Opiate-prostaglandin interactions in the regulation of insulin secretion from rat islets of Langerhans in vitro

The inadequate insulin secretory response to glucose stimulation in non-insulin dependent diabetes has been attributed to many factors including high PGE2 levels blunting the secretory response, and to the existence of inhibitory opiate activity in vivo. The purpose of the present work was to see if there was a connection between these two independent theories. Radioimmunoassayable PGE2 in islets of Langerhans was found to be proportional to islet number and protein content and was typically 4 to 5pg/micrograms islet protein. Indomethacin (2.8 X 10(-5) M), sodium salicylate (1.25 X 10(-3) M) and chlorpropamide (7.2 X 10(-5) M) all lowered islet PGE2 levels and stimulated insulin release in vitro. Dynorphin (1-13), stimulated insulin release at a concentration of 6 X 10(-9) M, while lowering islet PGE2. Conversely, at a higher concentration, (6 X 10(-7) M), dynorphin had no stimulatory effect on insulin secretion and did not lower PGE2 levels in islets or in the incubation media. The stimulatory effects of dynorphin and sodium salicylate on insulin secretion were blocked by exogenous PGE2 (10(-5) M). PGE2 at a lower concentration (10(-9) M) did not exert any inhibitory effect on dynorphin- or sodium salicylate-induced insulin release. This concentration of exogenous PGE2 stimulated insulin release in the presence of 6mM glucose. Results from these experiments suggest that since an opioid peptide can lower endogenous PGE2 production in islets and since the stimulatory effects of the opioid peptide are reversed by exogenous PGE2 there may be interactions between these two modulators of insulin secretion.     

Effect of calcitonin gene-related peptide on glucose and gastric inhibitory polypeptide-stimulated insulin release from cultured newborn and adult rat islet cells

Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide that is present in peripheral cells of islets and in nerves around and within islets. CGRP can inhibit insulin secretion in vitro and in vivo. Whether the inhibitory action of CGRP is mediated by somatostatin or by nerve terminals is, however, not known. The objective of this study was to examine the effect of CGRP on insulin secretion, using cultured newborn and adult rat islet cells which did not contain nerve terminals. In adult rat islet cells, CGRP (10(-10) to 10(-8) M) significantly inhibited glucose-stimulated and gastric inhibitory polypeptide (GIP)-potentiated insulin secretion, but in newborn rat islet cells, CGRP did not inhibit glucose-stimulated insulin secretion. Inhibition of glucose-stimulated and GIP-potentiated insulin release was dependent on the glucose concentration during the prestimulation period. CGRP did not stimulate release of somatostatin. These findings suggest that rat CGRP can act directly on beta-cells through a specific receptor that is absent in newborn rat beta-cells.     

Glucagon and insulin secretion by dispersed islet cells: possible paracrine relationships

The ability of dispersed islet cells in a perifusion system to secret glucagon and insulin in response to physiologic stimuli was investigated. Normal hamster islets were isolated by collagenase digestion and the cells dispersed by sequential digestion with collagenase and trypsin. Following a 50-min period of equilibrium in buffer with high glucose concentrations (5.0 mg/ml), glucagon secretion was stimulated by glucopenia and subsequently, inhibited by increasing the concentration of glucose. The responsiveness to glucose inhibition was significantly less in dispersed islet cells than in intact islets. However, the dispersed islet cells showed significantly greater response to arginine. Glucagon secretion by dispersed islet cells was stimulated to tolbutamide and epinephrine but somatostatin had no effect. Dispersed islet cell preparations did not augment insulin secretion in response to glucose but did secrete more insulin in response to arginine. Intact islets secreted insulin in response to glucose but not arginine. We conclude that A cells in cell suspension do not need direct contact or an intact intra-islet environment in order to respond to glucose, arginine, epinephrine, or tolbutamide but the extent of response may be influenced by paracrine effects. However, paracrine relationships may be important in determining the response of B cells to secretagogues.     

Response of pancreatic immunoreactive somatostatin to arginine

Perfusion of isolated dog pancreases with arginine (20 mM) was associated with a prompt and sustained increase in immunoreactive somatostatin (IRS) in the venous effluent while insulin and glucagon rose promptly but soon receded from their peak levels. These results are compatible with a postulated feedback relationship between somatostatin-, glucagon-, and perhaps insulin-secreting cells of the islets in which somatostatin, stimulated by local glucagon, restrains glucagon secretion and perhaps glucagon-mediated insulin release as well.The demonstration that D-cells of the pancreatic islets contain immunoreactive somatostatin (1, 2, 3) which is probably biologically active (4), and are situated topographically between the A-cells and B-cells in the heterocellular region of the islet (5) has suggested a functional role for these components of the islet of Langerhans (6). In view of the inhibitory action of somatostatin upon both insulin and glucagon secretion (7, 8, 9), it was postulated that the D-cell might serve to restrain glucagon and/or insulin secretion (6). We have since reported that the release of IRS from the isolated dog pancreas increases promptly during the perfusion of high concentrations of glucagon whereas high concentrations of insulin do not appear to stimulate IRS release (10). In this study we examine the effect of perfusion with arginine, a potent stimulus of both glucagon and insulin secretion, upon pancreatic IRS release.     

Effects of porcine diazepam-binding inhibitor on insulin and glucagon secretion in vitro from the rat endocrine pancreas.

Porcine diazepam-binding inhibitor (pDBI) is a novel peptide that has been isolated from the small bowel of the pig, and that occurs also in the islet D-cells. We have studied its effects on hormone release in vitro from the endocrine pancreas of the rat. In isolated islets, pDBI (10(-9)-10(-6)M) did not affect basal insulin release at 3.3 mM glucose, whereas stimulated release at 8.3 mM glucose was dose-dependently suppressed by 32-69% (P less than 0.01). Furthermore, insulin secretion stimulated by either 16.7 mM glucose or 1 mM IBMX (3-isobutyl-1-methylxanthine) or 1 micrograms/ml glibenclamide was suppressed by pDBI at 10(-8) M (by 28-30%, P less than 0.05) and 10(-7) M (by 43-47%, P less than 0.01). In contrast, islet insulin secretion induced by 20 mM arginine was unaffected by these concentrations of pDBI. In the perfused rat pancreas, pDBI (10(-8) M) enhanced by 30% (P less than 0.05) the first phase (0-5 min) of arginine-stimulated insulin release, whereas the second phase (5-20 min) was unchanged. Moreover, pDBI suppressed by 28% (P less than 0.05) the second phase of arginine-induced glucagon release. Arginine-induced somatostatin release was not significantly affected by the peptide. Since pDBI immunoreactivity has been localized also to islet D-cells, the present results suggest that pDBI may act as a local modulator of islet hormone release.     

Effect of C-terminal tetrapeptide cholecystokinin (CCK-4) on the function of the islands of Langerhans and the adenohypophysis

A study was made of the action of C-terminal tetrapeptide cholecystokinin (CCK-4) on the secretory function of A-, B- and D-cells of the islets of Langerhans and on the lactotropic function of the hypophysis. Intravenous injection into rats of CCK-4 in doses of 5 and 50 micrograms/kg bw resulted within 2 min in increased blood immunoreactive insulin. Tetrapeptide also exerted a stimulant dose-dependent action on the function of insulin-, glucagon- and somatostatin-secreting pancreatic cells of the pancreatic islets in culture at concentrations ranging within 10(-9)-10(-6)M. When given in the same doses CCK-4 did not affect basal or dopamine-inhibited prolactin secretion by cultured adenohypophyseal cells. It is concluded that CCK-4 stimulates insulin, glucagon and somatostatin secretion by direct contact with target cells.     

Vitrification of human islets of Langerhans

Cryopreservation of human islets of Langerhans by vitrification was studied. Isolated islets were divided into four groups: (1) control islets which were cultured for 6 days, (2) islets which were vitrified after 2 days of culture, (3) control islets which were cultured for 10-13 days, and (4) islets which were vitrified after 6-9 days of culture. After warming, islets from groups 2 and 4 were cultured for 4 days. The thus treated islets were investigated with respect to insulin secretion in the presence of 2.5 or 25 mM glucose, capacity to survive during postwarming culture, and morphology. The insulin secretion in islets from all groups could be stimulated by an increase of the concentration of glucose from 2.5 to 25 mM. No significant differences were observed between the insulin secretions of the vitrified and control islets or between the islets vitrified after 2 and 6-9 days of culture. It is concluded that human islets of Langerhans cryopreserved by vitrification are functional in vitro.     

Attenuation of insulin secretion by insulin-like growth factor binding protein-1 in pancreatic beta-cells

IGFBP-1 is involved in glucohomeostasis, but the direct action of IGFBP-1 on the beta-cell remains unclear. Incubation of dispersed mouse beta-cells with IGFBP-1 for 30min inhibited insulin secretion stimulated by glucose, glucagon-like peptide 1 (GLP-1) or tolbutamide without changes in basal release of insulin and in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and NAD(P)H evoked by glucose. In contrast, IGFBP-1 augmented glucose-stimulated insulin secretion in intact islets, associated with a reduced somatostatin secretion. These results suggest a suppressive action of IGFBP-1 on insulin secretion in isolated beta-cells through a mechanism distal to energy generating steps and not involving regulation of [Ca(2+)](i). In contrast, IGFBP-1 amplifies glucose-stimulated insulin secretion in intact islets, possibly by suppressing somatostatin secretion. These direct modulatory influences of IGFBP-1 on insulin secretion may imply an important regulatory role of IGFBP-1 in vivo and in the pathogenesis of type 2 diabetes, in which loss of insulin release is an early pathogenetic event.     

Proghrelin-derived peptides influence the secretion of insulin, glucagon, pancreatic polypeptide and somatostatin: a study on isolated islets from mouse and rat pancreas

Proghrelin, the precursor of the orexigenic and adipogenic peptide hormone ghrelin, is synthetized in endocrine (A-like) cells in the gastric mucosa. During its cellular processing, proghrelin gives rise to the 28-amino acid peptide desacyl ghrelin, which after octanoylation becomes active acyl ghrelin, and to the 23-amino acid peptide obestatin, claimed to be a physiological opponent of acyl ghrelin. This study examines the effects of the proghrelin products, alone and in combinations, on the secretion of insulin, glucagon, pancreatic polypeptide (PP) and somatostatin from isolated islets of mice and rats. Surprisingly, acyl ghrelin and obestatin had almost identical effects in that they stimulated the secretion of glucagon and inhibited that of PP and somatostatin from both mouse and rat islets. Obestatin inhibited insulin secretion more effectively than acyl ghrelin. In mouse islets, acyl ghrelin inhibited insulin secretion at low doses and stimulated at high. In rat islets, acyl ghrelin inhibited insulin secretion in a dose-dependent manner but the IC(50) for the acyl ghrelin-induced inhibition of insulin release was 7.5 x 10(-8) M, while the EC(50) and IC(50) values, with respect to stimulation of glucagon release and to inhibition of PP and somatostatin release, were in the 3 x 10(-12)-15 x 10(-12) M range. The corresponding EC(50) and IC(50) values for obestatin ranged from 5 x 10(-12) to 20 x 10(-12) M. Desacyl ghrelin per se did not affect islet hormone secretion. However, at a ten times higher concentration than acyl ghrelin (corresponding to the ratio of the two peptides in circulation), desacyl ghrelin abolished the effects of acyl ghrelin but not those of obestatin. Acyl ghrelin and obestatin affected the secretion of glucagon, PP and somatostatin at physiologically relevant concentrations; with obestatin this was the case also for insulin secretion. The combination of obestatin, acyl ghrelin and desacyl ghrelin in concentrations and proportions similar to those found in plasma resulted in effects that were indistinguishable from those induced by obestatin alone. From the data it seems that the effects of endogenous, circulating acyl ghrelin may be overshadowed by obestatin or blunted by desacyl ghrelin.     

The effect of calcium on somatostatin inhibition of insulin release and cyclic AMP production in mouse pancreatic islets.

The effect of somatostatin on glucose-induced insulin secretion and cyclic AMP accumulation in isolated islets from obese, hyperglycemic ob/ob mice was studied in a microperifusion system. The normal biphasic pattern of insulin release as well as the inhibitory pattern of insulin release produced by somatostatin (0.5--1 microgram/ml) was matched by similar changes in the intracellular concentration of cyclic AMP. When islets were stimulated by glucose (3 mg/ml) plus 3-isobutyl-1-methylxanthine (0.1 mM), somatostatin (0.5 microgram/ml) failed to inhibit insulin secretion or cyclic AMP formation in the second phase whereas in the first phase both parameters were significantly reduced by somatostatin (0.5 microgram/ml). In batch-type incubations it was shown that addition of excess calcium (to 6 mM) reversed this inhibition. In the second phase calcium potentiated the (glucose + 3-isobutyl-1-methylxanthine)-stimulated insulin secretion without affecting the cyclic AMP production. This potentiation was inhibited by somatostatin (0.1 microgram/ml). Somatostatin (1 microgram/ml) inhibited adenylate cyclase activity in islet homogenates. No effect of somatostatin on islet glucose utilization could be demonstrated. The results indicate a dual action of somatostatin in the inhibition of insulin release, one involving the islet adenylate cyclase and one affecting the islet uptake of calcium.     

The effect of calcium on somatostatin inhibition of insulin release and cyclic AMP production in mouse pancreatic islets

The effect of somatostatin on glucose-induced insulin secretion and cyclic AMP accumation in isolated islets from obese, hyperglycemic ob/ob mice was studied in a microperifusion system. The normal biphasic pattern of insulin release as well as the inhibitory pattern of insulin release produced by somatostatin (0.5–1 μg/ml) was matched by similar changes in the intracellular concentration of cyclic AMP. When islets were stimulated by glucose (3 mg/ml) plus 3-isobutyl-1-methylxanthine (0.1 mM), somatostatin (0.5 μg/ml) failed to inhibit insulin secretion or cyclic AMP formation in the second phase whereas in the first phase both parameters were significantly reduced by somatostatin (0.5 μg/ml). In batch-type incubations it was shown that addition of excess calcium (to 6 mM) reversed this inhibition. In the second phase calcium potentiated the (glucose + 3-isobutyl-1-methylxanthine)-stimulated insulin secretion without affecting the cyclic AMP production. This potentiation was inhibited by somatostatin (0.1 μg/ml). Somatostatin (1 μg/ml) inhibited adenylate cyclase activity in islet homogenates. No effect of somatostatin on islet glucose utilization could be demonstrated.The results indicate a dual action of somatostatin in the inhibition of insulin release, one involving the islet adenylate cyclase and one affecting the islet uptake of calcium.     

Somatostatin inhibition of insulin release from freshly isolated and organ cultured rat islets of langerhans in vitro

1×10^?6M somatostatin causes a 37–44% inhibition of glucose induced insulin release from freshly isolated rat islets of Langerhans. A 81 to 95% inhibition is observed when the isolated islets are maintained in organ culture for 2 days prior to the somatostatin treatment. The dose curve of somatostatin on cultured islets shows an apparent K_I of 1.4×10^?9. The tetradecapeptide also causes a reversible inhibition of the stimulation of insulin release by 5 mM theophylline and 23 mM K⁺.     

Entrapment of dispersed pancreatic islet cells in CultiSpher-S macroporous gelatin microcarriers: Preparation, in vitro characterization, and microencapsulation.

Immunoprotection of pancreatic islets for successful allo- or xenotransplantation without chronic immunosuppression is an attractive, but still elusive, approach for curing type 1 diabetes. It was recently shown that, even in the absence of fibrotic overgrowth, other factors, mainly insufficient nutrition to the core of the islets, represent a major barrier for long-term survival of intraperitoneal microencapsulated islet grafts. The use of dispersed cells might contribute to solve this problem due to the conceivably easier nutritional support to the cells. In the present study, purified bovine islets, prepared by collagenase digestion and density gradient purification, and dispersed bovine islet cells, obtained by trypsin and DNAsi (viability > 90%), were entrapped into either 2% (w/v) sodium alginate (commonly used for encapsulation purposes) or (dispersed islet cells only) macroporous gelatin microcarriers (CulthiSpher-S, commonly used for the production of biologicals by animal cells). Insulin release studies in response to glucose were performed within 1 week and after 1 month from preparation of the varying systems and showed no capability of dispersed bovine islet cells within sodium alginate microcapsules to sense glucose concentration changes. On the contrary, bovine islet cells entrapped in CulthiSpher-S microcarriers showed maintained capacity of increasing insulin secretion upon enhanced glucose concentration challenge. In this case, insulin release was approximately 60% of that from intact bovine islets within sodium alginate microcapsules. MTT and hematoxylineosin staining of islet cell-containing microcarriers showed the presence of viable and metabolically active cells throughout the study period. This encouraging functional data prompted us to test whether the microcarriers could be immunoisolated for potential use in transplantation. The microcarriers were embedded within 3% sodium alginate, which was then covered with a poly-L-lysine layer and a final outer alginate layer. Maintained insulin secretion function of this system was observed, which raises the possibility of using microencapsulated CulthiSpher-S microcarriers, containing dispersed pancreatic islet cells, in experimental transplantation studies.     

Islet amyloid polypeptide (IAPP;amylin) influences the endocrine but not the exocrine rat pancreas.

The effect of synthetic rat amylin (10,100,1000 pmol/l) on glucose (10 mmol/) and arginine (10 mmol/l) -stimulated islet hormone release from the isolated perfused rat pancreas and on amylase release from isolated pancreatic acini was investigated. Amylin stimulated the insulin release during the first (+76%) and the second secretion period (+42%) at 1 nmol/l. The first phase of the glucagon release was inhibited concentration dependently by amylin and completely suppressed during the second phase. Amylin diminished the somatostatin release in a concentration dependent manner. This effect was more pronounced at the first than the second secretion period (1 nmol amylin: 1 phase: -60%, 2.phase: -22%). Amylin was without any effect on basal and CCK stimulated amylase release from isolated rat pancreatic acini. Our data suggest amylin, a secretory product of pancreatic B-cells, as a peptide with approximately strong paracrine effects within the Langerhans islet. Therefore, amylin might be involved in the regulation of glucose homeostasis.     

Glucose regulates glucokinase activity in cultured islets from rat pancreas

In this study, we have used isolated pancreatic islets cultured for 7 days in 3 or 30 mM glucose to explore whether glucokinase is induced or activated by high glucose concentrations and has related enzyme activity to glucose-stimulated insulin release. Islets cultured in low glucose medium or low glucose medium plus 350 ng/ml insulin did not respond to high glucose stimulation. Islets cultured in medium containing high glucose concentrations showed a high rate of basal insulin secretion when perifused with 5 mM glucose, and the insulin release was greatly augmented in a biphasic secretion profile when the glucose concentration was raised to 16 mM. Islet glucokinase and hexokinase activities were determined by a sensitive and specific fluorometric method. Glucokinase activity was reduced to approximately 50% in islets cultured in low glucose medium with or without insulin present compared to results with fresh islets. However, islets cultured in 30 mM glucose showed that glucokinase activity was elevated to 236% compared to results with fresh islets. It is concluded that (a) glucose is the physiological regulator of glucokinase in the islet of Langerhans and that (b) the activity of glucokinase plays a crucial role in glucose-induced insulin secretion.     

Insulin, glucagon, and somatostatin secretion by cultured rat islet cell tumor and its clones

Cells derived from rat islet tumor and grown in culture (parent cells-RIN-m) and two clones obtained from them were used to study the effect of various secretagogues on insulin, glucagon, and somatostatin secretion. Parent cells secreted all three hormones in various quantities, while clone 5F secreted predominantly insulin and clone 14B secreted predominantly somatostatin. The secretory behavior of these cells were compared to each other and to that of normal islets. In general, as in the case of normal islets, insulin secretion was stimulated by calcium, potassium, tolbutamide, theophylline, and glucagon. It was inhibited by somatostatin. Glucagon secretion was stimulated by calcium, arginine, and theophylline. Somatostatin secretion was stimulated in clone 14B by arginine, tolbutamide, theophylline, and insulin. These cells differ from normal islets, in that they do not respond to glucose or arginine with increased insulin secretion. Also somatostatin failed to inhibit glucagon secretion. The similarity in insulin secretory responses of parent cells and clone 5F suggests that local or paracrine islet hormone secretion plays only a negligible role in the control of other hormone secretion in these cells.     

KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion

Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in β-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS–1 cells. Taking advantage of hemicannels’opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 μM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 μM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1). ATP acts independently of K_ATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion.     

Effect of fibroblast growth factor and nerve growth factor on the cellular proliferation and functional activity of isolated islands of Langerhans

The effects of fibroblast growth factor (FGF) and nerve growth factor (NGF) on DNA synthesis and insulin secretion were studied in 4-5-day cultures of the isolated neonatal rat islets. FGF (0.1 ng/ml) stimulated significantly the incorporation of 3H-thymidine into DNA of the isolated islets, but failed to change either insulin content in the islets or the rate of insulin secretion. NGF (0.1-1000 ng/ml) did not affect the above parameters. The responses of the islets of Langerhans to increasing concentrations of glucose and isobutylmethylxanthine were not modified after prolonged exposure to NGF. The role of FGF and NGF in the regulation of proliferation and secretory process in pancreatic islet cells is discussed.     

Isolated mouse islets respond to glucose with an initial peak of glucagon release followed by pulses of insulin and somatostatin in antisynchrony with glucagon

Recent studies of isolated human islets have shown that glucose induces hormone release with repetitive pulses of insulin and somatostatin in antisynchrony with those of glucagon. Since the mouse is the most important animal model we studied the temporal relation between hormones released from mouse islets. Batches of 5-10 islets were perifused and the hormones measured with radioimmunoassay in 30s fractions. At 3mM glucose, hormone secretion was stable with no detectable pulses of glucagon, insulin or somatostatin. Increase of glucose to 20mM resulted in an early secretory phase with a glucagon peak followed by peaks of insulin and somatostatin. Subsequent hormone secretion was pulsatile with a periodicity of 5min. Cross-correlation analyses showed that the glucagon pulses were antisynchronous to those of insulin and somatostatin. In contrast to the marked stimulation of insulin and somatostatin secretion, the pulsatility resulted in inhibition of overall glucagon release. The cytoarchitecture of mouse islets differs from that of human islets, which may affect the interactions between the hormone-producing cells. Although indicating that paracrine regulation is important for the characteristic patterns of pulsatile hormone secretion, the mouse data mimic those of human islets with more than 20-fold variations of the insulin/glucagon ratio. The data indicate that the mouse serves as an appropriate animal model for studying the temporal relation between the islet hormones controlling glucose production in the liver.     

Somatostatin-induced inhibition of insulin secretion from isolated islets of rat pancreas in presence of glucagon.

In order to study the oeffect of somatostatin on the endocrine pancreas directly, islets isolated from rat pancreas by collagenase were incubated for 2 hrs 1) at 50 and 200 mg/100 ml glucose in the absence and presence of somatostatin (1, 10 and 100 mg/ml) and2) at 200 mg/100 ml glucose together with glucagon (5 mug/ml), with or without somatostatin (100 ng/ml). Immunologically measurable insulin was determined in the incubation media at 0, 1 and 2 hrs. Insulin release was not statistically affected by any concentration stomatostatin. On the other hand, somatostatin exerted a significant inhibitory action on glucagon-potentiated insulin secretion (mean +/- SEM, mu1/2 hrs/10 islets: glucose and glucagon: 1253 +/- 92; glucose, glucagon and somatostatin: 786 +/- 76). The insulin output in th epresence of glucose, glucagon and somatostatin was also significantly smaller than in thepresence of glucose alone (1104 +/- 126) or of glucose and somatostatin (1061 +/- 122). The failure of somatostatin to affect glucose-stimulated release of insulin from isolated islets contrasts its inhibitory action on insulin secretion as observed in the isolated perfused pancreas and in vivo. This discrepancy might be ascribed to the isolation procedure using collagenase. However, somatostatin inhibited glucagon-potentiated insulin secretion in isolated islets which resulted in even lower insulin levels than obtained in the parallel experiments without glucagon. It is concluded that the hormone of the alpha cells, or the cyclic AMP system, might play a part in the machanism of somatostatin-induced inhibition of insulin release from the beta-cell.