G972R IRS-1 Variant Impairs Insulin Regulation of Endothelial Nitric Oxide Synthase in Cultured Human Endothelial Cells

Massimo Federici, Assunta Pandolfi, Elena Anna De Filippis, Giuliana Pellegrini, Rossella Menghini, Davide Lauro, Marina Cardellini, Mario Romano, Giorgio Sesti, Renato Lauro, Agostino Consoli

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)399-405
Number of pages7
JournalCirculation
Volume109
Issue number3
DOIs
StatePublished - Jan 27 2004
Externally publishedYes

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Nitric Oxide Synthase Type III
Human Umbilical Vein Endothelial Cells
Endothelial Cells
Insulin
Nitric Oxide Synthase
Phosphorylation
Nitric Oxide
Citrulline
Genetic Predisposition to Disease
Phosphatidylinositol 3-Kinases
Vasodilation
Restriction Fragment Length Polymorphisms
Reverse Transcription
Blood Vessels
Arginine
Cardiovascular Diseases
Western Blotting

Keywords

  • Endothelium
  • Insulin
  • Nitric oxide synthase

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

G972R IRS-1 Variant Impairs Insulin Regulation of Endothelial Nitric Oxide Synthase in Cultured Human Endothelial Cells. / Federici, Massimo; Pandolfi, Assunta; De Filippis, Elena Anna; Pellegrini, Giuliana; Menghini, Rossella; Lauro, Davide; Cardellini, Marina; Romano, Mario; Sesti, Giorgio; Lauro, Renato; Consoli, Agostino.

In: Circulation, Vol. 109, No. 3, 27.01.2004, p. 399-405.

Research output: Contribution to journalArticle

Federici, M, Pandolfi, A, De Filippis, EA, Pellegrini, G, Menghini, R, Lauro, D, Cardellini, M, Romano, M, Sesti, G, Lauro, R & Consoli, A 2004, 'G972R IRS-1 Variant Impairs Insulin Regulation of Endothelial Nitric Oxide Synthase in Cultured Human Endothelial Cells', Circulation, vol. 109, no. 3, pp. 399-405. https://doi.org/10.1161/01.CIR.0000109498.77895.6F
Federici, Massimo ; Pandolfi, Assunta ; De Filippis, Elena Anna ; Pellegrini, Giuliana ; Menghini, Rossella ; Lauro, Davide ; Cardellini, Marina ; Romano, Mario ; Sesti, Giorgio ; Lauro, Renato ; Consoli, Agostino. / G972R IRS-1 Variant Impairs Insulin Regulation of Endothelial Nitric Oxide Synthase in Cultured Human Endothelial Cells. In: Circulation. 2004 ; Vol. 109, No. 3. pp. 399-405.
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abstract = "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.",
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AU - De Filippis, Elena Anna

AU - Pellegrini, Giuliana

AU - Menghini, Rossella

AU - Lauro, Davide

AU - Cardellini, Marina

AU - Romano, Mario

AU - Sesti, Giorgio

AU - Lauro, Renato

AU - Consoli, Agostino

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N2 - 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.

AB - 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.

KW - Endothelium

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