ATP-sensitive K+ channel-dependent regulation of glucagon release and electrical activity by glucose in wild-type and SUR1-/- mouse alpha-cells.

Patch-clamp recordings and glucagon release measurements were combined to determine the role of plasma membrane ATP-sensitive K+ channels (KATP channels) in the control of glucagon secretion from mouse pancreatic alpha-cells. In wild-type mouse islets, glucose produced a concentration-dependent (half-maximal inhibitory concentration [IC50]=2.5 mmol/l) reduction of glucagon release. Maximum inhibition (approximately 50%) was attained at glucose concentrations >5 mmol/l. The sulfonylureas tolbutamide (100 micromol/l) and glibenclamide (100 nmol/l) inhibited glucagon secretion to the same extent as a maximally inhibitory concentration of glucose. In mice lacking functional KATP channels (SUR1-/-), glucagon secretion in the absence of glucose was lower than that observed in wild-type islets and both glucose (0-20 mmol/l) and the sulfonylureas failed to inhibit glucagon secretion. Membrane potential recordings revealed that alpha-cells generate action potentials in the absence of glucose. Addition of glucose depolarized the alpha-cell by approximately 7 mV and reduced spike height by 30% Application of tolbutamide likewise depolarized the alpha-cell (approximately 17 mV) and reduced action potential amplitude (43%). Whereas insulin secretion increased monotonically with increasing external K+ concentrations (threshold 25 mmol/l), glucagon secretion was paradoxically suppressed at intermediate concentrations (5.6-15 mmol/l), and stimulation was first detectable at >25 mmol/l K+. In alpha-cells isolated from SUR1-/- mice, both tolbutamide and glucose failed to produce membrane depolarization. These effects correlated with the presence of a small (0.13 nS) sulfonylurea-sensitive conductance in wild-type but not in SUR1-/- alpha-cells. Recordings of the free cytoplasmic Ca2+ concentration ([Ca2+]i) revealed that, whereas glucose lowered [Ca2+]i to the same extent as application of tolbutamide, the Na+ channel blocker tetrodotoxin, or the Ca2+ channel blocker Co2+ in wild-type alpha-cells, the sugar was far less effective on [Ca2+]i in SUR1-/- alpha-cells. We conclude that the KATP channel is involved in the control of glucagon secretion by regulating the membrane potential in the alpha-cell in a way reminiscent of that previously documented in insulin-releasing beta-cells. However, because alpha-cells possess a different complement of voltage-gated ion channels involved in action potential generation than the beta-cell, moderate membrane depolarization in alpha-cells is associated with reduced rather than increased electrical activity and secretion.

The imidazoline NNC77-0020 affects glucose-dependent insulin, glucagon and somatostatin secretion in mouse pancreatic islets.

The effect of the novel imidazoline compound 2-[2-(4,5-dihydro-1H-imidazol-2-yl)-1-(5-methyl-2,3-dihydrobenzofuran-7-yl)-ethyl]-pyridine (NNC77-0020) on stimulus-secretion coupling and hormone secretion was investigated in mouse pancreatic islets and isolated alpha- and beta-cells. In the presence of elevated glucose concentrations NNC77-0020 stimulated insulin secretion concentration dependently (EC(50) 64 nM) by 200% without affecting the whole-cell K(+) current or cytoplasmic Ca(2+) levels. Capacitance measurements in single mouse beta-cells showed that intracellular application of NNC77-0020 via the recording pipette enhanced Ca(2+)-dependent exocytosis. This action was dependent on protein kinase C (PKC) and cytoplasmic phospholipase A(2) (cPLA(2)) activity and required functional granular ClC-3 Cl(-) channels. In intact islets NNC77-0020 stimulated glucose-dependent somatostatin secretion, an effect that was also dependent on PKC and cPLA(2) activity. NNC77-0020 also inhibited glucagon secretion. In single mouse alpha-cells this action was not associated with a change in spontaneous electrical activity and resulted from a reduction in the rate of Ca(2+)-dependent exocytosis. Inhibition of exocytosis by NNC77-0020 was pertussis toxin sensitive and mediated by activation of the protein phosphatase calcineurin. In conclusion, our data suggest that the imidazoline compound NNC77-0020 modulates pancreatic hormone secretion in a complex fashion, comprising glucose-dependent stimulation of insulin and somatostatin secretion and inhibition of glucagon release. These mechanisms of action constitute an ideal basis for the development of novel imidazoline-containing anti-diabetic compounds.

Rapid association of protein kinase C-epsilon with insulin granules is essential for insulin exocytosis.

Glucose-dependent exocytosis of insulin requires activation of protein kinase C (PKC). However, because of the great variety of isoforms and their ubiquitous distribution within the beta-cell, it is difficult to predict the importance of a particular isoform and its mode of action. Previous data revealed that two PKC isoforms (alpha and epsilon) translocate to membranes in response to glucose (Zaitzev, S. V., Efendic, S., Arkhammar, P., Bertorello, A. M., and Berggren, P. O. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9712-9716). Using confocal microscopy, we have now established that in response to glucose, PKC-epsilon but not PKC-alpha associates with insulin granules and that green fluorescent protein-tagged PKC-epsilon changes its distribution within the cell periphery upon stimulation of beta-cells with glucose. Definite evidence of PKC-epsilon requirement during insulin granule exocytosis was obtained by using a dominant negative mutant of this isoform. The presence of this mutant abolished glucose-induced insulin secretion, whereas transient expression of the wild-type PKC-epsilon led to a significant increase in insulin exocytosis. These results suggest that association of PKC-epsilon with insulin granule membranes represents an important component of the secretory network because it is essential for insulin exocytosis in response to glucose.

Involvement of protein kinase C-epsilon in inositol hexakisphosphate-induced exocytosis in mouse pancreatic beta-cells.

Inositolhexakisphosphate (InsP6) plays a pivotal role in the pancreatic beta-cell stimulus-secretion coupling. We have used capacitance measurements to study the effects of InsP6 on Ca2+-dependent exocytosis in single mouse pancreatic beta-cells. In the presence of inhibitors of the protein phosphatase calcineurin to block endocytosis, intracellular application of InsP6 produced a dose-dependent stimulation of exocytosis, and half-maximal effect was observed at 22 microM. The stimulatory effect of InsP6 was dependent on protein kinase C (PKC) activity. Antisense oligonucleotides directed against specific PKC isoforms (alpha, beta II, delta, epsilon, xi) revealed the involvement of PKC-epsilon in InsP6-induced exocytosis. Furthermore, expression of dominant negative PKC-epsilon abolished InsP6-evoked exocytosis, whereas expression of wild-type PKC-epsilon led to a significant stimulation of InsP6-induced exocytosis. These data demonstrate that PKC-epsilon is involved in InsP6-induced exocytosis in pancreatic beta-cells.

Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic beta cells.

Insulin secretion is controlled by the beta cell's metabolic state, and the ability of the secretory granules to undergo exocytosis increases during glucose stimulation in a membrane potential-independent fashion. Here, we demonstrate that exocytosis of insulin-containing secretory granules depends on phosphatidylinositol 4-kinase (PI 4-kinase) activity and that inhibition of this enzyme suppresses glucose-stimulated insulin secretion. Intracellular application of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] stimulated exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in a readily releasable pool. Reducing the cytoplasmic ADP concentration in a way mimicking the effects of glucose stimulation activated PI 4-kinase and increased exocytosis whereas changes of the ATP concentration in the physiological range had little effect. The PI(4,5)P(2)-binding protein Ca(2+)-dependent activator protein for secretion (CAPS) is present in beta cells, and neutralization of the protein abolished both Ca(2+)- and PI(4,5)P(2)-induced exocytosis. We conclude that ADP-induced changes in PI 4-kinase activity, via generation of PI(4,5)P(2), represents a metabolic sensor in the beta cell by virtue of its capacity to regulate the release competence of the secretory granules.

Imidazoline NNC77-0074 stimulates Ca2+-evoked exocytosis in INS-1E cells by a phospholipase A2-dependent mechanism.

We have previously demonstrated that the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)-pyridine (NNC77-0074) increases insulin secretion from pancreatic beta-cells by stimulation of Ca(2+)-dependent exocytosis. Using capacitance measurements, we now show that NNC77-0074 stimulates exocytosis in clonal INS-1E cells. NNC77-0074-stimulated exocytosis was antagonised by the cytoplasmic phospholipase A(2) (cPLA(2)) inhibitors ACA and AACOCF(3) and in cells treated with antisense oligonucleotide against cPLA(2)alpha. NNC77-0074-evoked insulin secretion was likewise inhibited by ACA, AACOCF(3), and cPLA(2)alpha antisense oligonucleotide treatment. In pancreatic islets NNC77-0074 stimulated PLA(2) activity. We propose that cPLA(2)alpha plays an important role in the regulation of NNC77-0074-evoked exocytosis in insulin secreting beta-cells.

Imidazoline NNC77-0074 stimulates insulin secretion and inhibits glucagon release by control of Ca(2+)-dependent exocytosis in pancreatic alpha- and beta-cells.

We have investigated the effects of the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)-pyridine (NNC77-0074) on stimulus-secretion coupling in isolated pancreatic alpha- and beta-cells. NNC77-0074 stimulated glucose-dependent insulin secretion in intact mouse pancreatic islets. No effect was observed at

cPLA2alpha-evoked formation of arachidonic acid and lysophospholipids is required for exocytosis in mouse pancreatic beta-cells.

Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A2 (cPLA2alpha) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca2+-dependent exocytosis in single mouse pancreatic beta-cells. cPLA2alpha dose dependently (EC50 = 86 nM) stimulated depolarization-evoked exocytosis by 450% without affecting the whole cell Ca2+ current or cytoplasmic Ca2+ levels. The stimulatory effect involved priming of secretory granules as reflected by an increase in the size of the readily releasable pool of granules from 70-80 to 280-300. cPLA2alpha-stimulated exocytosis was antagonized by the specific cPLA2 inhibitor AACOCF3. Ca2+-evoked exocytosis was reduced by 40% in cells treated with AACOCF3 or an antisense oligonucleotide against cPLA2alpha. The action of cPLA2alpha was mimicked by a combination of arachidonic acid and lysophosphatidylcholine (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve Cl- uptake through ClC-3 Cl- channels. Accordingly, the stimulatory action of cPLA2alpha was inhibited by the Cl- channel inhibitor DIDS and in cells pretreated with ClC-3 Cl- channel antisense oligonucleotides. We propose that cPLA2alpha has an important role in controlling the rate of exocytosis in beta-cells. This effect of cPLA2alpha reflects an enhanced transgranular Cl- flux, leading to an increase in the number of granules available for release, and requires the combined actions of arachidonic acid and lysophosphatidylcholine.

Increase in cellular glutamate levels stimulates exocytosis in pancreatic beta-cells.

Glutamate has been implicated as an intracellular messenger in the regulation of insulin secretion in response to glucose. Here we demonstrate by measurements of cell capacitance in rat pancreatic beta-cells that glutamate (1 mM) enhanced Ca2+-dependent exocytosis. Glutamate (1 mM) also stimulated insulin secretion from permeabilized rat beta-cells. The effect was dose-dependent (half-maximum at 5.1 mM) and maximal at 10 mM glutamate. Glutamate-induced exocytosis was stronger in rat beta-cells and clonal INS-1E cells compared to beta-cells isolated from mice and in parental INS-1 cells, which correlated with the expressed levels of glutamate dehydrogenase. Glutamate-induced exocytosis was inhibited by the protonophores FCCP and SF6847, by the vacuolar-type H+-ATPase inhibitor bafilomycin A(1) and by the glutamate transport inhibitor Evans Blue. Our data provide evidence that exocytosis in beta-cells can be modulated by physiological increases in cellular glutamate levels. The results suggest that stimulation of exocytosis is associated with accumulation of glutamate in the secretory granules, a process that is dependent on the transgranular proton gradient.

Sulfonylurea receptor type 1 knock-out mice have intact feeding-stimulated insulin secretion despite marked impairment in their response to glucose.

The ATP-sensitive potassium channel is a key molecular complex for glucose-stimulated insulin secretion in pancreatic beta cells. In humans, mutations in either of the two subunits for this channel, the sulfonylurea type 1 receptor (Sur1) or Kir6.2, cause persistent hyperinsulinemic hypoglycemia of infancy. We have generated and characterized Sur1 null mice. Interestingly, these animals remain euglycemic for a large portion of their life despite constant depolarization of membrane, elevated cytoplasmic free Ca(2+) concentrations, and intact sensitivity of the exocytotic machinery to Ca(2+). A comparison of glucose- and meal-stimulated insulin secretion showed that, although Sur1 null mice do not secrete insulin in response to glucose, they secrete nearly normal amounts of insulin in response to feeding. Because Sur1 null mice lack an insulin secretory response to GLP-1, even though their islets exhibit a normal rise in cAMP by GLP-1, we tested their response to cholinergic stimulation. We found that perfused Sur1 null pancreata secreted insulin in response to the cholinergic agonist carbachol in a glucose-dependent manner. Together, these findings suggest that cholinergic stimulation is one of the mechanisms that compensate for the severely impaired response to glucose and GLP-1 brought on by the absence of Sur1, thereby allowing euglycemia to be maintained.

Sulfatide controls insulin secretion by modulation of ATP-sensitive K(+)-channel activity and Ca(2+)-dependent exocytosis in rat pancreatic beta-cells.

The glycosphingolipid sulfatide is present in secretory granules and at the surface of pancreatic beta-cells, and antisulfatide antibodies (ASA; IgG1) are found in serum from the majority of patients with newly diagnosed type 1 diabetes. Here we demonstrate that sulfatide produced a glucose- and concentration-dependent inhibition of insulin release from isolated rat pancreatic islets. This inhibition of insulin secretion was due to activation of ATP-sensitive K(+)-(K(ATP)) channels in single rat beta-cells. No effect of sulfatide was observed on whole-cell Ca(2+)-channel activity or glucose-induced elevation of cytoplasmic Ca(2+) concentration. It is interesting that sulfatide stimulated Ca(2+)-dependent exocytosis determined by capacitance measurements and depolarized-induced insulin secretion from islets exposed to diazoxide and high external KCl. The monoclonal sulfatide antibody Sulph I as well as ASA-positive serum reduced glucose-induced insulin secretion by inhibition of Ca(2+)-dependent exocytosis. Our data suggest that sulfatide is important for the control of glucose-induced insulin secretion and that both an increase and a decrease in the sulfatide content have an impact on the secretory capacity of the individual beta-cells.

Nateglinide, but not repaglinide, stimulates growth hormone release in rat pituitary cells by inhibition of K channels and stimulation of cyclic AMP-dependent exocytosis.

OBJECTIVE: GH causes insulin resistance, impairs glycemic control and increases the risk of vascular diabetic complications. Sulphonylureas stimulate GH secretion and this study was undertaken to investigate the possible stimulatory effect of repaglinide and nateglinide, two novel oral glucose regulators, on critical steps of the stimulus-secretion coupling in single rat somatotrophs. METHODS: Patch-clamp techniques were used to record whole-cell ATP-sensitive K(+) (K(ATP)) and delayed outward K(+) currents, membrane potential and Ca(2+)-dependent exocytosis. GH release was measured from perifused rat somatotrophs. RESULTS: Both nateglinide and repaglinide dose-dependently suppressed K(ATP) channel activity with half-maximal inhibition being observed at 413 nM and 13 nM respectively. Both compounds induced action potential firing in the somatotrophs irrespective of whether GH-releasing hormone was present or not. The stimulation of electrical activity by nateglinide, but not repaglinide, was associated with an increased mean duration of the action potentials. The latter effect correlated with a reduction of the delayed outward K(+) current, which accounts for action potential repolarization. The latter effect had a K(d) of 19 microM but was limited to 38% inhibition. When applied at concentrations similar to those required to block K(ATP) channels, nateglinide in addition potentiated Ca(2+)-evoked exocytosis 3.3-fold (K(d)=3 microM) and stimulated GH release 4.5-fold. The latter effect was not shared by repaglinide. The stimulation of exocytosis by nateglinide was mimicked by cAMP and antagonized by the protein kinase A inhibitor Rp-cAMPS. CONCLUSION: Nateglinide stimulates GH release by inhibition of plasma membrane K(+) channels, elevation of cytoplasmic cAMP levels and stimulation of Ca(2+)-dependent exocytosis. By contrast, the effect of repaglinide was confined to inhibition of the K(ATP) channels.

Inositol hexakisphosphate promotes dynamin I- mediated endocytosis.

Membrane homeostasis is maintained by exocytosis and endocytosis. The molecular mechanisms regulating the interplay between these two processes are not clear. Inositol hexakisphosphate (InsP(6)) is under metabolic control and serves as a signal in the pancreatic beta cell stimulus-secretion coupling by increasing Ca(2+)-channel activity and insulin exocytosis. We now show that InsP(6) also promotes dynamin I-mediated endocytosis in the pancreatic beta cell. This effect of InsP(6) depends on calcineurin-induced dephosphorylation and is accounted for by both activation of protein kinase C and inhibition of the phosphoinositide phosphatase synaptojanin and thereby formation of phosphatidylinositol 4,5-bisphosphate. In regulating both exocytosis and endocytosis, InsP(6) thus may have an essential integral role in membrane trafficking.