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.


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 approximately 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.
Zeng
G
G
Hypertension-Endocrine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1755, USA.
Nystrom
F H
FH
Ravichandran
L V
LV
Cong
L N
LN
Kirby
M
M
Mostowski
H
H
Quon
M J
MJ
D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000502:physiology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D020794:Receptor Protein-Tyrosine Kinases / Q000502:physiology; D011972:Receptor, Insulin / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D018631:ras Proteins / Q000502:physiology
D007328:Insulin; D009569:Nitric Oxide; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D020794:Receptor Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D018631:ras Proteins; C494918:AKT1 protein, human