EphA4 Receptor Forward Signaling Inhibits Glucagon Secretion From α-Cells.
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ABSTRACT:
SUBMITTER: Hutchens T
PROVIDER: S-EPMC4613968 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cytoplasmic ATP and Ca(2+) are implicated in current models of glucose's control of glucagon and insulin secretion from pancreatic α- and β-cells, respectively, but little is known about ATP and its relation to Ca(2+) in α-cells. We therefore expressed the fluorescent ATP biosensor Perceval in mouse pancreatic islets and loaded them with a Ca(2+) indicator. With total internal reflection fluorescence microscopy, we recorded subplasma membrane concentrations of Ca(2+) and ATP ([Ca(2+)]pm; [ATP]pm) in superficial α- and β-cells of intact islets and related signaling to glucagon and insulin secretion by immunoassay. Consistent with ATP's controlling glucagon and insulin secretion during hypo- and hyperglycemia, respectively, the dose-response relationship for glucose-induced [ATP]pm generation was left shifted in α-cells compared to β-cells. Both cell types showed [Ca(2+)]pm and [ATP]pm oscillations in opposite phase, probably reflecting energy-consuming Ca(2+) transport. Although pulsatile insulin and glucagon release are in opposite phase, [Ca(2+)]pm synchronized in the same phase between α- and β-cells. This paradox can be explained by the overriding of Ca(2+) stimulation by paracrine inhibition, because somatostatin receptor blockade potently stimulated glucagon release with little effect on Ca(2+). The data indicate that an α-cell-intrinsic mechanism controls glucagon in hypoglycemia and that paracrine factors shape pulsatile secretion in hyperglycemia.
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Project description:Glucagon receptor (GCGR) is a potential target for diabetes therapy. Several emerging GCGR antagonism-based therapies are under pre-clinical and clinical development. However, the GCGR antagonism as well as GCGR deficient animal accompanied with α-cell hyperplasia and hyperglucagonemia, which may limit the application of GCGR antagonism. To better understand the physiological changes in the α cells during the GCGR disruption, we performed the single cell sequencing of α cells isolated from control and gcgr-/- zebrafish. We found that α cells in gcgr-/- zebrafish dramatically increased glucagon (both gcga and gcgb) expression, we also found that several transcriptional factors that regulate glucagon expression were also increased. Based on the sequencing data, we further experimentally confirmed that gcgr-/- up-regulated glucagon mRNA level by in situ hybridization, and the gcgr-/- increased glucagon promoter activity indicated by reporter line Tg(gcga: GFP). Moreover, our results also revealed that α cells increased glucagon granule population and glucagon level in gcgr-/- zebrafish. These data suggested that hyperglucagonemia in the organism of GCGR antagonism not only contributed by the α-cell hyperplasia but also contributed by the increased glucagon expression and secretion from α cells. Our study provided more comprehensive understanding of physiological changes of α-cell during the GCGR disruption.
2022-10-26 | GSE179894 | GEO
Project description:Inhibition of glucagon hypersecretion from pancreatic α-cells is an appealing strategy for the treatment of diabetes. Our hypothesis is that proteins that associate with glucagon within alpha cell secretory granules will regulate glucagon secretion, and may provide druggable targets for controlling abnormal glucagon secretion in diabetes. Recently, we identified a dynamic glucagon interactome within the secretory granules of the α cell line, αTC1-6, and showed that select proteins within the interactome could modulate glucagon secretion. In the present study, we show that one of these interactome proteins, the neuronal protein stathmin-2, is expressed in αTC1-6 cells and in mouse pancreatic alpha cells, and is a novel regulator of glucagon secretion. The secretion of both glucagon and Stmn2 was significantly enhanced in response to 55 mM K+, and immunofluorescence confocal microscopy showed co-localization of stathmin-2 with glucagon and the secretory granule markers chromogranin A and VAMP-2 in αTC1-6 cells. In mouse pancreatic islets, Stathmin-2 co-localized with glucagon, but not with insulin, and co-localized with secretory pathway markers. To show a function for stathmin-2 in regulating glucagon secretion, we showed that siRNA-mediated depletion of stathmin-2 in αTC1-6 cells caused glucagon secretion to become constitutive without any effect on proglucagon mRNA levels, while overexpression of stathmin-2 completely abolished both basal and K+-stimulated glucagon secretion. Overexpression of stathmin-2 increased the localization of glucagon into the endosomal-lysosomal compartment, while depletion of stathmin-2 reduced the endosomal localization of glucagon. Therefore, we describe stathmin-2 as having a novel role as an alpha cell secretory granule protein that modulates glucagon secretion via trafficking through the endosomal-lysosomal system. These findings describe a potential new pathway for the regulation of glucagon secretion, and may have implications for controlling glucagon hypersecretion in diabetes.
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Project description:Previous work has demonstrated that the peptide hormone ghrelin raises blood glucose. Such has been attributed to ghrelin's ability to enhance GH secretion, restrict insulin release, and/or reduce insulin sensitivity. Ghrelin's reported effects on glucagon have been inconsistent. Here, both animal- and cell-based systems were used to determine the role of glucagon in mediating ghrelin's effects on blood glucose. The tissue and cell distribution of ghrelin receptors (GHSR) was evaluated by quantitative PCR and histochemistry. Plasma glucagon levels were determined following acute acyl-ghrelin injections and in pharmacological and/or transgenic mouse models of ghrelin overexpression and GHSR deletion. Isolated mouse islets and the α-cell lines αTC1 and InR1G9 were used to evaluate ghrelin's effects on glucagon secretion and the role of calcium and ERK in this activity. GHSR mRNA was abundantly expressed in mouse islets and colocalized with glucagon in α-cells. Elevation of acyl-ghrelin acutely (after sc administration, such that physiologically relevant plasma ghrelin levels were achieved) and chronically (by slow-releasing osmotic pumps and as observed in transgenic mice harboring ghrelinomas) led to higher plasma glucagon and increased blood glucose. Conversely, genetic GHSR deletion was associated with lower plasma glucagon and reduced fasting blood glucose. Acyl-ghrelin increased glucagon secretion in a dose-dependent manner from mouse islets and α-cell lines, in a manner requiring elevation of intracellular calcium and phosphorylation of ERK. Our study shows that ghrelin's regulation of blood glucose involves direct stimulation of glucagon secretion from α-cells and introduces the ghrelin-glucagon axis as an important mechanism controlling glycemia under fasting conditions.
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Project description:The insulin resistance state of pancreatic α-cells seems to be related to glucagon hypersecretion in type 2 diabetes. Treatment that can improve the insulin sensitivity of α-cells could control glucagon levels in patients with diabetes mellitus. The aim of this study was to investigate the preventive role of D-chiro-inositol (DCI), which has insulin receptor-sensitizer effects on insulin signaling pathways and glucagon secretion in pancreatic α-TC1 clone 6 cells. Cells were chronically treated with palmitate to induce insulin resistance in the presence/absence of DCI. DCI treatment improved the insulin signaling pathway and restored insulin-mediated glucagon suppression in α-TC1-6 cells exposed to palmitate. These results indicate that DCI treatment prevents the insulin resistance of α-TC1-6 cells chronically exposed to palmitate. Our data provide evidence that DCI could be useful to improve the insulin sensitivity of pancreatic α-cells in diabetes treatment.
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Project description:Glucose-dependent insulinotropic polypeptide (GIP) is an intestinally derived peptide that is secreted in response to feeding. The GIP receptor (GIPR) is expressed in many cell types involved in the regulation of metabolism, including α- and β-cells. Glucagon and insulin exert tremendous control over glucose metabolism. Thus, GIP action in islets strongly dictates metabolic control in the postprandial state. Loss of GIPR activity in β-cells is a characteristic of type 2 diabetes (T2D) which associates with reduced postprandial insulin secretion and hyperglycemia. Less is known about GIPR activity in α-cells or the control of glucagon secretion. GIP stimulates glucagon secretion in a glucose-dependent manner in healthy people, with enhanced activity at lower glycemia. However, GIP stimulates glucagon secretion even at hyperglycemia in people with T2D, suggesting that inappropriate GIPR activity in α-cells contributes to the pathogenesis of T2D. Here, we review the literature describing GIP action and GIPR activity in the α-cell, detailing the basic science that has shaped the view of how GIP regulates glucagon secretion. We also contrast the effects of GIP on glucagon secretion in healthy and T2D people. Finally, we contextualize these observations in light of recent work that redefines the role of glucagon in glucose homeostasis, suggesting that hyperglucagonemia per se does not drive hyperglycemia. As new medications for T2D that incorporate GIPR activity are being developed, it is clear that a better understanding of GIPR activity beyond the β-cell is necessary. This work highlights the importance of focusing on the GIPR in α-cells.
| S-EPMC7580028 | biostudies-literature
Project description:ObjectiveTo document the properties of the voltage-gated ion channels in human pancreatic alpha-cells and their role in glucagon release.Research design and methodsGlucagon release was measured from intact islets. [Ca(2+)](i) was recorded in cells showing spontaneous activity at 1 mmol/l glucose. Membrane currents and potential were measured by whole-cell patch-clamping in isolated alpha-cells identified by immunocytochemistry.ResultGlucose inhibited glucagon secretion from human islets; maximal inhibition was observed at 6 mmol/l glucose. Glucagon secretion at 1 mmol/l glucose was inhibited by insulin but not by ZnCl(2). Glucose remained inhibitory in the presence of ZnCl(2) and after blockade of type-2 somatostatin receptors. Human alpha-cells are electrically active at 1 mmol/l glucose. Inhibition of K(ATP)-channels with tolbutamide depolarized alpha-cells by 10 mV and reduced the action potential amplitude. Human alpha-cells contain heteropodatoxin-sensitive A-type K(+)-channels, stromatoxin-sensitive delayed rectifying K(+)-channels, tetrodotoxin-sensitive Na(+)-currents, and low-threshold T-type, isradipine-sensitive L-type, and omega-agatoxin-sensitive P/Q-type Ca(2+)-channels. Glucagon secretion at 1 mmol/l glucose was inhibited by 40-70% by tetrodotoxin, heteropodatoxin-2, stromatoxin, omega-agatoxin, and isradipine. The [Ca(2+)](i) oscillations depend principally on Ca(2+)-influx via L-type Ca(2+)-channels. Capacitance measurements revealed a rapid (<50 ms) component of exocytosis. Exocytosis was negligible at voltages below -20 mV and peaked at 0 mV. Blocking P/Q-type Ca(2+)-currents abolished depolarization-evoked exocytosis.ConclusionsHuman alpha-cells are electrically excitable, and blockade of any ion channel involved in action potential depolarization or repolarization results in inhibition of glucagon secretion. We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.
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Project description:Aims/hypothesisGlucagon 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.MethodsSubplasmalemmal 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.ResultsGlucose 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/interpretationGlucose 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.
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Project description:Eph receptors are widely expressed during cerebral cortical development, yet a role for Eph signaling in the generation of cells during corticogenesis has not been shown. Cortical progenitor cells selectively express one receptor, EphA4, and reducing EphA4 signaling in cultured progenitors suppressed proliferation, decreasing cell number. In vivo, EphA4(-/-) cortex had a reduced area, fewer cells and less cell division compared with control cortex. To understand the effects of EphA4 signaling in corticogenesis, EphA4-mediated signaling was selectively depressed or elevated in cortical progenitors in vivo. Compared with control cells, cells with reduced EphA4 signaling were rare and mitotically inactive. Conversely, overexpression of EphA4 maintained cells in their progenitor states at the expense of subsequent maturation, enlarging the progenitor pool. These results support a role for EphA4 in the autonomous promotion of cell proliferation during corticogenesis. Although most ephrins were undetectable in cortical progenitors, ephrin B1 was highly expressed. Our analyses demonstrate that EphA4 and ephrin B1 bind to each other, thereby initiating signaling. Furthermore, overexpression of ephrin B1 stimulated cell division of neighboring cells, supporting the hypothesis that ephrin B1-initiated forward signaling of EphA4 promotes cortical cell division.
| S-EPMC2729353 | biostudies-literature
Project description:Glucagon plays a major role in the regulation of glucose homeostasis during fed and fasting states. However, the mechanisms responsible for the regulation of pancreatic α cell mass and function are not completely understood. In the current study, we identified mTOR complex 1 (mTORC1) as a major regulator of α cell mass and glucagon secretion. Using mice with tissue-specific deletion of the mTORC1 regulator Raptor in α cells (αRaptorKO), we showed that mTORC1 signaling is dispensable for α cell development, but essential for α cell maturation during the transition from a milk-based diet to a chow-based diet after weaning. Moreover, inhibition of mTORC1 signaling in αRaptorKO mice and in WT animals exposed to chronic rapamycin administration decreased glucagon content and glucagon secretion. In αRaptorKO mice, impaired glucagon secretion occurred in response to different secretagogues and was mediated by alterations in KATP channel subunit expression and activity. Additionally, our data identify the mTORC1/FoxA2 axis as a link between mTORC1 and transcriptional regulation of key genes responsible for α cell function. Thus, our results reveal a potential function of mTORC1 in nutrient-dependent regulation of glucagon secretion and identify a role for mTORC1 in controlling α cell-mass maintenance.
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