The alpha2-adrenergic receptor is more effective than the galanin receptor in activating G-proteins in RINm5F beta-cell membranes.

Activated receptors for galanin and norepinephrine, and for several other agonists, inhibit insulin release from pancreatic beta-cells via pertussis toxin-sensitive Gi- and Go-proteins and by acting on at least four cellular mechanisms. These mechanisms include repolarization via activation of the ATP-sensitive potassium (K ATP) channel, inhibition of adenylyl cyclase, and inhibition by unknown mechanism at a "distal" site. For norepinephrine and galanin there is also inhibition of the L-type Ca2+ channel. Consequently, during simultaneous activation by multiple agonists, the effectiveness with which a receptor interacts with the G-proteins will, to some extent, determine the responses. This could have important consequences for the beta-cell. Therefore, the G-protein interactions of two activated receptors, those for norepinephrine and galanin, were compared in the same beta-cell membranes. Measurements were made of the rates of receptor-G-protein interaction (by GTPgammaS binding) and of the rates of turnover of G-proteins (by GTPase activity). A comparison was also made of the ability of norepinephrine and galanin to facilitate ADP ribosylation of the alpha-subunits of Gi and Go by cholera toxin (CTX). Such CTX-induced ADP ribosylation of Gi and Go occurs during G-protein interaction with an activated receptor. By measurement of the number of receptors in the membrane preparation used, the relative effectiveness of the two receptors was assessed. The alpha2-adrenergic receptor was found to be markedly more effective than the galanin receptor in activating G-proteins.

Glucose augmentation of mastoparan-stimulated insulin secretion in rat and human pancreatic islets.

Mastoparan, a tetradecapeptide component of wasp venom, activates heterotrimeric G-proteins and stimulates exocytosis in several cell types, including the pancreatic beta-cell. In this study, its effects on insulin secretion were assessed in both rat and human pancreatic islets, along with the ability of glucose and alpha-ketoisocaproate (alpha-KIC) to augment mastoparan-stimulated release. In Ca2+-free Krebs-Ringer bicarbonate buffer containing 2.8 mmol/l glucose, 20 micromol/l mastoparan stimulated insulin secretion 12- and 14-fold in rat and human islets, respectively. The inactive analog mastoparan-17 had no effect on release. Under the same Ca2+-free conditions, 11.1 mmol/l glucose had no effect on insulin release alone, but augmented mastoparan-stimulated release by 74% in both rat and human islets. Stimulation of release by mastoparan and augmentation of release by glucose were unaffected by treatment with pertussis toxin. The effect of cellular GTP depletion on the mastoparan stimulation of release and augmentation by alpha-KIC was studied by culturing rat islets in the presence of 25 microg/ml mycophenolic acid for 20 h. In the control islets, alpha-KIC augmented mastoparan-stimulated insulin release by 80%. In the GTP-depleted rat islets, mastoparan-stimulated insulin release was not changed, while the augmentation by alpha-KIC was eliminated. Mannoheptulose completely blocked the augmentation by glucose. In conclusion, mastoparan stimulates insulin release by activation of a signal transduction pathway that can be augmented by nutrients such as glucose and alpha-KIC. Nutrient augmentation of this pathway is heavily dependent on GTP.

Identification of the docked granule pool responsible for the first phase of glucose-stimulated insulin secretion.

The mechanisms underlying the first phase of glucose-stimulated insulin release, the deterioration of which marks the early stages of both type 1 and type 2 diabetes, are essentially unknown. Among many hypotheses, one holds that the first phase is due to a readily releasable pool of insulin-containing granules. We used current knowledge of the mechanisms of exocytosis and the proteins involved in docking granules at the plasma membrane to test this hypothesis. A docked pool of readily releasable granules was identified by immunoprecipitation of the plasma membrane protein syntaxin with a specific antibody and by co-immunoprecipitation of soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25) and the granule proteins synaptobrevin and synaptotagmin. The four SNARE proteins co-immunoprecipitated each other, thus identifying the core complex associated with docked granules. Using co-immunoprecipitation as a marker for docked granules, we found that the docked pool was rapidly discharged during the first phase of glucose-stimulated insulin release and refilled during the second phase. Other secretagogues also released the pool, whereas the physiological inhibitor norepinephrine blocked its release. Further studies on the nature of this pool of granules should shed light on the causes of its deterioration in the early stages of diabetes and the reasons for deficient insulin release.

Glucose activates both K(ATP) channel-dependent and K(ATP) channel-independent signaling pathways in human islets.

Insulin secretion by isolated islets of Langerhans from 19 human donors (9 women and 10 men) was studied in vitro to test the hypothesis that human islets contain both the K(ATP) channel-dependent and the K(ATP) channel-independent signaling pathways. The results demonstrated the presence of both of these major pathways of glucose signaling. Thus, insulin secretion was stimulated by high glucose concentrations, by the sulfonylurea tolbutamide, and by a depolarizing concentration of potassium chloride. Diazoxide, which activates the K(ATP) channel, completely blocked the stimulation of release by glucose. Stimulation of insulin release by tolbutamide, which inhibits the K(ATP) channel and depolarizes the beta-cell, and inhibition of glucose-stimulated release by diazoxide, which activates the channel and repolarizes the beta-cell, confirm the involvement of the K(ATP) channel-dependent pathway in glucose signaling. The participation of the K(ATP) channel-independent pathway in the stimulation of insulin release by glucose was demonstrated for the first time in human islets. This was done in two ways. The first method, in the presence of diazoxide, blocked the action of glucose on the K(ATP) channel in combination with a depolarizing concentration of KCl to raise [Ca2+]i. Under these conditions, glucose stimulated insulin release. A second method to demonstrate the involvement of the K(ATP) channel-independent pathway was to close the K(ATP) channels with tolbutamide. Again, with no possibility of further action on the K(ATP) channel, glucose stimulated insulin release. In a final series of experiments, glucose-stimulated insulin release was profoundly inhibited by somatostatin, clonidine, and prostaglandin E2, but not by galanin.

Augmentation of insulin release by glucose in the absence of extracellular Ca2+: new insights into stimulus-secretion coupling.

Glucose stimulates insulin secretion in the pancreatic beta-cell by means of a synergistic interaction between at least two signaling pathways. One, the K(ATP) channel-dependent pathway, increases the entry of Ca2+ through voltage-gated channels by closure of the K(ATP) channels and depolarization of the beta-cell membrane. The resulting increase in [Ca2+]i stimulates insulin exocytosis. The other, a K(ATP) channel-independent pathway, requires that [Ca2+]i be elevated and augments the Ca2+-stimulated release. These mechanisms are in accord with the belief that glucose-stimulated insulin secretion has an essential requirement for extracellular Ca2+ and increased [Ca2+]i. However, when protein kinases A and C are activated simultaneously, a large effect of glucose to augment insulin release can be seen in the absence of extracellular Ca2+, under conditions in which [Ca2+]i is not increased, and even when [Ca2+]i is decreased to low levels by intracellular chelation with BAPTA. In the presence or absence of Ca2+, there are similarities in the characteristics of augmentation of insulin release that suggest that only one augmentation mechanism may be involved. These similarities include time course, glucose dose-responses, augmentation by nutrients other than glucose such as alpha-ketoisocaproate (alpha-KIC), and augmentation by the fatty acids palmitate and myristate. However, augmentation in the presence and absence of Ca2+ is distinctly different in GTP dependency. Therefore, exocytosis under these two conditions appears to be triggered differently-one by Ca2+ and the other by GTP or a GTP-dependent mechanism. The augmentation pathways are likely responsible for time-dependent potentiation of secretion and for the second phase of glucose-stimulated insulin release.

Cerulenin, an inhibitor of protein acylation, selectively attenuates nutrient stimulation of insulin release: a study in rat pancreatic islets.

Nutrients such as glucose stimulate insulin release from pancreatic beta-cells through both ATP-sensitive K+ channel-independent and -dependent mechanisms, which are most likely interrelated. Although little is known of the molecular basis of ATP-sensitive K+ channel-independent insulinotropic nutrient actions, mediation by cytosolic long-chain acyl-CoA has been implicated. Because protein acylation might be a sequel of cytosolic long-chain acyl-CoA accumulation, we examined if this reaction is engaged in nutrient stimulation of insulin release, using cerulenin, an inhibitor of protein acylation. In isolated rat pancreatic islets, cerulenin inhibited the glucose augmentation of Ca2+-stimulated insulin release evoked by a depolarizing concentration of K+ in the presence of diazoxide and Ca2+-independent insulin release triggered by a combination of forskolin and phorbol ester under stringent Ca2+-free conditions. Cerulenin inhibition of glucose effects was concentration dependent, with a 50% inhibitory concentration (IC50) of 5 microg/ml and complete inhibition at 100 microg/ml. Cerulenin also inhibited augmentation of insulin release by alpha-ketoisocaproate, a mitochondrial fuel. Furthermore, cerulenin abolished augmentation of both Ca2+-stimulated and Ca2+-independent insulin release by 10 micromol/l palmitate, which causes palmitoylation of cellular proteins. In contrast, cerulenin did not attenuate insulin release elicited by nonnutrient secretagogues, such as a depolarizing concentration of K+, activators of protein kinases A and C, and mastoparan. Glucose oxidation, ATP content in islets, and palmitate oxidation were not affected by cerulenin. In conclusion, cerulenin inhibits nutrient augmentation of insulin release with a high selectivity. The finding is consistent with a prominent role of protein acylation in the process of beta-cell nutrient sensing.

Hyperinsulinism of infancy: the regulated release of insulin by KATP channel-independent pathways.

Hyperinsulinism of infancy (HI) is a congenital defect in the regulated release of insulin from pancreatic beta-cells. Here we describe stimulus-secretion coupling mechanisms in beta-cells and intact islets of Langerhans isolated from three patients with a novel SUR1 gene defect. 2154+3 A to G SUR1 (GenBank accession number L78207) is the first report of familial HI among nonconsanguineous Caucasians identified in the U.K. Using patch-clamp methodologies, we have shown that this mutation is associated with both a decrease in the number of operational ATP-sensitive K+ channels (KATP channels) in beta-cells and impaired ADP-dependent regulation. There were no apparent defects in the regulation of Ca2+- and voltage-gated K+ channels or delayed rectifier K+ channels. Intact HI beta-cells were spontaneously electrically active and generating Ca2+ action currents that were largely insensitive to diazoxide and somatostatin. As a consequence, when intact HI islets were challenged with glucose and tolbutamide, there was no rise in intracellular free calcium ion concentration ([Ca2+]i) over basal values. Capacitance measurements used to monitor exocytosis in control and HI beta-cells revealed that there were no defects in Ca2+-dependent exocytotic events. Finally, insulin release studies documented that whereas tolbutamide failed to cause insulin secretion as a consequence of impaired [Ca2+]i signaling, glucose readily promoted insulin release. Glucose was also found to augment the actions of protein kinase C- and protein kinase A-dependent agonists in the absence of extracellular Ca2+. These findings document the relationship between SUR1 gene defects and insulin secretion in vivo and in vitro and describe for the first time KATP channel-independent pathways of regulated insulin secretion in diseased human beta-cells.

Mechanisms of action of VIP and PACAP in the stimulation of insulin release.

VIP and PACAP stimulate insulin release by interaction with the VIP-2/PACAP-3 receptor on the beta cell. Activation of the receptor results in Gs-mediated stimulation of adenylyl cyclase and increased cellular cyclic AMP levels. Increased cyclic AMP results in a small and transient increase in [Ca2+]i, which is likely to have only a small and transient effect on the secretion rate. Cyclic AMP also potentiates insulin secretion by an as yet unknown action at a distal site. A third action of VIP and PACAP is responsible for the continued stimulation of insulin secretion after the levels of cyclic AMP and [Ca2+]i have returned to basal values. This third pathway, which is identified at present only by its sensitivity to low concentrations of wortmannin, plays a major role in the prolonged stimulation of insulin release by VIP and PACAP.

Progesterone inhibits insulin secretion by a membrane delimited, non-genomic action.

In rat islets, progesterone caused a prompt concentration-dependent inhibition of glucose-stimulated insulin release with an IC50 of 10 microM at 8.4mM glucose. The inhibition was specific since both testosterone and 17beta-estradiol had no such effect. The degree of inhibition was similar in islets from male and female rats. The inhibition was not blocked in PTX-treated islets thus ruling out the Gi/Go proteins as mediators of the inhibition. Progesterone inhibited both glucose- and BayK-8644-stimulated insulin secretion in HIT-T15 cells and the IC50 vs. 10 mM glucose was also 10 microM. There was no effect on intracellular cyclic AMP concentration in the presence 0.2 and 10 mM glucose. Progesterone decreased [Ca2+]i under all conditions tested. The decrease in [Ca2+]i was due to blockade of the L-type voltage-dependent Ca2+ channels. Under Ca(2+)-free conditions, progesterone did not inhibit the stimulation of insulin release due to the combination of glucose, phorbol ester and forskolin. Thus blockade of Ca2+ entry appears to be the sole mechanism by which progesterone inhibits insulin release. As progesterone covalently linked to albumin had a similar inhibitory effect as progesterone itself, it is concluded that the steroid acts at the outer surface of the beta-cell plasma membrane. These effects would be classified as either AI or AIIb in the Mannheim classification of nongenomically initiated steroid actions.

Glucose-dependent insulinotropic polypeptide stimulates insulin secretion via increased cyclic AMP and [Ca2+]1 and a wortmannin-sensitive signalling pathway.

The effect of wortmannin, a fungal metabolite which is known to inhibit phosphatidylinositol 3-kinase (PI3-kinase) at low concentrations, has been examined for its effect on insulin secretion stimulated by glucose-dependent insulinotropic polypeptide (GIP). Using a hamster derived clonal beta-cell line, the HIT-T15 cell, wortmannin inhibited GIP-stimulated insulin secretion under both static incubation and perfusion conditions. In contrast, wortmannin did not inhibit glucose-stimulated or forskolin-stimulated insulin secretion. The inhibitory effect was of large magnitude, although always partial, and occurred within a few minutes of the onset of stimulation by GIP. Thus GIP, like vasoactive intestinal polypeptide (VIP) and pituitary adenylyl cyclase activating polypeptide (PACAP), exerts some of its stimulatory effect on insulin release via a wortmannin-sensitive signal transduction pathway.

A wortmannin-sensitive signal transduction pathway is involved in the stimulation of insulin release by vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptide.

Vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27), and PACAP-38 stimulated insulin release with EC50 values of 0.15, 0.15, and 0.06 nM respectively, as expected for the VIP2/PACAP3 receptor subtype. Secretion was stimulated promptly and peaked at 6-10 min. At 30 min, the secretion rate was still 2-3-fold higher than the control rate. The peptides increased cyclic AMP and [Ca2+]i transiently so that at 30 min they had returned to control values. Therefore, an additional signal is required to explain the prolonged stimulation of release. The prolonged effects, but not the acute effects of VIP and PACAP on insulin release were inhibited by low concentrations of wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. While wortmannin inhibited PI 3-kinase activity in cell lysates, no activation by the peptides was seen. Therefore, the wortmannin-sensitive pathway is either dependent on basal PI 3-kinase activity, or another target for wortmanin is responsible for inhibition of the peptide-stimulated secretion. It is concluded that the acute stimulation of insulin release by VIP and PACAP is mediated by increased cyclic AMP and [Ca2+]i, whereas the sustained release is mediated by a novel wortmannin-sensitive pathway.

Vasoactive intestinal polypeptide-augmented insulin release: actions on ionic fluxes and electrical activity of mouse islets.

Vasoactive intestinal polypeptide is a pancreatic neurotransmitter which augments insulin release. To obtain more detailed information on its mode of action on the pancreatic beta cell we studied the effect of vasoactive intestinal polypeptide on 86Rb+ efflux, 45Ca2+ uptake, electrical activity and second messenger systems of isolated mouse islets. Vasoactive intestinal polypeptide enhanced insulin release and 45Ca2+ uptake in a concentration-dependent manner, and was effective at non-stimulatory and stimulatory glucose levels. It increased glucose-induced electrical activity but was without effect on either glucose-mediated changes of 86Rb+ efflux, cAMP or inositol-1,4,5-trisphosphate content. It is suggested that vasoactive intestinal polypeptide augments insulin release by increasing the uptake of Ca2+ into the cell by as yet undefined mechanisms.

The calcimimetic R-467 potentiates insulin secretion in pancreatic beta cells by activation of a nonspecific cation channel.

The extracellular, G protein-linked Ca(2+)-sensing receptor (CaSR), first identified in the parathyroid gland, is expressed in several tissues and cells and can be activated by Ca(2+) and some other inorganic cations and organic polycations. Calcimimetics such as NPS (R)-N-(3-phenylpropyl)-alpha-methyl-3-methoxybenzylamine hydrochloride (R-467), a phenylalkylamine, are thought to activate CaSR by allosterically increasing the affinity of the receptor for Ca(2+). When tested for its effect on insulin release in C57BL/6 mice, R-467 had no effect under basal conditions but enhanced both phases of glucose-stimulated release. The betaHC9 cell also responded to R-467 and to the enantiomer S-467 with a stimulation of insulin release. In subsequent studies with the betaHC9 cell, it was found that the stimulatory effect was due to activation of a nonspecific cation channel, depolarization of the beta-cell, and increased Ca(2+) entry. No other stimulatory mechanism was uncovered. The depolarization of the cell induced by the calcimimetic could be due to a direct action on the channel or via the CaSR. However, it appeared not to be mediated by G(i), G(o), G(q/11), or G(s). The novel mode of action of the calcimimetic, combined with the glucose-dependence of the stimulation on islets, raises the possibility of a totally new class of drugs that will stimulate insulin secretion during hyperglycemia but which will not cause hypoglycemia.

Pituitary adenylate cyclase-activating peptide, carbachol, and glucose stimulate insulin release in the absence of an increase in intracellular Ca2+.

Insulin secretion from the pancreatic beta cell line HIT-T15 was examined under conditions in which the elevation of intracellular free Ca2+ concentration ([Ca2+]i) was inhibited by nitrendipine or diazoxide or by severe Ca2+ deprivation. Glucose-induced insulin release was completely abolished under these conditions. However, in the presence of 12-O-tetradecanoyl-phorbol-13-acetate or forskolin, 10 mM glucose significantly enhanced insulin release, even in the presence of 5 microM nitrendipine or 150 microM diazoxide. The [Ca2+]i was not increased under these conditions. Even under Ca(2+)-deprived conditions, achieved by 60-min preincubation in Ca(2+)-free buffer containing 1 mM ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), glucose in the complete absence of extracellular Ca2+ significantly enhanced insulin release when the cells were treated also with 12-O-tetradecanoylphorbol-13-acetate and forskolin. Because of these findings, additional studies were performed with pituitary adenylate cyclase-activating peptide (PACAP) and carbachol to see whether physiological stimulation via receptor activation could stimulate insulin release in the absence of a rise in [Ca2+]i. Under normal Ca(2+)-containing conditions, PACAP and carbachol stimulated insulin release and markedly potentiated glucose-stimulated release. In the presence of nitrendipine and thapsigargin, glucose failed to stimulate insulin release. Also, neither glucose in combination with PACAP nor glucose with carbachol was able to stimulate release. However, under the same conditions, the combination of glucose, PACAP, and carbachol did stimulate release while being unable to elevate [Ca2+]i. Thus, simultaneous activation of the beta cell by PACAP, carbachol, and glucose can stimulate insulin release even when [Ca2+]i is not elevated.