Effect of GIP on the secretion of insulin and somatostatin and the accumulation of cyclic AMP in vitro in the rat

The effects of gastric inhibitory polypeptide (GIP) on insulin secretion as well as on the intra-islet accumulation of [3H]cyclic AMP were investigated in isolated pancreatic islets of the rat. In the presence of 6.7 mmol/l of glucose, 3.0 and 30 nmol/l of GIP induced both insulin and [3H]cyclic AMP responses, while lower and higher concentrations of the peptide were ineffective. A coupling of the two parameters was also found with regard to interaction between glucose and GIP. Thus while 30 nmol/l of GIP was stimulatory together with 6.7, 16.7 or 33.3 mmol/l of glucose, the peptide stimulated neither insulin release, nor the accumulation of [3H]cyclic AMP in the presence of a low concentration of glucose (3.3 mmol/l). The concomittant release of insulin and somatostatin was studied in the perfused pancreas in order to assess a possible influence by somatostatin on the dose-response pattern for GIP-induced insulin release. In this preparation 1.0 to 10 nmol/l of GIP stimulated insulin and somatostatin secretion; however while these concentrations were equipotent on insulin release, 10 nmol/l of GIP stimulated somatostatin release more than 1 nmol/l, indicating differences in dose-response curves for the GIP-induced stimulation of the two hormones. It is concluded that 1) modulation of GIP-induced insulin release is coupled to changes in cyclc AMP response in the islet, 2) GIP-induced somatostatin secretion may influence the concomittant insulin response.     

Effects of galanin on islet cell secretory responses to VIP, GIP, 8-CCK, and glucagon by the perfused rat pancreas

Exogenous galanin has been shown to suppress insulin secretion as elicited by a number of secretagogues such as glucose, arginine, tolbutamide, carbachol, and oral nutrients. To achieve further insight into the influence of galanin on the endocrine pancreas, we have investigated the effect of synthetic porcine galanin (a 200 ng bolus followed by constant infusion at a concentration of 16.8 ng/mL for 16 to 24 minutes) on unstimulated insulin, glucagon, and somatostatin release, as well as on the responses of these hormones to 1 nmol/L vasoactive intestinal peptide (VIP), 1 nmol/L gastric inhibitory peptide (GIP), 1 nmol/L 26 to 33 octapeptide form of cholecystokinin (8-CCK) or 10 nmol/L glucagon in the perfused rat pancreas. Galanin infusion reduced unstimulated insulin secretion by 60% without modifying glucagon and somatostatin output. Galanin also blocked insulin release elicited by VIP, GIP, and 8-CCK, it did not affect the glucagon responses to VIP and GIP, or the somatostatin responses to VIP, GIP, and 8-CCK. Finally, galanin inhibited the insulin output, but not the somatostatin release induced by glucagon. In conclusion, in the perfused rat pancreas, galanin appears to behave as a general inhibitor of insulin secretion. Since this neuropeptide does not modify glucagon or somatostatin release, a direct effect of galanin on the B-cell seems plausible.     

The interaction of Vasoactive Intestinal Polypeptide (VIP), glucose and arginine on the secretion of insulin,glucagon and somatostatin in the perfused rat pancreas

Summary Vasoactive Intestinal Polypeptide (VIP) increased the release of insulin, glucagon and somatostatin from the perfused rat pancreas. The amount of these hormones released was dependent upon the prevailing glucose concentration. VIP stimulated glucagon release in the absence of glucose, while insulin and somatostatin release were increased by VIP only in the presence of glucose concentrations of 4.4 mmol/l and above. Glucagon secretion stimulated by arginine in the presence of 4.4 mmol/l glucose was potentiated by VIP. In contrast, VIP did not induce any further increase in the secretion of insulin and somatostatin over that stimulated by arginine. At higher concentrations of glucose (6.7, 16.7, and 33.3 mmol/l) VIP continued to stimulate insulin and somatostatin release, this effect being synergistic on early-phase insulin release. The effects of VIP on islet cells thus depend on the levels of modulating nutrients.     

Effects of gastric inhibitory polypeptide, vasoactive intestinal polypeptide and peptide histidine isoleucine on the secretion of hormones by isolated mouse pancreatic islets

The release of insulin, glucagon, somatostatin and pancreatic polypeptide (PP) by isolated mouse pancreatic islets was determined during 30-min incubations at 5.6 and 16.7 mmol glucose/l in the absence and presence of gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) at concentrations of 1-1000 nmol/l. Insulin release was enhanced (greater than 50%) by GIP (100-1000 nmol/l) and VIP (1 mumol/l) at 5.6 mmol glucose/l, but not at 16.7 mmol glucose/l. Glucagon release was increased by GIP (100-1000 nmol/l), and by VIP and PHI (1-1000 nmol/l) at both glucose concentrations in a dose-related manner (maximum increases greater than tenfold). Somatostatin release was similarly increased by GIP (10-1000 nmol/l) at both glucose concentrations. Only the highest concentration (1 mumol/l) of PHI tested increased somatostatin release (twofold) at 5.6 mmol glucose/l, whereas PHI and VIP (1-1000 nmol/l) reduced (greater than 37%) somatostatin release at 16.7 mmol glucose/l. PP release was increased (49-58%) by 100-1000 nmol GIP/l, but was not significantly altered by VIP, and was reduced (39-56%) by PHI. The results indicate that GIP, VIP and PHI each stimulate glucagon release in a dose-related manner, but they exert discretely different effects on other islet hormones depending upon the dose and the prevailing glucose concentration.     

Pancreastatin inhibits insulin secretion as induced by glucagon, vasoactive intestinal peptide, gastric inhibitory peptide, and 8-cholecystokinin in the perfused rat pancreas

Pancreastatin is a 49-amino acid straight chain molecule isolated from porcine pancreatic extracts. In the perfused rat pancreas, this peptide has been shown to inhibit unstimulated insulin release and the insulin responses to glucose, arginine, and tolbutamide. To further explore the influence of pancreastatin on islet cell secretion, the effect of synthetic porcine pancreastatin (a 2-micrograms priming dose, followed by constant infusion at a concentration of 15.7 nmol/L) was studied on the insulin, glucagon, and somatostatin responses to 1 nmol/L vasoactive intestinal peptide (VIP), 1 nmol/L gastric inhibitory peptide (GIP), and 1 nmol/L 26 to 33 octapeptide form of cholecystokinin (8-CCK). The effect of pancreastatin on the insulin and somatostatin secretion elicited by glucagon (20 nmol/L) was also examined. Pancreastatin infusion consistently reduced the insulin responses to VIP, GIP, and 8-CCK without modifying glucagon or somatostatin release. It also inhibited the insulin release but not the somatostatin output induced by glucagon. These observations broaden the spectrum of pancreastatin as an inhibitor of insulin release. The finding that pancreastatin does not alter glucagon or somatostatin secretion supports the concept that it influences the B cell directly, and not through an A cell or D cell paracrine effect.     

Interaction of glucagon-like peptide-1(7-36) amide and gastric inhibitory polypeptide or cholecystokinin on insulin and glucagon secretion from the isolated perfused rat pancreas.

The interaction of three incretin candidates, glucagon-like peptide-1(7-36)amide (t-GLP-1), gastric inhibitory polypeptide (GIP), and sulfated COOH-terminal octapeptide of cholecystokinin (CCK-8-S), on insulin and glucagon release from the isolated perfused rat pancreas was studied. Under the perfusate condition of 8.3 mmol/L glucose, coinfusion of 0.1 nmol/L t-GLP-1 and 0.1 nmol/L GIP resulted in an augmented insulin release greater than that obtained by the same dose of each peptide alone. The degree of stimulation elicited by t-GLP-1 and GIP reached a plateau at 0.3 nmol/L for both infusates, and no cooperative effect was observed by coinfusion at 0.3 nmol/L. Coinfusion of 0.1 nmol/L t-GLP-1 and and 0.1 nmol/L CCK-8-S also resulted in an augmented insulin release greater than that obtained by the same dose of each peptide alone. A similar cooperative effect was observed by coinfusion at 0.3 nmol/L, 1 nmol/L, and 3 nmol/L. With the same perfusion experiments, glucagon release was not significantly affected by any peptide at concentrations of 0.1, 0.3, 1, or 3 nmol/L. The coinfusion of 1 nmol/L t-GLP-1 and GIP elicited a transient, but significant, increase in glucagon release. A similar result was obtained by the coinfusion of 0.3 nmol/L and 3 nmol/L t-GLP-1 and GIP, respectively. The coinfusion of t-GLP-1 and CCK-8-S did not affect the glucagon release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cholecystokinin (CCK)-8, CCK-33, and gastric inhibitory polypeptide (GIP) on basal and meal-stimulated pancreatic hormone secretion in man.

Gastrointestinal hormones with insulinotropic effects, like cholecystokinin (CCK) and gastric inhibitory polypeptide (GIP) might tentatively be used in the treatment of non-insulin-dependent diabetes mellitus. We therefore examined the effects of intravenous injection of pharmacological dose levels of CCK-8 (100 and 300 pmol/kg), CCK-33 (100 pmol/kg), GIP (100 pmol/kg), and CCK-8 plus GIP (100 pmol/kg of each) on plasma levels of glucose, insulin, somatostatin, glucagon, and pancreatic polypeptide (PP) in healthy human volunteers. The peptides were given under basal conditions or in combination with a mixed meal. CCK-8, CCK-33, and GIP were all found to increase the basal plasma levels of insulin, somatostatin, and PP; the increases were observed already in samples taken at 2 min after the injection. In contrast, the plasma glucagon levels were unaffected by the peptides. CCK-8, CCK-33, and GIP (100 pmol/kg) all potentiated the meal-induced plasma responses of insulin and PP, whereas plasma levels of glucagon after the meal were not affected. Plasma somatostatin levels after the meal were increased by GIP but not affected by CCK-8 or CCK-33. CCK-8 and GIP together (100 pmol/kg for both) increased plasma levels of insulin, PP and somatostatin as much as each of the peptides given alone, both under basal conditions and after the meal intake. Plasma levels of glucagon were not affected by CCK-8 and GIP together. We conclude that in man, both CCK-8, CCK-33, and GIP moderately stimulate basal and meal related insulin release without any synergistic effects and that the peptides do not inhibit the secretion of glucagon.     

Effect of oleic and octanoic acids on glucose-induced insulin release in vitro.

The isolated perfused rat pancreas was utilized to characterize the effect of 1.5 mM oleic acid and 1.5 mM octanoic acid on insulin release in response to glucose (4.4, 8.3, 11.1, 16.7 and 27.7 mM). Both oleic and octanoic acids potentiated glucose-induced insulin release without modification of the pattern of secretion. At 4.4 mM glucose, the potentiation of insulin release by fatty acids was clearcut for the first phase but not significant with respect to the total amount of insulin released during the stimulation period. Maximal potentiation was observed at 11.1 mM glucose both with oleic and octanoic acids. At all glucose concentrations, octanoic acid was more effective than oleic acid in the potentiation of the insulin secretion.     

Interleukin-1 potentiates glucose stimulated insulin release in the isolated perfused pancreas

The acute effects of recombinant human interleukin-1 beta (rIL-1) on basal and glucose-stimulated insulin release were investigated in the isolated perfused pancreas. At a concentration of 20 micrograms/l rIL-1 had no effect on basal insulin release, but increased the total amount of insulin released during first and second phase insulin release in response to 20 mmol/l D-glucose in the rat pancreas (P less than 0.05). In addition, 26 micrograms/l of rIL-1 potentiated insulin release in response to square wave infusions of stimulatory concentrations of glucose (11 mmol/l) in the porcine pancreas. We hypothesize that IL-1 in the systemic circulation may affect B cell function in vivo.     

Pancreatic polypeptide,glucagon and insulin secretion from the isolated perfused canine pancreas

Summary The release of pancreatic polypeptide (PP) by gut hormones, acetyl choline and adrenaline was investigated in an isolated perfused pancreas preparation. PP was potently released by 1 nmol/l caerulein (186±12%, p<0.001) and gastric inhibitory peptide (GIP) (211±31%, p<0.005) as well as by 1 [mol/l acetyl choline (1097±59%, p<0.001). A significant two-fold release of PP was also evoked by 1 nmol/l vasoactive intestinal peptide (VIP) (129±38%, p<0.02 and gastrin (108±25% p<0.01). Insulin release, induced by high glucose concentration was enhanced by both GIP (210 ±38%, p<(0.01) and VIP (48±5%, p<0.001). In addition GIP enhanced the release of glucagon by 179±18% (p<0.001) at 1.4 mmol/l glucose and by 127±24% (p<0.005) at 8.3 mmol/l glucose. Thus no simple inter-relationship appears to exist between the control of the three circulating islet hormones.     

Thyroxine treatment and insulin secretion in the rat

Thyroxine treatment increases blood glucose and plasma insulin levels in the rat. The hypoglycemic effect of tolbutamide is more pronounced in treated animals. The immediate insulin secretory response of the isolated perfused pancreas to maximal, but not to submaximal, glucose stimuli was increased after thyroxine treatment, especially in the lower dose range. However, as thyroxine treatment reduces insulin release during the prolonged late phase, the total amount of insulin released from the pancreas is reduced. Both the early response to tolbutamide and the subsequent basal secretion were increased after thyroxine treatment. When the pancreas of treated rats was exposed to glucose plus pyruvate the inhibition of the late phase was reversed. Isoprenaline did not overcome the inhibitory effect of thyroxine treatment on the late phase of glucose-induced insulin release. Thyroxine induces a selective inhibition of glucose induced insulin release which is reversed by pyruvate; this indicates that thyroxine interferes with the glycolysis in the beta cell.     

Effects of hydrocortisone on glucose- and cholecystokinin-induced insulin release from the isolated perfused rat pancreas

The acute and chronic effects of hydrocortisone on insulin secretion were examined in the isolated perfused rat pancreas. In the first part of this study, the chronic effects of hydrocortisone on insulin release were examined using isolated perfused pancreas prepared from rats that had been given subcutaneous injections of hydrocortisone at doses of 1.25, 2.5, 5.0, and 10.0 mg/kg body weight once daily for 7 days. Hydrocortisone treatment led to a dose-dependent increase in insulin secretion in response to 8.3 mM glucose. The insulin response to 100 pM cholecystokinin (CCK-8) was also significantly higher in the hydrocortisone-treated rats than in the control group. However, the increment of insulin level over the value before CCK-8 addition in rats treated with hydrocortisone was not significantly different from that in the control rats. In the second part, the acute effects of hydrocortisone on insulin release were studied. Hydrocortisone (17-hydroxycorticosterone) at a concentration of 100 microM caused significant inhibition of the stimulatory effect of CCK-8 on insulin secretion. The inhibition started within 1 min of the beginning of hydrocortisone administration and ceased immediately after the termination of its infusion. We have demonstrated in this study a dual effect of hydrocortisone on insulin release: first, the potentiation of the insulin secretion stimulated by glucose but not by CCK-8 and, second, the inhibition of CCK-8-stimulated insulin secretion.     

Gliclazide at a lower concentration than therapeutic dose increases the sensitivity of insulin secretion to glucose in perfused rat pancreas

OBJECTIVES: We studied the difference between effects of therapeutic dose and sub-therapeutic dose of gliclazide on the glucose-induced insulin secretion. METHODS: The normal rat pancreas was isolated and perfused with Krebs-Ringer buffer containing 1-14 mmol/l glucose. Influcences of 0.25 and 2.5 microg/ml gliclazide on the glucose concentration-insulin secretion curve was examined. RESULTS: Gliclazide at 0.25 microg/ml significantly potentiated 5-8 mmol/l glucose-induced insulin secretion (2.5 +/- 0.5 vs 1.0 +/- 0.3 mU for 15 min at 6.5 mmol/l glucose, P<0.01), but did not give influence on either 1-3 or 10-14 mmol/l glucose-induced insulin secretion. The glucose concentration, at which half-maximal insulin secretion was observed, was lower with gliclazide (5.9 mmol/l) than in the control (7.5 mmol/l). Gliclazide at 2.5 microg/ml markedly increased the maximally glucose-stimulated insulin secretion from 3.9 +/- 0.5 mU for 15 min in the control to 6.6 +/- 0.7 mU for 15 min (P<0.01). The half-maximal insulin secretion was observed at a lower glucose concentration (5.0 mmol/l) than in the absence of gliclazide. CONCLUSION: Gliclazide in sub-therapeutically low dose has different effects on insulin secretion from in therapeutic dose, namely sharpens the insulin secretion sensitivity to glucose with no influence on the maximal insulin secretion. It is possible that low doses of gliclazide might be of interest in some type 2 diabetics whose main pathophysiology is the blunting of insulin secretion response to hyperglycemia.     

Defective amplification of the late phase insulin response to glucose by GIP in obese Type II diabetic patients

AIMS/HYPOTHESIS: Glucagon-like-peptide-1 (GLP-1) is strongly insulinotropic in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas glucose-dependent insulinotropic polypeptide (GIP) is less effective. Our investigation evaluated "early" (protocol 1) - and "late phase" (protocol 2) insulin and C-peptide responses to GLP-1 and GIP stimulation in patients with Type II diabetes. METHODS: Protocol 1: eight Type II diabetic patients and eight matched healthy subjects received i.v. bolus injections of GLP-1(2.5 nmol) or GIP(7.5 nmol) concomitant with an increase of plasma glucose to 15 mmol/l. Protocol 2: eight Type II diabetic patients underwent a hyperglycaemic clamp (15 mmol/l) with infusion (per kg body weight/min) of either: 1 pmol GLP-1 (7-36) amide (n=8), 4 pmol GIP (n=8), 16 pmol GIP (n=4) or no incretin hormone (n=5). For comparison, six matched healthy subjects were examined. RESULTS: Protocol 1: Type II diabetic patients were characterised by a decreased "early phase" response to both stimuli, but their relative response to GIP versus GLP-1 stimulation was exactly the same as in healthy subjects [insulin (C-peptide): patients 59+/-9% (74+/-6%) and healthy subjects 62+/-5% (71+/-9%)]. Protocol 2, "Early phase" (0-20 min) insulin response to glucose was delayed and reduced in the patients, but enhanced slightly and similarly by GIP and GLP-1. GLP-1 augmented the "late phase" (20-120 min) insulin secretion to levels similar to those observed in healthy subjects. In contrast, the "late phase" responses to both doses of GIP were not different from those obtained with glucose alone. Accordingly, glucose infusion rates required to maintain the hyperglycaemic clamp in the "late phase" period (20-120 min) were similar with glucose alone and glucose plus GIP, whereas a doubling of the infusion rate was required during GLP-1 stimulation. CONCLUSION/INTERPRETATION: Lack of GIP amplification of the late phase insulin response to glucose, which contrasts markedly to the normalising effect of GLP-1, could be a key defect in insulin secretion in Type II diabetic patients.     

GIP potentiates CCK stimulated pancreatic enzyme secretion: correlation of anatomical structures with the effects of GIP and CCK on amylase secretion

Utilizing scanning electron microscopy and isolated, vascularly perfused rat pancreas, we studied the angioarchitecture and the effect of the insulinotropic hormone, gastric inhibitory polypeptide (GIP), on cholecystokinin (CCK)-stimulated exocrine secretion. We tried to correlate the microcirculation with the physiological effect of insulin on the pancreatic secretion in the same species. We found a vascular pattern consisting of direct insuloacinar connections, a venous drainage system of islets, and a direct arterial supply of acini. GIP at a concentration as low as 2 ng/ml or exogenous rat insulin potentiated CCK-stimulated pancreatic enzyme secretion. To have similar effects as insulin which is endogenously released by GIP, about 30 times more exogenous rat insulin has to be infused. We conclude that based on the angioarchitecture, insulinotropic hormones like GIP could link the metabolic and the digestive function of the pancreas.     

Effects of vasoactive intestinal polypeptide (VIP), secretin and gastrin on insulin secretion in the mouse

Summary The in vivo effects of vasoactive intestinal polypeptide (VIP), secretin and two different molecular forms of gastrin, gastrin 17 and pentagastrin, on basal and stimulated insulin secretion have been investigated in the mouse. All these peptides induced a moderate dose-dependent increase in basal insulin secretion. The different polypeptides showed complex effects on insulin release stimulated by glucose, the cholinergic agonist carbachol or the adrenergic agonist L-isopropylnoradrenaline (LIPNA), these effects being dependent on the nature of the secretagogue. VIP and secretin both potentiated glucose-induced insulin release. Secretin inhibited insulin secretion induced by carbachol and LIPNA, whereas VIP potentiated L-IPNA-induced insulin secretion and had no influence on the effect of carbachol. Gastrin 17 and pentagastrin did not affect glucose- or carbachol-induced insulin release, whereas they inhibited L-IPNA-induced insulin secretion. The results suggest that VIP, secretin and gastrin display their effects on insulin secretion through different mechanisms. The results indirectly suggest the existence of separate insulin secretory pathways which operate differently, or at least partly differently, after glucose stimulation, cholinergic stimulation, and -adrenergic stimulation.     

Influence of an absorbable alpha-glucosidase inhibitor (Bay o 1248) on the endocrine and exocrine pancreas of the rat

The influence of an absorbable glucosidase inhibitor (Bay o 1248) on the endocrine and exocrine rat pancreas was evaluated. Rats fed a standard diet containing Bay o 1248 over 10 days consumed less food, gained about 30% less body weight than controls and showed meteorism. In these animals postprandial plasma insulin and glucose levels were decreased, but the total pancreatic insulin content was not different versus controls. The early insulin secretory response studied by pancreas perfusions was found reduced after a stimulatory glucose load (10 mM). Addition of the glucosidase inhibitor (1 mM) to the incubation medium diminished the glucose-induced insulin release from isolated islets of rats fed a standard diet. The compound added to the perfusion medium (10 microM) induced a slight reduction of half-maximal glucose-induced (10 mM) insulin release from the perfused pancreas. This inhibitory effect disappeared during maximal stimulation (20 mM glucose) of insulin secretion. The compound neither altered basal nor arginine-induced (15 mM) insulin release from the perfused organ. The exocrine pancreas was studied after feeding a Bay o 1248-enriched standard diet over 10 days. Amylase and trypsin concentration and total output into the biliary-pancreatic juice in response to CCK and secretin (20 IU or CU/kg body weight each) were diminished. The pancreatic enzyme content did not differ compared to controls. A significant role of carbohydrate maldigestion, systemic effects of the glucosidase inhibitor, and endocrine-exocrine pancreatic interrelations are discussed to account for the effects of the compound on the rat pancreas.     

Interaction of glucagon-like peptide-1 (7-36)amide and cholecystokinin-8 in the endocrine and exocrine rat pancreas.

The synergistic impact of glucagon-like peptide-1 (GLP-1) (7-36)amide and cholecystokinin-8 (CCK-8) was studied in the rat pancreas. The GLP-1 (7-36)amide (1 pM-1 microM) had no effect on the basal or CCK-stimulated (1 nM-1 pM) amylase release from isolated pancreatic acini. The insulinotropic action of 0.5 nM GLP-1 (7-36)amide, which weakly stimulated the glucose-induced (6.7 mM) insulin release from the isolated perfused rat pancreas, was strongly potentiated by the addition of CCK-8 (20, 50, and 100 pM) to the perfusate. In concentrations as they occur physiologically after a meal, CCK-8 alone had no significant effect on basal or glucose-stimulated (6.7 mM) insulin secretion. Our data support the assumption that the nutrient-regulated intestinal release of various peptides represents a regulatory system to ensure an adequate insulin response to food intake, at least in rats.     

Effect of gastrin-releasing peptide on the secretion of mouse islet hormones in vitro

Collagenase-isolated mouse islets were incubated with gastrin-releasing peptide (GRP). At 5.6 mmol glucose/l. 10 nmol GRP/l increased the release of insulin (by 50%) and glucagon (by twofold), decreased the release of pancreatic polypeptide (by 35%), but did not significantly affect the release of somatostatin. At 16.7 mmol glucose/l, 10 nmol GRP/l increased glucagon release (by fivefold) and decreased pancreatic polypeptide release (by 46%), without significantly altering insulin and somatostatin release. GRP (200 nmol/l) did not affect insulin release by perifused mouse islets at 2.8 mmol glucose/l, but increased both first and second phase insulin release after a square wave increase in the glucose concentration to 11.1 mmol/l. At 5.6 mmol glucose/l, GRP (100 pmol/1-100 nmol/l) increased (by 50-70%) insulin release by the RINm5F clonal cell line. GRP did not affect glucose oxidation or the cyclic adenosine monophosphate content of RINm5F cells. However, the intracellular free Ca2+ concentration of RINm5F cells was rapidly and transiently increased by GRP (maximum increase of 64% about 10 s after exposure to 1 mumol GRP/l). The rise of intracellular free Ca2+ was approximately halved in the absence of extracellular Ca2+. The results suggest that GRP may contribute to the normal regulation of the endocrine pancreas. The insulin-releasing effect of GRP is mediated via increased cytosolic free Ca2+, derived both from an increased net influx of extracellular Ca2+ and from mobilization of intracellular Ca2+ stores.