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Aims/hypothesis Glucagon is critical for normal glucose homeostasis and aberrant secretion of the hormone aggravates dysregulated glucose control in diabetes. However, the mechanisms by which glucose controls glucagon secretion from pancreatic alpha cells remain elusive. The aim of this study was to investigate the role of the intracellular messenger cAMP in alpha-cell-intrinsic glucose regulation of glucagon release. Methods Subplasmalemmal cAMP and Ca2+ concentrations were recorded in isolated and islet-located alpha cells using fluorescent reporters and total internal reflection microscopy. Glucagon secretion from mouse islets was measured using ELISA. Results Glucose induced Ca2+-independent alterations of the subplasmalemmal cAMP concentration in alpha cells that correlated with changes in glucagon release. Glucose-lowering-induced stimulation of glucagon secretion thus corresponded to an elevation in cAMP that was independent of paracrine signalling from insulin or somatostatin. Imposed cAMP elevations stimulated glucagon secretion and abolished inhibition by glucose elevation, while protein kinase A inhibition mimicked glucose suppression of glucagon release. Conclusions/interpretation Glucose concentrations in the hypoglycaemic range control glucagon secretion by directly modulating the cAMP concentration in alpha cells independently of paracrine influences. These findings define a novel mechanism for glucose regulation of glucagon release that underlies recovery from hypoglycaemia and may be disturbed in diabetes. Electronic supplementary material The online version of this article (10.1007/s00125-019-4857-6) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- (Rapgef4-/-) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis., eLife digest Insulin is the hormone that signals to the body to take up sugar from the blood. Specialized cells in the pancreas – known as β-cells – release insulin after a meal. Before that, insulin molecules are stored in tiny granules inside the β-cells; these granules must fuse with the cells’ surface membranes to release their contents. The first step in this process creates a narrow pore that allows small molecules, but not the larger insulin molecules, to seep out. The pore then widens to release the insulin. Since the small molecules are known to act locally in the pancreas, it is possible that this “molecular sieve” is biologically important. Yet it is not clear how the pore widens. One of the problems for people with type 2 diabetes is that they release less insulin into the bloodstream. Two kinds of drugs used to treat these patients work by stimulating β-cells to release their insulin. One way to achieve this is by raising the levels of a small molecule called cAMP, which is well known to help prepare insulin granules for release. The cAMP molecule also seems to slow the widening of the pore, and Gucek et al. have now investigated how this happens at a molecular level. By observing individual granules of human β-cells using a special microscope, Gucek et al. could watch how different drugs affect pore widening and content release. They also saw that cAMP activated a protein called Epac2, which then recruited two other proteins – amisyn and dynamin – to the small pores. These two proteins together then closed the pore, rather than expanding it to let insulin out. Type 2 diabetes patients sometimes have high levels of amisyn in their β-cells, which could explain why they do not release enough insulin. The microscopy experiments also revealed that two common anti-diabetic drugs activate Epac2 and prevent the pores from widening, thereby counteracting their positive effect on insulin release. The combined effect is likely a shift in the balance between insulin and the locally acting small molecules. These findings suggest that two common anti-diabetic drugs activate a common mechanism that may lead to unexpected outcomes, possibly even reducing how much insulin the β-cells can release. Future studies in mice and humans will have to investigate these effects in whole organisms.

PKC signaling has been implicated in the regulation of many cell functions, including metabolism, cell death, proliferation, and secretion. Activation of conventional and novel PKC isoforms is associated with their Ca2+- and/or diacylglycerol (DAG)-dependent translocation to the plasma membrane. In 13 cells, exocytosis of insulin granules evokes brief (

The Src homology-2 domain containing protein B (SHB) has previously been shown to function as a pleiotropic adapter protein, conveying signals from receptor tyrosine kinases to intracellular signaling intermediates. The overexpression ofShbin β-cells promotes β-cell proliferation by increased insulin receptor substrate (IRS) and focal adhesion kinase (FAK) activity, whereasShbdeficiency causes moderate glucose intolerance and impaired first-peak insulin secretion. Using an array of techniques, including live-cell imaging, patch-clamping, immunoblotting, and semi-quantitative PCR, we presently investigated the causes of the abnormal insulin secretory characteristics inShb-knockout mice.Shb-knockout islets displayed an abnormal signaling signature with increased activities of FAK, IRS, and AKT. β-catenin protein expression was elevated and it showed increased nuclear localization. However, there were no major alterations in the gene expression of various proteins involved in the β-cell secretory machinery. Nor wasShbdeficiency associated with changes in glucose-induced ATP generation or cytoplasmic Ca2+handling. In contrast, the glucose-induced rise in cAMP, known to be important for the insulin secretory response, was delayed in theShb-knockout compared with WT control. Inhibition of FAK increased the submembrane cAMP concentration, implicating FAK activity in the regulation of insulin exocytosis. In conclusion,Shbdeficiency causes a chronic increase in β-cell FAK activity that perturbs the normal insulin secretory characteristics of β-cells, suggesting multi-faceted effects of FAK on insulin secretion depending on the mechanism of FAK activation.

Aims/hypothesis Beta cells situated in the islet of Langerhans respond more vigorously to glucose than do dissociated beta cells. Mechanisms for this discrepancy were studied by comparing insulin-producing MIN6 cells aggregated into pseudoislets with MIN6 monolayer cells and mouse and human islets. Methods MIN6 monolayers, pseudoislets and mouse and human islets were exposed to glucose, α-ketoisocaproic acid (KIC), pyruvate, KIC plus glutamine and the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 or wortmannin. Insulin secretion (ELISA), cytoplasmic Ca2+ concentration ([Ca2+]c; microfluorometry), glucose oxidation (radiolabelling), the expression of genes involved in mitochondrial metabolism (PCR) and the phosphorylation of insulin receptor signalling proteins (western blotting) were measured. Results Insulin secretory responses to glucose, pyruvate, KIC and glutamine were higher in pseudoislets than monolayers and comparable to those of human islets. Glucose oxidation and genes for mitochondrial metabolism were upregulated in pseudoislets compared with single cells and monolayers, respectively. Phosphorylation at the inhibitory S636/639 site of IRS-1 was significantly higher in monolayers and dispersed human and mouse cells than pseudoislets and intact human and mouse islets. PI3K inhibition only slightly attenuated glucose-stimulated insulin secretion from monolayers, but substantially reduced that from pseudoislets and human and mouse islets without suppressing the glucose-induced [Ca2+]c response. Conclusions/interpretation We propose that islet architecture is critical for proper beta cell mitochondrial metabolism and IRS-1 signalling, and that PI3K regulates insulin secretion at a step distal to the elevation of [Ca2+]c. Electronic supplementary material The online version of this article (doi:10.1007/s00125-013-2903-3) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Vascular permeability is a hallmark response to the main angiogenic factor VEGF-A and we have previously described a reduction of this response in Shb knockout mice. To characterize the molecular mechanisms responsible for this effect, endothelial cells were isolated from lungs and analyzed in vitro. Shb deficient endothelial cells exhibited less migration in a scratch wound-healing assay both under basal conditions and after vascular endothelial growth factor-A (VEGF-A) stimulation, suggesting a functional impairment of these cells in vitro. Staining for VE-cadherin and vascular endothelial growth factor receptor-2 (VEGFR-2) showed co-localization in adherens junctions and in intracellular sites such as the perinuclear region in wild-type and Shb knockout cells. VEGF-A decreased the VE-cadherin/VEGFR-2 co-localization in membrane structures resembling adherens junctions in wild-type cells whereas no such response was noted in the Shb knockout cells. VE-cadherin/VEGFR-2 co-localization was also recorded using spinning-disc confocal microscopy and VEGF-A caused a reduced association in the wild-type cells whereas the opposite pattern was observed in the Shb knockout cells. The latter expressed slightly more of cell surface VEGFR-2. VEGF-A stimulated extracellular-signal regulated kinase, Akt and Rac1 activities in the wild-type cells whereas no such responses were noted in the knockout cells. We conclude that aberrant signaling characteristics with respect to ERK, Akt and Rac1 are likely explanations for the observed altered pattern of VE-cadherin/VEGFR-2 association. The latter is important for understanding the reduced in vivo vascular permeability response in Shb knockout mice, a phenomenon that has patho-physiological relevance. De 2 första författarna delar förstaförfattarskapet.

Aims/hypothesis ATP links changes in glucose metabolism to electrical activity, Ca2+ signalling and insulin secretion in pancreatic beta cells. There is evidence that beta cell metabolism oscillates, but little is known about ATP dynamics at the plasma membrane, where regulation of ion channels and exocytosis occur. Methods The sub-plasma-membrane ATP concentration ([ATP]pm) was recorded in beta cells in intact mouse and human islets using total internal reflection microscopy and the fluorescent reporter Perceval. Results Glucose dose-dependently increased [ATP]pm with half-maximal and maximal effects at 5.2 and 9 mmol/l, respectively. Additional elevations of glucose to 11 to 20 mmol/l promoted pronounced [ATP]pm oscillations that were synchronised between neighbouring beta cells. [ATP]pm increased further and the oscillations disappeared when voltage-dependent Ca2+ influx was prevented. In contrast, K+-depolarisation induced prompt lowering of [ATP]pm. Simultaneous recordings of [ATP]pm and the sub-plasma-membrane Ca2+ concentration ([Ca2+]pm) during the early glucose-induced response revealed that the initial [ATP]pm elevation preceded, and was temporarily interrupted by the rise of [Ca2+]pm. During subsequent glucose-induced oscillations, the increases of [Ca2+]pm correlated with lowering of [ATP]pm. Conclusions/interpretation In beta cells, glucose promotes pronounced oscillations of [ATP]pm, which depend on negative feedback from Ca2+. The bidirectional interplay between these messengers in the sub-membrane space generates the metabolic and ionic oscillations that underlie pulsatile insulin secretion. Electronic supplementary material The online version of this article (doi:10.1007/s00125-013-2894-0) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

The events leading to the activation of store-operated Ca2+ entry (SOCE) involve Ca2+ depletion of the endoplasmic reticulum (ER) resulting in translocation of the transmembrane Ca2+ sensor protein, stromal interaction molecule 1 (STIM1), to the junctions between ER and the plasma membrane where it binds to the Ca2+ channel protein Orai1 to activate Ca2+ influx. Using confocal and total internal reflection fluorescence microscopy, we studied redistribution kinetics of fluorescence-tagged STIM1 and Orai1 as well as SOCE in insulin-releasing beta-cells and glucagon-secreting alpha-cells within intact mouse and human pancreatic islets. ER Ca2+ depletion triggered accumulation of STIM1 puncta in the subplasmalemmal ER where they co-clustered with Orai1 in the plasma membrane and activated SOCE. Glucose, which promotes Ca2+ store filling and inhibits SOCE, stimulated retranslocation of STIM1 to the bulk ER. This effect was evident at much lower glucose concentrations in alpha-than in beta-cells consistent with involvement of SOCE in the regulation of glucagon secretion. Epinephrine stimulated subplasmalemmal translocation of STIM1 in beta-cells and retranslocation in beta-cells involving raising and lowering of cAMP, respectively. The cAMP effect was mediated both by protein kinase A and exchange protein directly activated by cAMP. However, the cAMP-induced STIM1 puncta did not co-cluster with Orai1, and there was no activation of SOCE. STIM1 translocation can consequently occur independently of Orai1 clustering and SOCE.