Insulin release from collagenase-isolated islets of rat pancreas in the presence of cyclic AMP and somatostatin.

In order to study the role of cyclic AMP in the inhibition by somatostatin of glucose-induced insulin release, the effect of somatostatin on the potentiation by dibutyryl-cyclic AMP (db-cAMP) of insulin release from isolated pancreatic islets of rats was examined. Isolated islets were obtained from the rat pancreas by the collagenase method. Ten islets were incubated for periods of 30 min in Krebs-Ringer bicarbonate buffer containg albumin and glucose 2.0 mg/ml in the presence or absence of somatostatin (1 microgram/ml or 100 ng/ml) and/or db-cAMP 1 mM. Glucose-induced insulin release was reduced by somatostatin in concentrations of 1 microgram/ml. Somatostatin in a concentration of 100 ng/ml significantly abolished the potentiation by db-cAMP of insulin release (p less than 0;01), in spite of exerting no inhibition of glucose-induced insulin release. However, in the presence of theophylline 5 mM, somatostatin 100 ng/ml did not show that inhibitory effect on the potentiated insulin release.     

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.     

Islet-activating protein. Enhanced insulin secretion and cyclic AMP accumulation in pancreatic islets due to activation of native calcium ionophores.

The mechanism whereby "islet-activating protein" (IAP) purified from the culture medium of Bordetella pertussis potentiates insulin secretion was studied by experiments in vitro with islets of rats once injected with IAP (0.5 micrograms/100 g body weight, 3 days before killing) or with islets that had been exposed to IAP (0.1 to 100 ng/ml) for 24 h. The IAP treatment markedly enhanced insulin secretory responses and cAMP accumulation in islets, facilitated the efflux of 45Ca through the cell membrane, and abolished the alpha-adrenergic action of epinephrine (and somatostatin) to inhibit glucose-induced insulin release, cAMP accumulation, and 45Ca uptake. These effects of the IAP treatment were reduced when islets were incubated in a low calcium medium. Based on these results, it was concluded that IAP interacts directly but slowly with the islet B cell in such a manner as to render more calcium available to the stimulus-secretion coupling mechanism as a result of sustained activation of native calcium ionophores on the cell membrane.     

Pulses of somatostatin release are slightly delayed compared with insulin and antisynchronous to glucagon

It was early proposed that somatostatin-producing delta-cells in pancreatic islets have local inhibitory effects on the release of insulin and glucagon. Recent observations that pulses of insulin and glucagon are antisynchronous make it important to examine the temporal characteristics of glucose-induced somatostatin release. Analysis of 30 s fractions from the perfused rat pancreas indicated that increase of glucose from 3 to 20 mmol/l results in initial suppression of somatostatin release followed by regular 4-5 min pulses. During continued exposure to 20 mmol/l glucose, the pulses of somatostatin overlapped those of insulin with a delay of 30 s. Somatostatin and glucagon pulses were coupled in antisynchronous fashion (phase shift 2.4+/-0.2 min), supporting the idea that the delta-cells have a local inhibitory effect on glucagon release. It was possible to remove the pulses of somatostatin and glucagon with maintenance of the insulin rhythmicity by addition of 1 micromol/l of the P2Y(1) receptor antagonist MRS 2179.     

Modulation of somatostatin release by endogenous glucagon and insulin: physiological relationship between A, B and D cells in rat pancreatic islets

In order to understand the physiological role of endogenous insulin or glucagon in somatostatin release, isolated rat pancreatic islets were treated with antiinsulin or antiglucagon antiserum in the presence of physiological amounts of glucose. The release of somatostatin was unchanged by treatment with antiinsulin antiserum which neutralized insulin released by 3.3, 8.3 and 16.7 mM of glucose. However, somatostatin release after treatment with antiglucagon antiserum was much reduced at all concentrations of glucose when compared with the release from control serum. Exogenous rat insulin (0.11, 1.11 micrograms/ml) had no effect, but exogenous glucagon (1, 5 micrograms/ml) resulted in a significant increase. Somatostatin release was stimulated by glucose, but the effect was insignificant. These results clearly indicate the physiological role of endogenous glucagon in the modulation of somatostatin release from the islets of Langerhans. Furthermore, the physiological relationship between A, B and D cells may be mediated through the paracrine mechanism.     

Somatostatin and insulin release from isolated rat pancreatic islets in response to D-glucose, l-leucine, alpha-ketoisocaproic acid or D-glyceraldehyde: evidence for a regulatory role of adenosine-3' ,5'-cyclic monophosphate.

Somatostatin and insulin release from isolated rat pancreatic islets was stimulated by glucose, leucine or α-ketoisocaproic acid. D-glyceraldehyde stimulated insulin release but diminished the secretion of somatostatin. Glucagon and theophylline amplified the glucose-induced somatostatin release.A regulatory role of the D-cell's adenylate cyclase/phosphodiesterase system for the release of somatostatin is suggested. Furthermore, stimulation as well as inhibition of somatostatin release might be of significance for the secretory function of the B-cell.     

Interaction of somatostatin and calcium in regulating insulin release from isolated pancreatic islets of rats.

The effect of synthetic somatostatin on insulin release was studied in vitro by using isolated islets of rats. Somatostatin, with concentrations from 10 ng/ml to 10μg/ml, inhibited insulin release induced by 16.7 mM glucose. Insulin release elicited by 10 μg/ml glucagon or 2 mM dibutyryl cyclic AMP was likewise inhibited by 100ng/ml somatostatin. By raising the calcium concentration of the incubation medium to 6 mM, glucose-induced insulin release was fully restored even in the presence of somatostatin.However, the same maneuver only partially counteracted the somatostatin inhibition of dibutyryl cyclic AMP-induced insulin release. These results suggest the involvement of calcium mobilization process in the inhibitory action of somatostatin.     

Mathematical modeling of stimulus-secretion coupling in the pancreatic beta-cell. III. Glucose-induced inhibition of calcium efflux.

The inhibitory effect of glucose upon 45Ca efflux from prelabeled pancreatic islets was simulated in a mathematical model for Ca2+-cyclic AMP interaction in the process of glucose-induced insulin release. At variance with a previous interpretation, it was postulated that glucose inhibits 45Ca efflux by facilitating the uptake of the cation by the vacuolar system. The latter facilitation did not hinder glucose from provoking a rapid accumulation of cytosolic Ca2+ and, hence, insulin release. The postulated facilitation was also suitable in simulating the effect of glucose upon 45Ca efflux, uptake, and intracellular distribution in the pancreatic islets.     

Insulin release from pancreatic islets of fetal rats mediated by leucine b-BCH, tolbutamide, glibenclamide, arginine, potassium chloride, and theophylline does not require stimulation of Ca2+ net uptake

In pancreatic islets of fetal rats the effect of glucose (3 and 16.7 mM), glyceraldehyde (10 mM), leucine (20 mM), b-BCH (20 mM), tolbutamide (100 micrograms/ml), glibenclamide (0.5 and 5.0 micrograms/ml) arginine (20 mM), KCl (20 mM) and theophylline (2.5 mM) on 45Ca2+ net uptake and secretion of insulin was studied. All compounds tested failed to stimulate 45Ca2+ net uptake. However, in contrast to glucose and glyceraldehyde, leucine, b-BCH, tolbutamide, glibenclamide, arginine, KCl and theophylline significantly stimulated release of insulin. This effect could not be inhibited by the calcium antagonist verapamil (20 microM). Elevation of the glucose concentration from 3 to 5.6 mM did not alter 86Rb+ efflux of fetal rat islets but inhibited 86Rb+ efflux of adult rat islets. Stimulation of 86Rb+ efflux with tolbutamide (100 micrograms/ml), leucine (20 mM) or b-BCH (20 mM) in the presence of 3 mM glucose was also ineffective in fetal rat islets. Our data suggest that stimulation of calcium uptake via the voltage dependent calcium channel is not possible in the fetal state. They also provide evidence that stimulators of insulin release which are thought not to act through their metabolism, initiate insulin secretion from fetal islets by a mechanism which is different from stimulation of calcium influx.     

The stimulus-secretion coupling of glucose-induced insulin release. Cationic and secretory effects of menadione in the endocrine pancreas.

1. Menadione (2-methyl-1,4-naphthoquinone) inhibits insulin release evoked in the rat endocrine pancreas by glucose or glyceraldehyde, but fails to affect the secretory response to Ca2+, Ba2+, theophylline or gliclazide. The inhibitory effect of menadione upon glucose-induced insulin release is a dose-related, rapid and reversible phenomenon, menadione and glucose acting apparently as competitive antagonists. Menadione affects both the early and late phase of the secretory response to glucose. Menadione also antagonizes in a dose-related fashion the ability of glucose to reduce 86Rb efflux, to provoke 86Rb accumulation, to cause biphasic changes in 45 Ca efflux and to stimulate 45 Ca net uptake in pancreatic islets. 2. It is concluded that menadione impairs the insulinotropic action of glucose and other nutrients by impeding the remodelling of cationic fluxes normally provoked by these secretagogues in islet cells. Menadione, however, does not affect the capacity of divalent cations to activate the effector system which controls the release of secretory granules. Menadione may therefore represent a valuable tool to elucidate the mechanism by which glucose normally modifies the movement of cations in the pancreatic B-cell.     

Effect of thioloxidant diazene dicarboxylic acid bis-(N'-methylpiperazide) (DIP) on 45Ca2+ net uptake into rat pancreatic islets

The effect of DIP (an oxidant of glutathione) on 45Ca2+ net uptake induced by a variety of stimulators of insulin secretion was studied in rat pancreatic islets. In addition the effect of exogenous glutathione (GSH) on 45Ca2+ net uptake in response to glucose was tested. DIP (0.1 mM) inhibited the increase of 45Ca2+ net uptake in the presence of glucose (16.7 mM) and glyceraldehyde (10 mM). A similar inhibitory effect could be demonstrated, when 45Ca2+ net uptake was enhanced by tolbutamide (100 micrograms/ml), glibenclamide (0.5 micrograms/ml), b-BCH (20 mM), 2-ketoisocaproate (20 mM), arginine (20 mM) in the presence of 3 mM glucose or by high extracellular potassium (20 mM). The increase of 45Ca2+ net uptake stimulated by leucine (20 mM) plus glucose (3 mM) was further augmented by DIP. Exogenous GSH did not affect 45Ca2+ net uptake in the presence of (5.6-16.7 mM) glucose. It is suggested that 45Ca2+ net uptake of pancreatic islets depends on the redox state of islet thiols regardless of whether uptake is promoted via inhibition of potassium efflux (nutrients, sulfonylureas) or by high potassium and arginine. The voltage sensitive calcium-channel is the site of action of critical thiols. It is possible that these thiols are localized at the inner side of the plasma membrane.     

Effect of perchlorate on calcium uptake and insulin secretion in mouse pancreatic islets.

Microdissected beta-cell-rich pancreatic islets of non-inbred ob/ob mice were used in studies of how perchlorate (CIO4-) affects stimulus-secretion coupling in beta-cells. CIO4- at 16 mM potentiated D-glucose-induced insulin release, without inducing secretion at non-stimulatory glucose concentrations. The potentiation mainly applied to the first phase of stimulated insulin release. In the presence of 20 mM-glucose, the half-maximum effect of CIO4- was reached at 5.5 mM and maximum effect at 12 mM of the anion. The potentiation was reversible and inhibitable by D-mannoheptulose (20 mM) or Ca2+ deficiency. CIO4- at 1-8 mM did not affect glucose oxidation. The effects on secretion were paralleled by a potentiation of glucose-induced 45Ca2+ influx during 3 min. K+-induced insulin secretion and 45Ca2+ uptake were potentiated by 8-16 mM-CIO4-. The spontaneous inactivation of K+-induced (20.9 mM-K+) insulin release was delayed by 8 mM-CIO4-. The anion potentiated the 45Ca2+ uptake induced by glibenclamide, which is known to depolarize the beta-cell. Insulin release was not affected by 1-10 mM-trichloroacetate. It is suggested that CIO4- stimulates the beta-cell by affecting the gating of voltage-controlled Ca2+ channels.     

R-type Ca(2+)-channel-evoked CICR regulates glucose-induced somatostatin secretion

Pancreatic islets have a central role in blood glucose homeostasis. In addition to insulin-producing beta-cells and glucagon-secreting alpha-cells, the islets contain somatostatin-releasing delta-cells. Somatostatin is a powerful inhibitor of insulin and glucagon secretion. It is normally secreted in response to glucose and there is evidence suggesting its release becomes perturbed in diabetes. Little is known about the control of somatostatin release. Closure of ATP-regulated K(+)-channels (K(ATP)-channels) and a depolarization-evoked increase in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) have been proposed to be essential. Here, we report that somatostatin release evoked by high glucose (>or=10 mM) is unaffected by the K(ATP)-channel activator diazoxide and proceeds normally in K(ATP)-channel-deficient islets. Glucose-induced somatostatin secretion is instead primarily dependent on Ca(2+)-induced Ca(2+)-release (CICR). This constitutes a novel mechanism for K(ATP)-channel-independent metabolic control of pancreatic hormone secretion.     

Modification of mouse islet function by 5-hydroxytryptamine, dopamine and their precursors

5-Hydroxytryptamine (5-HT) and dopamine were found to inhibit glucose-induced insulin release and 45Ca2+ net uptake in islets microdissected from ob/ob-mice. Dopamine was more potent than 5-HT. L-DOPA, the precursor of dopamine, had an effect similar to that of dopamine and this effect was reduced by benserazide. L-5-hydroxytryptophan, the precursor of 5-HT, potentiated glucose-induced insulin release and stimulated 45Ca2+ uptake. This effect was also blocked by benserazide. It is concluded that dopamine is a stronger inhibitor than 5-HT and that the different actions of 5-HTP and L-DOPA might be explained by this difference in the magnitude of inhibition.     

Mechanisms of action of cyclosporin A on islet alpha- and beta-cells. Effects on cAMP- and calcium-dependent pathways.

The involvement of cAMP- and calcium-dependent pathways on the inhibitory effect of CsA (0.5 micrograms/ml) on insulin and glucagon release was studied in collagenase-isolated islets. CsA suppressed by 50% the release of insulin in pertussis toxin treated islets stimulated by 20 mM D-glucose. CsA blocked glucagon and insulin release induced by 0.2 mM IBMX (80% and 50% respectively). Similarly it inhibited glucagon and insulin release induced by 1 microM A23187 (53% and 40% respectively). CsA also abolished 0.1 microM glucagon-induced insulin release and 10 ng/ml VIP-induced glucagon release (70% and 38% respectively). The glucagon response to 2 mM D-glucose and to 10 mM arginine was decreased 25% and 45% respectively by CsA. The inhibitory effect of 0.1 microM somatostatin on insulin release was significantly abolished by CsA (p less than 0.001 vs control). On the other hand 1 microM forskolin induced insulin and glucagon release was not modified by CsA. Rats treated with CsA (10 mg/kg body wt) during 10 days showed hyperglycaemia, hypoglucagonemia and higher contents of pancreatic glucagon. It is concluded that CsA affects alpha- and beta-cell function, in vivo and in vitro, acting through calcium and cAMP-dependent pathways. This latter pathway involves the Ca(2+)-calmodulin dependent phosphodiesterase and the regulatory proteins Gs and Gi.     

Fasting-induced dissociation of cationic and secretory events in pancreatic islets

In pancreatic islets removed from 48 h-fasted rats, as distinct from fed animals, the release of insulin evoked by D-glucose is more severely impaired than that evoked by 2-ketoisocaproate. This decreased secretory response to D-glucose contrasts with an unimpaired cationic response to the sugar in terms of the glucose-induced decrease in both 86Rb and 45Ca outflow from pre-labelled islets. Likewise, fasting only causes a modest decrease of the secondary rise in 45Ca outflow evoked by D-glucose in islets perifused at normal Ca2+ concentration. The latter decrease appears more marked, however, if the cationic response to glucose is expressed relative to that evoked by 2-ketoisocaproate in islets removed from rats in the same nutritional state. It is concluded that, in the process of nutrient-stimulated insulin release, neither the decrease in K+ conductance (inhibition of 86Rb outflow) nor the sequestration of Ca2+ by intracellular organelles and/or direct inhibition of Ca2+ outward transport (decrease in 45Ca outflow) represent the sole determinant(s) of the subsequent gating of Ca2+ channels (secondary rise in 45Ca efflux).     

Studies on the mechanism of inhibition of glucose-stimulated insulin secretion by noradrenaline in rat islets of Langerhans.

Noradrenaline (norepinephrine) was shown to be a potent inhibitor of glucose-induced insulin release from rat pancreatic islets, with half-maximal inhibition of the secretory response to 20 mM-glucose occurring at approx. 0.3 microM, and complete suppression of the response occurring at 4 microM-noradrenaline. Inhibition of insulin secretion by noradrenaline was antagonized by the alpha 2-adrenergic antagonist yohimbine (half maximally effective dose approximately 1 microM), but was largely unaffected by the alpha 1-adrenergic antagonist prazosin at concentrations up to 50 microM, suggesting that the response was mediated by alpha 2-adrenergic receptors. Noradrenaline significantly reduced the extent of 45Ca2+ accumulation in glucose-stimulated islets, although as much as 5 microM-noradrenaline was required for 50% inhibition of this response. The ability of noradrenaline to inhibit islet-cell 45Ca2+ uptake was totally abolished in media containing 1 mM-dibutyryl cyclic AMP, suggesting that the response may have been secondary to lowering of islet cyclic AMP. Under these conditions, however, noradrenaline was still able to inhibit insulin secretion maximally. The data suggest that the site(s) at which noradrenaline acts to mediate inhibition of insulin secretion in rat islets lies distal to both islet-cell cyclic AMP accumulation and Ca2+ uptake.     

Studies on the interaction of staphylococcal delta-haemolysin with isolated islets of Langerhans.

delta-Haemolysin, a small surface-active polypeptide purified from the culture media of Staphylococcus aureus, was observed to stimulate the release of insulin from isolated rat islets of Langerhans. This effect was dose-dependent and saturable, with the half-maximal response elicited by a delta-haemolysin concentration of 10 micrograms/ml. Stimulation of insulin release by delta-haemolysin (10 micrograms/ml) was not dependent on the presence of glucose in the incubation medium, but was augmented by increasing concentrations of the sugar. The release of insulin in response to delta-haemolysin could be inhibited by depletion of extracellular Ca2+ or by adrenaline (epinephrine) (10 microM) and was readily reversible when delta-haemolysin was removed from the medium. In addition, the response was potentiated by incubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mM). These observations suggest that delta-haemolysin induced a true activation of the beta-cell secretory mechanism. Stimulation of islets of Langerhans with delta-haemolysin was found to be associated with a modest increase in intracellular cyclic AMP levels, although the adenylate cyclase activity of islet homogenates was not increased by delta-haemolysin. delta-Haemolysin was observed to induce a dose-dependent net accumulation of 45Ca2+ by islet cells and to stimulate the efflux of 45Ca2+ from preloaded islets. The efflux of 45Ca2+ was modest in size and short-lived, but dramatically increased in medium depleted fo 40Ca2+. Incubation in the presence of verapamil augmented delta-haemolysin-induced 45Ca2+ efflux and insulin secretion. delta-Haemolysin was found to be a potent 45Ca2+-translocating ionophore in an artificial system. This response was dose-dependent and could be augmented by verapamil. In addition, phosphatidylcholine (25 micrograms/ml) was found to inhibit both delta-haemolysin induced 45Ca2+ translocation and insulin release in a precisely parallel manner. These studies suggest that the ability of delta-haemolysin to stimulate insulin release may be due, in part, to the facilitation of Ca2+ entry into the beta-cells of islets of Langerhans, mediated directly by an ionophoretic mechanism.     

Chronic exposure to beta-hydroxybutyrate inhibits glucose-induced insulin release from pancreatic islets by decreasing NADH contents

To investigate the effects of chronic exposure to ketone bodies on glucose-induced insulin secretion, we evaluated insulin release, intracellular Ca2+ and metabolism, and Ca2+ efficacy of the exocytotic system in rat pancreatic islets. Fifteen-hour exposure to 5 mM d-beta-hydroxybutyrate (HB) reduced high glucose-induced insulin secretion and augmented basal insulin secretion. Augmentation of basal release was derived from promoting the Ca2+-independent and ATP-independent component of insulin release, which was suppressed by the GDP analog. Chronic exposure to HB affected mostly the second phase of glucose-induced biphasic secretion. Dynamic experiments showed that insulin release and NAD(P)H fluorescence were lower, although the intracellular Ca2+ concentration ([Ca2+](i)) was not affected 10 min after exposure to high glucose. Additionally, [Ca2+](i) efficacy in exocytotic system at clamped concentrations of ATP was not affected. NADH content, ATP content, and ATP-to-ADP ratio in the HB-cultured islets in the presence of high glucose were lower, whereas glucose utilization and oxidation were not affected. Mitochondrial ATP production shows that the respiratory chain downstream of complex II is not affected by chronic exposure to HB, and that the decrease in ATP production is due to decreased NADH content in the mitochondrial matrix. Chronic exposure to HB suppresses glucose-induced insulin secretion by lowering the ATP level, at least partly by inhibiting ATP production by reducing the supply of NADH to the respiratory chain. Glucose-induced insulin release in the presence of aminooxyacetate was not reduced, which implies that chronic exposure to HB affects the malate/aspartate shuttle and thus reduces NADH supply to mitochondria.