Glucose-dependent insulinotropic polypeptide activates the Raf-Mek1/2-ERK1/2 module via a cyclic AMP/cAMP-dependent protein kinase/Rap1-mediated pathway

The gastrointestinal hormone, glucose-dependent insulinotropic polypeptide (GIP), is one of the most important regulators of insulin secretion following ingestion of a meal. GIP stimulates insulin secretion from the pancreatic beta-cell via its G protein-coupled receptor activation of adenylyl cyclase and other signal transduction pathways, but there is little known regarding subsequent protein kinase pathways that are activated. A screening technique was used to determine the relative abundance of 75 protein kinases in CHO-K1 cells expressing the GIP receptor and in two pancreatic beta-cell lines (betaTC-3 and INS-1 (832/13) cells). This information was used to identify kinases that are potentially regulated following GIP stimulation, with a focus on GIP regulation of the ERK1/2 MAPK pathway. In CHO-K1 cells, GIP induced phosphorylation of Raf-1 (Ser-259), Mek1/2 (Ser-217/Ser-221), ERK1/2 (Thr-202 and Tyr-204), and p90 RSK (Ser-380) in a concentration-dependent manner. Activation of ERK1/2 was maximal at 4 min and was cAMP-dependent protein kinase-dependent and protein kinase C-independent. Studies using a beta-cell line (INS-1 clone 832/13) corroborated these findings, and it was also demonstrated that the ERK1/2 module could be activated by GIP in the absence of glucose. Finally, we have shown that GIP regulation of the ERK1/2 module is via Rap1 but does not involve Gbetagamma subunits nor Src tyrosine kinase, and we propose that cAMP-based regulation occurs via B-Raf in both CHO-K1 and beta-cells. These results establish the importance of GIP in the cellular regulation of the ERK1/2 module and identify a role for cAMP in coupling its G protein-coupled receptors to ERK1/2 activity in pancreatic beta-cells.     

Oleanolic acid enhances insulin secretion in pancreatic beta-cells

We investigated the effect of oleanolic acid, a plant-derived triterpenoid, on insulin secretion and content in pancreatic beta-cells and rat islets. Oleanolic acid significantly enhanced insulin secretion at basal and stimulatory glucose concentrations in INS-1 832/13 cells and enhanced acute glucose-stimulated insulin secretion in isolated rat islets. In the cell line the effects of oleanolic acid on insulin secretion were comparable to that of the sulfonylurea tolbutamide at basal glucose levels and with the incretin mimetic Exendin-4 under glucose-stimulated conditions, yet neither Ca(2+) nor cAMP rose in response to oleanolic acid. Chronic treatment with oleanolic acid increased total cellular insulin protein and mRNA levels. These effects may contribute to the anti-diabetic properties of this natural product.     

Important role of phosphodiesterase 3B for the stimulatory action of cAMP on pancreatic beta-cell exocytosis and release of insulin

Cyclic AMP potentiates glucose-stimulated insulin release and mediates the stimulatory effects of hormones such as glucagon-like peptide 1 (GLP-1) on pancreatic beta-cells. By inhibition of cAMP-degrading phosphodiesterase (PDE) and, in particular, selective inhibition of PDE3 activity, stimulatory effects on insulin secretion have been observed. Molecular and functional information on beta-cell PDE3 is, however, scarce. To provide such information, we have studied the specific effects of the PDE3B isoform by adenovirus-mediated overexpression. In rat islets and rat insulinoma cells, approximate 10-fold overexpression of PDE3B was accompanied by a 6-8-fold increase in membrane-associated PDE3B activity. The cAMP concentration was significantly lowered in transduced cells (INS-1(832/13)), and insulin secretion in response to stimulation with high glucose (11.1 mm) was reduced by 40% (islets) and 50% (INS-1). Further, the ability of GLP-1 (100 nm) to augment glucose-stimulated insulin secretion was inhibited by approximately 30% (islets) and 70% (INS-1). Accordingly, when stimulating with cAMP, a substantial decrease (65%) in exocytotic capacity was demonstrated in patch-clamped single beta-cells. In untransduced insulinoma cells, application of the PDE3-selective inhibitor OPC3911 (10 microm) was shown to increase glucose-stimulated insulin release as well as cAMP-enhanced exocytosis. The findings suggest a significant role of PDE3B as an important regulator of insulin secretory processes.     

Beta-cell PDE3B regulates Ca2+-stimulated exocytosis of insulin

cAMP signaling is important for the regulation of insulin secretion in pancreatic beta-cells. The level of intracellular cAMP is controlled through its production by adenylyl cyclases and its breakdown by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE3B is involved in the regulation of nutrient-stimulated insulin secretion. Here, aiming at getting deeper functional insights, we have examined the role of PDE3B in the two phases of insulin secretion as well as its localization in the beta-cell. Depolarization-induced insulin secretion was assessed and in models where PDE3B was overexpressed [islets from transgenic RIP-PDE3B/7 mice and adenovirally (AdPDE3B) infected INS-1 (832/13) cells], the first phase of insulin secretion, occurring in response to stimulation with high K(+) for 5 min, was significantly reduced ( approximately 25% compared to controls). In contrast, in islets from PDE3B(-/-) mice the response to high K(+) was increased. Further, stimulation of isolated beta-cells from RIP-PDE3B/7 islets, using successive trains of voltage-clamped depolarizations, resulted in reduced Ca(2+)-triggered first phase exocytotic response as well as reduced granule mobilization-dependent second phase, compared to wild-type beta-cells. Using sub-cellular fractionation, confocal microscopy and transmission electron microscopy of isolated mouse islets and INS-1 (832/13) cells, we show that endogenous and overexpressed PDE3B is localized to insulin granules and plasma membrane. We conclude that PDE3B, through hydrolysis of cAMP in pools regulated by Ca(2+), plays a regulatory role in depolarization-induced insulin secretion and that the enzyme is associated with the exocytotic machinery in beta-cells.     

Activation of adenylyl cyclases, regulation of insulin status, and cell survival by G(alpha)olf in pancreatic beta-cells

Because we recently identified the G(alpha)olf subunit in rat pancreatic beta-cells, we investigated the downstream effectors and the biological functions of this G protein in HEK-293T cells and the insulin-secreting mouse betaTC-3 cell line. With the use of transient transfection of HEK-293T cells with constitutively activated G(alpha)olf (G(alpha)olfQ214L, i.e., AG(alpha)olf), together with expression vectors encoding the adenylyl cyclase (AC) isoforms (AC-I to -VIII and soluble AC), compared with cotransfections using AG(alphas) (G(alphas)R201C), we observed that AG(alpha)olf preferentially activates AC-I and -VIII, which are also expressed in beta-cells. Stable overexpression of wild-type or AG(alpha)olf in betaTC-3 cells resulted in partial attenuation of insulin secretion and biosynthesis, suggesting that chronic activation of the G(alpha)olf-signaling pathway is associated with beta-cell desensitization. In agreement, transfected betaTC-3 cells present a decreased insulin content with respect to parental cells, whereas the proinsulin convertases PC-1 and PC-2 were unaffected. Furthermore, betaTC-3-AG(alpha)olf cells are resistant to serum starvation-induced apoptosis. Our findings suggest that G(alpha)olf is involved in insulin status, cell survival, and regeneration of the insulin-secreting beta-cells during development and diabetes.     

Extracts of Rehmanniae radix, Ginseng radix and Scutellariae radix improve glucose-stimulated insulin secretion and β-cell proliferation through IRS2 induction

Recent studies have revealed that beta-cell dysfunction is an important factor in developing type 2 diabetes. beta-cell dysfunction is related to impairment of the insulin/IGF-1 signaling cascade through insulin receptor substrate-2 (IRS2). The induction of IRS2 in beta-cells plays an important role in potentiating beta-cell function and mass. In this study, we investigated whether herbs used for treating diabetes in Chinese medicine-Galla rhois, Rehmanniae radix, Machilus bark, Ginseng radix, Polygonatum radix, and Scutellariae radix-improved IRS2 induction in rat islets, glucose-stimulated insulin secretion and beta-cell survival. R. radix, Ginseng radix and S. radix significantly enhanced glucose-stimulated insulin secretion compared to the control, i.e., by 49, 67 and 58%, respectively. These herbs induced the expression of IRS2, pancreas duodenum homeobox-1 (PDX-1), and glucokinase. The increased level of glucokinase could explain the enhancement of glucose-stimulated insulin secretion with these extracts. Increased PDX-1 expression was associated with beta-cell proliferation, which was consistent with the cell viability assay. In conclusion, R. radix, Ginseng radix and S. radix had an insulinotropic action similar to that of exendin-4.     

Deletion of GαZ protein protects against diet-induced glucose intolerance via expansion of β-cell mass

Insufficient plasma insulin levels caused by deficits in both pancreatic β-cell function and mass contribute to the pathogenesis of type 2 diabetes. This loss of insulin-producing capacity is termed β-cell decompensation. Our work is focused on defining the role(s) of guanine nucleotide-binding protein (G protein) signaling pathways in regulating β-cell decompensation. We have previously demonstrated that the α-subunit of the heterotrimeric G(z) protein, Gα(z), impairs insulin secretion by suppressing production of cAMP. Pancreatic islets from Gα(z)-null mice also exhibit constitutively increased cAMP production and augmented glucose-stimulated insulin secretion, suggesting that Gα(z) is a tonic inhibitor of adenylate cyclase, the enzyme responsible for the conversion of ATP to cAMP. In the present study, we show that mice genetically deficient for Gα(z) are protected from developing glucose intolerance when fed a high fat (45 kcal%) diet. In these mice, a robust increase in β-cell proliferation is correlated with significantly increased β-cell mass. Further, an endogenous Gα(z) signaling pathway, through circulating prostaglandin E activating the EP3 isoform of the E prostanoid receptor, appears to be up-regulated in insulin-resistant, glucose-intolerant mice. These results, along with those of our previous work, link signaling through Gα(z) to both major aspects of β-cell decompensation: insufficient β-cell function and mass.     

Alkali pH directly activates ATP-sensitive K+ channels and inhibits insulin secretion in beta-cells

Glucose stimulation of pancreatic beta-cells is reported to lead to sustained alkalization, while extracellular application of weak bases is reported to inhibit electrical activity and decrease insulin secretion. We hypothesize that beta-cell K(ATP) channel activity is modulated by alkaline pH. Using the excised patch-clamp technique, we demonstrate a direct stimulatory action of alkali pH on recombinant SUR1/Kir6.2 channels due to increased open probability. Bath application of alkali pH similarly activates native islet beta-cell K(ATP) channels, leading to an inhibition of action potentials, and hyperpolarization of membrane potential. In situ pancreatic perfusion confirms that these cellular effects of alkali pH are observable at a functional level, resulting in decreases in both phase 1 and phase 2 glucose-stimulated insulin secretion. Our data are the first to report a stimulatory effect of a range of alkali pH on K(ATP) channel activity and link this to downstream effects on islet beta-cell function.     

Lysophosphatidylcholine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled receptor

A lysophospholipid series, such as lysophosphatidic acid, lysophosphatidylserine, and lysophosphatidylcholine (LPC), is a bioactive lipid mediator with diverse physiological and pathological functions. LPC has been reported to induce insulin secretion from pancreatic beta-cells, however, the precise mechanism has remained elusive to date. Here we show that an orphan G-protein-coupled receptor GPR119 plays a pivotal role in this event. LPC potently enhances insulin secretion in response to high concentrations of glucose in the perfused rat pancreas via stimulation of adenylate cyclase, and dose-dependently induces intracellular cAMP accumulation and insulin secretion in a mouse pancreatic beta-cell line, NIT-1 cells. The Gs-protein-coupled receptor for LPC was identified as GPR119, which is predominantly expressed in the pancreas. GPR119-specific siRNA significantly blocked LPC-induced insulin secretion from NIT-1 cells. Our findings suggest that GPR119, which is a novel endogenous receptor for LPC, is involved in insulin secretion from beta-cells, and is a potential target for anti-diabetic drug development.     

Insulin receptor substrate 1-induced inhibition of endoplasmic reticulum Ca2+ uptake in beta-cells. Autocrine regulation of intracellular ca2+ homeostasis and insulin secretion.

To understand the role of the insulin receptor pathway in beta-cell function, we have generated stable beta-cells (betaIRS1-A) that overexpress by 2-fold the insulin receptor substrate-1 (IRS-1) and compared them to vector-expressing controls. IRS-1 overexpression dramatically increased basal cytosolic Ca2+ levels from 81 to 278 nM, but it did not affect Ca2+ response to glucose. Overexpression of the insulin receptor also caused an increase in cytosolic Ca2+. Increased cytosolic Ca2+ was due to inhibition of Ca2+ uptake by the endoplasmic reticulum, because endoplasmic reticulum Ca2+ uptake and content were reduced in betaIRS1-A cells. Fractional insulin secretion was significantly increased 2-fold, and there was a decrease in betaIRS1-A insulin content and insulin biosynthesis. Steady-state insulin mRNA levels and glucose-stimulated ATP were unchanged. High IRS-1 levels also reduced beta-cell proliferation. These data demonstrate a direct link between the insulin receptor signaling pathway and the Ca2+-dependent pathways regulating insulin secretion of beta-cells. We postulate that during regulated insulin secretion, released insulin binds the beta-cell insulin receptor and activates IRS-1, thus further increasing cytosolic Ca2+ by reducing Ca2+ uptake. We suggest the existence of a novel pathway of autocrine regulation of intracellular Ca2+ homeostasis and insulin secretion in the beta-cell of the endocrine pancreas.     

Pancreatic insulin content regulation by the estrogen receptor ER alpha

The function of pancreatic beta-cells is the synthesis and release of insulin, the main hormone involved in blood glucose homeostasis. Estrogen receptors, ER alpha and ER beta, are important molecules involved in glucose metabolism, yet their role in pancreatic beta-cell physiology is still greatly unknown. In this report we show that both ER alpha and ER beta are present in pancreatic beta-cells. Long term exposure to physiological concentrations of 17beta-estradiol (E2) increased beta-cell insulin content, insulin gene expression and insulin release, yet pancreatic beta-cell mass was unaltered. The up-regulation of pancreatic beta-cell insulin content was imitated by environmentally relevant doses of the widespread endocrine disruptor Bisphenol-A (BPA). The use of ER alpha and ER beta agonists as well as ER alphaKO and ER betaKO mice suggests that the estrogen receptor involved is ER alpha. The up-regulation of pancreatic insulin content by ER alpha activation involves ERK1/2. These data may be important to explain the actions of E2 and environmental estrogens in endocrine pancreatic function and blood glucose homeostasis.