The pathophysiological states with insulin resistance, including obesity and type 2 diabetes, are associated with increased expression of inducible nitric oxide synthase (iNOS) and oxidative stress. The diverse actions of nitric oxide (NO) can be classified in two categories; the direct effects of NO (e.g., cGMP-dependent vasodilation), and the indirect, redox state-dependent effects that are mediated by reactive nitrogen species (e.g., NO+, OONO-) through nitrosative protein modifications such as S-nitrosylation and tyrosine nitration. Nitrosative posttranslational modifications modulate functions of the variety of proteins, and are associated with iNOS induction. iNOS induction and resultant nitrosative protein modifications have been implicated in the development of many disorders, including hypertension, atherosclerosis, neurodegenerative disease, as well as type 1 diabetes and chronic diabetic complications. iNOS induction leads to prolonged, exaggerated production of NO and accelerates oxidative stress. Conversely, concomitant oxidative stress promotes nitrosative protein modifications. Our preliminary data both in vivo and in vitro indicate that (1) Akt/PKB, which plays a pivotal role in insulin action, is inactivated by NO+ (nitrosonium ion) donors both in intact cells and in vitro through nitrosative cysteine (thiol) modifications, (2) glycogen synthase kinase-3beta (GSK-3beta) activity is up- regulated by NO donor in a redox state-dependent manner, (3) iNOS-/- ob/ob mice are protected from developing hyperglycemia and exhibit relatively lower plasma insulin level compared with iNOS+/+ ob/ob mice, and (4) acute and chronic treatment of genetically obese, diabetic (ob/ob) mice with iNOS inhibitors, L- NIL and aminoguanidine results in decreases in blood glucose levels with concomitant glycogen synthase activation and glycogen phosphorylase inactivation. Based on these compelling and convincing preliminary data, we wish to test the hypothesis that iNOS plays a critical role in the pathogenesis of insulin resistance. The immediate goal of this proposal is, therefore, to confirm this hypothesis and clarify the molecular mechanisms by which iNOS induces insulin resistance. With this in mind, the following specific aims will be investigated.
Specific Aim 1 and 2 will determine the inhibitory and stimulatory effects of NO+ on Akt/PKB and GSK 3beta activity and the molecular mechanism for this process. The importance of nitrosative modification of cysteine residue(s) of Akt/PKB and GSK-3beta will be clarified by examining the effects of NO donor on these kinases with Cysteine to Serine substitutions. The biological relevance of RNS-mediated inactivation of Akt/PKB and activation of GSK-3beta will be confirmed with tissue samples from iNOS-/- ob/ob and iNOS+/+ ob/ob mice.
Specific Aim 3 will determine whether iNOS is required for insulin resistance related to obesity in vivo . This hypothesis will be tested by examining the effects of iNOS deficiency in ob/ob background. We have mated iNOS-/-and ob/ob mice and generated both iNOS-/- ob/ob and NOS+/+ ob/ob mice. Insulin sensitivity will be evaluated by insulin tolerance test. The long-term objectives of this proposal are, therefore, to develop novel interventions for treatment of insulin resistance and type 2 diabetes. In the short-term, the proposed studies will provide novel significant insights into the pathogenesis of insulin resistance, and also a scientific basis for development of novel therapeutic strategies to prevent and/or treat insulin resistance, and type 2 diabetes.
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