Alexander M. Efanov

Fibroblast Growth Factor-21 Improves Pancreatic β-Cell Function and Survival by Activation of Extracellular Signal–Regulated Kinase 1/2 and Akt Signaling Pathways

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.

Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic beta cells.

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)P2] 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)P2-binding protein Ca2+-dependent activator protein for secretion (CAPS) is present in β cells, and neutralization of the protein abolished both Ca2+- and PI(4,5)P2-induced exocytosis. We conclude that ADP-induced changes in PI 4-kinase activity, via generation of PI(4,5)P2, represents a metabolic sensor in the β cell by virtue of its capacity to regulate the release competence of the secretory granules.

Munc-18 associates with syntaxin and serves as a negative regulator of exocytosis in the pancreatic β-cell

The Munc-18 protein (mammalian homologue of theunc-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 β-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 Ca2+ channel activity. The findings taken together show that the Munc-18 protein is present in insulin-secreting β-cells and implicate Munc-18 as a negative regulator of the insulin secretory machinery via a mechanism that does not involve syntaxin-associated Ca2+ channels.

Increase in cellular glutamate levels stimulates exocytosis in pancreatic β‐cells

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.

GPR142 Controls Tryptophan-Induced Insulin and Incretin Hormone Secretion to Improve Glucose Metabolism

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.

Activation of prostaglandin E receptor 4 triggers secretion of gut hormone peptides GLP-1, GLP-2, and PYY.

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 PDZ/coiled-coil domain containing protein PIST modulates insulin secretion in MIN6 insulinoma cells by interacting with somatostatin receptor subtype 5

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.

GPR142 prompts glucagon-like Peptide-1 release from islets to improve β cell function

Objective GPR142 agonists are being pursued as novel diabetes therapies by virtue of their insulin secretagogue effects. But it is undetermined whether GPR142s functions in pancreatic islets are limited to regulating insulin secretion. The current study expands research on its action. Methods and Results We demonstrated by in situ hybridization and immunostaining that GPR142 is expressed not only in β cells but also in a subset of α cells. Stimulation of GPR142 by a selective agonist increased glucagon secretion in both human and mouse islets. More importantly, the GPR142 agonist also potentiated glucagon-like peptide-1 (GLP-1) production and its release from islets through a mechanism that involves upregulation of prohormone convertase 1/3 expression. Strikingly, stimulation of insulin secretion and increase in insulin content via GPR142 engagement requires intact GLP-1 receptor signaling. Furthermore, GPR142 agonist increased β cell proliferation and protected both mouse and human islets against stress-induced apoptosis. Conclusions Collectively, we provide here evidence that local GLP-1 release from α cells defines GPR142s beneficial effects on improving β cell function and mass, and we propose that GPR142 agonism may have translatable and durable efficacy for the treatment of type 2 diabetes.