Search

Phenotypic screening is a neoclassical approach for drug discovery. We conducted phenotypic screening for insulin secretion enhancing agents using INS-1E insulinoma cells as a model system for pancreatic beta-cells. A principal regulator of insulin secretion in beta-cells is the metabolically regulated potassium channel Kir6.2/SUR1 complex. To characterize hit compounds, we developed an assay to quantify endogenous potassium channel activity in INS-1E cells. We quantified ligand-regulated potassium channel activity in INS-1E cells using fluorescence imaging and thallium flux. Potassium channel activity was metabolically regulated and coupled to insulin secretion. The pharmacology of channel opening agents (diazoxide) and closing agents (sulfonylureas) was used to validate the applicability of the assay. A precise high-throughput assay was enabled, and phenotypic screening hits were triaged to enable a higher likelihood of discovering chemical matter with novel and useful mechanisms of action.

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 Ca(2+) 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.

Oxytocin (OXT) has been shown to suppress appetite, induce weight loss, and improve glycemic control and lipid metabolism in several species, including humans, monkeys, and rodents. However, OXT’s short half-life in circulation and lack of receptor selectivity limit its application and efficacy. In this study, we report an OXT peptide analog (OXTGly) that is potent and selective for the OXT receptor (OXTR). OXT, but not OXTGly, activated vasopressin receptors in vitro and acutely increased blood pressure in vivo when administered IP. OXT suppressed food intake in mice, whereas OXTGly had a moderate effect on food intake when administered IP or intracerebroventricularly. Both OXT (IP) and OXTGly (IP) improved glycemic control in glucose tolerance tests. Additionally, both OXT (IP) and OXTGly (IP) stimulated insulin, glucagon-like peptide 1, and glucagon secretion in mice. We generated lipid-conjugated OXT (acylated-OXT) and OXTGly (acylated-OXTGly) and demonstrated that these molecules have significantly extended half-lives in vivo. Compared with OXT, 2-week treatment of diet-induced obese mice with acylated-OXT [subcutaneous(ly) (SC)] resulted in enhanced body weight reduction, an improved lipid profile, and gene expression changes consistent with increased lipolysis and decreased gluconeogenesis. Treatment with acylated-OXTGly (SC) also resulted in a statistically significant weight loss, albeit to a lesser degree compared with acylated-OXT treatment. In conclusion, we demonstrate that selective activation of the OXTR pathway results in both acute and chronic metabolic benefits, whereas potential activation of vasopressin receptors by nonselective OXT analogs causes physiological stress that contributes to additional weight loss.

Significance Both type 1 and type 2 diabetes are associated with reduced β-cell mass or function, resulting from decreased proliferation and increased apoptosis. Understanding the signals governing β-cell survival and regeneration is critical for developing strategies to maintain healthy populations of these cells in individuals. Both forms of diabetes are associated with hyperglucagonemia and an increased plasma glucagon:insulin ratio. Glucagon excess contributes to metabolic dysregulation of the diabetic state and glucagon receptor antagonism is a potential target area for the treatment and prevention of diabetes. Our studies presented here suggest that blockade of glucagon signaling lowers glycemia in mouse models of type 1 diabetes while enhancing formation of functional β-cell mass and production of insulin-positive cells from α-cell precursors., 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.

Incretin and insulin responses to nutrient loads are suppressed in persons with diabetes, resulting in decreased glycemic control. Agents including sulfonylureas and dipeptidyl peptidase-4 inhibitors (DPP4i) partially reverse these effects and provide therapeutic benefit; however, their modes of action limit efficacy. Because somatostatin (SST) has been shown to suppress insulin and glucagonlike peptide-1 (GLP-1) secretion through the Gi-coupled SST receptor 5 (SSTR5) isoform in vitro, antagonism of SSTR5 may improve glycemic control via intervention in both pathways. Here, we show that a potent and selective SSTR5 antagonist reverses the blunting effects of SST on insulin secretion from isolated human islets, and demonstrate that SSTR5 antagonism affords increased levels of systemic GLP-1 in vivo. Knocking out Sstr5 in mice provided a similar increase in systemic GLP-1 levels, which were not increased further by treatment with the antagonist. Treatment of mice with the SSTR5 antagonist in combination with a DPP4i resulted in increases in systemic GLP-1 levels that were more than additive and resulted in greater glycemic control compared with either agent alone. In isolated human islets, the SSTR5 antagonist completely reversed the inhibitory effect of exogenous SST-14 on insulin secretion. Taken together, these data suggest that SSTR5 antagonism should increase circulating GLP-1 levels and stimulate insulin secretion (directly and via GLP-1) in humans, improving glycemic control in patients with diabetes.

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.

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 insulin-secreting 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 in HEK293 cells 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.

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.

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.

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.

The effect of the imidazoline compound LY374284 has been studied in pancreatic islets of db/db mice, a progressive model of diabetes. In perifusion experiments, pancreatic islets of db/db mice showed a progressive deterioration of glucose-induced insulin release with increasing age, whereby the first phase of insulin secretion was almost completely abolished and the second phase was substantially decreased by 15 weeks of age. LY374284 restored the first phase of glucose-induced insulin secretion in islets of 16-week-old db/db mice to 70% of that observed in islets isolated from age-matched nondiabetic db/1 mice. LY374284 did not affect insulin secretion at a low glucose concentration (3.3 mmol/L). A similar restoration of first phase insulin secretion was observed after application of glucagon-like peptide-1, whereas a sulfonylurea agent, tolbutamide, was inactive. LY374284 did not affect cytosolic Ca(2+) concentration or cellular ATP content. Furthermore, LY374284 strongly enhanced insulin secretion in islets of db/db and db/1 mice maximally depolarized by 30 mmol/L K(+) and 250 micromol/L diazoxide. The present data suggest that the imidazoline compound LY374284 restores biphasic insulin secretion in islets of diabetic db/db mice by amplifying glucose-induced insulin secretion at a site distal to Ca(2+)-influx.

The release of sPLA(2) from single mouse pancreatic beta-cells was monitored using a fluorescent substrate of the enzyme incorporated in the outer leaflet of the plasma membrane. Stimulation of beta-cells with agents that increased cytosolic free Ca(2+) concentration ([Ca(2+)](i)) induced a rapid release of sPLA(2) to the extracellular medium. Exogenous sPLA(2) strongly stimulated insulin secretion in mouse pancreatic islets at both basal and elevated glucose concentrations. The stimulation of insulin secretion by sPLA(2) was mediated via inhibition of ATP-dependent K(+) channels and an increase in [Ca(2+)](i). Measurements of cell capacitance in single beta-cells revealed that sPLA(2) did not modify depolarisation-induced exocytosis. Our data suggest that a positive feedback regulation of insulin secretion by co-released sPLA(2) is operational in pancreatic beta-cells and point to this enzyme as an autocrine regulator of insulin secretion.

Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A2(cPLA2α) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca2+-dependent exocytosis in single mouse pancreatic β-cells. cPLA2α dose dependently (EC50= 86 nM) stimulated depolarization-evoked exocytosis by 450% without affecting the whole cell Ca2+current or cytoplasmic Ca2+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. cPLA2α-stimulated exocytosis was antagonized by the specific cPLA2inhibitor AACOCF3. Ca2+-evoked exocytosis was reduced by 40% in cells treated with AACOCF3or an antisense oligonucleotide against cPLA2α. The action of cPLA2α 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-uptake through ClC-3 Cl-channels. Accordingly, the stimulatory action of cPLA2α was inhibited by the Cl-channel inhibitor DIDS and in cells pretreated with ClC-3 Cl-channel antisense oligonucleotides. We propose that cPLA2α has an important role in controlling the rate of exocytosis in β-cells. This effect of cPLA2α reflects an enhanced transgranular Cl-flux, leading to an increase in the number of granules available for release, and requires the combined actions of arachidonic acid and lysophosphatidylcholine.

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 insulin-containing 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 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 2 -binding protein Ca 2+ -dependent activator protein for secretion (CAPS) is present in β cells, and neutralization of the protein abolished both Ca 2+ - and PI(4,5)P 2 -induced exocytosis. We conclude that ADP-induced changes in PI 4-kinase activity, via generation of PI(4,5)P 2 , represents a metabolic sensor in the β cell by virtue of its capacity to regulate the release competence of the secretory granules.

The ATP-sensitive potassium channel is a key molecular complex for glucose-stimulated insulin secretion in pancreatic beta cells. In humans, mutations in either of the two subunits for this channel, the sulfonylurea type 1 receptor (Sur1) or Kir6.2, cause persistent hyperinsulinemic hypoglycemia of infancy. We have generated and characterized Sur1 null mice. Interestingly, these animals remain euglycemic for a large portion of their life despite constant depolarization of membrane, elevated cytoplasmic free Ca(2+) concentrations, and intact sensitivity of the exocytotic machinery to Ca(2+). A comparison of glucose- and meal-stimulated insulin secretion showed that, although Sur1 null mice do not secrete insulin in response to glucose, they secrete nearly normal amounts of insulin in response to feeding. Because Sur1 null mice lack an insulin secretory response to GLP-1, even though their islets exhibit a normal rise in cAMP by GLP-1, we tested their response to cholinergic stimulation. We found that perfused Sur1 null pancreata secreted insulin in response to the cholinergic agonist carbachol in a glucose-dependent manner. Together, these findings suggest that cholinergic stimulation is one of the mechanisms that compensate for the severely impaired response to glucose and GLP-1 brought on by the absence of Sur1, thereby allowing euglycemia to be maintained.

The Munc-18 protein (mammalian homologue of the unc-18 gene; also called nSec1 or rbSec1) has been identified as an essential component of the synaptic vesicle fusion protein complex. The cellular and subcellular localization and functional role of Munc-18 protein in pancreatic beta-cells was investigated. Subcellular fractionation of insulin-secreting HIT-T15 cells revealed a 67-kDa protein in both cytosol and membrane fractions. Immunohistochemistry showed punctate Munc-18 immunoreactivity in the cytoplasm of rat pancreatic islet cells. Direct double-labeling immunofluorescence histochemistry combined with confocal laser microscopy revealed the presence of Munc-18 immunoreactivity in insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing cells. Syntaxin 1 immunoreactivity was detected in extracts of HIT-T15 cells, which were immunoprecipitated using Munc-18 antiserum, suggesting an intimate association of Munc-18 with syntaxin 1. Administration of Munc-18 peptide or Munc-18 antiserum to streptolysin O-permeabilized HIT-T15 cells resulted in significantly increased insulin release, but did not have any significant effect on voltage-gated Ca(2+) channel activity. The findings taken together show that the Munc-18 protein is present in insulin-secreting beta-cells and implicate Munc-18 as a negative regulator of the insulin secretory machinery via a mechanism that does not involve syntaxin-associated Ca(2+) channels.

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 insulin-secreting 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.

Prostaglandins E1 and E2 are synthesized in the intestine and mediate a range of gastrointestinal functions via activation of the prostanoid E type (EP) family of receptors. We examined the potential role of EP receptors in the regulation of gut hormone secretion from L cells. Analysis of mRNA expression in mouse enteroendocrine GLUTag cells demonstrated the abundant expression of EP4 receptor, whereas expression of other EP receptors was much lower. Prostaglandin E1 and E2, nonselective agonists for all EP receptor subtypes, triggered glucagon like peptide 1 (GLP-1) secretion from GLUTag cells, as did the EP4-selective agonists CAY10580 and TCS2510. The effect of EP4 agonists on GLP-1 secretion was blocked by incubation of cells with the EP4-selective antagonist L161,982 or by down-regulating EP4 expression with specific small interfering RNA. Regulation of gut hormone secretion with EP4 agonists was further studied in mice. Administration of EP4 agonists to mice produced a significant elevation of plasma levels of GLP-1, glucagon like peptide 2 (GLP-2) and peptide YY (PYY), whereas gastric inhibitory peptide (GIP) levels were not increased. Thus, our data demonstrate that activation of the EP4 receptor in enteroendocrine L cells triggers secretion of gut hormones.

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 uptake.

Liver X receptors (LXRs) form functional heterodimers with the retinoid X receptors (RXRs) and regulate cholesterol, lipid, and glucose metabolism. We demonstrated previously that activation of LXR modulates insulin secretion in MIN6 cells and pancreatic islets. In this study we investigated the effects of the LXR agonist T0901317 and the RXR agonist 9-cis-retinoic acid (9cRA) on cell proliferation and apoptosis in MIN6 cells. Whereas T0901317 showed no effect on proliferation of MIN6 cells, combination of T0901317 with 9cRA inhibited cell proliferation. Flow cytometry analysis of cell cycle demonstrated that activation of LXR/RXR prevented MIN6 cells from G1 to G2 phase progression. Combination of T0901317 and 9cRA increased apoptosis rate and caspase-3/7 activity in MIN6 cells. Moreover, T0901317 or its combination with 9cRA significantly increased the cell susceptibility to free fatty acid- and cytokine-induced apoptosis. Treatment of MIN6 cells with LXR and RXR agonists produced a strong increase in expression of mothers against decapentaplegic homolog 3, a protein known to inhibit cell cycle G1/S phase progression and induce apoptosis. In isolated rat islets, the effect of palmitic acid on caspase-3/7 activity was increased with T0901317 alone and even more with the combination of T0901317 and 9cRA. Thus, activation of LXR/RXR signaling inhibits cell proliferation and induces apoptosis in pancreatic beta-cells.

Liver X receptors (LXRalpha and LXRbeta) regulate glucose and lipid metabolism. Pancreatic beta-cells and INS-1E insulinoma cells express only the LXRbeta isoform. Activation of LXRbeta with the synthetic agonist T0901317 increased glucose-induced insulin secretion and insulin content, whereas deletion of the receptor in LXRbeta knockout mice severely blunted insulin secretion. Analysis of gene expression in LXR agonist-treated INS-1E cells and islets from LXRbeta-deficient mice revealed that LXRbeta positively regulated expression of ATP-binding cassette transporter A1 (ABCA1), sterol regulatory element-binding protein 1 (SREBP-1), insulin, PDX-1, glucokinase, and glucose transporter 2 (Glut2). Down-regulation of SREBP-1 expression with the specific small interfering RNA blocked basal and LXRbeta-induced expression of pancreatic duodenal homeobox 1 (PDX-1), insulin, and Glut2 genes. SREBP-1 small interfering RNA also prevented an increase in insulin secretion and insulin content induced by T0901317. Moreover, 5-(tetradecyloxy)-2-furoic acid, an inhibitor of the SREBP-1 target gene acetyl-coenzyme A carboxylase, blocked T0901317-induced stimulation of insulin secretion. In conclusion, activation of LXRbeta in pancreatic beta-cells increases insulin secretion and insulin mRNA expression via SREBP-1-regulated pathway. These data support the role of LXRbeta, SREBP-1, and cataplerosis/anaplerosis pathways in the control of insulin secretion in pancreatic beta-cells.

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.

The glucose-sensing enzyme glucokinase (GK) plays a key role in glucose metabolism. We report here the effects of a novel glucokinase activator, LY2121260. The activator enhanced GK activity via binding to the allosteric site located in the hinge region of the enzyme. LY2121260 stimulated insulin secretion in a glucose-dependent manner in pancreatic beta-cells and increased glucose use in rat hepatocytes. In addition, incubation of beta-cells with the GK activator resulted in increased GK protein levels, suggesting that enhanced insulin secretion on chronic treatment with a GK activator may be due to not only changed enzyme kinetics but also elevated enzyme levels. Animals treated with LY2121260 showed an improved glucose tolerance after oral glucose challenge. These results support the concept that GK activators represent a new class of compounds that increase both insulin secretion and hepatic glucose use and in doing so may prove to be effective agents for the control of blood glucose levels in patients with type 2 diabetes.

Cytosolic free Ca 2+ plays an important role in the molecular mechanisms leading to regulated insulin secretion by the pancreatic β cell. A number of Ca 2+ -binding proteins have been implicated in this process. Here, we define the role of the Ca 2+ -binding protein neuronal Ca 2+ 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 (PI) 4-kinase β activity and the generation of phosphoinositides, specifically PI 4-phosphate and PI 4,5-bisphosphate. In turn, PI 4,5-bisphosphate controls exocytosis through the Ca 2+ -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 downstream target, PI 4-kinase β, are critical players in this process by virtue of their capacity to regulate the release competence of the secretory granules.

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.

The respiratory burst induced by phorbol myristate acetate in mouse macrophages was inhibited by ultra-low doses (10−15 −10−13 M) of an opioid peptide [d-Ala2] methionine enkephalinamide. The effect disappeared at concentrations above and below this range. The inhibition approached 50% and was statistically significant (P < 0.001). Increasing the time of the opioid incubation with cells brought about a shift in the maximal effect to lower concentrations of the opioid (from 10−13 to 5 · 10−15 M) and led to a decrease in the value of the effect, fully in accord with the previously proposed adaptation mechanism of the action of ultra-low doses.

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 Ca 2+ -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 A 1 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.

Membrane homeostasis is maintained by exocytosis and endocytosis. The molecular mechanisms regulating the interplay between these two processes are not clear. Inositol hexakisphosphate (InsP 6 ) is under metabolic control and serves as a signal in the pancreatic β cell stimulus-secretion coupling by increasing Ca 2+ -channel activity and insulin exocytosis. We now show that InsP 6 also promotes dynamin I-mediated endocytosis in the pancreatic β cell. This effect of InsP 6 depends on calcineurin-induced dephosphorylation and is accounted for by both activation of protein kinase C and inhibition of the phosphoinositide phosphatase synaptojanin and thereby formation of phosphatidylinositol 4,5-bisphosphate. In regulating both exocytosis and endocytosis, InsP 6 thus may have an essential integral role in membrane trafficking.

The insulinotropic activity of the imidazoline derivative RX871024 was compared in pancreatic islets from nondiabetic Wistar rats and spontaneously diabetic Goto-Kakizaki (GK) rats. RX871024 significantly stimulated insulin secretion in islets from both animal groups. The insulinotropic activity of RX871024 was higher than that of the sulfonylurea glibenclamide. This difference was more pronounced in islets from GK rats compared with Wistar rat islets. More importantly, RX871024 substantially improved glucose sensitivity in diabetic β-cells, whereas glibenclamide stimulated insulin secretion about twofold over a broad range of glucose concentrations in nondiabetic and diabetic rats. RX871024 induced a faster increase in cytosolic free Ca2+concentration and faster inhibition of ATP-dependent K+channel activity in GK rat islets compared with Wistar rat islets. RX871024 also induced a more pronounced increase in diacylglycerol concentration in GK rat islets. These data support the idea that imidazoline compounds can form the basis for the development of novel drugs for treatment of type 2 diabetes, which can restore glucose sensitivity in diabetic β-cells.

Effects of the imidazoline compound RX871024 on cytosolic free Ca(2+) concentration ([Ca(2+)]i) and insulin secretion in pancreatic beta-cells from SUR1 deficient mice have been studied. In beta-cells from wild-type mice RX871024 increased [Ca(2+)]i by blocking ATP-dependent K(+)-current (K(ATP)) and inducing membrane depolarization. In beta-cells lacking a component of the K(ATP)-channel, SUR1 subunit, RX871024 failed to increase [Ca(2+)]i. However, insulin secretion in these cells was strongly stimulated by the imidazoline. Thus, a major component of the insulinotropic activity of RX871024 is stimulation of insulin exocytosis independently from changes in K(ATP)-current and [Ca(2+)]i. This means that effects of RX871024 on insulin exocytosis are partly mediated by interaction with proteins distinct from those composing the K(ATP)-channel.

The insulinotropic activity of the novel imidazoline compound BL11282 was investigated. Intravenous administration of BL11282 (0.3 mg · kg–1 · 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 μmol/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.

Imidazoline compound RX871024 and carbamylcholine (CCh) stimulate insulin secretion in isolated rat pancreatic islets. Combination of CCh and RX871024 induces a synergetic effect on insulin secretion. RX871024 and CCh produce twofold increases in diacylglycerol (DAG) concentration. The combination of two compounds has an additive effect on DAG concentration. Effects of RX871024 on insulin secretion and DAG concentration are not dependent on the presence of D609, an inhibitor of phosphatidylcholine-specific phospholipase C. It is concluded that as in case with CCh the increase in DAG concentration induced by imidazoline RX871024 contributes to the insulinotropic activity of the compound.

The effects of the imidazoline compound RX871024 on arginine-induced insulin, glucagon, and somatostatin secretion in the isolated perfused rat pancreas have been investigated. Arginine induced biphasic insulin, glucagon, and somatostatin release when infused for 20 min at 20 mM concentration and 3.3 mM glucose in the medium. RX871024, at 10 microM, did not influence basal hormone secretion but enhanced arginine-stimulated insulin and somatostatin release. In contrast, glucagon secretion was markedly inhibited by 10 microM imidazoline. RX871024 (1 microM) did not significantly affect arginine-induced insulin and somatostatin secretion but had an inhibitory effect on the second phase of glucagon release. In conclusion, RX871024 exerts a complex effect on the endocrine pancreas challenged by arginine, comprising stimulation of insulin and somatostatin release and inhibition of glucagon release. These effects on hormone release probably constitute the main mechanism of the antidiabetogenic action of the imidazolines.

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 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 beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 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 beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.