TY - JOUR
T1 - Acute insulin signaling in pancreatic beta-cells is mediated by multiple Raf-1 dependent pathways
AU - Alejandro, Emilyn U.
AU - Kalynyak, Tatyana B.
AU - Taghizadeh, Farnaz
AU - Gwiazda, Kamila S.
AU - Rawstron, Erin K.
AU - Jacob, Karen J.
AU - Johnson, James D.
PY - 2010
Y1 - 2010
N2 - 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.
AB - 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.
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U2 - 10.1210/en.2009-0678
DO - 10.1210/en.2009-0678
M3 - Article
C2 - 20056832
AN - SCOPUS:74949133820
SN - 0013-7227
VL - 151
SP - 502
EP - 512
JO - Endocrinology
JF - Endocrinology
IS - 2
ER -