Palmitate-Induced β-Cell Dysfunction Is Associated with Excessive NO Production and Is Reversed by Thiazolidinedione-Mediated Inhibition of GPR40 Transduction Mechanisms

BACKGROUND: Type 2 diabetes often displays hyperlipidemia. We examined palmitate effects on pancreatic islet function in relation to FFA receptor GPR40, NO generation, insulin release, and the PPARgamma agonistic thiazolidinedione, rosiglitazone. PRINCIPAL FINDINGS: Rosiglitazone suppressed acute palmitate-stimulated GPR40-transduced PI hydrolysis in HEK293 cells and insulin release from MIN6c cells and mouse islets. Culturing islets 24 h with palmitate at 5 mmol/l glucose induced beta-cell iNOS expression as revealed by confocal microscopy and increased the activities of ncNOS and iNOS associated with suppression of glucose-stimulated insulin response. Rosiglitazone reversed these effects. The expression of iNOS after high-glucose culturing was unaffected by rosiglitazone. Downregulation of GPR40 by antisense treatment abrogated GPR40 expression and suppressed palmitate-induced iNOS activity and insulin release. CONCLUSION: We conclude that, in addition to mediating acute FFA-stimulated insulin release, GPR40 is an important regulator of iNOS expression and dysfunctional insulin release during long-term exposure to FFA. The adverse effects of palmitate were counteracted by rosiglitazone at GPR40, suggesting that thiazolidinediones are beneficial for beta-cell function in hyperlipidemic type 2 diabetes

Excessive Islet NO Generation in Type 2 Diabetic GK Rats Coincides with Abnormal Hormone Secretion and Is Counteracted by GLP-1

BACKGROUND: A distinctive feature of type 2 diabetes is inability of insulin-secreting beta-cells to properly respond to elevated glucose eventually leading to beta-cell failure. We have hypothesized that an abnormally increased NO production in the pancreatic islets might be an important factor in the pathogenesis of beta-cell dysfunction. PRINCIPAL FINDINGS: We show now that islets of type 2 spontaneous diabetes in GK rats display excessive NO generation associated with abnormal iNOS expression in insulin and glucagon cells, increased ncNOS activity, impaired glucose-stimulated insulin release, glucagon hypersecretion, and impaired glucose-induced glucagon suppression. Pharmacological blockade of islet NO production by the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) greatly improved hormone secretion from GK islets suggesting islet NOS activity being an important target to inactivate for amelioration of islet cell function. The incretin hormone GLP-1, which is used in clinical practice suppressed iNOS and ncNOS expression and activity with almost full restoration of insulin release and partial restoration of glucagon release. GLP-1 suppression of iNOS expression was reversed by PKA inhibition but unaffected by the proteasome inhibitor MG132. Injection of glucose plus GLP-1 in the diabetic rats showed that GLP-1 amplified the insulin response but induced a transient increase and then a poor depression of glucagon. CONCLUSION: The results suggest that abnormally increased NO production within islet cells is a significant player in the pathogenesis of type 2 diabetes being counteracted by GLP-1 through PKA-dependent, nonproteasomal mechanisms

Uridine diphosphate (UDP) stimulates insulin secretion by activation of P2Y(6) receptors.

We examined the transcriptional expression and functional effects of receptors for the extracellular pyrimidines uridine triphosphate (UTP) and uridine diphosphate (UDP), on insulin and glucagon secretion in isolated mouse pancreatic islets and purified beta-cells. Using real-time PCR, the UDP receptor P2Y(6) was found to be highly expressed in both whole islets and beta-cells purified by repeated counter-flow elutriation, whereas no mRNA expression for UTP receptors P2Y(4) and P2Y(2) could be detected. Functional in vitro experiments revealed that the P2Y(6) agonist UDPbetaS dose-dependently enhanced insulin and glucagon release during short-term incubation (1h), while P2Y(6) activation during a longer period (24h), selectively increased insulin release, especially at high glucose levels. The corresponding EC(50) value for UDPbetaS ranged from 3.2x10(-8)M to 1.6x10(-8)M for both glucose concentrations. The P2Y(6) antagonist MRS2578 inhibited the effects of UDPbetaS, supporting a P2Y(6) specific effect. In addition to negative RT-PCR results, the lack of response to UTPgammaS a selective P2Y(2/4) agonist further rule out the involvement of P2Y(2/4) receptors in the islet hormone release. Our results suggest a modulatory role for UDP via a functional active P2Y(6) receptor in the regulation of islet hormone release

Correction: Palmitate-Induced β-Cell Dysfunction Is Associated with Excessive NO Production and Is Reversed by Thiazolidinedione-Mediated Inhibition of GPR40 Transduction Mechanisms.

https://researchrepository.wvu.edu/wvu_herbwvfern/2768/thumbnail.jp

Confocal microscopy and fluorescence intensity after 24 h culturing.

<p>Confocal microscopy and fluorescence intensity measurements of murine islets cultured for 24h at 7 mmol/l glucose (7G) ± 5 μmol/l glibenclamide (Glib) or 20 μmol/l metformin (Met) or both as well as at 20 mmol/l glucose (20G) ± 20 μmol/l metformin (Met). The islets were double-immunolabelled for insulin (A, D, G, J, M, P) and iNOS protein (B, E, H, K, N, Q). Insulin staining appears as green and iNOS as red staining, respectively. Co-localization of insulin/iNOS is seen as orange-yellowish fluorescence (C, F, I, L, O, R). Bars indicate 20 μm Lower part of the figure shows fluorescence intensity measurements quantified pixel by pixel using Zen 2009 software. Means ± SEM are shown for 19–34 observations in each group. ***p<0.001</p

Effect of metformin on the NOS-NO system and glibenclamide-induced insulin release.

<p>ncNOS, iNOS and total NOS activities as well as insulin release in murine islets incubated at 7 mmol/l glucose (7G) for 60 min in the absence and presence of 20 μmol/l metformin (Met) or 5 μmol/l glibenclamide (Glib) or both. Means ± SEM for 6–9 batches of islets in each group are shown. *p<0.05; **p<0.01; ***p<0.001</p