on January 5, 2004
Published online before print January 5, 2004, doi: 10.1161/01.CIR.0000109498.77895.6F
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Submitted on February 26, 2003
From the Department of Internal Medicine University of Tor Vergata, Rome (M.F., R.M., D.L., M.C., R.L.); the Departments of Biomorphology (A.P.), Medicine and Aging Sciences (E.A.D., G.P., A.C.), and Biomedical Sciences (M.R.), Centro Scienze Invecchiamento, University d’Annunzio, Chieti; and the Department of Clinical and Experimental Medicine, University of Catanzaro (G.S.), Italy. * To whom correspondence should be addressed. E-mail: consoli{at}unich.it.
Background--Impaired insulin-mediated vasodilation might contribute to vascular damage in insulin-resistant states. Little is known about insulin regulation of nitric oxide (NO) synthesis in insulin-resistant cells. The aim of this work was to investigate insulin regulation of NO synthesis in human umbilical vein endothelial cells (HUVECs) carrying the IRS-1 gene G972R variant, known to be associated with impaired insulin activation of the PI3-kinase (PI3-K) pathway in transfected cells. Methods and Results--HUVECs were screened for the presence of the G972R-IRS-1 (HUVEC-G972R) variant by restriction fragment length polymorphisms. After 24-hour exposure to 10-7 mol/L insulin, endothelial NO synthase (eNOS) mRNA (reverse transcription-polymerase chain reaction), eNOS protein levels (Western blotting), and NOS activity (conversion of [3H]arginine into [3H]citrulline) were increased in wild-type HUVECs (HUVEC-WT), whereas they did not change from baseline in HUVEC-G972R. Compared with HUVEC-WT, in HUVEC-G972R after 2 and 10 minutes of insulin stimulation, IRS-1-associated PI3-K activity was reduced by 47% and 32%, respectively; Akt phosphorylation was decreased by 40% at both time points; and eNOS-Ser1177 phosphorylation was reduced by 38% and 51%, respectively. In HUVEC-WT, eNOS-Thr495 phosphorylation decreased after insulin stimulation. In contrast, in HUVEC-G972R, eNOS-Thr495 phosphorylation increased after insulin stimulation and was 40% greater than in HUVEC-WT. Conclusions--Our data demonstrate that genetic impairment of the (IRS)-1/PI3-K/PDK-1/Akt insulin signaling cascade determines impaired insulin-stimulated NO release and suggest that the G972R-IRS-1 polymorphism, through a direct impairment of Akt/eNOS activation in endothelial cells, may contribute to the genetic predisposition to develop endothelial dysfunction and cardiovascular disease.
Revised on September 22, 2003
Accepted on September 22, 2003
G972R IRS-1 Variant Impairs Insulin Regulation of Endothelial Nitric Oxide Synthase in Cultured Human Endothelial Cells
Massimo Federici MD,
Key words: endothelium • insulin • nitric oxide synthase
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