Published online before print January 5, 2004, doi: 10.1161/01.CIR.0000109498.77895.6F
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(Circulation. 2004;109:399-405.)
© 2004 American Heart Association, Inc.
Basic Science Reports |
G972R IRS-1 Variant Impairs Insulin Regulation of Endothelial Nitric Oxide Synthase in Cultured Human Endothelial Cells
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.
Correspondence to Prof Agostino Consoli, MD, Department of Medicine and Aging Sciences, Edificio CeSi, Room 271, University of Chieti, Via dei Vestini, 1, 66100 Chieti, Italy. E-mail consoli{at}unich.it
Received February 26, 2003; de novo received July 16, 2003; revision received September 22, 2003; accepted September 22, 2003.
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.
Key Words: endothelium • insulin • nitric oxide synthase
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