Insulin receptor substrate-1 and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial cells.
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-3T3(IR) 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 insulin-stimulated 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 insulin-signaling pathways.
Montagnani
Monica
M
Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1755, USA.
Ravichandran
Lingamanaidu V
LV
Chen
Hui
H
Esposito
Diana L
DL
Quon
Michael J
MJ
D064413:3-Phosphoinositide-Dependent Protein Kinases; D016475:3T3 Cells; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D009154:Mutation; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology
D064413:3-Phosphoinositide-Dependent Protein Kinases; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011972:Receptor, Insulin; D011994:Recombinant Proteins; C526292:IRS1 protein, human; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; C496314:NOS3 protein, human; C496315:Nos3 protein, mouse; C496316:Nos3 protein, rat