJU/10 min) showed no s i g n i f i c a n t d i f f e r e n c e . However, when IRI s e c r e t i o n was s t i m u l a t e d by 8.9 mM glucose (Figure 14), i n f u s i o n of e i t h e r SS-14 (1412 ± 207 v s . 911 ± 93 pU/10 min) or SMS (1412 ± 207 vs. 802 ± 70 j_U/10 min) produced a s i g n i f i c a n t i n h i b i t i o n . As i l l u s t r a t e d i n Figure 15, both agents a l s o suppressed the IRI response to 2.0 nM GIP i n the presence of 8.9 mM glucose, from a c o n t r o l value of 12515 ± 911 / IU/ IO min to 8272 ± 501 ^U/10 min during SS-14 i n f u s i o n and 7389 ± 627 ^U/10 min during i n f u s i o n of SMS. F i n a l l y , Figure 16 shows that a potent i n h i b i t i o n of 1.0 ;_M ACh-stimulated IRI s e c r e t i o n was observed during i n f u s i o n of e i t h e r SS-14 (1219 + 132 vs. 427 ± 50 pU/10 min) or SMS (1219 ± 3 2 vs. 391 ± 65 jiU/10 min). Upon c e s s a t i o n of SS-14 or SMS i n f u s i o n , ACh-stimulated IRI s e c r e t i o n remained suppressed compared to c o n t r o l s . The mean i n t e g r a t e d IRI responses to each s t i m u l u s , during i n f u s i o n of SS-14, SMS, or a c o n t r o l , are summarized i n Figure 17. 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 the i n h i b i t o r y e f f e c t s of 6.1 nM SS-14 and 9.4 nM SMS on the IRI response to glucose, GIP, and ACh were observed. 67 Figure 13: THE EFFECT OF SS-14 AND SMS ON THE IRI RESPONSE TO 17.8 mM GLUCOSE. The glucose concentration was inc r e a s e d from 4.4 to 17.8 mM at minute 5. E i t h e r 6.1 nM SS-14 (A) or 9.4 nM SMS (B) was i n f u s e d during minutes 25-35. Each point represents the mean IRI conc e n t r a t i o n (juU/min). E r r o r bars represent the S.E.M.. Open t r i a n g l e s : 17.8 mM glucose (n=6); closed t r i a n g l e s : 17.8 mM glucose + SS-14 (n=6) or SMS (n-6). 68 1 7 . 8 m M GlucosQ S S - 1 4 (6.1 nM) 1 7 . 8 m M G l u Glu + S S - 1 4 B 0 5 10 15 20 25 30 35 40 45 50 3000 -| 2500 -| 2000 -\ 1500 -I 1 7 . 8 m M Glucose ^ 1000 -I ~ 500 -I 0 SMS (9.4 n M ) 1 7 . 8 m M Glu Glu + S M S 0 5 10 15 20 25 30 35 40 45 50 T i m Q ( m i n ) 69 Figure 14: THE EFFECT OF SS-14 AND SMS ON THE IRI RESPONSE TO 8.9 mM GLUCOSE. The glucose concentration was increased from 4.4 to 8.9 mM at minute 5. E i t h e r 6.1 nM SS-14 (A) or 9.4 nM SMS (B) was i n f u s e d during minutes 25-35. Each point represents the mean IRI concentration (jiU/min). E r r o r bars represent the S.E.M.. Open t r i a n g l e s : 8.9 mM glucose (n=6); closed t r i a n g l e s : 17.8 mM glucose + SS-14 (n=6) or SMS (n=6). 70 8 . 9 m M Glucose 350 -I 300 -250 -i — \ 200 -) 3 1 5 0 -100 -1 —' 5 0 -0 -B E ZD 3 350 -j 300 -250 200 -1 5 0 -100 50 0 0 S S - 1 4 (6.1 n M ) 8 . 9 m M Glu Glu + S S - 1 4 10 20 30 40 8 . 9 mM Glucose 50 SMS ( 9 . 4 nM) 8 . 9 m M Glu Glu + SMS 10 20 30 T i m Q (m i n ) 40 50 71 Figure 15: THE EFFECT OF SS-14 AND SMS ON THE IRI RESPONSE TO GIP. The glucose concentration was inc r e a s e d from 4.4 to 8.9 mM at minute 5. GIP (2.0 nM) was i n f u s e d during minutes 20-45, and e i t h e r 6.1 nM SS-14 (A) or 9.4 nM SMS (B) was i n f u s e d during minutes 25-35. Each point represents the mean IRI con c e n t r a t i o n (jaU/min). E r r o r bars represent the S.E.M.. Open t r i a n g l e s : GIP (n=6); c l o s e d t r i a n g l e s : GIP + SS-14 (n=6) or SMS (n=6). 72 8.9 mM glucose 2500 -| _ 2000 -c: J 1500 ZD 1000 = 500 -1 0 B 3000 -j 2500 -2000 -E ZD 1500 -;—• 1000 -500 -o i 0 GIP (2.0 n M ) S S - 1 4 (B.1 n M ) GIP GIP + S S - 1 4 10 20 30 40 50 GIP (2.0 nM) | SMS (9.4 nM) | GIP GIP + SMS 10 20 30 40 50 Timg (min) 73 Figure 16: THE EFFECT OF SS-14 AND SMS ON THE IRI RESPONSE TO ACH. ACh (1.0 ;uM) was i n f u s e d during minutes 5-40 i n the presence of 4.4 mM glucose, and e i t h e r 6.1 nM SS-14 (A) or 9.4 nM SMS (B) was i n f u s e d during minutes 20-30. Each point represents the mean IRI concentration (uU/min). E r r o r bars represent the S.E.M.. Open t r i a n g l e s : ACh (n=5); closed t r i a n g l e s : ACh + SS-14 (n=5) or SMS (n=5). 74 400 -j 350 -300 -E 250 -Z D 3 200 -150 -100 -5 0 -0 -B I J J M Ach 400 n 350 -c 300 -E 250 -Z D 3 200 -150 -1 0 0 -50 : 0 -SS-14 (6.1 n M ) Ach Ach + SS-14 0 ~5 10 15 20 25 30 35 40 45 Ach (ljuM) SMS (9.4 nM) Ach + SMS Ach -i 1 1 1 1 1 1 i i 0 5 10 15 20 25 30 35 40 45 TimQ (m in) 75 Figure 17: SUMMARY OF THE EFFECTS OF SS-14 AND SMS ON THE IRI RESPONSE TO GLUCOSE, GIP, AND ACH. The data represents the mean (+ S.E.M.) integrated IRI output (mU/10 min) for the 10 min period during i n f u s i o n of SS-14 (closed bars), SMS (hatched bars) or a control (open bars). *Denotes s i g n i f i c a n t difference (p<0.05) of SS-14 or SMS compared to controls. 76 77 V. E f f e c t of Glucose and GIP on SLI S e c r e t i o n The s e c r e t i o n of pan c r e a t i c SLI was measured during s t i m u l a t i o n by glucose (8.9 mM) or GIP (2.0 nM) to i n v e s t i g a t e a p o s s i b l e r o l e f o r endogenously released somatostatin. Figure 18 i l l u s t r a t e s the e f f e c t of 8.9 mM glucose, i n the presence and absence of 2.0 nM GIP, on pancreatic SLI s e c r e t i o n . The mean SLI l e v e l s during i n f u s i o n of glucose (49 + 22 pg/min) or glucose plus GIP (64 + 14 pg/min) 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 p r e - i n f u s i o n l e v e l s (70 + 21 and 62 ± 25 pg/min, r e s p e c t i v e l y ) . 78 Figure 18: THE EFFECT OF GLUCOSE AND GIP ON SLI SECRETION. The glucose concentration was increased from 4.4 to 8.9 mM at minute 5. A. 8.9 mM glucose alone (n=4); B. 8.9 mM glucose + 2.0 nM GIP i n f u s e d during minutes 5-40 ( n=4). Each point represents the mean SLI concentration (pg/min). E r r o r bars represent the S.E.M.. 79 80 DISCUSSION Previous s t u d i e s have shown that the enhanced i n s u l i n s e c r e t i o n associated w i t h meal i n g e s t i o n was mediated by the parasympathetic nervous system (Ahren et a l , 1986; Lucey et a l , 1985), i n a d d i t i o n to increases i n c i r c u l a t i n g n u t r i e n t s , i n p a r t i c u l a r glucose and g a s t r o i n t e s t i n a l hormones (Berthoud, 1984). The present study demonstrated that glucose, GIP and SS-14, which have been shown to be present i n increased concentrations i n the plasma p o s t p r a n d i a l l y (Berthoud, 1984; Kuzio et a l , 1974; Shaughnessy et a l , 1985), a l l had potent and d i r e c t e f f e c t s on i n s u l i n s e c r e t i o n s t i m u l a t e d by ACh. A dose-dependent s t i m u l a t o r y e f f e c t of parasympathomimetics on i n s u l i n s e c r e t i o n i n v i t r o had been demonstrated i n s e v e r a l s t u d i e s (Loubatieres-Mariani et a l , 1973; M u l l e r et a l , 1986; Sharp et a l , 1974). The concentration of ACh used throughout the present study was 1.0 u^M, which was shown p r e v i o u s l y 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 from the perfused r a t pancreas (Loubatieres-Mariani et a l , 1973; Trus et a l , 1978). The glucose dependency of c h o l i n e r g i c a l l y s t i m u l a t e d i n s u l i n s e c r e t i o n was c l e a r l y demonstrated i n t h i s study. When glucose l e v e l s were i n the mid to high p h y s i o l o g i c a l range (4.4 - 8.9 mM), ACh p o t e n t l y 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 , however, no e f f e c t was observed i n the presence of 2.2 mM glucose. Other s t u d i e s have a l s o found the threshold glucose concentration f o r c h o l i n e r g i c s t i m u l a t i o n of i n s u l i n s e c r e t i o n to be approximately 3 - 4 mM (Loubatieres-Mariani et a l , 1973; M u l l e r et a l , 1986). The demonstration that a glucose concentration (4.4 mM) below the t h r e s h o l d f o r glucose-stimulated i n s u l i n r e l e a s e (approximately 5.5 mM) was s u f f i c i e n t f o r c h o l i n e r g i c s t i m u l a t i o n of the B - c e l l suggested that the parasympathetic nervous system could 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 i n the presence of b a s a l glucose l e v e l s . This was supported by i n . v i v o s t u d i e s , i n which i t was suggested that the c e p h a l i c phase of the i n s u l i n response to a meal was mediated by the parasympathetic nervous system. The i n s u l i n i n c r e a s e which occurred p r i o r to p o s t p r a n d i a l i n c r e a s e s i n blood glucose was abolished by atropine or vagotomy ( S t o r l i e n , 1985). The p h y s i o l o g i c a l f u n c t i o n of t h i s a n t i c i p a t o r y r e l e a s e of i n s u l i n may have been to prepare the metabolic pathways f o r incoming n u t r i e n t s (Ahren et a l , 1986). I t has been suggested that most of the i n s u l i n response during the f i r s t 10 min f o l l o w i n g meal i n g e s t i o n was mediated by the vagus nerves (Berthoud, 1984). However, the c o n t r i b u t i o n of the parasympathetic nervous system to the i n s u l i n response f o l l o w i n g p o s t p r a n d i a l increases i n glucose and g a s t r o i n t e s t i n a l hormones has not yet been determined. In the present study, the B - c e l l was found to be s e n s i t i v e to c h o l i n e r g i c s t i m u l i i n the presence of higher glucose l e v e l s (6.6 and 8.9 mM), which have been shown to be i n the range of blood glucose l e v e l s seen p o s t p r a n d i a l l y i n the r a t (Berthoud, 1984). Several studies have suggested that at l e a s t part of the i n s u l i n response to hyperglycemia observed i n v i v o may have been mediated v i a the vagus nerves. N i i j i m a (1975) showed an i n c r e a s e i n f i r i n g of e f f e r e n t f i b e r s i n the p a n c r e a t i c branch of the vagus nerve i n response to intravenous glucose. I n a d d i t i o n , Bloom and Edwards (1980) found that atropine reduced the i n s u l i n response to intravenous glucose i n the conscious c a l f . The present r e s u l t s demonstrated the potent s t i m u l a t o r y e f f e c t of ACh on insulin secretion during moderate hyperglycemia. These results were supportive of a role for the vagus nerves in the postprandial homeostasis of glucose levels. However, the B-cell was found to be less sensitive to cholinergic stimulation in the presence of very high glucose levels (17.8 mM). This could not be attributed to a maximal stimulation of the B-cell by 17.8 mM glucose and ACh, since a greater insulin response has been observed in the presence of 17.8 mM glucose and GIP (Pederson and Brown, 1976). Therefore, the results indicated that the vagus nerves were of importance in the regulation of insulin secretion under normal physiological conditions, but not during more profound metabolic alterations such as extreme hypo- or hyperglycemia, where i t has been suggested that glucose may have been the prime determinant of B-cell secretion (Gerich et a l , 1976). Glucose was thus shown to have an obligatory, permissive effect on ACh-stimulated insulin secretion. The mechanism of the permissive action of glucose on cholinergically stimulated insulin secretion has not been elucidated. It has been suggested that the presence of glucose was required to maintain the energy state of the B-cell , allowing the stimulation of insulin secretion by ACh (Meglasson et a l , 1986). However, whether other fuel molecules such as amino acids were capable of substituting for glucose as permissive agents for ACh-induced insulin release has not been investigated. Such a role has been demonstrated for arginine in allowing GIP-stimulated insulin secretion in the presence of subthreshold levels of glucose (Pederson and Brown, 1978). Since the stimulatory effect of ACh has been shown to be dependent on Ca influx, which was stimulated only in the presence of a sufficient glucose concentration, the permissive role of glucose has been suggested to be due to depolarization of the B-cell membrane, which allowed the cholinergic stimulation of Ca influx (Nenquin et a l , 1984). Such a mechanism may explain the weaker insulin response to ACh observed in the presence of 17.8 mM glucose, since i t was shown that, at a high glucose concentration (16.7 mM), ACh failed to stimulate "^*Ca uptake in islets and had only a weak stimulatory effect on insulin secretion (Wollheim et a l , 1980). The insulin response to 17.8 mM glucose and ACh may have been inconclusive. Robbins et al (1981) showed that 16.5 mM glucose potently stimulated somatostatin secretion from the perfused pancreas, but inhibition of this response by muscimol had no effect on insulin release. In contrast, glucose-stimulated insulin secretion has been shown to be enhanced in islets isolated from rats treated with cysteamine, which depleted pancreatic somatostatin (Kanatsuka et a l , 1984). Although somatostatin release in the presence of 17.8 mM glucose was not measured in the present study, 8.9 mM glucose was found to have no effect on pancreatic SLI secretion. However, the inability of glucose to stimulate SLI release from the perfused pancreas in this study would not preclude an effect of pancreatic somatostatin on insulin secretion, since the paracrine release of somatostatin could not be measured. It has been suggested that the effect of islet somatostatin on B-cell secretion would probably have been mediated by a paracrine mechanism, since the direction of blood flow within the islet indicated that the majority of B-cells would not be exposed to somatostatin released into islet capillaries (Bonner-Weir and Orci, 1982). The effect of ACh on insulin secretion may also have been influenced by ACh-induced changes in islet glucagon or somatostatin secretion. The release of glucagon, a potent stimulator of insulin release (Samols et al , 1965), has been shown to be increased by either parasympathetic or sympathetic stimulation of the pancreas. In the perfused pancreas, infusion of ACh (Iversen, 1973) and stimulation of the vagus nerves (Kaneto et a l , 1967) have both been found to enhance glucagon release. An effect of parasympathomimetics on islet somatostatin release has not been conclusively demonstrated, although i t has been suggested that ACh produced a slight stimulation of somatostatin secretion which was mediated by D-cell muscarinic receptors (Miller, 1981). The potent inhibitory effect of atropine on ACh-stimulated insulin secretion suggested that the stimulation of insulin release by parasympathomimetics was mediated mostly by muscarinic receptors on the B-cell. The presence of muscarinic receptors on the B-cell membrane had 3 been previously indicated by the binding of [H]-methylscopolamine to rat islets (Gri l l and Ostenson, 1983). The importance of this mechanism has been demonstrated in vivo, since the insulin response to a meal has been shown to be strongly suppressed by atropine in rat (Berthoud 1984) and in man (Lucey et a l , 1985). In the present study, the concentration of atropine used (1.0 uM), which had been previously shown to abolish vagally stimulated insulin secretion in the perfused canine pancreas (Bergman and Miller, 1973), produced a potent yet incomplete blockade of ACh-stimulated insulin secretion, suggesting that part of the insulin response to ACh may have occurred via indirect, non-muscarinic mechanisms. The inhibition of Ach-stimulated insulin secretion by hexamethonium indicated that at least part of the response was mediated by nicotinic receptors on intrapancreatic ganglia. The inability of hexamethonium to completely block Ach-stimulated insulin release was probably due to the direct action of ACh on B-cell muscarinic receptors. A role for the pancreatic ganglia in the control of insulin secretion had been previously suggested by Chapal and Loubatieres-Mariani (1978), who showed that nicotine stimulated insulin secretion from the perfused pancreas. In the present study, stimulation of nicotinic receptors by ACh could have caused the release of a number of different postganglionic neurotransmitters, including ACh and various peptides. Stimulation of the vagus nerves in pigs has been shown to cause the release of VIP and GRP from the pancreatic nerves, and these peptides have been found in the pancreatic nerves of rats as well (Ahren et a l , 1986). Therefore, the mechanism of stimulation of insulin release by ACh in the perfused pancreas could have involved the release of peptidergic postganglionic neurotransmitters, as well as the direct effect of ACh on the B-cell. The recent identification of sympathetic ganglia in the pancreas (Luiten et a l , 1986) suggested that the effects of ACh seen in the perfused pancreas may also have been partly mediated by sympathetic postganglionic nerve fibers. Although the primary effect of sympathetic stimulation on insulin secretion has been shown to be inhibitory, a stimulatory effect of low concentrations of norepinephrine via beta-adrenergic receptors has been demonstrated (Miller, 1981). However, the effect of adrenergic antagonists on the insulin response to ACh or nicotine has not been studied. In addition, the stimulation of glucagon release by ACh, which has been demonstrated in the perfused pancreas (Iversen, 1973), could have occurred via the stimulation of sympathetic ganglia. Therefore, in the present study, the insulin response to ACh infusion may have been mediated partly by the sympathetic stimulation of endogenous glucagon release. Although ACh-stimulated glucagon secretion was not measured in this study, the close proximity of A-cells and B-cells within the islet suggested possible paracrine interactions between these two peptides (Samols et a l , 1986). The possible importance of glucagon in mediating the insulin response to islet stimuli was recently suggested by Pipeleers et al (1985), who showed that exogenous glucagon was required for glucose-stimulated insulin secretion from purified B-cells. The demonstration that neither atropine nor hexamethonium affected glucose-stimulated insulin secretion indicated that the insulin response to glucose occurred via direct stimulation of the B-cell , and was not mediated by cholinergic receptors. The direct and potent effect of glucose on insulin secretion, in the absence of cholinergic input, has been demonstrated in numerous studies in vitro (Hedeskov, 1980; Pipeleers et al , 1982). In apparent conflict with these results was the study of Sharp et al (1974), who found that the insulin response to 16.4 mM glucose in perifused islets was inhibited by atropine. The present results suggested that the site of interaction between glucose and ACh in the stimulation of insulin release was not at the muscarinic receptor. In support of this idea, G r i l l and Ostenson (1983) 3 demonstrated that glucose did not affect the binding of [H]-methylscopolamine to B-cell muscarinic receptors. In addition, i t has been established that the stimulus-secretion coupling of glucose-induced insulin secretion occurred within the B-cell and did not involve a glucoreceptor on the B-cell membrane (Malaisse, 1979). Therefore, the synergistic interaction between glucose and ACh in the stimulation of insulin secretion must have occurred at a site distal to the internalization of glucose. The present study also demonstrated a potentiating effect of physiological concentrations of GIP on the insulin response to ACh, indicating that these two agents interacted in the control of insulin secretion. These results suggested that postprandial increases in GIP modulated the parasympathetic stimulation of insulin secretion. The most marked potentiation of ACh-stimulated insulin secretion was seen in the presence of 1.0 nM GIP, whereas the observed effect of 0.2 nM GIP and ACh was only slightly greater than additive. The postprandial plasma levels of GIP have been shown to reach approximately 0.2 nM in man (Kuzio et a l , 1974), suggesting that the dose of 1.0 nM GIP may have been pharmacological. However, Jorde et al (1985) recently demonstrated the considerable variation in the measurement of GIP levels in human plasma by radioimmunoassay, indicating that the actual postprandial concentration of plasma GIP could not be accurately assessed. Previously, GIP had been shown to potentiate carbachol-induced glucagon release in isolated islets of the mouse (Ahren and Lundquist, 1982), suggesting a similar interaction between GIP and the vagus nerves in the regulation of A-cell secretion. A stimulatory effect of endogenously released glucagon on the B-cell may have affected the results described here. One recent study has also examined the interaction between ACh and GIP in the control of insulin secretion in the perfused rat pancreas (Muller et a l , 1986). It was found that the - 88 insulinotropic effects of similar concentrations of ACh (0.25 and 2.5 juM) and GIP (1.0 nM) were slightly less than additive, suggesting that the stimulation of insulin secretion by these two agents was by separate mechanisms. However, the insulin response to ACh in that study may have been blunted by the very high glucose concentration used (15.8 mM), since similar levels of glucose were shown in the present experiment to produce a weaker response to ACh. An interaction between GIP and the parasympathetic nervous system in the regulation of B-cell secretion had also been suggested by McCullough et al (1985), who demonstrated that the presence of carbachol was required for the stimulation of insulin release by GIP from rat pancreatic lobules, and that this response was abolished by atropine. It was reasoned that an intact cholinergic innervation was required for the insulinotropic action of GIP, and that the interaction between GIP and cholinergic agonists was mediated at muscarinic receptors. However, a cholinergic dependence of GIP-stimulated insulin release had not been previously demonstrated in the perfused pancreas (Pederson and Brown, 1976). In the present study, atropine was shown to have no significant effect on the insulin response to two different concentrations of GIP (0.2 and 1.0 nM). In addition, GIP-stimulated insulin secretion was not affected by hexamethonium. These results suggested that GIP had a direct stimulatory effect on the B-cell , and that the insulin response to GIP was not cholinergically mediated. This idea has been supported by two other studies. First , Amland et al (1985) showed that the increase in plasma insulin levels in man during intravenous administration of GIP was not affected by infusion of atropine. Second, Muller et al (1986), in the perfused rat pancreas, found that the insulin response to concomitant infusion of GIP, ACh, and atropine was approximately equal to the response to GIP alone, suggesting that the GIP stimulated component of insulin secretion was insensitive to atropine. However, studies have not yet conclusively shown that an intact cholinergic innervation of the B-cell was not a requirement for GIP-stimulated insulin secretion, since in the perfused pancreas, the cholinergic innervation of the pancreas would have remained intact (Pipeleers, 1984) whereas in pancreatic lobules the cholinergic nerve fibers impinging on the islets may have been removed. In addition, in the present study, a slight, transient suppression of insulin secretion in the presence of both GIP concentrations was observed when atropine infusion commenced. This could have been an inhibition of insulin release stimulated by endogenously released ACh. However, a partial dependence of GIP-stimulated insulin secretion on the presence of an intact cholinergic innervation could not be discounted. Thus, although the present studies have suggested a potentiating interaction between GIP and ACh in the regulation of insulin release, the mechanism and physiological relevance of this interaction has not been determined. Future investigations should examine the nature of the GIP-ACh interaction in isolated islets. A direct inhibitory action of SS-14 on ACh-stimulated insulin secretion in vitro had not been demonstrated prior to this study. Bloom et al (1980) had shown that the in vivo insulin response to 2-deoxyglucose infusion, which was thought to be mediated by the parasympathetic nervous system, was abolished by the intravenous administration of SS-14. It was suggested that SS-14 was a physiological regulator of vagally stimulated insulin secretion. A role for SS-14 in the regulation of the insulin response to a meal was demonstrated by O'Shaughnessy et al (1985) who showed that infusion of physiological doses of SS-14 (approximately 5 nM) inhibited postprandial insulin secretion. In the present study, a similar dose of SS-14 almost abolished the insulin response to ACh, suggesting that SS-14 had a direct and potent inhibitory effect on the cholinergic stimulation of the B-cell. A close relationship between the parasympathetic nervous system and SS-14 has also been demonstrated in the stomach (Mcintosh et a l , 1981). It was found that GIP-stimulated SLI release from the perfused rat stomach was inhibited by infusion of ACh or stimulation of the vagus nerves. However, studies on the effect of cholinergic agonists on the secretion of pancreatic somatostatin have yielded equivocal results (Miller, 1981). Following the cessation of infusion of SS-14 during ACh-stimulated insulin secretion, the insulin response remained suppressed, whereas in the presence of other stimuli insulin secretion returned to control levels after removal of SS-14. The inhibitory effect of SS-14 on the cholinergic stimulation of the B-cell thus persisted in the absence of SS-14. The mechanism of this long-lasting inhibition could not be determined. However, this observation suggested that the mechanism of inhibition of ACh-stimulated by SS-14 was different from the inhibitory mechanism of SS-14 on other B-cell stimuli. Also, the results were supportive of an important role for SS-14 in the regulation of vagally stimulated insulin secretion. Somatostatin-14 also inhibited the insulin response to 8.9 mM glucose, whereas no effect was observed in the presence of 17.8 mM glucose. It had been previously demonstrated that the inhibitory effect of SS-14 on glucose-stimulated insulin secretion was overcome at higher levels of glucose (Efendic et al , 1978). In the perfused rat pancreas, Curry et al (1974) had shown that large doses of SS-14 (> 30 nM) were required to inhibit the insulin response to 16.5 mM glucose. This B-cell insensitivity to the inhibitory effects of SS-14 in the presence of high levels of glucose may have been of physiological importance in the regulation of blood glucose levels during hyperglycemia. The stimulation of insulin secretion by GIP was also suppressed by SS-14. Since the magnitude of the insulin response to GIP and to 17.8 mM glucose were similar, and SS-14 only inhibited the insulin response to GIP, the inhibitory effects of SS-14 appeared to be more specific to GIP-stimulated insulin secretion. Elevated postprandial plasma levels levels of both GIP (Kuzio et a l , 1974) and SS-14 (0'Shaughnessy et a l , 1985) have been demonstrated in man. In addition, an inhibitory effect of SS-14 on the insulinotropic action of GIP has been shown in the dog in vivo (Pederson et a l , 1975c). These results therefore suggested that GIP and SS-14, released in response to meal ingestion, interacted at the B-cell and were involved in the regulation of postprandial insulin secretion. An important relationship between GIP and SS-14 had been previously suggested in the control of gastric acid secretion, which has been shown to be inhibited by SS-14 (Creutzfeldt and Arnold, 1978). Using the isolated, perfused rat stomach, Mcintosh et al (1981) showed that infusion of GIP potently stimulated the release of gastric SLI, and that this effect could be abolished by infusion of ACh or stimulation of the vagus nerves. It was suggested that the inhibitory effect of GIP on gastric acid secretion was mediated by SS-14. However, whether a similar relationship exists in the control of insulin and pancreatic SS-14 secretion has not been shown. Ipp et al (1977) demonstrated the stimulation of SLI release from the perfused dog pancreas by a pharmacological dose of GIP (11.6 nM). In the present experiment, GIP (2.0 nM) failed to stimulate the release of pancreatic SLI. However, a possible paracrine secretion of SS-14 influencing GIP-stimulated insulin release should not be discounted. The physiological function and control of secretion of islet SS-14 thus requires further study. The role of GIP and the parasympathetic nervous system should be of particular interest. In general, the inhibitory effects of SS-14 depended on the nature of the insulin stimulus. In the presence of weak insulin stimuli (8.9 mM glucose or ACh), the inhibitory effect of SS-14 was strongest. When insulin release was strongly stimulated (17.8 mM glucose or GIP), the inhibitory effect of SS-14 was weaker. This was most evident in the inhibition of glucose-stimulated insulin release. Thus, B-cell sensitivity to SS-14 was found to be decreased in situations where a maximal insulin response was required, such as during profound hyperglycemia. The present data therefore suggested a physiological role for SS-14 in producing an appropriate insulin response to various metaboli c condi t i ons. The inhibitory effects of the somatostatin analog, SMS, were found to be quite similar to the inhibitory effects of SS-14. Like the native peptide, the analog also inhibited the insulin response to 8.9 mM glucose, GIP, and ACh, but not 17.8 mM glucose. The insensitivity of the B-cell to SMS in the presence of high glucose levels was also recently demonstrated by Lembcke et al (1987). The late insulin response to meal ingestion, which was thought to be due to increases in blood glucose, was less affected by SMS than the in i t ia l insulin response, which has been shown to be due to neural and hormonal mechani sms. Previous studies (Pless et a l , 1982; Takemura et a l , 1986) compared the inhibitory effects of SMS and SS-14 on a weight basis. The concentrations of SMS (9.4 nM) and SS-14 (6.1 nM) used in the present experiment were equivalent by weight (10 ng/ml). It was demonstrated that SMS and SS-14 were approximately equipotent (weight basis) in their inhibition of insulin secretion in vitro. Previously, Pless et al (1982) had shown that SMS was approximately 3 times more potent than SS-14 in inhibiting insulin secretion stimulated by intravenous glucose, suggesting that the analog had a more potent direct effect on the B-cell than SS-14. However, SMS was administered Intramuscularly in that experiment, and the Insulin levels were measured 15 min after the glucose bolus. Therefore, the increased potency of the analog was probably due to its longer half-life in plasma (approximately 90 min) compared to the native peptide (2-3 min), since the results of the present study suggested that the direct inhibition by SMS or SS-14 of the B-cell response to various stimuli was the same. This idea was supported by Reubi et al (1984), who showed that SMS and SS-14 bound equally well to somatostatin receptors on a hamster insulinoma. In contrast, in the pituitary, somatostatin receptors showed a slightly higher affinity for SMS than for SS-14. These observations suggested that the increased specificity of the analog for the inhibition of growth hormone secretion may have been partly due to a more potent direct effect on the pituitary. In summary, these results demonstrated that many of the metabolic, hormonal, and nervous factors involved in the regulation of the postprandial insulin response interacted directly at the B-cell. It was shown that the cholinergic stimulation of insulin release was enhanced in the presence of physiological levels of glucose or GIP, and inhibited by SS-14. The results indicated a role for the parasympathetic nervous system in the regulation of insulin secretion under normal physiological conditions, but not during extreme hypo- or hyperglycemia. It was also demonstrated that the inhibitory effects of SS-14 on the B-cell were dependent on the nature of the insulin stimulus, suggesting that the degree of SS-14 inhibition of insulin secretion depended on the prevailing metabolic situation. 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