Insulin-stimulated Activation of eNOS Is Independent of Ca2+ but Requires Phosphorylation by Akt at Ser1179

doi:10.1074/jbc.M103702200

Insulin-stimulated Activation of eNOS Is Independent of Ca²⁺ but Requires Phosphorylation by Akt at Ser¹¹⁷⁹*

From the Cardiology Branch, NHLBI, and^‡Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

Abstract

Vasodilator actions of insulin are mediated by activation of endothelial nitric-oxide synthase (eNOS) and subsequent production of NO. Phosphatidylinositol 3-kinase and Akt play important roles in insulin-signaling pathways leading to production of NO in vascular endothelium. Here we dissected mechanisms whereby insulin activates eNOS by using the fluorescent dye DAF-2 to directly measure NO production in single cells. Insulin caused a rapid increase in intracellular NO in NIH-3T3^IR cells transiently transfected with eNOS. The stimulation of NO production by lysophosphatidic acid (LPA) was abrogated by pretreatment of cells with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. Remarkably, in the same cells, insulin-stimulated production of NO was unaffected. However, cells expressing the eNOS-S1179A mutant (disrupted Akt phosphorylation site) did not produce detectable NO in response to insulin, whereas the response to LPA was similar to that observed in cells expressing wild-type eNOS. Moreover, production of NO in response to insulin was blocked by coexpression of an inhibitory mutant of Akt, whereas the response to LPA was unaffected. Phosphorylation of eNOS at Ser¹¹⁷⁹ was observed only in response to treatment with insulin, but not with LPA. Interestingly, platelet-derived growth factor treatment of cells activated Akt but not eNOS. Results from human vascular endothelial cells were qualitatively similar to those obtained in transfected NIH-3T3^IR cells, although the magnitude of the responses was smaller. We conclude that insulin regulates eNOS activity using a Ca²⁺-independent mechanism requiring phosphorylation of eNOS by Akt. Importantly, phosphorylation-dependent mechanisms that enhance eNOS activity can operate independently from Ca²⁺-dependent mechanisms.

Footnotes

↵* This work was supported in part by a Research Award from the American Diabetes Association (to M. J. Q.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ To whom correspondence should be addressed: Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bldg. 10, Rm. 8C-218, 10 Center Dr., MSC 1755, Bethesda, MD 20892-1755. Tel.: 301-496-6269; Fax: 301-402-1679; E-mail: quonm@nih.gov.
Published, JBC Papers in Press, June 11, 2001, DOI 10.1074/jbc.M103702200
Abbreviations:

PI

phosphatidylinositol

eNOS

endothelial nitric-oxide synthase

WT

wild type

HUVEC

human umbilical vein endothelial cells

DAF-2

4,5-diaminofluorescein

DAF-2 DA

DAF-2 diacetate

BAPTA

1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

RFP

red fluorescent protein

LPA

lysophosphatidic acid

PDGF

platelet-derived growth factor
- Received April 25, 2001.
The American Society for Biochemistry and Molecular Biology, Inc.