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Roles of Raf-1 kinase in pancreatic beta-cells Alejandro, Emilyn Uy

Abstract

A decrease in functional β-cell mass is key in the pathogenesis of both type 1 and type 2 diabetes and in the failure of transplanted islet grafts. Knowledge of the endogenous regulators of β-cell proliferation and survival are important for understanding the physiological regulation of β-cell mass. We have shown that physiological concentrations of the insulin hormone act directly on β-cells to promote proliferation and survival, but its mechanisms remain unclear. We hypothesized that Raf-1, a kinase upstream of both ERK1/2 and Bad, is a critical target of insulin in β-cells. To test this hypothesis, we treated primary β-cells and MIN6 β-cells with multiple insulin concentrations and examined putative downstream targets. Low concentrations of insulin rapidly activated Raf-1 and ERK1/2 in primary islets and MIN6 cells. The phosphorylation of ERK1/2 by insulin was eliminated by exposure to a Raf inhibitor or transfection with a dominant-negative Raf-1 mutant. Insulin enhanced the interaction between mitochondrial Raf-1 and Bad, promoting the inactivation of pro-apoptotic Bad. Over-expression of Raf-1 was sufficient to increase proliferation in the absence of insulin, whereas a dominant-negative Raf-1 reduced proliferation in the presence of insulin. We also tested if Raf-1 signalling plays an important role in β-cell survival both in vitro and in vivo. We utilized a Raf inhibitor and dominant-negative Raf-1 mutants to block basal Raf-1 signalling in serum free conditions in vitro and the Cre-lox recombination system to obtain a β-cell specific deletion of the Raf-1 gene in vivo. Our data show that blocking basal Raf-1 signalling in vitro caused apoptosis. Preliminary data indicate that β-cell specific Raf-1 knockout mice are viable, have increased fasting basal blood glucose levels and have impaired glucose tolerance compared to littermate controls, consistent with the concept that Raf-1 plays an important role in β-cell survival. Together, these findings have significant implications for the understanding of insulin signalling pathway in β-cells and the regulation of β-cell mass.

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