Acute insulin signaling in pancreatic beta-cells is mediated by multiple Raf-1 dependent pathways.
Insulin enhances the proliferation and survival of pancreatic beta-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 beta-cells. To test this hypothesis, we treated human and mouse islets as well as MIN6 beta-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 Ca(2+) signaling resulted in nonadditive beta-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 beta-cells. Activation of Raf-1 leads to both an ERK-dependent pathway that involves nicotinic acid adenine dinucleotide phosphate-sensitive Ca(2+) stores and Ca(2+)-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 beta-cells and shed light on insulin's antiapoptotic and mitogenic mechanisms.
Alejandro
Emilyn U
EU
Department of Cellular and Physiological Sciences, University of British Columbia, 5358 Life Sciences Building, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada.
Kalynyak
Tatyana B
TB
Taghizadeh
Farnaz
F
Gwiazda
Kamila S
KS
Rawstron
Erin K
EK
Jacob
Karen J
KJ
Johnson
James D
JD
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000502:physiology*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007328:Insulin / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D010636:Phenols / Q000494:pharmacology; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D007211:Indoles; D007328:Insulin; D010636:Phenols; D019908:Proto-Oncogene Proteins c-raf; C489251:5-iodo-3-((3,5-dibromo-4-hydroxyphenyl)methylene)-2-indolinone; D002118:Calcium