(Circulation. 2000;101:1539.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
Roles for Insulin Receptor, PI3-Kinase, and Akt in Insulin-Signaling Pathways Related to Production of Nitric Oxide in Human Vascular Endothelial Cells
From the Hypertension-Endocrine Branch (G.Z., F.H.N., L.V.R., L.-N.C., M.J.Q.) and Hematology Branch (M.K.), National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md; and the Center for Biological Experimental Research (H.M.), Food and Drug Administration, Bethesda, Md.
Correspondence to Michael J. Quon, MD, PhD, Hypertension-Endocrine Branch, NHLBI, NIH, Bldg 10, Room 8C-218, 10 Center Dr MSC 1755, Bethesda, MD 20892-1755. E-mail quonm{at}nih.gov
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-stimulated production of
NO. Overexpression of an inhibitory mutant of
Ras had a much smaller effect.
Conclusions—Receptor kinase activity is necessary to mediate production of NO through the insulin receptor. Both PI3K and Akt contribute importantly to this process, whereas the contribution of Ras is small.
Key Words: insulin • endothelium • signal transduction • nitric oxide • receptors
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