MIN6 beta-cell-beta-cell interactions influence insulin secretory responses to nutrients and non-nutrients

Insulin-secreting MIN6 cells show greatly enhanced secretory responsiveness to nutrients when grown as islet-like structures (pseudoislets). Since beta-cells use different mechanisms to respond to nutrient and non-nutrient stimuli, we have now investigated the role of homotypic beta-cell interactions in secretory responses to pharmacological or receptor-operated non-nutrient stimuli in MIN6 pseudoislets. In addition to an enhanced secretory responsiveness to glucose, insulin secretion from MIN6 pseudoislets was also enhanced by non-nutrients, including carbachol, tolbutamide, PMA, and forskolin. The improved secretory responsiveness was dependent on the cells being configured as pseudoislets and was lost on dispersal of the pseudoislets into single cells and regained on the re-formation of pseudoislet structures. These observations emphasise the importance of islet anatomy on secretory responsiveness, and demonstrate that homotypic beta-cell interactions play an important role in generating physiologically appropriate insulin secretory responses to both nutrient and non-nutrient stimuli.     

Normalization of intracellular Ca(2+) induces a glucose-responsive state in glucose-unresponsive beta-cells

Although intracellular Ca(2+) in pancreatic beta-cells is the principal signal for insulin secretion, the effect of chronic elevation of the intracellular Ca(2+) concentration ([Ca(2+)](i)) on insulin secretion is poorly understood. We recently established two pancreatic beta-cell MIN6 cell lines that are glucose-responsive (MIN6-m9) and glucose-unresponsive (MIN6-m14). In the present study we have determined the cause of the glucose unresponsiveness in MIN6-m14. Initially, elevated [Ca(2+)](i) was observed in MIN6-m14, but normalization of the [Ca(2+)](i) by nifedipine, a Ca(2+) channel blocker, markedly improved the intracellular Ca(2+) response to glucose and the glucose-induced insulin secretion. The expression of subunits of ATP-sensitive K(+) channels and voltage-dependent Ca(2+) channels were increased at both mRNA and protein levels in MIN6-m14 treated with nifedipine. As a consequence, the functional expression of these channels at the cell surface, both of which are decreased in MIN6-m14 without nifedipine treatment, were increased significantly. Contrariwise, Bay K8644, a Ca(2+) channel agonist, caused severe impairment of glucose-induced insulin secretion in glucose-responsive MIN6-m9 due to decreased expression of the channel subunits. Chronically elevated [Ca(2+)](i), therefore, is responsible for the glucose unresponsiveness of MIN6-m14. The present study also suggests normalization of [Ca(2+)](i) in pancreatic beta-cells as a therapeutic strategy in treatment of impaired insulin secretion.     

E-cadherin and cell adhesion: a role in architecture and function in the pancreatic islet.

BACKGROUND/AIMS: The efficient secretion of insulin from beta-cells requires extensive intra-islet communication. The cell surface adhesion protein epithelial (E)-cadherin (ECAD) establishes and maintains epithelial tissues such as the islets of Langerhans. In this study, the role of ECAD in regulating insulin secretion from pseudoislets was investigated. METHODS: The effect of an immuno-neutralising ECAD on gross morphology, cytosolic calcium signalling, direct cell-to-cell communication and insulin secretion was assessed by fura-2 microfluorimetry, Lucifer Yellow dye injection and insulin ELISA in an insulin-secreting model system. RESULTS: Antibody blockade of ECAD reduces glucose-evoked changes in [Ca(2+)](i) and insulin secretion. Neutralisation of ECAD causes a breakdown in the glucose-stimulated synchronicity of calcium oscillations between discrete regions within the pseudoislet, and the transfer of dye from an individual cell within a cell cluster is attenuated in the absence of ECAD ligation, demonstrating that gap junction communication is disrupted. The functional consequence of neutralising ECAD is a significant reduction in insulin secretion. CONCLUSION: Cell adhesion via ECAD has distinct roles in the regulation of intercellular communication between beta-cells within islets, with potential repercussions for insulin secretion.     

Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet beta-cells: mediation by PLC and L-type Ca2+ channel and link to insulin release

It has long been thought that long-chain free fatty acids (FFAs) stimulate insulin secretion via mechanisms involving their metabolism in pancreatic beta-cells. Recently, it was reported that FFAs function as endogenous ligands for GPR40, a G protein-coupled receptor, to amplify glucose-stimulated insulin secretion in an insulinoma cell line and rat islets. However, signal transduction mechanisms for GPR40 in beta-cells are little known. The present study was aimed at elucidating GPR40-linked Ca(2+) signaling mechanisms in rat pancreatic beta-cells. We employed oleic acid (OA), an FFA that has a high affinity for the rat GPR40, and examined its effect on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in single beta-cells by fura 2 fluorescence imaging. OA at 1-10 microM concentration-dependently increased [Ca(2+)](i) in the presence of 5.6, 8.3, and 11.2 mM, but not 2.8 mM, glucose. OA-induced [Ca(2+)](i) increases at 11.2 mM glucose were inhibited in beta-cells transfected with small interfering RNA targeted to rat GPR40 mRNA. OA-induced [Ca(2+)](i) increases were also inhibited by phospholipase C (PLC) inhibitors, U73122 and neomycin, Ca(2+)-free conditions, and an L-type Ca(2+) channel blocker, nitrendipine. Furthermore, OA increased insulin release from isolated islets at 8.3 mM glucose, and it was markedly attenuated by PLC and L-type Ca(2+) channel inhibitors. These results demonstrate that OA interacts with GPR40 to increase [Ca(2+)](i) via PLC- and L-type Ca(2+) channel-mediated pathway in rat islet beta-cells, which may be link to insulin release.     

Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic beta-cells (MIN6)

We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001) J. Cell Biol. 155, 41-51) that ryanodine receptors (RyR) are present on insulin-containing secretory vesicles. Here we show that pancreatic islets and derived beta-cell lines express type I and II, but not type III, RyRs. Purified by subcellular fractionation and membrane immuno-isolation, dense core secretory vesicles were found to possess a similar level of type I RyR immunoreactivity as Golgi/endoplasmic reticulum (ER) membranes but substantially less RyR II than the latter. Monitored in cells expressing appropriately targeted aequorins, dantrolene, an inhibitor of RyR I channels, elevated free Ca(2+) concentrations in the secretory vesicle compartment from 40.1 +/- 6.7 to 90.4 +/- 14.8 microm (n = 4, p < 0.01), while having no effect on ER Ca(2+) concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca(2+)-mobilizing agent, decreased dense core secretory vesicle but not ER free Ca(2+) concentrations in permeabilized MIN6 beta-cells, and flash photolysis of caged NAADP released Ca(2+) from a thapsigargin-insensitive Ca(2+) store in single MIN6 cells. Because dantrolene strongly inhibited glucose-stimulated insulin secretion (from 3.07 +/- 0.51-fold stimulation to no significant glucose effect; n = 3, p < 0.01), we conclude that RyR I-mediated Ca(2+)-induced Ca(2+) release from secretory vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion.     

Co-ordinated Ca(2+)-signalling within pancreatic islets: does beta-cell entrainment require a secreted messenger

Isolated beta-cells are heterogeneous in sensory, biosynthetic and secretory capabilities, however, to enable efficient and appropriate secretion, cellular activity within the intact islet is synchronised. Historically, the entrainment of activity to a common pattern has been attributed to gap-junction mediated cell-to-cell communication. Although clearly influential, the possibility remains for other local synchronising mechanisms. In this study, we have used small clusters of insulin-secreting MIN6 cells to assess how contact-dependent, homotypic interactions between cells influences nutrient- and non-nutrient- evoked Ca(2+)-handling and insulin secretion, and to determine whether a secreted product plays a role in the synchronisation of oscillatory activity. Tolbutamide evoked a concentration-dependent recruitment of active cells within cell clusters, both in terms of numbers of cells and amplitude of the evoked Ca(2+)-response. The change in [Ca(2+)](i) was characteristically oscillatory above a mean elevated plateau, and was in phase between member cells of an individual cluster. Even at maximal concentrations (100 microM) some cells within a cluster responded before their immediate neighbours. Subsequent oscillatory behaviour then became entrained between member cells within that cluster. Inhibiting exocytosis using the microtubule inhibitors vincristine and nocodazole, or the adrenergic agent noradrenaline, did not prevent tolbutamide-evoked oscillatory changes in [Ca(2+)](i) but did reduce the probability of obtaining synchronous activity within an individual cluster. Above a threshold glucose concentration, the number of cells secreting insulin increased, without a commensurate change in secretory efficiency. This recruitment of cells secreting insulin mirrored Ca(2+) data that showed a glucose-dependent increase in cell number, without a change in the mean basal-to-peak change in [Ca(2+)](i). Together these data suggest that synchronised behaviour in MIN6 cells is dependent, in part, on a secreted factor that acts in a local paracrine fashion to recruit heterogeneous individual cellular activity into an organised group response.     

Prolonged culture in low glucose induces apoptosis of rat pancreatic beta-cells through induction of c-myc

Prolonged culture in low-glucose concentrations (相似文献   

Homotypic cell contact enhances insulin but not glucagon secretion

Intra-islet interactions influence beta-cell function, and disruption of islet architecture results in a reduction in glucose-induced insulin secretion, whereas re-aggregation improves secretory responsiveness. Our studies on MIN6 cells have shown that by configuring beta-cells as three-dimensional islet-like structures there is a marked improvement in glucose-induced insulin secretion compared to that of their monolayer equivalents. In the present study, we have used the mouse glucagon-secreting alphaTC1 cell line to see whether homotypic interactions are important in the regulation of glucagon secretion from alpha-cells. We found no significant difference in the secretory responses of alphaTC1 cells maintained as monolayers or as cell clusters. We also found that different cell adhesion molecules are involved in cell interactions between alpha- and beta-cells; MIN6 cells express ECAD, whereas alphaTC1 cells express NCAM. ECAD is necessary for cell cluster formation by MIN6 cells but not by alphaTC1 cells, whereas NCAM is not needed for the formation of cell clusters in either cell line.     

Blockage of cell-to-cell communication within pancreatic acini is associated with increased basal release of amylase

To assess whether junctional coupling is involved in the secretory activity of pancreatic acinar cells, dispersed rat acini were incubated for 30 min in the presence of either heptanol (3.5 mM) or octanol (1.0 mM). Exposure to either alkanol caused a marked uncoupling of the acinar cells which, in control acini, were extensively coupled. Uncoupling was associated with an increased basal release of amylase that was at least twice that of controls. By contrast, carbamylcholine (10(-5) M)-induced maximal amylase secretion, cytosolic pH, and free Ca2+, as well as the structure of gap junctions joining the acinar cells, were unaffected. Both uncoupling and the alteration of basal secretion were already observed after only 5 min of exposure to heptanol, they both persisted throughout the 30-min exposure to the alkanols, and were reversible after removal of either heptanol or octanol. Since neither of the two uncouplers appeared to alter unspecifically the secretory machinery and the nonjunctional membrane of acinar cells, the data are consistent with the view that junctional coupling participates in the control of the basal secretion of acinar cells.     

Bidirectional insulin granule turnover in the submembrane space during K(+) depolarization-induced secretion

Like primary mouse islets, MIN6 pseudoislets responded to the depolarization by 40 mm KCl and the resulting increase in the free cytosolic Ca(2+) concentration ([Ca(2+) ](i) ) with a massive increase in insulin secretion, whereas 15 mm KCl had little effect in spite of a clear increase in [Ca(2+) ](i) . Analysis of insulin-enhanced green fluorescent protein (EGFP)-labeled granules in MIN6 cells by total internal reflection fluorescence (TIRF) microscopy showed that 40 mm KCl increased the number of short-term resident granules (<1 second presence in the submembrane space), while the total granule number and the number of long-term resident granules decreased. The rates of granule arrival at and departure from the submembrane space changed in parallel and were two orders of magnitude higher than the release rates, suggesting a back-and-forth movement of the granules as the primary determinant of the submembrane granule number. The effect of 15 mm KCl resembled that of 40 mm but did not achieve significance. Both 15 and 40 mm KCl evoked a [Ca(2+) ](i) increase, which was antagonized by 10 μm nifedipine. Nifedipine also antagonized the effect on secretion and on granule number and mobility. In conclusion, during KCl depolarization L-type Ca(2+) channels seem to regulate two processes, insulin granule turnover in the submembrane space and granule exocytosis.     

The characterization of mitochondrial permeability transition in clonal pancreatic beta-cells. Multiple modes and regulation

Mitochondrial permeability transition (MPT), which contributes substantially to the regulation of normal mitochondrial metabolism, also plays a crucial role in the initiation of cell death. It is known that MPT is regulated in a tissue-specific manner. The importance of MPT in the pancreatic beta-cell is heightened by the fact that mitochondrial bioenergetics serve as the main glucose-sensing regulator and energy source for insulin secretion. In the present study, using MIN6 and INS-1 beta-cells, we revealed that both Ca(2+)-phosphate- and oxidant-induced MPT is remarkably different from other tissues. Ca(2+)-phosphate-induced transition is accompanied by a decline in mitochondrial reactive oxygen species production related to a significant potential dependence of reactive oxygen species formation in beta-cell mitochondria. Hydroperoxides, which are indirect MPT co-inducers active in liver and heart mitochondria, are inefficient in beta-cell mitochondria, due to the low mitochondrial ability to metabolize them. Direct cross-linking of mitochondrial thiols in pancreatic beta-cells induces the opening of a low conductance ion permeability of the mitochondrial membrane instead of the full scale MPT opening typical for liver mitochondria. Low conductance MPT is independent of both endogenous and exogenous Ca(2+), suggesting a novel type of nonclassical MPT in beta-cells. It results in the conversion of electrical transmembrane potential into DeltapH instead of a decrease in total protonmotive force, thus mitochondrial respiration remains in a controlled state. Both Ca(2+)- and oxidant-induced MPTs are phosphate-dependent and, through the "phosphate flush" (associated with stimulation of insulin secretion), are expected to participate in the regulation in beta-cell glucose-sensing and secretory activity.     

Ca(2+)-induced Ca(2+) release via inositol 1,4,5-trisphosphate receptors is amplified by protein kinase A and triggers exocytosis in pancreatic beta-cells

Hormones, such as glucagon and glucagon-like peptide-1, potently amplify nutrient stimulated insulin secretion by raising cAMP. We have studied how cAMP affects Ca(2+)-induced Ca(2+) release (CICR) in pancreatic beta-cells from mice and rats and the role of CICR in secretion. CICR was observed as pronounced Ca(2+) spikes on top of glucose- or depolarization-dependent rise of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)). cAMP-elevating agents strongly promoted CICR. This effect involved sensitization of the receptors underlying CICR, because many cells exhibited the characteristic Ca(2+) spiking at low or even in the absence of depolarization-dependent elevation of [Ca(2+)](i). The cAMP effect was mimicked by a specific activator of protein kinase A in cells unresponsive to activators of cAMP-regulated guanine nucleotide exchange factor. Ryanodine pretreatment, which abolishes CICR mediated by ryanodine receptors, did not prevent CICR. Moreover, a high concentration of caffeine, known to activate ryanodine receptors independently of Ca(2+), failed to mobilize intracellular Ca(2+). On the contrary, a high caffeine concentration abolished CICR by interfering with inositol 1,4,5-trisphosphate receptors (IP(3)Rs). Therefore, the cell-permeable IP(3)R antagonist 2-aminoethoxydiphenyl borate blocked the cAMP-promoted CICR. Individual CICR events in pancreatic beta-cells were followed by [Ca(2+)](i) spikes in neighboring human erythroleukemia cells, used to report secretory events in the beta-cells. The results indicate that protein kinase A-mediated promotion of CICR via IP(3)Rs is part of the mechanism by which cAMP amplifies insulin release.     

Synchronization and entrainment of cytoplasmic Ca2+ oscillations in cell clusters prepared from single or multiple mouse pancreatic islets

In contrast to pancreatic islets, isolated beta-cells stimulated by glucose display irregular and asynchronous increases in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)). Here, clusters of 5-30 cells were prepared from a single mouse islet or from pools of islets, loaded with fura-2, and studied with a camera-based system. [Ca(2+)](i) oscillations were compared in pairs of clusters by computing the difference in period and a synchronization index lambda. During perifusion with 12 mM glucose, the clusters exhibited regular [Ca(2+)](i) oscillations that were quasi-perfectly synchronized (Delta period of 1.4% and index lambda close to 1.0) between cells of each cluster. In contrast, separate clusters were not synchronized, even when prepared from one single islet. Pairs of clusters neighboring on the same coverslip were not better synchronized than pairs of clusters examined separately (distinct coverslips). We next attempted to synchronize clusters perifused with 12 mM glucose by applying external signals. A single pulse of 20 mM glucose, 10 mM amino acids, or 10 microM tolbutamide transiently altered [Ca(2+)](i) oscillations but did not reset the clusters to oscillate synchronously. On a background of 12 mM glucose, repetitive applications (1 min/5 min) of 10 microM tolbutamide, but not of 20 mM glucose, synchronized separate clusters. Our results identify a level of beta-cell heterogeneity intermediate between single beta-cells and the whole islet. They do not support the idea that substances released by islet cells serve as paracrine synchronizers. However, synchronization can be achieved by an external signal, if this signal has a sufficient strength to overwhelm the intrinsic rhythm of glucose-induced oscillations and is repetitively applied.     

Increased glucose sensitivity of both triggering and amplifying pathways of insulin secretion in rat islets cultured for 1 wk in high glucose

Chronic hyperglycemia has been shown to induce either a lack of response or an increased sensitivity to glucose in pancreatic beta-cells. We reinvestigated this controversial issue in a single experimental model by culturing rat islets for 1 wk in 10 or 30 mmol/l glucose (G10, Controls; or G30, High-glucose islets) before testing the effect of stepwise glucose stimulation from G0.5 to G20 on key beta-cell stimulus-secretion coupling events. Compared with Controls, the glucose sensitivity of High-glucose islets was markedly increased, leading to maximal stimulation of oxidative metabolism and both triggering and amplifying pathways of insulin secretion in G6 rather than G20, hence to loss of glucose effect above G6. This enhanced glucose sensitivity occurred despite an approximately twofold increase in islet uncoupling protein 2 mRNA expression. Besides this increased glucose sensitivity, the maximal glucose stimulation of insulin secretion in High-glucose islets was reduced by approximately 50%, proportionally to the reduction of insulin content. In High-glucose islets, changes in (45)Ca(2+) influx induced by glucose and diazoxide were qualitatively similar but quantitatively smaller than in Control islets and, paradoxically, did not lead to detectable changes in the intracellular Ca(2+) concentration measured by microspectrofluorimetry (fura PE 3). In conclusion, after 1 wk of culture in G30, the loss of glucose stimulation of insulin secretion in the physiological range of glucose concentrations (G5-G10) results from the combination of an increased sensitivity to glucose of both triggering and amplifying pathways of insulin secretion and an approximately 50% reduction in the maximal glucose stimulation of insulin secretion.     

Dynamics of glucose-induced membrane recruitment of protein kinase C beta II in living pancreatic islet beta-cells

The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet beta-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (betaII), novel (delta), or atypical (zeta) PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 beta-cells. Examined by laser scanning confocal or total internal reflection fluorescence microscopy, elevated glucose concentrations induced oscillatory translocations of PKCbetaII to spatially confined regions of the plasma membrane. Suggesting that increases in free cytosolic Ca(2+) concentrations ([Ca(2+)](c)) were primarily responsible, prevention of [Ca(2+)](c) increases with EGTA or diazoxide completely eliminated membrane recruitment, whereas elevation of cytosolic [Ca(2+)](c) with KCl or tolbutamide was highly effective in redistributing PKCbetaII both to the plasma membrane and to the surface of dense core secretory vesicles. By contrast, the distribution of PKCdelta.EGFP, which binds diacylglycerol but not Ca(2+), was unaffected by glucose. Measurement of [Ca(2+)](c) immediately beneath the plasma membrane with a ratiometric "pericam," fused to synaptic vesicle-associated protein-25, revealed that depolarization induced significantly larger increases in [Ca(2+)](c) in this domain. These data demonstrate that nutrient stimulation of beta-cells causes spatially and temporally complex changes in the subcellular localization of PKCbetaII, possibly resulting from the generation of Ca(2+) microdomains. Localized changes in PKCbetaII activity may thus have a role in the spatial control of insulin exocytosis.     

Attenuation of expression of gamma-glutamylcysteine synthetase by ribozyme transfection enhance insulin secretion by pancreatic beta cell line, MIN6

Low levels of intracellular antioxidant enzyme activities as well as glutathione (GSH) concentrations have been described in pancreatic beta cells. We examined the effects of intracellular GSH depletion on insulin secretion and the role of intracellular GSH in signal transduction in beta cell line, MIN6 cells. Anti-gamma-glutamylcysteine synthetase (gamma-GCS) heavy subunit ribozyme was stably transfected to MIN6 cells to reduce intracellular GSH concentration. In the presence of 10 mM glucose, ribozyme-transfected cells (RTC) increased insulin secretion from 0.58 microg/10(6) cells/h in control cells (CC) to 1.48 microg/10(6) cells/h. This was associated with increased intracellular Ca(2+) concentration in RTC, detected by fluo-3 staining. Our results demonstrated that intracellular GSH concentration might influence insulin secretion by MIN6 cells, and suggest that enhanced insulin secretion by beta cells conditioned by chronic depletion of GSH is mediated by increased intracellular Ca(2+) concentration.     

Regulation of cAMP dynamics by Ca2+ and G protein-coupled receptors in the pancreatic beta-cell: a computational approach

In this report we describe a mathematical model for the regulation of cAMP dynamics in pancreatic beta-cells. Incretin hormones such as glucagon-like peptide 1 (GLP-1) increase cAMP and augment insulin secretion in pancreatic beta-cells. Imaging experiments performed in MIN6 insulinoma cells expressing a genetically encoded cAMP biosensor and loaded with fura-2, a calcium indicator, showed that cAMP oscillations are differentially regulated by periodic changes in membrane potential and GLP-1. We modeled the interplay of intracellular calcium (Ca(2+)) and its interaction with calmodulin, G protein-coupled receptor activation, adenylyl cyclases (AC), and phosphodiesterases (PDE). Simulations with the model demonstrate that cAMP oscillations are coupled to cytoplasmic Ca(2+) oscillations in the beta-cell. Slow Ca(2+) oscillations (<1 min(-1)) produce low-frequency cAMP oscillations, and faster Ca(2+) oscillations (>3-4 min(-1)) entrain high-frequency, low-amplitude cAMP oscillations. The model predicts that GLP-1 receptor agonists induce cAMP oscillations in phase with cytoplasmic Ca(2+) oscillations. In contrast, observed antiphasic Ca(2+) and cAMP oscillations can be simulated following combined glucose and tetraethylammonium-induced changes in membrane potential. The model provides additional evidence for a pivotal role for Ca(2+)-dependent AC and PDE activation in coupling of Ca(2+) and cAMP signals. Our results reveal important differences in the effects of glucose/TEA and GLP-1 on cAMP dynamics in MIN6 beta-cells.     

The elevation of glutamate content and the amplification of insulin secretion in glucose-stimulated pancreatic islets are not causally related

Glucose increases insulin secretion by raising cytoplasmic Ca(2+) ([Ca(2+)](i)) in beta-cells (triggering pathway) and augmenting the efficacy of Ca(2+) on exocytosis (amplifying pathway). It has been suggested that glutamate formed from alpha-ketoglutarate is a messenger of the amplifying pathway (Maechler, P., and Wollheim, C. B. (1999) Nature 402, 685-689). This hypothesis was tested with mouse islets depolarized with 30 mm KCl (+ diazoxide) or with a saturating concentration of sulfonylurea. Because [Ca(2+)](i) was elevated under these conditions, insulin secretion was stimulated already in 0 mm glucose. The amplification of secretion produced by glucose was accompanied by an increase in islet glutamate. However, glutamine (0.5-2 mm) markedly augmented islet glutamate without affecting insulin secretion, whereas glucose augmented secretion without influencing glutamate levels when these were elevated by glutamine. Allosteric activation of glutamate dehydrogenase by BCH (2-amino 2-norbornane carboxylic acid) lowered islet glutamate but increased insulin secretion. Similar insulin secretion thus occurred at very different cellular glutamate levels. Glutamine did not affect islet [Ca(2+)](i) and pH(i), whereas glucose and BCH slightly raised pH(i) and either slightly decreased (30 mm KCl) or increased (tolbutamide) [Ca(2+)](i). The general dissociation between changes in islet glutamate and insulin secretion refutes a role of beta-cell glutamate in the amplification of insulin secretion by glucose.     

Inhibition of Kv2.1 voltage-dependent K+ channels in pancreatic beta-cells enhances glucose-dependent insulin secretion

Voltage-dependent (Kv) outward K(+) currents repolarize beta-cell action potentials during a glucose stimulus to limit Ca(2+) entry and insulin secretion. Dominant-negative "knockout" of Kv2 family channels enhances glucose-stimulated insulin secretion. Here we show that a putative Kv2.1 antagonist (C-1) stimulates insulin secretion from MIN6 insulinoma cells in a glucose- and dose-dependent manner while blocking voltage-dependent outward K(+) currents. C-1-blocked recombinant Kv2.1-mediated currents more specifically than currents mediated by Kv1, -3, and -4 family channels (Kv1.4, 3.1, 4.2). Additionally, C-1 had little effect on currents recorded from MIN6 cells expressing a dominant-negative Kv2.1 alpha-subunit. The insulinotropic effect of acute Kv2.1 inhibition resulted from enhanced membrane depolarization and augmented intracellular Ca(2+) responses to glucose. Immunohistochemical staining of mouse pancreas sections showed that expression of Kv2.1 correlated highly with insulin-containing beta-cells, consistent with the ability of C-1 to block voltage-dependent outward K(+) currents in isolated mouse beta-cells. Antagonism of Kv2.1 in an ex vivo perfused mouse pancreas model enhanced first- and second-phase insulin secretion, whereas glucagon secretion was unaffected. The present study demonstrates that Kv2.1 is an important component of beta-cell stimulus-secretion coupling, and a compound that enhances, but does not initiate, beta-cell electrical activity by acting on Kv2.1 would be a useful antidiabetic agent.