Insulin resistance does not diminish eNOS expression, phosphorylation, or binding to HSP-90.
Previously, using an animal model of syndrome X, the obese Zucker rat (OZR), we documented impaired endothelium-dependent vasodilation. The aim of this study was to determine whether reduced expression or altered posttranslational regulation of endothelial nitric oxide synthase (eNOS) underlies the vascular dysfunction in OZR rats. There was no significant difference in the relative abundance of eNOS in hearts, aortas, or skeletal muscle between lean Zucker rats (LZR) and OZR regardless of age. There was no difference in eNOS mRNA levels, as determined by real-time PCR, between LZR and OZR. The inability of insulin resistance to modulate eNOS expression was also documented in two additional in vivo models, the ob/ob mouse and the fructose-fed rat, and in vitro via adenoviral expression of protein tyrosine phosphatase 1B in endothelial cells. We next investigated whether changes in the acute posttranslational regulation of eNOS occurs with insulin resistance. Phosphorylation of eNOS at S632 (human S633) and T494 was not different between LZR and OZR; however, phosphorylation of S1176 was significantly enhanced in OZR. Phosphorylation of S1176 was not different in the ob/ob mouse or in fructose-fed rats. The association of heat shock protein 90 with eNOS, a key regulatory step controlling nitric oxide and aberrant O2- production, was not different between OZR and LZR. Taken together, these results suggest that changes in eNOS expression or posttranslation regulation do not underlie the vascular dysfunction seen with insulin resistance and that other mechanisms, such as altered localization, reduced availability of cofactors, substrates, and the elevated production of O2-, may be responsible.
Fulton
David
D
Vascular Biology Center, Medical College of Georgia, 1459 Laney Walker Blvd., Augusta, GA 30912, USA. dfulton@mcg.edu
Harris
M Brennan
MB
Kemp
Bruce E
BE
Venema
Richard C
RC
Marrero
Mario B
MB
Stepp
David W
DW
D000818:Animals; D004730:Endothelium, Vascular / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D007333:Insulin Resistance / Q000502:physiology*; D009569:Nitric Oxide / Q000097:blood; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011924:Rats, Zucker; D014664:Vasodilation / Q000502:physiology
D018841:HSP90 Heat-Shock Proteins; D012333:RNA, Messenger; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat