Roles for Insulin Receptor, PI3-Kinase, and Akt in Insulin-Signaling Pathways Related to Production of Nitric Oxide in Human Vascular Endothelial Cells

Background—Previously, we demonstrated that insulin stimulates production of nitric oxide (NO) in endothelial cells. However, specific insulin-signaling pathways mediating production of NO have not been elucidated. Methods and Results—We developed methods for transfection of human umbilical vein endothelial cells (HUVECs) and direct measurement of NO to begin defining insulin-signaling pathways related to NO production. HUVECs were cotransfected with enhanced Green Fluorescent Protein (eGFP) and another gene of interest. Transfection efficiencies >95% were obtained by selecting cells expressing eGFP. Overexpression of insulin receptors in HUVECs resulted in an ≈3-fold increase in production of NO in response to insulin. In contrast, HUVECs overexpressing a tyrosine kinase–deficient mutant insulin receptor had a dose-response curve similar to that of control cells. Overexpression of inhibitory mutants of either phosphatidylinositol 3-kinase (PI3K) or Akt resulted in nearly complete inhibition of insulin-st...