Glucose Controls Glucagon Secretion by Regulating Fatty Acid Oxidation in Pancreatic α-Cells.

Whole-body glucose homeostasis is coordinated through secretion of glucagon and insulin from pancreatic islets. When glucose is low, glucagon is released from α-cells to stimulate hepatic glucose production. However, the mechanisms that regulate glucagon secretion from pancreatic α-cells remain unclear. Here we show that in α-cells, the interaction between fatty acid oxidation and glucose metabolism controls glucagon secretion. The glucose-dependent inhibition of glucagon secretion relies on pyruvate dehydrogenase and carnitine palmitoyl transferase 1a activity and lowering of mitochondrial fatty acid oxidation by increases in glucose. This results in reduced intracellular ATP and leads to membrane repolarization and inhibition of glucagon secretion. These findings provide a new framework for the metabolic regulation of the α-cell, where regulation of fatty acid oxidation by glucose accounts for the stimulation and inhibition of glucagon secretion. ARTICLE HIGHLIGHTS: It has become clear that dysregulation of glucagon secretion and α-cell function plays an important role in the development of diabetes, but we do not know how glucagon secretion is regulated. Here we asked whether glucose inhibits fatty acid oxidation in α-cells to regulate glucagon secretion. We found that fatty acid oxidation is required for the inhibitory effects of glucose on glucagon secretion through reductions in ATP. These findings provide a new framework for the regulation of glucagon secretion by glucose.

Hindbrain insulin controls feeding behavior.

OBJECTIVE: Pancreatic insulin was discovered a century ago, and this discovery led to the first lifesaving treatment for diabetes. While still controversial, nearly one hundred published reports suggest that insulin is also produced in the brain, with most focusing on hypothalamic or cortical insulin-producing cells. However, specific function for insulin produced within the brain remains poorly understood. Here we identify insulin expression in the hindbrain's dorsal vagal complex (DVC), and determine the role of this source of insulin in feeding and metabolism, as well as its response to diet-induced obesity in mice. METHODS: To determine the contribution of Ins2-producing neurons to feeding behavior in mice, we used the cross of transgenic RipHER-cre mouse and channelrhodopsin-2 expressing animals, which allowed us to optogenetically stimulate neurons expressing Ins2 in vivo. To confirm the presence of insulin expression in Rip-labeled DVC cells, in situ hybridization was used. To ascertain the specific role of insulin in effects discovered via optogenetic stimulation a selective, CNS applied, insulin receptor antagonist was used. To understand the physiological contribution of insulin made in the hindbrain a virogenetic knockdown strategy was used. RESULTS: Insulin gene expression and presence of insulin-promoter driven fluorescence in rat insulin promoter (Rip)-transgenic mice were detected in the hypothalamus, but also in the DVC. Insulin mRNA was present in nearly all fluorescently labeled cells in DVC. Diet-induced obesity in mice altered brain insulin gene expression, in a neuroanatomically divergent manner; while in the hypothalamus the expected obesity-induced reduction was found, in the DVC diet-induced obesity resulted in increased expression of the insulin gene. This led us to hypothesize a potentially divergent energy balance role of insulin in these two brain areas. To determine the acute impact of activating insulin-producing neurons in the DVC, optic stimulation of light-sensitive channelrhodopsin 2 in Rip-transgenic mice was utilized. Optogenetic photoactivation induced hyperphagia after acute activation of the DVC insulin neurons. This hyperphagia was blocked by central application of the insulin receptor antagonist S961, suggesting the feeding response was driven by insulin. To determine whether DVC insulin has a necessary contribution to feeding and metabolism, virogenetic insulin gene knockdown (KD) strategy, which allows for site-specific reduction of insulin gene expression in adult mice, was used. While chow-fed mice failed to reveal any changes of feeding or thermogenesis in response to the KD, mice challenged with a high-fat diet consumed less food. No changes in body weight were identified, possibly resulting from compensatory reduction in thermogenesis. CONCLUSIONS: Together, our data suggest an important role for hindbrain insulin and insulin-producing cells in energy homeostasis.

A critical role of the mechanosensor PIEZO1 in glucose-induced insulin secretion in pancreatic ß-cells.

Glucose-induced insulin secretion depends on ß-cell electrical activity. Inhibition of ATP-regulated potassium (KATP) channels is a key event in this process. However, KATP channel closure alone is not sufficient to induce ß-cell electrical activity; activation of a depolarizing membrane current is also required. Here we examine the role of the mechanosensor ion channel PIEZO1 in this process. Yoda1, a specific PIEZO1 agonist, activates a small membrane current and thereby triggers ß-cell electrical activity with resultant stimulation of Ca2+-influx and insulin secretion. Conversely, the PIEZO1 antagonist GsMTx4 reduces glucose-induced Ca2+-signaling, electrical activity and insulin secretion. Yet, PIEZO1 expression is elevated in islets from human donors with type-2 diabetes (T2D) and a rodent T2D model (db/db mouse), in which insulin secretion is reduced. This paradox is resolved by our finding that PIEZO1 translocates from the plasmalemma into the nucleus (where it cannot influence the membrane potential of the ß-cell) under experimental conditions emulating T2D (high glucose culture). ß-cell-specific Piezo1-knockout mice show impaired glucose tolerance in vivo and reduced glucose-induced insulin secretion, ß-cell electrical activity and Ca2+ elevation in vitro. These results implicate mechanotransduction and activation of PIEZO1, via intracellular accumulation of glucose metabolites, as an important physiological regulator of insulin secretion.

Gs/Gq signaling switch in ß cells defines incretin effectiveness in diabetes.

By restoring glucose-regulated insulin secretion, glucagon-like peptide-1-based (GLP-1-based) therapies are becoming increasingly important in diabetes care. Normally, the incretins GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) jointly maintain normal blood glucose levels by stimulation of insulin secretion in pancreatic ß cells. However, the reason why only GLP-1-based drugs are effective in improving insulin secretion after presentation of diabetes has not been resolved. ATP-sensitive K+ (KATP) channels play a crucial role in coupling the systemic metabolic status to ß cell electrical activity for insulin secretion. Here, we have shown that persistent membrane depolarization of ß cells due to genetic (ß cell-specific Kcnj11-/- mice) or pharmacological (long-term exposure to sulfonylureas) inhibition of the KATP channel led to a switch from Gs to Gq in a major amplifying pathway of insulin secretion. The switch determined the relative insulinotropic effectiveness of GLP-1 and GIP, as GLP-1 can activate both Gq and Gs, while GIP only activates Gs. The findings were corroborated in other models of persistent depolarization: a spontaneous diabetic KK-Ay mouse and nondiabetic human and mouse ß cells of pancreatic islets chronically treated with high glucose. Thus, a Gs/Gq signaling switch in ß cells exposed to chronic hyperglycemia underlies the differential insulinotropic potential of incretins in diabetes.

'Resistance is futile?' - paradoxical inhibitory effects of KATP channel closure in glucagon-secreting α-cells.

By secreting insulin and glucagon, the ß- and α-cells of the pancreatic islets play a central role in the regulation of systemic metabolism. Both cells are equipped with ATP-regulated potassium (KATP ) channels that are regulated by the intracellular ATP/ADP ratio. In ß-cells, KATP channels are active at low (non-insulin-releasing) glucose concentrations. An increase in glucose leads to KATP channel closure, membrane depolarization and electrical activity that culminates in elevation of [Ca2+ ]i and initiation of exocytosis of the insulin-containing secretory granules. The α-cells are also equipped with KATP channels but they are under strong tonic inhibition at low glucose, explaining why α-cells are electrically active under hypoglycaemic conditions and generate large Na+ - and Ca2+ -dependent action potentials. Closure of residual KATP channel activity leads to membrane depolarization and an increase in action potential firing but this stimulation of electrical activity is associated with inhibition rather than acceleration of glucagon secretion. This paradox arises because membrane depolarization reduces the amplitude of the action potentials by voltage-dependent inactivation of the Na+ channels involved in action potential generation. Exocytosis in α-cells is tightly linked to the opening of voltage-gated P/Q-type Ca2+ channels, the activation of which is steeply voltage-dependent. Accordingly, the inhibitory effect of the reduced action potential amplitude exceeds the stimulatory effect resulting from the increased action potential frequency. These observations highlight a previously unrecognised role of the action potential amplitude as a key regulator of pancreatic islet hormone secretion.

A method for the generation of human stem cell-derived alpha cells.

The generation of pancreatic cell types from renewable cell sources holds promise for cell replacement therapies for diabetes. Although most effort has focused on generating pancreatic beta cells, considerable evidence indicates that glucagon secreting alpha cells are critically involved in disease progression and proper glucose control. Here we report on the generation of stem cell-derived human pancreatic alpha (SC-alpha) cells from pluripotent stem cells via a transient pre-alpha cell intermediate. These pre-alpha cells exhibit a transcriptional profile similar to mature alpha cells and although they produce proinsulin protein, they do not secrete significant amounts of processed insulin. Compound screening identified a protein kinase c activator that promotes maturation of pre-alpha cells into SC-alpha cells. The resulting SC-alpha cells do not express insulin, share an ultrastructure similar to cadaveric alpha cells, express and secrete glucagon in response to glucose and some glucagon secretagogues, and elevate blood glucose upon transplantation in mice.

Glucose stimulates somatostatin secretion in pancreatic δ-cells by cAMP-dependent intracellular Ca2+ release.

Somatostatin secretion from pancreatic islet δ-cells is stimulated by elevated glucose levels, but the underlying mechanisms have only partially been elucidated. Here we show that glucose-induced somatostatin secretion (GISS) involves both membrane potential-dependent and -independent pathways. Although glucose-induced electrical activity triggers somatostatin release, the sugar also stimulates GISS via a cAMP-dependent stimulation of CICR and exocytosis of somatostatin. The latter effect is more quantitatively important and in mouse islets depolarized by 70 mM extracellular K+ , increasing glucose from 1 mM to 20 mM produced an ∼3.5-fold stimulation of somatostatin secretion, an effect that was mimicked by the application of the adenylyl cyclase activator forskolin. Inhibiting cAMP-dependent pathways with PKI or ESI-05, which inhibit PKA and exchange protein directly activated by cAMP 2 (Epac2), respectively, reduced glucose/forskolin-induced somatostatin secretion. Ryanodine produced a similar effect that was not additive to that of the PKA or Epac2 inhibitors. Intracellular application of cAMP produced a concentration-dependent stimulation of somatostatin exocytosis and elevation of cytoplasmic Ca2+ ([Ca2+]i). Both effects were inhibited by ESI-05 and thapsigargin (an inhibitor of SERCA). By contrast, inhibition of PKA suppressed δ-cell exocytosis without affecting [Ca2+]i Simultaneous recordings of electrical activity and [Ca2+]i in δ-cells expressing the genetically encoded Ca2+ indicator GCaMP3 revealed that the majority of glucose-induced [Ca2+]i spikes did not correlate with δ-cell electrical activity but instead reflected Ca2+ release from the ER. These spontaneous [Ca2+]i spikes are resistant to PKI but sensitive to ESI-05 or thapsigargin. We propose that cAMP links an increase in plasma glucose to stimulation of somatostatin secretion by promoting CICR, thus evoking exocytosis of somatostatin-containing secretory vesicles in the δ-cell.

Diabetes causes marked inhibition of mitochondrial metabolism in pancreatic ß-cells.

Diabetes is a global health problem caused primarily by the inability of pancreatic ß-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of ß-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic ßV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 ß-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in ß-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of ß-cells in diabetes.

PYY plays a key role in the resolution of diabetes following bariatric surgery in humans.

BACKGROUND: Bariatric surgery leads to early and long-lasting remission of type 2 diabetes (T2D). However, the mechanisms behind this phenomenon remain unclear. Among several factors, gut hormones are thought to be crucial mediators of this effect. Unlike GLP-1, the role of the hormone peptide tyrosine tyrosine (PYY) in bariatric surgery in humans has been limited to appetite regulation and its impact on pancreatic islet secretory function and glucose metabolism remains under-studied. METHODS: Changes in PYY concentrations were examined in obese patients after bariatric surgery and compared to healthy controls. Human pancreatic islet function was tested upon treatment with sera from patients before and after the surgery, in presence or absence of PYY. Alterations in intra-islet PYY release and insulin secretion were analysed after stimulation with short chain fatty acids (SCFAs), bile acids and the cytokine IL-22. FINDINGS: We demonstrate that PYY is a key effector of the early recovery of impaired glucose-mediated insulin and glucagon secretion in bariatric surgery. We establish that the short chain fatty acid propionate and bile acids, which are elevated after surgery, can trigger PYY release not only from enteroendocrine cells but also from human pancreatic islets. In addition, we identify IL-22 as a new factor which is modulated by bariatric surgery in humans and which directly regulates PYY expression and release. INTERPRETATION: This study shows that some major metabolic benefits of bariatric surgery can be emulated ex vivo. Our findings are expected to have a direct impact on the development of new non-surgical therapy for T2D correction.

Dysregulation of Glucagon Secretion by Hyperglycemia-Induced Sodium-Dependent Reduction of ATP Production.

Diabetes is a bihormonal disorder resulting from combined insulin and glucagon secretion defects. Mice lacking fumarase (Fh1) in their ß cells (Fh1ßKO mice) develop progressive hyperglycemia and dysregulated glucagon secretion similar to that seen in diabetic patients (too much at high glucose and too little at low glucose). The glucagon secretion defects are corrected by low concentrations of tolbutamide and prevented by the sodium-glucose transport (SGLT) inhibitor phlorizin. These data link hyperglycemia, intracellular Na+ accumulation, and acidification to impaired mitochondrial metabolism, reduced ATP production, and dysregulated glucagon secretion. Protein succination, reflecting reduced activity of fumarase, is observed in α cells from hyperglycemic Fh1ßKO and ß-V59M gain-of-function KATP channel mice, diabetic Goto-Kakizaki rats, and patients with type 2 diabetes. Succination is also observed in renal tubular cells and cardiomyocytes from hyperglycemic Fh1ßKO mice, suggesting that the model can be extended to other SGLT-expressing cells and may explain part of the spectrum of diabetic complications.

GLP-1 suppresses glucagon secretion in human pancreatic alpha-cells by inhibition of P/Q-type Ca2+ channels.

Glucagon is the body's main hyperglycemic hormone, and its secretion is dysregulated in type 2 diabetes mellitus (T2DM). The incretin hormone glucagon-like peptide-1 (GLP-1) is released from the gut and is used in T2DM therapy. Uniquely, it both stimulates insulin and inhibits glucagon secretion and thereby lowers plasma glucose levels. In this study, we have investigated the action of GLP-1 on glucagon release from human pancreatic islets. Immunocytochemistry revealed that only <0.5% of the α-cells possess detectable GLP-1R immunoreactivity. Despite this, GLP-1 inhibited glucagon secretion by 50-70%. This was due to a direct effect on α-cells, rather than paracrine signaling, because the inhibition was not reversed by the insulin receptor antagonist S961 or the somatostatin receptor-2 antagonist CYN154806. The inhibitory effect of GLP-1 on glucagon secretion was prevented by the PKA-inhibitor Rp-cAMPS and mimicked by the adenylate cyclase activator forskolin. Electrophysiological measurements revealed that GLP-1 decreased action potential height and depolarized interspike membrane potential. Mathematical modeling suggests both effects could result from inhibition of P/Q-type Ca2+ channels. In agreement with this, GLP-1 and ω-agatoxin (a blocker of P/Q-type channels) inhibited glucagon secretion in islets depolarized by 70 mmol/L [K+ ]o , and these effects were not additive. Intracellular application of cAMP inhibited depolarization-evoked exocytosis in individual α-cells by a PKA-dependent (Rp-cAMPS-sensitive) mechanism. We propose that inhibition of glucagon secretion by GLP-1 involves activation of the few GLP-1 receptors present in the α-cell membrane. The resulting small elevation of cAMP leads to PKA-dependent inhibition of P/Q-type Ca2+ channels and suppression of glucagon exocytosis.

CPT1a-Dependent Long-Chain Fatty Acid Oxidation Contributes to Maintaining Glucagon Secretion from Pancreatic Islets.

Glucagon, the principal hyperglycemic hormone, is secreted from pancreatic islet α cells as part of the counter-regulatory response to hypoglycemia. Hence, secretory output from α cells is under high demand in conditions of low glucose supply. Many tissues oxidize fat as an alternate energy substrate. Here, we show that glucagon secretion in low glucose conditions is maintained by fatty acid metabolism in both mouse and human islets, and that inhibiting this metabolic pathway profoundly decreases glucagon output by depolarizing α cell membrane potential and decreasing action potential amplitude. We demonstrate, by using experimental and computational approaches, that this is not mediated by the KATP channel, but instead due to reduced operation of the Na+-K+ pump. These data suggest that counter-regulatory secretion of glucagon is driven by fatty acid metabolism, and that the Na+-K+ pump is an important ATP-dependent regulator of α cell function.

Adrenaline Stimulates Glucagon Secretion by Tpc2-Dependent Ca2+ Mobilization From Acidic Stores in Pancreatic α-Cells.

Adrenaline is a powerful stimulus of glucagon secretion. It acts by activation of ß-adrenergic receptors, but the downstream mechanisms have only been partially elucidated. Here, we have examined the effects of adrenaline in mouse and human α-cells by a combination of electrophysiology, imaging of Ca2+ and PKA activity, and hormone release measurements. We found that stimulation of glucagon secretion correlated with a PKA- and EPAC2-dependent (inhibited by PKI and ESI-05, respectively) elevation of [Ca2+]i in α-cells, which occurred without stimulation of electrical activity and persisted in the absence of extracellular Ca2+ but was sensitive to ryanodine, bafilomycin, and thapsigargin. Adrenaline also increased [Ca2+]i in α-cells in human islets. Genetic or pharmacological inhibition of the Tpc2 channel (that mediates Ca2+ release from acidic intracellular stores) abolished the stimulatory effect of adrenaline on glucagon secretion and reduced the elevation of [Ca2+]i Furthermore, in Tpc2-deficient islets, ryanodine exerted no additive inhibitory effect. These data suggest that ß-adrenergic stimulation of glucagon secretion is controlled by a hierarchy of [Ca2+]i signaling in the α-cell that is initiated by cAMP-induced Tpc2-dependent Ca2+ release from the acidic stores and further amplified by Ca2+-induced Ca2+ release from the sarco/endoplasmic reticulum.

α-cell glucokinase suppresses glucose-regulated glucagon secretion.

Glucagon secretion by pancreatic α-cells is triggered by hypoglycemia and suppressed by high glucose levels; impaired suppression of glucagon secretion is a hallmark of both type 1 and type 2 diabetes. Here, we show that α-cell glucokinase (Gck) plays a role in the control of glucagon secretion. Using mice with α-cell-specific inactivation of Gck (αGckKO mice), we find that glucokinase is required for the glucose-dependent increase in intracellular ATP/ADP ratio and the closure of KATP channels in α-cells and the suppression of glucagon secretion at euglycemic and hyperglycemic levels. αGckKO mice display hyperglucagonemia in the fed state, which is associated with increased hepatic gluconeogenic gene expression and hepatic glucose output capacity. In adult mice, fed hyperglucagonemia is further increased and glucose intolerance develops. Thus, glucokinase governs an α-cell metabolic pathway that suppresses secretion at or above normoglycemic levels; abnormal suppression of glucagon secretion deregulates hepatic glucose metabolism and, over time, induces a pre-diabetic phenotype.

Biphasic voltage-dependent inactivation of human NaV 1.3, 1.6 and 1.7 Na+ channels expressed in rodent insulin-secreting cells.

KEY POINTS: Na+ current inactivation is biphasic in insulin-secreting cells, proceeding with two voltage dependences that are half-maximal at ∼-100 mV and -60 mV. Inactivation of voltage-gated Na+ (NaV ) channels occurs at ∼30 mV more negative voltages in insulin-secreting Ins1 and primary ß-cells than in HEK, CHO or glucagon-secreting αTC1-6 cells. The difference in inactivation between Ins1 and non-ß-cells persists in the inside-out patch configuration, discounting an involvement of a diffusible factor. In Ins1 cells and primary ß-cells, but not in HEK cells, inactivation of a single NaV subtype is biphasic and follows two voltage dependences separated by 30-40 mV. We propose that NaV channels adopt different inactivation behaviours depending on the local membrane environment. ABSTRACT: Pancreatic ß-cells are equipped with voltage-gated Na+ channels that undergo biphasic voltage-dependent steady-state inactivation. A small Na+ current component (10-15%) inactivates over physiological membrane potentials and contributes to action potential firing. However, the major Na+ channel component is completely inactivated at -90 to -80 mV and is therefore inactive in the ß-cell. It has been proposed that the biphasic inactivation reflects the contribution of different NaV α-subunits. We tested this possibility by expression of TTX-resistant variants of the NaV subunits found in ß-cells (NaV 1.3, NaV 1.6 and NaV 1.7) in insulin-secreting Ins1 cells and in non-ß-cells (including HEK and CHO cells). We found that all NaV subunits inactivated at 20-30 mV more negative membrane potentials in Ins1 cells than in HEK or CHO cells. The more negative inactivation in Ins1 cells does not involve a diffusible intracellular factor because the difference between Ins1 and CHO persisted after excision of the membrane. NaV 1.7 inactivated at 15--20 mV more negative membrane potentials than NaV 1.3 and NaV 1.6 in Ins1 cells but this small difference is insufficient to solely explain the biphasic inactivation in Ins1 cells. In Ins1 cells, but never in the other cell types, widely different components of NaV inactivation (separated by 30 mV) were also observed following expression of a single type of NaV α-subunit. The more positive component exhibited a voltage dependence of inactivation similar to that found in HEK and CHO cells. We propose that biphasic NaV inactivation in insulin-secreting cells reflects insertion of channels in membrane domains that differ with regard to lipid and/or membrane protein composition.

Fumarate Hydratase Deletion in Pancreatic ß Cells Leads to Progressive Diabetes.

We explored the role of the Krebs cycle enzyme fumarate hydratase (FH) in glucose-stimulated insulin secretion (GSIS). Mice lacking Fh1 in pancreatic ß cells (Fh1ßKO mice) appear normal for 6-8 weeks but then develop progressive glucose intolerance and diabetes. Glucose tolerance is rescued by expression of mitochondrial or cytosolic FH but not by deletion of Hif1α or Nrf2. Progressive hyperglycemia in Fh1ßKO mice led to dysregulated metabolism in ß cells, a decrease in glucose-induced ATP production, electrical activity, cytoplasmic [Ca2+]i elevation, and GSIS. Fh1 loss resulted in elevated intracellular fumarate, promoting succination of critical cysteines in GAPDH, GMPR, and PARK 7/DJ-1 and cytoplasmic acidification. Intracellular fumarate levels were increased in islets exposed to high glucose and in islets from human donors with type 2 diabetes (T2D). The impaired GSIS in islets from diabetic Fh1ßKO mice was ameliorated after culture under normoglycemic conditions. These studies highlight the role of FH and dysregulated mitochondrial metabolism in T2D.

Mutant Mice With Calcium-Sensing Receptor Activation Have Hyperglycemia That Is Rectified by Calcilytic Therapy.

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor that plays a pivotal role in extracellular calcium homeostasis. The CaSR is also highly expressed in pancreatic islet α- and ß-cells that secrete glucagon and insulin, respectively. To determine whether the CaSR may influence systemic glucose homeostasis, we characterized a mouse model with a germline gain-of-function CaSR mutation, Leu723Gln, referred to as Nuclear flecks (Nuf). Heterozygous- (CasrNuf/+) and homozygous-affected (CasrNuf/Nuf) mice were shown to have hypocalcemia in association with impaired glucose tolerance and insulin secretion. Oral administration of a CaSR antagonist compound, known as a calcilytic, rectified the glucose intolerance and hypoinsulinemia of CasrNuf/+ mice and ameliorated glucose intolerance in CasrNuf/Nuf mice. Ex vivo studies showed CasrNuf/+ and CasrNuf/Nuf mice to have reduced pancreatic islet mass and ß-cell proliferation. Electrophysiological analysis of isolated CasrNuf/Nuf islets showed CaSR activation to increase the basal electrical activity of ß-cells independently of effects on the activity of the adenosine triphosphate (ATP)-sensitive K+ (KATP) channel. CasrNuf/Nuf mice also had impaired glucose-mediated suppression of glucagon secretion, which was associated with increased numbers of α-cells and a higher α-cell proliferation rate. Moreover, CasrNuf/Nuf islet electrophysiology demonstrated an impairment of α-cell membrane depolarization in association with attenuated α-cell basal KATP channel activity. These studies indicate that the CaSR activation impairs glucose tolerance by a combination of α- and ß-cell defects and also influences pancreatic islet mass. Moreover, our findings highlight a potential application of targeted CaSR compounds for modulating glucose metabolism.

A role of PLC/PKC-dependent pathway in GLP-1-stimulated insulin secretion.

Glucagon-like peptide-1 (GLP-1) is an endogenous glucose-lowering hormone and GLP-1 receptor agonists are currently being used as antidiabetic drugs clinically. The canonical signalling pathway (including cAMP, Epac2, protein kinase A (PKA) and KATP channels) is almost universally accepted as the main mechanism of GLP-1-stimulated insulin secretion. This belief is based on in vitro studies that used nanomolar (1-100 nM) concentrations of GLP-1. Recently, it was found that the physiological concentrations (1-10 pM) of GLP-1 also stimulate insulin secretion from isolated islets, induce membrane depolarization and increase of intracellular [Ca2+] in isolated ß cells/pancreatic islets. These responses were unaffected by PKA inhibitors and occurred without detectable increases in intracellular cAMP and PKA activity. These PKA-independent actions of GLP-1 depend on protein kinase C (PKC), involve activation of the standard GLP-1 receptor (GLP1R) and culminate in activation of phospholipase C (PLC), leading to an elevation of diacylglycerol (DAG), increased L-type Ca2+ and TRPM4/TRPM5 channel activities. Here, we review these recent data and contrast them against the effects of nanomolar concentrations of GLP-1. The differential intracellular signalling activated by low and high concentrations of GLP-1 could provide a clue to explain how GLP-1 exerts different function in the central nervous system and peripheral organs.

PYY-Dependent Restoration of Impaired Insulin and Glucagon Secretion in Type 2 Diabetes following Roux-En-Y Gastric Bypass Surgery.

Roux-en-Y gastric bypass (RYGB) is a weight-reduction procedure resulting in rapid resolution of type 2 diabetes (T2D). The role of pancreatic islet function in this restoration of normoglycemia has not been fully elucidated. Using the diabetic Goto-Kakizaki (GK) rat model, we demonstrate that RYGB restores normal glucose regulation of glucagon and insulin secretion and normalizes islet morphology. Culture of isolated islets with serum from RYGB animals mimicked these effects, implicating a humoral factor. These latter effects were reversed following neutralization of the gut hormone peptide tyrosine tyrosine (PYY) but persisted in the presence of a glucagon-like peptide-1 (GLP-1) receptor antagonist. The effects of RYGB on secretion were replicated by chronic exposure of diabetic rat islets to PYY in vitro. These findings indicate that the mechanism underlying T2D remission may be mediated by PYY and suggest that drugs promoting PYY release or action may restore pancreatic islet function in T2D.

Synaptotagmin-7 phosphorylation mediates GLP-1-dependent potentiation of insulin secretion from ß-cells.

Glucose stimulates insulin secretion from ß-cells by increasing intracellular Ca(2+). Ca(2+) then binds to synaptotagmin-7 as a major Ca(2+) sensor for exocytosis, triggering secretory granule fusion and insulin secretion. In type-2 diabetes, insulin secretion is impaired; this impairment is ameliorated by glucagon-like peptide-1 (GLP-1) or by GLP-1 receptor agonists, which improve glucose homeostasis. However, the mechanism by which GLP-1 receptor agonists boost insulin secretion remains unclear. Here, we report that GLP-1 stimulates protein kinase A (PKA)-dependent phosphorylation of synaptotagmin-7 at serine-103, which enhances glucose- and Ca(2+)-stimulated insulin secretion and accounts for the improvement of glucose homeostasis by GLP-1. A phospho-mimetic synaptotagmin-7 mutant enhances Ca(2+)-triggered exocytosis, whereas a phospho-inactive synaptotagmin-7 mutant disrupts GLP-1 potentiation of insulin secretion. Our findings thus suggest that synaptotagmin-7 is directly activated by GLP-1 signaling and may serve as a drug target for boosting insulin secretion. Moreover, our data reveal, to our knowledge, the first physiological modulation of Ca(2+)-triggered exocytosis by direct phosphorylation of a synaptotagmin.