The trafficking/interaction of eNOS and caveolin-1 induced by insulin modulates endothelial nitric oxide production

Mol Endocrinol. 2009 Oct;23(10):1613-23. doi: 10.1210/me.2009-0115. Epub 2009 Jul 16.


Endothelial nitric oxide synthase (eNOS) activity is tightly regulated by posttranscriptional modification and its subcellular localization. Here we examined whether insulin modulates nitric oxide (NO) production by regulating eNOS subcellular localization. We used confocal microscopy and immunoblots to examine the time course for 1) subcellular targeting/association of eNOS and caveolin-1 (CAV-1); 2) eNOS Ser(1179) phosphorylation; and 3) NO production in cultured bovine aorta endothelial cells. Serum starvation increased eNOS/CAV-1 localization to the perinuclear region. Adding insulin provoked their prompt translocation to and association at the plasma membrane (PM). Specific monoclonal antibodies against either CAV-1 or eNOS coimmunoprecipitated the other from bovine aorta endothelial cell membrane extracts, and insulin increased this interaction. Insulin stimulated NO production transiently despite a persistent eNOS Ser(1179) phosphorylation. The decline of NO production correlated temporally to insulin-induced translocation of eNOS and CAV-1 to PM. Knockdown of CAV-1 expression with a specific small interfering RNA duplex resulted in eNOS redistributing to the perinuclear region and nearly doubled insulin-induced NO production. Inhibition of phosphatidylinositol 3-kinase activity with wortmannin not only significantly inhibited insulin-induced translocation of eNOS and CAV-1 to PM but also blocked insulin-induced interaction of CAV-1 with eNOS at PM. Insulin increased incorporation of [(3)H]palmitic acid into eNOS immunoprecipitates by approximately 140%. Insulin-induced translocation of eNOS and CAV-1 to PM was palmitoylation dependent. Inhibiting eNOS and CAV-1 palmitoylation enhanced the NO production while blocking the translocation of eNOS and CAV-1 to PM induced by insulin. These data show that insulin acutely regulates eNOS and CAV-1 trafficking to PM of vascular endothelial cells where their interaction can regulate eNOS activity.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / metabolism
  • Androstadienes / pharmacology
  • Animals
  • Cattle
  • Caveolin 1 / antagonists & inhibitors
  • Caveolin 1 / metabolism*
  • Cell Membrane / drug effects
  • Cell Membrane / enzymology
  • Endothelial Cells / cytology
  • Endothelial Cells / drug effects*
  • Endothelial Cells / enzymology*
  • Golgi Apparatus / drug effects
  • Golgi Apparatus / enzymology
  • Insulin / pharmacology*
  • Lipoylation / drug effects
  • Nitric Oxide / biosynthesis*
  • Nitric Oxide Synthase Type III / metabolism*
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphoserine / metabolism
  • Protein Binding / drug effects
  • Protein Transport / drug effects
  • RNA, Small Interfering / metabolism
  • Subcellular Fractions / drug effects
  • Subcellular Fractions / enzymology
  • Wortmannin


  • Androstadienes
  • Caveolin 1
  • Insulin
  • RNA, Small Interfering
  • Phosphoserine
  • Nitric Oxide
  • Nitric Oxide Synthase Type III
  • Acyltransferases
  • Phosphatidylinositol 3-Kinases
  • Wortmannin