Acute insulin signaling in pancreatic beta-cells is mediated by multiple Raf-1 dependent pathways

Emilyn U. Alejandro, Tatyana B. Kalynyak, Farnaz Taghizadeh, Kamila S. Gwiazda, Erin K. Rawstron, Karen J. Jacob, James D. Johnson

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

Insulin enhances the proliferation and survival of pancreatic β-cells, but its mechanisms remain unclear. We hypothesized that Raf-1, a kinase upstream of both ERK and Bad, might be a critical target of insulin in β-cells. To test this hypothesis, we treated human and mouse islets as well as MIN6 β-cells with multiple insulin concentrations and examined putative downstream targets using immunoblotting, immunoprecipitation, quantitative fluorescent imaging, and cell death assays. Low doses of insulin rapidly activated Raf-1 by dephosphorylating serine 259 and phosphorylating serine 338 in human islets, mouse islets, and MIN6 cells. The phosphorylation of ERK by insulin was eliminated by exposure to a Raf inhibitor (GW5074) or transfection with a dominant-negative Raf-1 mutant. Insulin also enhanced the interaction between mitochondrial Raf-1 and Bcl-2 agonist of cell death (Bad), promoting Bad inactivation via its phosphorylation on serine 112. Insulin-stimulated ERK phosphorylation was abrogated by calcium chelation,calcineurin and calmodulin-dependent protein kinase II inhibitors,and Ned-19, a nicotinic acid adenine dinucleotide phosphate receptor(NAADPR)antagonist.Blocking Raf-1 and Ca2+ signaling resulted in nonadditive β-cell death. Autocrine insulin signaling partly accounted for the effects of glucose on ERK phosphorylation. Our results demonstrate that Raf-1 is a critical target of insulin in primary β-cells. Activation of Raf-1 leads to both an ERK-dependent pathway that involves nicotinic acid adenine dinucleotide phosphate-sensitive Ca2+ stores and Ca2+-dependent phosphorylation events, and an ERK-independent pathway that involves Bad inactivation at the mitochondria. Together our findings identify a novel insulin signaling pathway in β-cells and shed light on insulin's antiapoptotic and mitogenic mechanisms.

Original languageEnglish (US)
Pages (from-to)502-512
Number of pages11
JournalEndocrinology
Volume151
Issue number2
DOIs
StatePublished - 2010

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