G972R IRS-1 variant impairs insulin regulation of endothelial nitric oxide synthase in cultured human endothelial cells.
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.


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 [(3)H]arginine into [(3)H]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.
Federici
Massimo
M
Department of Medicine and Aging Sciences, Edificio CeSi, Room 271, University of Chieti, Via dei Vestini, 1, 66100 Chieti, Italy. consoli@unich.it
Pandolfi
Assunta
A
De Filippis
Elena Anna
EA
Pellegrini
Giuliana
G
Menghini
Rossella
R
Lauro
Davide
D
Cardellini
Marina
M
Romano
Mario
M
Sesti
Giorgio
G
Lauro
Renato
R
Consoli
Agostino
A
D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011110:Polymorphism, Genetic; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction
D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D020928:Mitogen-Activated Protein Kinases; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; C526292:IRS1 protein, human; C496314:NOS3 protein, human; C494918:AKT1 protein, human