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GPR142, a putative amino acid receptor, is expressed in pancreatic islets and the gastrointestinal tract, but the ligand affinity and physiological role of this receptor remain obscure. In this study, we show that in addition to L-Tryptophan, GPR142 signaling is also activated by L-Phenylalanine but not by other naturally occurring amino acids. Furthermore, we show that Tryptophan and a synthetic GPR142 agonist increase insulin and incretin hormones and improve glucose disposal in mice in a GPR142-dependent manner. In contrast, Phenylalanine improves in vivo glucose disposal independently of GPR142. Noteworthy, refeeding-induced elevations in insulin and glucose-dependent insulinotropic polypeptide are blunted in Gpr142 null mice. In conclusion, these findings demonstrate GPR142 is a Tryptophan receptor critically required for insulin and incretin hormone regulation and suggest GPR142 agonists may be effective therapies that leverage amino acid sensing pathways for the treatment of type 2 diabetes. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor-21 (FGF-21) is a recently discovered metabolic regulator. Here, we investigated the effects of FGF-21 in the pancreatic β-cell. In rat islets and INS-1E cells, FGF-21 activated extracellular signal-regulated kinase 1/2 and Akt signaling pathways. In islets isolated from healthy rats, FGF-21 increased insulin mRNA and protein levels but did not potentiate glucose-induced insulin secretion. Islets and INS-1E cells treated with FGF-21 were partially protected from glucolipotoxicity and cytokine-induced apoptosis. In islets isolated from diabetic rodents, FGF-21 treatment increased islet insulin content and glucose-induced insulin secretion. Short-term treatment of normal or db/db mice with FGF-21 lowered plasma levels of insulin and improved glucose clearance compared with vehicle after oral glucose tolerance testing. Constant infusion of FGF-21 for 8 weeks in db/db mice nearly normalized fed blood glucose levels and increased plasma insulin levels. Immunohistochemistry of pancreata from db/db mice showed a substantial increase in the intensity of insulin staining in islets from FGF-21-treated animals as well as a higher number of islets per pancreas section and of insulin-positive cells per islet compared with control. No effect of FGF-21 was observed on islet cell proliferation. In conclusion, preservation of β-cell function and survival by FGF-21 may contribute to the beneficial effects of this protein on glucose homeostasis observed in diabetic animals. Diabetes 55:2470-2478, 2006 [ABSTRACT FROM AUTHOR]

Abstract: The multi-domain protein PIST (protein interacting specifically with Tc10) interacts with the SSTR5 (somatostatin receptor 5) and is responsible for its intracellular localization. Here, we show that PIST is expressed in pancreatic β-cells and interacts with SSTR5 in these cells. PIST expression in MIN6 insulinoma cells is reduced by somatostatin (SST). After stimulation with SST, SSTR5 undergoes internalization together with PIST. MIN6 cells over-expressing PIST display enhanced glucose-stimulated insulin secretion and a decreased sensitivity to SST-induced inhibition of insulin secretion. These data suggest that PIST plays an important role in insulin secretion by regulating SSTR5 availability at the plasma membrane. [Copyright &y& Elsevier]

Liver X receptors (LXRs) α and β, transcription factors of a nuclear hormone receptor family, are expressed in pancreatic islets as well as glucagon-secreting and insulin-secreting cell lines. Culture of pancreatic islets or insulin-secreting MIN6 cells with a LXR specific agonist T0901317 caused an increase in glucose-dependent insulin secretion and islet insulin content. The stimulatory effect of T0901317 on insulin secretion was observed only after >72 h of islet culture with the compound. In MIN6 cells, T0901317 increased protein expression of lipogenic enzymes, fatty acid synthase, and acetyl-CoA carboxylase. LXR activation also produced an increase in glucokinase protein and pyruvate carboxylase (PC) activity levels. The PC inhibitor phenylacetic acid abolished the increase in insulin secretion in cells treated with T0901317. The results suggest that LXRs can control insulin secretion and biosynthesis via regulation of glucose and lipid metabolism in pancreatic β-cells. Diabetes 53 (Suppl. 3):S75-S78, 2004 [ABSTRACT FROM AUTHOR]

The release of sPLA2 from single mouse pancreatic β-cells was monitored using a fluorescent substrate of the enzyme incorporated in the outer leaflet of the plasma membrane. Stimulation of β-cells with agents that increased cytosolic free Ca2+ concentration ([Ca2+]i) induced a rapid release of sPLA2 to the extracellular medium. Exogenous sPLA2 strongly stimulated insulin secretion in mouse pancreatic islets at both basal and elevated glucose concentrations. The stimulation of insulin secretion by sPLA2 was mediated via inhibition of ATP-dependent K+ channels and an increase in [Ca2+]i. Measurements of cell capacitance in single β-cells revealed that sPLA2 did not modify depolarisation-induced exocytosis. Our data suggest that a positive feedback regulation of insulin secretion by co-released sPLA2 is operational in pancreatic β-cells and point to this enzyme as an autocrine regulator of insulin secretion. [Copyright &y& Elsevier]

The imidazoline RX871024 increased basal- and glucose-stimulated insulin release in vitro and in vivo. The compound inhibited activity of ATP-sensitive K(+) channels as well as voltage-gated K(+) channels, which led to membrane depolarization, an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)), and insulin release. Importantly, RX871024 also enhanced the insulinotropic effect of glucose in cells with clamped [Ca(2+)](i) but in the presence of high ATP and Ca(2+)concentration inside the cell. We believe that the latter effect on insulin exocytosis was at least in part mediated by a rise in diacylglycerol, which then activated protein kinase C (PKC) and increased the generation of arachidonic acid (AA) metabolites. Activation of both the PKC and AA pathways resulted in potentiation of glucose effects on insulin secretion. Unlike RX871024, the novel imidazoline BL11282 did not block ATP-dependent K(+) channels, but similarly to RX871024, it stimulated insulin secretion in depolarized or permeabilized islets. Accordingly, BL11282 did not influence glucose and insulin levels under basal conditions either in vitro or in vivo, but it markedly enhanced the insulinotropic effects of glucose. BL11282 restored the impaired insulin response to glucose in islets from spontaneously diabetic GK rats. We conclude that BL11282 belongs to a new class of insulinotropic compounds that demonstrate a strong glucose-dependent effect on insulin exocytosis. [ABSTRACT FROM AUTHOR]

Examines the role of inositol (Ins) hexakisphosphate in improving dynamin-medicated endocytosis in the pancreatic cell. Molecular mechanism regulating the membrane homeostasis; Metabolic control of the Ins; Formation of the phosphatidylinositol biphosphate following activation of protein kinase C.

The insulinotropic activity of the novel imidazoline compound BL11282 was investigated. Intravenous administration of BL11282 (0.3 mg x kg(-1) x min(-1)) to anesthetized rats did not change blood glucose and insulin levels under basal conditions, but produced a higher increase in blood insulin levels and a faster glucose removal from the blood after glucose infusion. Similarly, in isolated Wistar rat pancreatic islets, 0.1-100 micromol/l BL11282 potently stimulated glucose-induced insulin secretion but did not modulate basal insulin secretion. Unlike previously described imidazolines, BL11282 did not block ATP-dependent K+ channels. Furthermore, the compound stimulated insulin secretion in islets depolarized with high concentrations of KCl or permeabilized with electric shock. Insulinotropic activity of BL11282 was dependent on activity of protein kinases A and C. In pancreatic islets from spontaneously diabetic GK rats, the imidazoline compound restored the impaired insulin response to glucose. In conclusion, the imidazoline BL11282 constitutes a new class of insulinotropic compounds that exerts an exclusive glucose-dependent insulinotropic activity in pancreatic islets by stimulating insulin exocytosis. [ABSTRACT FROM AUTHOR]

Shows that InsP6 can modulate insulin exocytosis in permeabilized insulin secreting cells. Cell culture; Measurements of protein kinase C activity; Array of inositol polyphosphates; Priming concentrations of INSP6.

Focuses on insulinotropic activity of imidazoline derivative in diabetic rat. Comparison of the insulinotropic activity of imidazoline derivative and sulfonylurea glibenclamide; Improvement in glucose sensitivity in diabetic cells due to imidazoline derivative; Development of novel drugs for treatment of type 2 diabetes with the help of imidazoline compounds.

Type 2 diabetes (T2D) is associated with defective insulin secretion and reduced β cell mass. Available treatments provide a temporary reprieve, but secondary failure rates are high, making insulin supplementation necessary. Reversibility of β cell failure is a key translational question. Here, we reverse engineered and interrogated pancreatic islet-specific regulatory networks to discover T2D-specific subpopulations characterized by metabolic inflexibility and endocrine progenitor/stem cell features. Single-cell gain- and loss-of-function and glucose-induced Ca2+ flux analyses of top candidate master regulatory (MR) proteins in islet cells validated transcription factor BACH2 and associated epigenetic effectors as key drivers of T2D cell states. BACH2 knockout in T2D islets reversed cellular features of the disease, restoring a nondiabetic phenotype. BACH2-immunoreactive islet cells increased approximately 4-fold in diabetic patients, confirming the algorithmic prediction of clinically relevant subpopulations. Treatment with a BACH inhibitor lowered glycemia and increased plasma insulin levels in diabetic mice, and restored insulin secretion in diabetic mice and human islets. The findings suggest that T2D-specific populations of failing β cells can be reversed and indicate pathways for pharmacological intervention, including via BACH2 inhibition. [ABSTRACT FROM AUTHOR]

Glutamate has been implicated as an intracellular messenger in the regulation of insulin secretion in response to glucose. Here we demonstrate by measurements of cell capacitance in rat pancreatic β-cells that glutamate (1 mM) enhanced Ca2+-dependent exocytosis. Glutamate (1 mM) also stimulated insulin secretion from permeabilized rat β-cells. The effect was dose-dependent (half-maximum at 5.1 mM) and maximal at 10 mM glutamate. Glutamate-induced exocytosis was stronger in rat β-cells and clonal INS-1E cells compared to β-cells isolated from mice and in parental INS-1 cells, which correlated with the expressed levels of glutamate dehydrogenase. Glutamate-induced exocytosis was inhibited by the protonophores FCCP and SF6847, by the vacuolar-type H+-ATPase inhibitor bafilomycin A1 and by the glutamate transport inhibitor Evans Blue. Our data provide evidence that exocytosis in β-cells can be modulated by physiological increases in cellular glutamate levels. The results suggest that stimulation of exocytosis is associated with accumulation of glutamate in the secretory granules, a process that is dependent on the transgranular proton gradient. [Copyright &y& Elsevier]

We evaluated the potential for a monoclonal antibody antagonist of the glucagon receptor (Ab-4) to maintain glucose homeostasis in type 1 diabetic rodents. We noted durable and sustained improvements in glycemia which persist long after treatment withdrawal. Ab-4 promoted β-cell survival and enhanced the recovery of insulin+ islet mass with concomitant increases in circulating insulin and C peptide. In PANIC-ATTAC mice, an inducible model of β-cell apoptosis which allows for robust assessment of β-cell regeneration following caspase-8-induced diabetes, Ab-4 drove a 6.7-fold increase in β-cell mass. Lineage tracing suggests that this restoration of functional insulin-producing cells was at least partially driven by α-cell-to-β-cell conversion. Following hyperglycemic onset in nonobese diabetic (NOD) mice, Ab-4 treatment promoted improvements in C-peptide levels and insulin+ islet mass was dramatically increased. Lastly, diabetic mice receiving human islet xenografts showed stable improvements in glycemic control and increased human insulin secretion. [ABSTRACT FROM AUTHOR]

Estrogen hormones play an important role in controlling glucose homeostasis and pancreatic β-cell function. Despite the significance of estrogen hormones for regulation of glucose metabolism, little is known about the roles of endogenous estrogen metabolites in modulating pancreatic β-cell function. In this study, we evaluated the effects of major natural estrogen metabolites, catechol estrogens, on insulin secretion in pancreatic β-cells. We show that catechol estrogens, hydroxylated at positions C2 and C4 of the steroid A ring, rapidly potentiated glucose-induced insulin secretion via a nongenomic mechanism. 2-Hydroxyestrone, the most abundant endogenous estrogen metabolite, was more efficacious in stimulating insulin secretion than any other tested catechol estrogens. In insulinsecreting cells, catechol estrogens produced rapid activation of calcium influx and elevation in cytosolic free calcium. Catechol estrogens also generated sustained elevations in cytosolic free calcium and evoked inward ion current inHEK293cells expressing the transient receptor potential A1 (TRPA1) cation channel. Calcium influx and insulin secretion stimulated by estrogen metabolites were dependent on the TRPA1 activity and inhibited with the channel-specific pharmacological antagonists or the siRNA. Our results suggest the role of estrogen metabolism in a direct regulation of TRPA1 activity with potential implications for metabolic diseases. [ABSTRACT FROM AUTHOR]

Extracellular ATP released from pancreatic β-cells acts as a potent insulinotropic agent through activation of P2 purinergic receptors. Ectonucleotidases, a family of membrane-bound nucleotide-metabolizing enzymes, regulate extracellular ATP levels by degrading ATP and related nucleotides. Ectonucleotidase activity affects the relative proportion of ATP and its metabolites, which in turn will impact the level of purinergic receptor stimulation exerted by extracellular ATP. Therefore, we investigated the expression and role of ectonucleotidases in pancreatic β-cells. Of the ectonucleotidases studied, only ENTPD3 (gene encoding the NTPDase3 enzyme) mRNA was detected at fairly abundant levels in human and mouse pancreatic islets as well as in insulinsecreting MIN6 cells. ARL67156, a selective ectonucleotidase inhibitor, blocked degradation of extracellular ATP that was added to MIN6 cells. The compound also decreased degradation of endogenous ATP released from cells. Measurements of insulin secretion in MIN6 cells as well as in mouse and human pancreatic islets demonstrated that ARL67156 potentiated glucose-dependent insulin secretion. Downregulation of NTPDase3 expression in MIN6 cells with the specific siRNA replicated the effects of ARL67156 on extracellular ATP hydrolysis and insulin secretion. Our results demonstrate that NTPDase3 is the major ectonucleotidase in pancreatic β-cells in multiple species and that it modulates insulin secretion by controlling activation of purinergic receptors. [ABSTRACT FROM AUTHOR]

The GPR119 receptor plays an important role in the secretion of incretin hormones in response to nutrient consumption. We have studied the ability of an array of naturally occurring endocannabinoid-like lipids to activate GPR119 and have identified several lipid receptor agonists. The most potent receptor agonists identified were three N-acylethanolamines: oleoylethanolamine (OEA), palmitoleoylethanolamine, and linoleylethanolamine (LEA), all of which displayed similar potency in activating GPR119. Another lipid, 2-oleoylglycerol (2-OG), also activated GPR119 receptor but with significantly lower potency. Endogenous levels of endocannabinoid-like lipids were measured in intestine in fasted and refed mice. Of the lipid GPR119 agonists studied, the intestinal levels of only OEA, LEA, and 2-OG increased significantly upon refeeding. Intestinal levels of OEA and LEA in the fasted mice were low. In the fed state, OEA levels only moderately increased, whereas LEA levels rose drastically. 2-OG was the most abundant of the three GPR119 agonists in intestine, and its levels were radically elevated in fed mice. Our data suggest that, in lean mice, 2-OG and LEA may serve as physiologically relevant endogenous GPR119 agonists that mediate receptor activation upon nutrient up-take. [ABSTRACT FROM AUTHOR]

Cytosolic free Ca2+ plays an important role in the molecular mechanisms leading to regulated insulin secretion by the pancreatic β cell. A number of Ca2+-binding proteins have been implicated in this process. Here, we define the role of the Ca2+-binding protein neuronal Ca2+ sensor-1 (NCS-1) in insulin secretion. In pancreatic β cells, NCS-1 increases exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in the readily releasable pool. The effect of NCS-1 on exocytosis is mediated through an increase in phosphatidylinositol (Pl) 4-kinase β activity and the generation of phosphoinositides, specifically Pl 4-phosphate and Pl 4,5-bisphosphate. In turn, Pl 4,5-bisphosphate controls exocytosis through the Ca2+ -dependent activator protein for secretion present in β cells. Our results provide evidence for an essential role of phosphoinositide synthesis in the regulation of glucose-induced insulin secretion by the pancreatic β cell. We also demonstrate that NCS-1 and its down- stream target Pl 4-kinase β, are critical players in this process by virtue of their capacity to regulate the release competence of the secretory granules. [ABSTRACT FROM AUTHOR]

An oscillatory increase in pancreatic β cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel β3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using β3 subunit-deficient β cells. β3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in β cells lacking the β3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the β3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the β3 subunit in the β cell may constitute the basis for a novel diabetes therapy. [Copyright &y& Elsevier]

Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A[sub 2] (cPLA[sub 2]α) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca[sup 2+]-dependent exocytosis in single mouse pancreatic β-cells, cPLA[sub 2]α dose dependently (EC[sub 50] = 86 nM) stimulated depolarization-evoked exocytosis by 450% without affecting the whole cell Ca[sup 2+] current or cytoplasmic Ca[sup 2+] levels. The stimulatory effect involved priming of secretory granules as reflected by an increase in the size of the readily releasable pool of granules from 70-80 to 280-300. cPLA[sub 2]αstimulated exocytosis was antagonized by the specific cPLA[sub 2] inhibitor AACOCF[sub 3]. Ca[sup 2+]-evoked exocytosis was reduced by 40% in cells treated with AACOCF[sub 3] or an antisense oligonucleotide against cPLA[sub 2]α. The action of cPLA[sub 2]α was mimicked by a combination of arachidonic acid and lysophosphatidylcholine (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve Cl[sup -] uptake through ClC-3 Cl[sup -] channels. Accordingly, the stimulatory action of cPLA[sub 2]α was inhibited by the Cl[sup -] channel inhibitor DIDS and in cells pretreated with ClC-3 Cl[sup -] channel antisense oligonucleotides. We propose that cPLA[sub 2]α has an important role in controlling the rate of exocytosis in β-cells. This effect of cPLA[sub 2]α reflects an enhanced transgranular Cl[sup -] flux, leading to an increase in the number of granules available for release, and requires the combined actions of arachidonic acid and lysophosphatidylcholine. [ABSTRACT FROM AUTHOR]

Insulin secretion is controlled by the β cell's metabolic state, and the ability of the secretory granules to undergo exocytosis increases during glucose stimulation in a membrane potential-independent fashion. Here, we demonstrate that exocytosis of insulincontaining secretory granules depends on phosphatidylinositol 4-kinase (PI 4-kinase) activity and that inhibition of this enzyme suppresses glucose-stimulated insulin secretion. Intracellular application of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P[sub 2]] stimulated exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in a readily releasable pool. Reducing the cytoplasmic ADP concentration in a way mimicking the effects of glucose stimulation activated PI 4-kinase and increased exocytosis whereas changes of the ATP concentration in the physiological range had little effect. The PI(4,5)P[sub 2]-binding protein Ca[sup 2+]-dependent activator protein for secretion (CAPS) is present in β cells, and neutralization of the protein abolished both Ca[sup 2+]- and PI(4,5)P[sub 2]-induced exocytosis. We conclude that ADPinduced changes in PI 4-kinase activity, via generation of PI(4,5)P[sub 2], represents a metabolic sensor in the β cell by virtue of its capacity to regulate the release competence of the secretory granules. [ABSTRACT FROM AUTHOR]

The discovery and optimization of a novel series of GPR142 agonists are described. These led to the identification of compound 21 (LY3325656), which demonstrated anti-diabetic benefits in pre-clinical studies and ADME/PK properties suitable for human dosing. Compound 21 is the first GPR142 agonist molecule advancing to phase 1 clinic trials for the treatment of Type 2 diabetes. [ABSTRACT FROM AUTHOR]

Agonists of G-protein-coupled receptor 142 (GPR142) are reported to increase insulin secretion and lower glucose in preclinical models. We identified a novel agonist, LY3325656 (LY) that selectively activates GPR142 in humans and mice. In preclinical models, LY increased glucose-dependent insulin secretion (GDIS) and secretion of glucagon and incretin hormones GLP-1 and GIP. To elucidate mechanisms of LY in humans, we designed a multi-part, single-dose study in 24 healthy volunteers (HV) and 12 patients with type 2 diabetes (T2D) (NCT03115099). LY was well tolerated through dose escalation up to 1950 mg in HV, with a median time to peak concentration of 4 hours and half-life of 4.59 hours. A graded glucose infusion evaluated GDIS, and LY demonstrated increased insulin secretion at higher doses (750-1950 mg). A 4-period, crossover design consisting of placebo (Pb), LY (1950mg), LY+DPP-IV inhibitor, and GLP-1R agonist treatment investigated glucose-lowering and incretin pharmacology in patients with T2D. LY significantly reduced post-meal glucose levels (2-hour glucose AUC) by >10% during a mixed meal tolerance test. Increases in post-meal GLP-1 AUC concentrations with LY compared to Pb were seen in a few, but not all patients with T2D. Serum glucagon levels were consistently increased by LY in patients with T2D and HV. We conclude that GPR142 activation in humans increases GDIS and augments glucagon secretion but does not substantively augment GIP or GLP-1 secretion. Our data suggest that acute stimulatory effects of GPR142 on insulin and glucagon secretion translate from preclinical models to man, unlike preclinical effects on gut peptide secretion. Innovative and efficient single-dose studies can provide key mechanistic understanding of the translatability of preclinical findings for the treatment of T2D. Disclosure: E.J. Pratt: Employee; Self; Eli Lilly and Company. T. Oura: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. X. Ma: Employee; Self; Eli Lilly and Company. H.V. Lin: Employee; Self; Eli Lilly and Company, Forkhead BioTherapeutics, Inc. Employee; Spouse/Partner; Novo Nordisk A/S. Stock/Shareholder; Self; Eli Lilly and Company, Forkhead BioTherapeutics, Inc. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. T. Ma: None. A.M. Efanov: Employee; Self; Eli Lilly and Company. M.K. Thomas: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. [ABSTRACT FROM AUTHOR]