GLP-1 metabolite GLP-1(9-36) is a systemic inhibitor of mouse and human pancreatic islet glucagon secretion.

AIMS/HYPOTHESIS: Diabetes mellitus is associated with impaired insulin secretion, often aggravated by oversecretion of glucagon. Therapeutic interventions should ideally correct both defects. Glucagon-like peptide 1 (GLP-1) has this capability but exactly how it exerts its glucagonostatic effect remains obscure. Following its release GLP-1 is rapidly degraded from GLP-1(7-36) to GLP-1(9-36). We hypothesised that the metabolite GLP-1(9-36) (previously believed to be biologically inactive) exerts a direct inhibitory effect on glucagon secretion and that this mechanism becomes impaired in diabetes. METHODS: We used a combination of glucagon secretion measurements in mouse and human islets (including islets from donors with type 2 diabetes), total internal reflection fluorescence microscopy imaging of secretory granule dynamics, recordings of cytoplasmic Ca2+ and measurements of protein kinase A activity, immunocytochemistry, in vivo physiology and GTP-binding protein dissociation studies to explore how GLP-1 exerts its inhibitory effect on glucagon secretion and the role of the metabolite GLP-1(9-36). RESULTS: GLP-1(7-36) inhibited glucagon secretion in isolated islets with an IC50 of 2.5 pmol/l. The effect was particularly strong at low glucose concentrations. The degradation product GLP-1(9-36) shared this capacity. GLP-1(9-36) retained its glucagonostatic effects after genetic/pharmacological inactivation of the GLP-1 receptor. GLP-1(9-36) also potently inhibited glucagon secretion evoked by ß-adrenergic stimulation, amino acids and membrane depolarisation. In islet alpha cells, GLP-1(9-36) led to inhibition of Ca2+ entry via voltage-gated Ca2+ channels sensitive to ω-agatoxin, with consequential pertussis-toxin-sensitive depletion of the docked pool of secretory granules, effects that were prevented by the glucagon receptor antagonists REMD2.59 and L-168049. The capacity of GLP-1(9-36) to inhibit glucagon secretion and reduce the number of docked granules was lost in alpha cells from human donors with type 2 diabetes. In vivo, high exogenous concentrations of GLP-1(9-36) (>100 pmol/l) resulted in a small (30%) lowering of circulating glucagon during insulin-induced hypoglycaemia. This effect was abolished by REMD2.59, which promptly increased circulating glucagon by >225% (adjusted for the change in plasma glucose) without affecting pancreatic glucagon content. CONCLUSIONS/INTERPRETATION: We conclude that the GLP-1 metabolite GLP-1(9-36) is a systemic inhibitor of glucagon secretion. We propose that the increase in circulating glucagon observed following genetic/pharmacological inactivation of glucagon signalling in mice and in people with type 2 diabetes reflects the removal of GLP-1(9-36)'s glucagonostatic action.

Theoretical study of the interactions between peptide tyrosine tyrosine [PYY (1-36)], a newly identified modulator in type 2 diabetes pathophysiology, with receptors NPY1R and NPY4R.

PURPOSE: Diabetes mellitus is a common condition in the clinically obese. Bariatric surgery is one of the ways to put type 2 diabetes in remission. Recent findings propose the appetite-regulator peptide tyrosine tyrosine (PYY) as a therapeutic option for patients with type 2 diabetes. This novel gut hormone restores impaired insulin and glucagon secretion in pancreatic islets and is implicated in type 2 diabetes reversal after bariatric surgery. The current study elucidates the interactions between PYY and the NPY1R and NPY4R receptors using computational methods. METHODS: Protein structure prediction, molecular docking simulation, and molecular dynamics (MD) simulation were performed to elucidate the interactions of PYY with NPY1R and NPY4R. RESULTS: The predicted binding models of PYY-NPY receptors are in agreement with those described in the literature, although different interaction partners are presented for the C-terminal tail of PYY. Non-polar interactions are predicted to drive the formation of the protein complex. The calculated binding energies show that PYY has higher affinity for NPY4R (ΔGGBSA = -65.08 and ΔGPBSA = -87.62 kcal/mol) than for NPY1R (ΔGGBSA = -23.11 and ΔGPBSA = -50.56 kcal/mol). CONCLUSIONS: Based on the constructed models, the binding conformations obtained from docking and MD simulation for both the PYY-NPY1R and PYY-NPY4R complexes provide a detailed map of possible interactions. The calculated binding energies show a higher affinity of PYY for NPY4R. These findings may help to understand the mechanisms behind the improvement of diabetes following bariatric surgery.

PYY, a Therapeutic Option for Type 2 Diabetes?

Metabolic surgery leads to rapid and effective diabetes reversal in humans, by weight-independent mechanisms. The crucial improvement in pancreatic islet function observed after surgery is induced by alteration in several factors, including gut hormones. In addition to glucagon-like peptide 1 (GLP-1), increasing lines of evidence show that peptide tyrosine tyrosine (PYY) plays a key role in the metabolic benefits associated with the surgery, ranging from appetite regulation to amelioration of islet secretory properties and survival. Here, we summarize the current knowledge and the latest advancements in the field, which pitch a strong case for the development of novel PYY-based therapy for the treatment of diabetes.

Somatostatin secretion by Na+-dependent Ca2+-induced Ca2+ release in pancreatic delta-cells.

Pancreatic islets are complex micro-organs consisting of at least three different cell types: glucagon-secreting α-, insulin-producing ß- and somatostatin-releasing δ-cells1. Somatostatin is a powerful paracrine inhibitor of insulin and glucagon secretion2. In diabetes, increased somatostatinergic signalling leads to defective counter-regulatory glucagon secretion3. This increases the risk of severe hypoglycaemia, a dangerous complication of insulin therapy4. The regulation of somatostatin secretion involves both intrinsic and paracrine mechanisms5 but their relative contributions and whether they interact remains unclear. Here we show that dapagliflozin-sensitive glucose- and insulin-dependent sodium uptake stimulates somatostatin secretion by elevating the cytoplasmic Na+ concentration ([Na+]i) and promoting intracellular Ca2+-induced Ca2+ release (CICR). This mechanism also becomes activated when [Na+]i is elevated following the inhibition of the plasmalemmal Na+-K+ pump by reductions of the extracellular K+ concentration emulating those produced by exogenous insulin in vivo 6. Islets from some donors with type-2 diabetes hypersecrete somatostatin, leading to suppression of glucagon secretion that can be alleviated by a somatostatin receptor antagonist. Our data highlight the role of Na+ as an intracellular second messenger, illustrate the significance of the intraislet paracrine network and provide a mechanistic framework for pharmacological correction of the hormone secretion defects associated with diabetes that selectively target the δ-cells.

A novel method for evaluating the effect of pollution on the human skin under controlled conditions.

BACKGROUND: Generally considered as a major risk factor for various respiratory diseases, air pollution can also have a significant impact on the skin. To date, there is a plethora of cosmetics products with "anti-pollution" claims. However, these claims have not been fully substantiated with robust scientific evidence and currently there is no standardized method in place for validating the anti-pollution efficacy of cosmetics products. MATERIALS AND METHODS: This article discusses an innovative Controlled Pollution Exposure System (CPES) which allows quantified administration of pollutants on the skin and analysis of their direct impact. Using CPES, human subjects were exposed to ambient dust and ozone and sebum were sampled and analyzed for biomarkers. RESULTS: Following exposure of human subjects' skin to either ambient dust(100-450 µg/cm3 ) or ozone(100-1000 ppb), analysis of sebum revealed a significant decrease in squalene concentration, and significant increases in squalene monohydroperoxide and malondialdehyde concentration. CONCLUSION: The findings demonstrate cutaneous oxidative stress induced by ambient dust and ozone. The findings also demonstrate the efficacy of CPES to accurately measure the direct effect of controlled gaseous and particulate pollutants on human skin and indicate that squalene, squalene monohydroperoxide and malondialdehyde may serve as potent biomarkers for evaluating potential anti-pollution claims of cosmetics products.

Loss of ZnT8 function protects against diabetes by enhanced insulin secretion.

A rare loss-of-function allele p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8), which is enriched in Western Finland, protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, particularly when compared with individuals matched for the genotype of a common T2D-risk allele in SLC30A8, p.Arg325. In genome-edited human induced pluripotent stem cell (iPSC)-derived ß-like cells, we establish that the p.Arg138* allele results in reduced SLC30A8 expression due to haploinsufficiency. In human ß cells, loss of SLC30A8 leads to increased glucose responsiveness and reduced KATP channel function similar to isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aimed at maintaining insulin secretion capacity in T2D.

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.

Insulin inhibits glucagon release by SGLT2-induced stimulation of somatostatin secretion.

Hypoglycaemia (low plasma glucose) is a serious and potentially fatal complication of insulin-treated diabetes. In healthy individuals, hypoglycaemia triggers glucagon secretion, which restores normal plasma glucose levels by stimulation of hepatic glucose production. This counterregulatory mechanism is impaired in diabetes. Here we show in mice that therapeutic concentrations of insulin inhibit glucagon secretion by an indirect (paracrine) mechanism mediated by stimulation of intra-islet somatostatin release. Insulin's capacity to inhibit glucagon secretion is lost following genetic ablation of insulin receptors in the somatostatin-secreting δ-cells, when insulin-induced somatostatin secretion is suppressed by dapagliflozin (an inhibitor of sodium-glucose co-tranporter-2; SGLT2) or when the action of secreted somatostatin is prevented by somatostatin receptor (SSTR) antagonists. Administration of these compounds in vivo antagonises insulin's hypoglycaemic effect. We extend these data to isolated human islets. We propose that SSTR or SGLT2 antagonists should be considered as adjuncts to insulin in diabetes therapy.

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.

New Approaches for Weight Loss: Experiments Using Animal Models.

The number of people who are overweight and obese are continuously increasing both in the adult and adolescent populations. Coinciding with this is the increased prevalence of health problems such as type 2 diabetes (T2D). Bariatric surgery is the only proven long-term treatment of obesity and may induce remission of T2D, although the underlying mechanisms are unknown. The translational studies presented here might provide insight on the mechanism of steady-state energy balance of the obese phenotype using a special time-restricted feeding regimen for weight loss during the steady-state energy balance; mechanism by vagal blocking therapy (vBLoc® therapy) as a new treatment for obesity; and possible mechanism behind the remission of T2D following gastric bypass surgery.

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.

Sitagliptin and Roux-en-Y gastric bypass modulate insulin secretion via regulation of intra-islet PYY.

AIMS: The gut hormone peptide tyrosine tyrosine (PYY) is critical for maintaining islet integrity and restoring islet function following Roux-en-Y gastric bypass (RYGB). The expression of PYY and its receptors (NPYRs) in islets has been documented but not fully characterized. Modulation of islet PYY by the proteolytic enzyme dipeptidyl peptidase IV (DPP-IV) has not been investigated and the impact of DPP-IV inhibition on islet PYY function remains unexplored. Here we have addressed these gaps and their effects on glucose-stimulated insulin secretion (GSIS). We have also investigated changes in pancreatic PYY in diabetes and following RYGB. METHODS: Immunohistochemistry and gene expression analysis were used to assess PYY, NPYRs and DPP-IV expression in rodent and human islets. DPP-IV activity inhibition was achieved by sitagliptin. Secretion studies were used to test PYY and the effects of sitagliptin on insulin release, and the involvement of GLP-1. Radioimmunoassays were used to measure hormone content in islets. RESULTS: PYY and DPP-IV localized in different cell types in islets while NPYR expression was confined to the beta-cells. Chronic PYY application enhanced GSIS in rodent and diabetic human islets. DPP-IV inhibition by sitagliptin potentiated GSIS; this was mediated by locally-produced PYY, and not GLP-1. Pancreatic PYY was markedly reduced in diabetes. RYGB strongly increased islet PYY content, but did not lead to full restoration of pancreatic GLP-1 levels. CONCLUSION: Local regulation of pancreatic PYY, rather than GLP-1, by DPP-IV inhibition or RYGB can directly modulate the insulin secretory response to glucose, indicating a novel role of pancreatic PYY in diabetes and weight-loss surgery.

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.

The Role of PYY in Pancreatic Islet Physiology and Surgical Control of Diabetes.

Bariatric surgery in obese individuals leads to rapid and lasting remission of type 2 diabetes (T2D). This phenomenon occurs independently of weight loss possibly via a combination of factors. The incretin hormone GLP-1 has so far been recognised as a critical factor. However, recent data have indicated that elevation in another gut hormone, peptide tyrosine tyrosine (PYY), may drive the beneficial effects of surgery. Here we discuss recent findings on PYY-mediated control of glucose homeostasis and its role in diabetes, in the context of what is known for GLP-1. Identification of factors that increase the expression of PYY following bariatric surgery and elucidation of its role in diabetes reversal may have clinical relevance as a nonsurgical therapy for T2D.

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.

GLP-1 stimulates insulin secretion by PKC-dependent TRPM4 and TRPM5 activation.

Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the KATP channel blocker tolbutamide, and the L-type Ca(2+) channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na(+). The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na(+)-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca(2+) from thapsigargin-sensitive Ca(2+) stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by ß cells.

High-content screening identifies a role for Na(+) channels in insulin production.

Insulin production is the central feature of functionally mature and differentiated pancreatic ß-cells. Reduced insulin transcription and dedifferentiation have been implicated in type 2 diabetes, making drugs that could reverse these processes potentially useful. We have previously established ratiometric live-cell imaging tools to identify factors that increase insulin promoter activity and promote ß-cell differentiation. Here, we present a single vector imaging tool with eGFP and mRFP, driven by the Pdx1 and Ins1 promoters, respectively, targeted to the nucleus to enhance identification of individual cells in a high-throughput manner. Using this new approach, we screened 1120 off-patent drugs for factors that regulate Ins1 and Pdx1 promoter activity in MIN6 ß-cells. We identified a number of compounds that positively modulate Ins1 promoter activity, including several drugs known to modulate ion channels. Carbamazepine was selected for extended follow-up, as our previous screen also identified this use-dependent sodium channel inhibitor as a positive modulator of ß-cell survival. Indeed, carbamazepine increased Ins1 and Ins2 mRNA in primary mouse islets at lower doses than were required to protect ß-cells. We validated the role of sodium channels in insulin production by examining Nav1.7 (Scn9a) knockout mice and remarkably islets from these animals had dramatically elevated insulin content relative to wild-type controls. Collectively, our experiments provide a starting point for additional studies aimed to identify drugs and molecular pathways that control insulin production and ß-cell differentiation status. In particular, our unbiased screen identified a novel role for a ß-cell sodium channel gene in insulin production.