Anchored phosphatases modulate glucose homeostasis.

Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic β-cells involves ion channels and mobilization of Ca(2+) and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-β-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca(2+) currents, and attenuates cytoplasmic accumulation of Ca(2+) and cAMP in β-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity

Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes. Pancreatic islets derived from stem cells are benchmarked against primary cells.Peer reviewe

Purinergic P2Y1 receptors take centre stage in autocrine stimulation of human beta cells

Insulin secretory vesicles contain high concentrations of adenine nucleotides, which are co-released with insulin during exocytosis. There is strong evidence that ATP and ADP serve as autocrine messengers in pancreatic beta cells, but the functional effects and detailed mechanisms of action are under debate. In this issue of Diabetologia, Khan and colleagues (DOI: 10.1007/s00125-014-3368-8 ) present the results of their study of autocrine purinergic signalling in isolated human beta cells. Using a combination of electrophysiological techniques, Ca(2+) imaging and measurements of insulin secretion, it is demonstrated that voltage-dependent Ca(2+) influx triggers release of ATP/ADP, which activates purinergic receptors of the Gq/11-coupled P2Y1 isoform. Activation of these receptors leads to membrane depolarisation and phospholipase C-mediated mobilisation of Ca(2+) from endoplasmic reticulum stores, which amplifies the exocytosis-triggering Ca(2+) signal. In contrast, there is little evidence for involvement of ionotropic P2X receptors in the autocrine stimulation of human beta cells. This commentary discusses these findings as well as various functional and therapeutic implications of the complex purinergic signalling network in the pancreatic islet

Autocrine Signaling Underlies Fast Repetitive Plasma Membrane Translocation of Conventional and Novel Protein Kinase C Isoforms in beta Cells

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 (<10 s) local DAG elevations ("spiking") at the plasma membrane because of autocrine activation of P2Y(1), purinoceptors by ATP co-released with insulin. Using total internal reflection microscopy, fluorescent protein -tagged PKCs, and signaling biosensors, we investigated whether DAG spiking causes membrane recruitment of PKCs and whether different classes of PKCs show characteristic responses. Glucose stimulation of MINE cells triggered DAG spiking with concomitant repetitive translocation of the novel isoforms PKCI, PKCE, and PKCirp The conventional PKCa, PKCI3I, and PKC beta II isoforms showed a more complex pattern with both rapid and slow translocation. K+ depolarization-induced PKCE translocation entirely mirrored DAG spiking, whereas PKC beta 1 translocation showed a sustained component, reflecting the subplasma membrane Ca2+ concentration ([Ca2+)pm), with additional effect during DAG spikes. Interference with DAG spiking by purinoceptor inhibition prevented intermittent translocation of PKCs and reduced insulin secretion but did not affect [Ca2+]{,1 elevation or sustained PKCAI translocation. The muscarinic agonist carbachol induced pronounced transient PKCi3I translocation and sustained recruitment of PKCE. When rise of [Ca2+](p), was prevented, the carbachol-induced DAG and PKCE responses were somewhat reduced, but PKCI3I translocation was completely abolished. We conclude that exocytosis-induced DAG spikes efficiently recruit both conventional and novel PKCs to the beta cell plasma membrane. PKC signaling is thus implicated in autocrine regulation of beta cell function

Oscillations of sub-membrane ATP in glucose-stimulated beta cells depend on negative feedback from Ca2+

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. 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. 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). 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

Feedback activation of phospholipase C via intracellular mobilization and store-operated influx of Ca2+ in insulin-secreting β-cells

Phospholipase C (PLC) regulates various cellular processes by catalyzing the formation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we have investigated the influence of Ca2+ on receptor-triggered PLC activity in individual insulin-secreting β-cells. Evanescent wave microscopy was used to record PLC activity using green fluorescent protein (GFP)-tagged PIP2/IP3-binding pleckstrin homology domain from PLCδ1, and the cytoplasmic Ca2+ concentration ([Ca2+]i) was simultaneously measured using the indicator Fura Red. Stimulation of MIN6 β-cells with the muscarinic-receptor agonist carbachol induced rapid and sustained PLC activation. By contrast, only transient activation was observed after stimulation in the absence of extracellular Ca2+ or in the presence of the non-selective Ca2+ channel inhibitor La3+. The Ca2+-dependent sustained phase of PLC activity did not require voltage-gated Ca2+ influx, as hyperpolarization with diazoxide or direct Ca2+ channel blockade with nifedipine had no effect. Instead, the sustained PLC activity was markedly suppressed by the store-operated channel inhibitors 2-APB and SKF96365. Depletion of intracellular Ca2+ stores with the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitors thapsigargin or cyclopiazonic acid abolished Ca2+ mobilization in response to carbachol, and strongly suppressed the PLC activation in Ca2+-deficient medium. Analogous suppressions were observed after loading cells with the Ca2+ chelator BAPTA. Stimulation of primary mouse pancreatic β-cells with glucagon elicited pronounced [Ca2+]i spikes, reflecting protein kinase A-mediated activation of Ca2+-induced Ca2+ release via IP3 receptors. These [Ca2+]i spikes were found to evoke rapid and transient activation of PLC. Our data indicate that receptor-triggered PLC activity is enhanced by positive feedback from Ca2+ entering the cytoplasm from intracellular stores and via store-operated channels in the plasma membrane. Such amplification of receptor signalling should be important in the regulation of insulin secretion by hormones and neurotransmitters

Glucose-induced cAMP elevation in β-cells involves amplification of constitutive and glucagon-activated GLP-1 receptor signalling

AIM: cAMP typically signals downstream of Gs -coupled receptors and regulates numerous cell functions. In β-cells, cAMP amplifies Ca2+ -triggered exocytosis of insulin granules. Glucose-induced insulin secretion is associated with Ca2+ - and metabolism-dependent increases of the sub-plasma-membrane cAMP concentration ([cAMP]pm ) in β-cells, but potential links to canonical receptor signalling are unclear. The aim of this study was to clarify the role of glucagon-like peptide-1 receptors (GLP1Rs) for glucose-induced cAMP signalling in β-cells. METHODS: Total internal reflection microscopy and fluorescent reporters were used to monitor changes in cAMP, Ca2+ and ATP concentrations as well as insulin secretion in MIN6 cells and mouse and human β-cells. Insulin release from mouse and human islets was also measured with ELISA. RESULTS: The GLP1R antagonist exendin-(9-39) (ex-9) prevented both GLP1- and glucagon-induced elevations of [cAMP]pm , consistent with GLP1Rs being involved in the action of glucagon. This conclusion was supported by lack of unspecific effects of the antagonist in a reporter cell-line. Ex-9 also suppressed IBMX- and glucose-induced [cAMP]pm elevations. Depolarization with K+ triggered Ca2+ -dependent [cAMP]pm elevation, an effect that was amplified by high glucose. Ex-9 inhibited both the Ca2+ and glucose-metabolism-dependent actions on [cAMP]pm . The drug remained effective after minimizing paracrine signalling by dispersing the islets and it reduced basal [cAMP]pm in a cell-line heterologously expressing GLP1Rs, indicating that there is constitutive GLP1R signalling. The ex-9-induced reduction of [cAMP]pm in glucose-stimulated β-cells was paralleled by suppression of insulin secretion. CONCLUSION: Agonist-independent and glucagon-stimulated GLP1R signalling in β-cells contributes to basal and glucose-induced cAMP production and insulin secretion

Glucose controls glucagon secretion by directly modulating cAMP in alpha cells

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