Catechol estrogens stimulate insulin secretion in pancreatic β-cells via activation of the transient receptor potential A1 (TRPA1) channel

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

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

AbstractGlutamate 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

Stimulation of insulin secretion independently from changes in cytosolic free Ca²+-concentration : studies with imidazolines and inositol polyphosphates

Insulin secretion from the islets of Langerhans is regulated by changes in glucose concentration. Glucose stimulates secretion by two mechanisms. First, it increases cytosolic free Ca2+ concentration ([Ca2+]i) by inhibition of ATP-dependent K+-channels (KATP) and induction of membrane depolarization. Second, glucose directly promotes insulin exocytosis. A number of other physiological and pharmacological stimuli, which modulate insulin secretion, can also influence either one or both of the two mentioned pathways. This study concentrates on mechanisms by which insulinotropic agents stimulate insulin exocytosis. For this purpose we have used two groups of compounds, imidazoline derivatives and inositol polyphosphates. The imidazoline compound, RX871024, stimulated insulin secretion through interaction with several molecular targets. First, the compound inhibited activity of KATP-channels as well as voltage-gated K+-channels, which in turn led to membrane depolarization and increase in [Ca2+]i, Elevated levels of [Ca2+]i stimulated insulin exocytosis. Second, RX871024 induced an increase in insulin secretion in cells with clamped [Ca2+]i, This effect required high both ATP and Ca2+ concentrations inside the cell. Third, the compound increased diacylglycerol (DAG) concentration in the islets. Finally, inhibition of protein kinase C (PKC) and cytochrome P-450 abolished the insulinotropic effect of RX871024. We interpreted these findings as indicating that the direct effect of RX871024 on insulin exocytosis was at least in part mediated by rise in DAG concentration, by the PKC inhibitor, and InsP6 also stimulated PKC activity in HIT T15 cell homogenate which then activated PKC and increased generation of arachidonic acid (AA) metabolites. Both PKC and AA pathway activation resulted in potentiation of insulin secretion. Comparison of the effects of RX871024 and the classical antidiabetic agent glibenclamide on insulin secretion demonstrated that the imidazoline exerted stronger stimulation of insulin secretion than glibenclamide. RX871024 also produced a potent increase in insulin secretion in islets from diabetic GK rats. In non-diabetic and diabetic rats, as well as non-diabetic humans, insulinotropic activity of RX871024 was highly dependent on glucose concentration and was stronger than that of glibenclamide. Inositol polyphosphates: myo-inositol 1,3,4,5,6-pentakisphosphate (InsP5) and myoinositol 1,2,3,4,5,6-hexakisphosphate (InsP6) stimulated basal (Ca2+-independent) insulin exocytosis in permeabilized insulin secreting HIT T15 cells. The effect of inositol polyphosphates was observed only in the presence of low free Ca2+ concentrations (30 nM) as InsP6 did not potentiate Ca2+-induced insulin exocytosis. However, when added before the concentration of Ca2+ was increased, InsP6 exerted a potent priming effect on Ca2+-induced insulin exocytosis. The effects of InsP6 on insulin exocytosis have been blocked. Thus, stimulation of Ca2+-independent insulin exocytosis and priming of Ca2+-dependent insulin exocytosis by inositol polyphosphates are mediated through activation of PKC. In addition to its effects on exocytosis, InsP6 increased rates of endocytosis in HIT T15 cells at both basal and elevated free Ca2+ concentrations. It is concluded that stimulation of protein kinases with subsequent phosphorylation of cellular proteins is responsible for the effects of imidazolines and inositol polyphosphates on insulin secretion. Compounds with effects on transport and fusion of insulin containing granules are therefore potential candidates for treatment of type 2 diabetes

Two Generations of Insulinotropic Imidazoline Compounds

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 Ca2+ concentration ([Ca2+]i), and insulin release. Importantly, RX871024 also enhanced the insulinotropic effect of glucose in cells with clamped [Ca2+]i but in the presence of high ATP and Ca2+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

Imidazoline RX871024 Raises Diacylglycerol Levels in Rat Pancreatic Islets

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 Imidazoline RX871024 Stimulates Insulin Secretion in Pancreatic β-Cells from Mice Deficient in KATP Channel Function

Effects of the imidazoline compound RX871024 on cytosolic free Ca2+ concentration ([Ca2+]i) and insulin secretion in pancreatic β-cells from SUR1 deficient mice have been studied. In β-cells from wild-type mice RX871024 increased [Ca2+]i by blocking ATP-dependent K+-current (KATP) and inducing membrane depolarization. In β-cells lacking a component of the KATP-channel, SUR1 subunit, RX871024 failed to increase [Ca2+]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 KATP-current and [Ca2+]i. This means that effects of RX871024 on insulin exocytosis are partly mediated by interaction with proteins distinct from those composing the KATP-channel

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