Inducible nitric-oxide synthase (iNOS) has been implicated in many human diseases including insulin resistance. However, how iNOS causes or exacerbates insulin resistance remains largely unknown. Protein S-nitrosylation is now recognized as a prototype of a redox-dependent, cGMP-independent signaling component that mediates a variety of actions of nitric oxide (NO). Here we describe the mechanism of inactivation of Akt/protein kinase B (PKB) in NO donor-treated cells and diabetic (db/db) mice. NO donors induced S-nitrosylation and inactivation of Akt/PKB in vitro and in intact cells. The inhibitory effects of NO donor were independent of phosphatidylinositol 3-kinase and cGMP. In contrast, the concomitant presence of oxidative stress accelerated S-nitrosylation and inactivation of Akt/PKB. In vitro denitrosylation with reducing agent reactivated recombinant and cellular Akt/PKB from NO donortreated cells. Mutated Akt1/PKB␣ (C224S), in which cysteine 224 was substituted by serine, was resistant to NO donor-induced S-nitrosylation and inactivation, indicating that cysteine 224 is a major S-nitrosylation acceptor site. In addition, S-nitrosylation of Akt/PKB was increased in skeletal muscle of diabetic (db/db) mice compared with wild-type mice. These data suggest that Snitrosylation-mediated inactivation may contribute to the pathogenesis of iNOS-and/or oxidative stress-involved insulin resistance. Nitric oxide (NO)1 is an endogenous cell signaling molecule involved in the regulation of many physiological functions and in the mediation of a variety of pathophysiological processes. NO and NO-related compounds function as both protective and cytotoxic, dependent on the cellular context and the nature of the NO group. The multifaceted actions of the NO group can be classified into two categories: 1) authentic NO-mediated, cGMPdependent, and 2) reactive nitrogen species-mediated, cGMPindependent actions. Nitrosative post-translational modifications, including protein S-nitrosylation and tyrosine nitration, are involved in the cGMP-independent actions. The cGMP-dependent actions play critical roles in a variety of physiological processes, including NO-mediated vasodilation. In contrast, cGMP-independent, nitrosative protein modifications are postulated to be involved in the pathological responses (1-4).Nitric-oxide synthases (NOSs) consist of three distinct genes, inducible nitric-oxide synthase (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). NO is generated by iNOS to a much greater extent, to over 1,000-fold, compared with that produced by the constitutive NOSs, eNOS and nNOS (2, 5). iNOS and nitrosative stress have been implicated in many human diseases, including insulin resistance (6, 7), atherosclerosis (8), inflammation, and neurodegenerative disorders (9). This is largely based on the evidence that iNOS deficiency results in significant amelioration of, or resistance to, these diseases. However, little is known about the molecular mechanisms by which iNOS causes and/or exacerbates these diseases. Furthe...

Chronic inflammation has been postulated to play an important role in the pathogenesis of insulin resistance. Inducible nitric oxide synthase (iNOS) has been implicated in many human diseases associated with inflammation. iNOS deficiency was shown to prevent high-fat diet-induced insulin resistance in skeletal muscle but not in the liver. A role for iNOS in fasting hyperglycemia and hepatic insulin resistance, however, remains to be investigated in obesity-related diabetes. To address this issue, we examined the effects of a specific inhibitor for iNOS, L-NIL, in obese diabetic (ob/ob) mice. iNOS expression was increased in the liver of ob/ob mice compared with wild-type mice. Treatment with iNOS inhibitor reversed fasting hyperglycemia with concomitant amelioration of hyperinsulinemia and improved insulin sensitivity in ob/ob mice. iNOS inhibitor also increased the protein expression of insulin receptor substrate (IRS)-1 and -2 1.5-and 2-fold, respectively, and enhanced IRS-1-and IRS-2-mediated insulin signaling in the liver of ob/ob mice. Exposure to NO donor and ectopically expressed iNOS decreased the protein expression of IRS-1 and -2 in cultured hepatocytes. These results suggest that iNOS plays a role in fasting hyperglycemia and contributes to hepatic insulin resistance in ob/ob mice. Diabetes 54:1340 -1348, 2005 C hronic low-grade inflammation has been proposed to be involved in the pathogenesis in obesity-related insulin resistance and type 2 diabetes. The expression of proinflammatory cytokines, including tumor necrosis factor-␣ (1) and interleukin-6 (2), is upregulated in animal models of and patients with type 2 diabetes. However, limited knowledge is thus far available about the molecular mechanisms by which chronic inflammation mediates insulin resistance and type 2 diabetes.The activation of inhibitor 〉 kinase ␤ (IKK␤)-nuclear factor-〉 (NF-〉), a crucial signaling cascade for inflammatory response, has been highlighted as a mediator of insulin resistance. The pharmacological inhibition or gene disruption of IKK␤ reversed obesity-related insulin resistance and fasting hyperglycemia in rodents and humans (3-5). However, little is known about genes that function as downstream effectors of the IKKb-NF-B pathway to mediate insulin resistance.Inducible nitric oxide synthase (iNOS; also termed NOS2), whose expression is regulated by IKK␤-NF-B (6), is assumed to be one of the candidates that mediate inflammation-involved insulin resistance. Accumulating evidence indicates a close link between iNOS and insulin resistance. Although iNOS was originally identified in macrophages, it is now known that it is widely expressed in many tissues, including insulin-sensitive organs such as skeletal muscle, adipose tissue, and liver, in normal rodents and humans. The expression of iNOS is upregulated by most, if not all, inducers of insulin resistance, including proinflammatory cytokines, obesity (7), free fatty acids (8), hyperglycemia (9,10), endotoxins (6,11), and oxidative stress. In fact, elevated expression of i...