Evidence demonstrates that exogenous nitric oxide (NO) and the NO produced by inducible nitric oxide synthase (iNOS) can induce insulin resistance in muscle. Here, we investigated whether this insulin resistance could be mediated by S-nitrosation of proteins involved in early steps of the insulin signal transduction pathway. Exogenous NO donated by S-nitrosoglutathione (GSNO) induced in vitro and in vivo S-nitrosation of the insulin receptor ␤ subunit (IR␤) and protein kinase B/Akt (Akt) and reduced their kinase activity in muscle. Insulin receptor substrate (IRS)-1 was also rapidly S-nitrosated, and its expression was reduced after chronic GSNO treatment. In two distinct models of insulin resistance associated with enhanced iNOS expression-diet-induced obesity and the ob/ob diabetic mice-we observed enhanced S-nitrosation of IR␤/IRS-1 and Akt in muscle. Reversal of S-nitrosation of these proteins by reducing iNOS expression yielded an improvement in insulin action in both animal models. Thus, S-nitrosation of proteins involved in insulin signal transduction is a novel molecular mechanism of iNOSinduced insulin resistance. Diabetes 54:959 -967, 2005

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S-Nitrosothiols (RSNOs) are considered to play important roles in storing, transporting, and releasing nitric oxide (nitrogen monoxide, NO) in vivo. Although tertiary RSNOs are known to be intrinsically more stable than primary RSNOs, the correlation between the structure of primary RSNOs and the kinetics of thermal NO release in solution has not been established yet. We have characterized the kinetics of thermal NO release from three primary RSNOs: S-nitrosocysteine (CySNO), S-nitroso-N-acetylcysteine (SNAC), and Snitrosoglutatione (GSNO) in aqueous solutions. It was found that the rates of NO release are strongly affected by the initial concentration of the solutions. Increasing the concentration of CySNO and SNAC from 1.0 × 10 -1 to 61.0 mmol L -1 led to 5.7-and 14.6-fold increases in their initial rates of decomposition, respectively, whereas GSNO was much less affected (a 2-fold increase). However, a smaller increase in concentration (0.1 to 1.0 mM) led to a 4.6-fold decrease, on average, in the rates of NO release in the three cases. This result was assigned to the combination of an autocatalytic effect promoted by the secondary reaction of thyil radicals with authentic RSNO molecules, which accelerates the decomposition reaction in concentrated solutions, and a nongeminate (diffusive, outside the cage) radical pair recombination effect that leads to a reduction in the rates of reaction in dilute solutions. In the low-concentration range, GSNO and SNAC were shown to be significantly more stable than CySNO. This result is in accordance with the conclusions derived from singlepoint energy calculations at the MP2/6-31G(2df,p)//MP2/6-31G(d) level of theory, which have shown that the acetamido group that is present in SNAC plays a key role in increasing the S-N bond strength. These results show that comparisons of stability among different S-nitrosothiols in solution must take the concentration effect carefully into account and indicate that the half-lives of primary RSNOs found in vivo can be partially determined by their intrinsic structural properties.

Topical RSNOs produce a consistent, sustained and biologically effective release of NO on human skin in vivo, which offers advantages over currently available topical NO donors. Dermal nitrite concentration--the oxidation product of NO--is directly correlated with blood flow at low and moderate levels of blood flow. At high levels of blood flow, there is a reduction in dermal nitrite, which is presumed to be due to increased blood scavenging.