Vasodilator actions of insulin are mediated by signaling pathways involving phosphatidylinositol 3-kinase (PI 3-kinase) and Akt that lead to activation of endothelial nitric oxide synthase (eNOS) in endothelium. Signaling molecules immediately upstream and downstream from PI 3-kinase involved with production of NO in response to insulin have not been previously identified. In this study, we evaluated roles of insulin receptor substrate 1 (IRS-1) and phosphoinositide- dependent kinase 1 (PDK-1) in production of NO. The fluorescent dye 4,5-diamine fluorescein diacetate was used to directly measure NO in NIH- 3T3IR cells transiently cotransfected with eNOS and various IRS-1 or PDK-1 constructs. In control cells, transfected with only eNOS, insulin stimulated a rapid dose-dependent increase in NO. Overexpression of wild-type IRS-1 increased the maximal insulin response 3-fold. Overexpression of IRS1-F6 (mutant that does not bind PI 3-kinase) or an antisense ribozyme against IRS-1 substantially inhibited insulinstimulated production of NO. Likewise, overexpression of wild-type PDK-1 enhanced insulin-stimulated production of NO, whereas a kinase-inactive mutant PDK-1 inhibited this action of insulin. Qualitatively similar results were observed in vascular endothelial cells. Production of NO by a calcium-dependent mechanism in response to lysophosphatidic acid was unaffected by either wild-type or mutant IRS-1 and PDK-1. We conclude that IRS-1 and PDK-1 play necessary roles in insulin-signaling pathways leading to activation of eNOS. Furthermore, classical Ca2- mediated pathways for activation of eNOS are separable from IRS-1- and PDK-1-dependent insulinsignaling pathways.
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|Titolo:||Insulin receptor substrate-1, and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial RID B-1970-2008|
|Data di pubblicazione:||2002|
|Citazione:||Insulin receptor substrate-1, and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial RID B-1970-2008 / Montagnani M; Ravichandran LV; Chen H; Esposito DL; Quon MJ. - In: MOLECULAR ENDOCRINOLOGY. - ISSN 0888-8809. - 16:8(2002), pp. 1931-1942.|
|Appare nelle tipologie:||1.1 Articolo in rivista|