Angiotensin II-induced NADPH Oxidase Activation Impairs Insulin Signaling in Skeletal Muscle Cells

doi:10.1074/jbc.M601320200

Angiotensin II-induced NADPH Oxidase Activation Impairs Insulin Signaling in Skeletal Muscle Cells^*

^‡Department of Internal Medicine and the ^¶Department of Pharmacology and Physiology, University of Missouri-Columbia 65212 and the ^§Harry S. Truman Memorial Veterans Medical Center, Columbia, Missouri 65201

2 To whom correspondence should be addressed: Dept. of Medicine, University of Arizona, Diabetes Research Center, 1656 E. Mabel St., P.O. 245218, Tucson, AZ 85724-5218. E-mail: stumpc{at}email.arizona.edu.

Abstract

The renin-angiotensin system (RAS) and reactive oxygen species (ROS) have been implicated in the development of insulin resistance and its related complications. There is also evidence that angiotensin II (Ang II)-induced generation of ROS contributes to the development of insulin resistance in skeletal muscle, although the precise mechanisms remain unknown. In the present study, we found that Ang II markedly enhanced NADPH oxidase activity and consequent ROS generation in L6 myotubes. These effects were blocked by the angiotensin II type 1 receptor blocker losartan, and by the NADPH oxidase inhibitor apocynin. Ang II also promoted the translocation of NADPH oxidase cytosolic subunits p47^phox and p67^phox to the plasma membrane within 15 min. Furthermore, Ang II abolished insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), activation of protein kinase B (Akt), and glucose transporter-4 (GLUT4) translocation to the plasma membrane, which was reversed by pretreating myotubes with losartan or apocynin. Finally, small interfering RNA (siRNA)-specific gene silencing targeted specifically against p47^phox (p47siRNA), in both L6 and primary myotubes, reduced the cognate protein expression, decreased NADPH oxidase activity, restored Ang II-impaired IRS1 and Akt activation as well as GLUT4 translocation by insulin. These results suggest a pivotal role for NADPH oxidase activation and ROS generation in Ang II-induced inhibition of insulin signaling in skeletal muscle cells.

Footnotes

↵2 The abbreviations used are: GLUT4, glucose transporter 4; ACE, angiotensin-converting enzyme; Akt, protein kinase B; Ang II, angiotensin II; AT₁R, angiotensin II-type 1 receptor; IL-6, interleukin-6; INS, insulin; NOS, nitric-oxide synthase; Nox, NADPH oxidase; PI3-K, phosphoinositol 3-kinase; RAS, renin-angiotensin system; REN-2, TG(mRen2)27 transgenic rat overexpressing the mouse renin gene; ROS, reactive oxygen species; T2DM, type 2 diabetes mellitus; TNF-α, tumor necrosis factor-α; PBS, phosphate-buffered saline; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium.
↵* This work was supported in part by VA VISN 15 and ARCD awards (to C. S. S.) and National Institutes of Health 5R01 HL073101-02 and VA Merit 0018 (to J. R. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 Present address: Dept. of Medicine, University of Arizona, Diabetes Research Center, 1656 E. Mabel St., P.O. 245218, Tucson, AZ 85724-5218. E-mail: sowers{at}email.arizona.edu.
- Received February 10, 2006.
- Revision received September 12, 2006.
The American Society for Biochemistry and Molecular Biology, Inc.