Insulin receptor substrate-1 and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial cells

Mol Endocrinol. 2002 Aug;16(8):1931-42. doi: 10.1210/me.2002-0074.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3-Phosphoinositide-Dependent Protein Kinases
  • 3T3 Cells
  • Animals
  • Cattle
  • Cells, Cultured
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / drug effects*
  • Endothelium, Vascular / metabolism*
  • Humans
  • Insulin / pharmacology*
  • Insulin Receptor Substrate Proteins
  • Mice
  • Mutation
  • Nitric Oxide / biosynthesis*
  • Nitric Oxide Synthase / genetics
  • Nitric Oxide Synthase / metabolism
  • Nitric Oxide Synthase Type II
  • Nitric Oxide Synthase Type III
  • Phosphatidylinositol 3-Kinases / genetics
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Rats
  • Receptor, Insulin / genetics
  • Receptor, Insulin / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Signal Transduction
  • Transfection
  • Vasodilation / drug effects
  • Vasodilation / physiology


  • IRS1 protein, human
  • Insulin
  • Insulin Receptor Substrate Proteins
  • Irs1 protein, mouse
  • Irs1 protein, rat
  • Phosphoproteins
  • Recombinant Proteins
  • Nitric Oxide
  • NOS3 protein, human
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II
  • Nitric Oxide Synthase Type III
  • Nos3 protein, mouse
  • Nos3 protein, rat
  • Phosphatidylinositol 3-Kinases
  • Receptor, Insulin
  • 3-Phosphoinositide-Dependent Protein Kinases
  • Protein-Serine-Threonine Kinases