Molecular Pharmacology of Insulin Resistance in Burns

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DESCRIPTION (provided by applicant) The hypermetabolic state of burns is associated with uncontrolled catabolism of proteins, fat and carbohydrates, and affects morbidity and mortality. The associated major metabolic anomaly is resistance to the effects of insulin, the pivotal anabolic hormone. Among the signaling cascades activated by insulin, the insulin receptor (IR), insulin receptor substrates (IRSs), phosphatidylinositol-3-phosphate kinase (PI 3-K) and Akt/PKB are central for energy metabolism and glucose homeostasis. Activated Akt/PKB in turn inhibits its downstream molecule, glucose synthase kinase-3 (GSK-3), resulting in increased protein and glycogen synthesis. Altered activation of all these signaling molecules occurs following burn injury, but the molecular mechanisms inducing these changes have not been elucidated. Many cytokines are expressed locally and systematically following burn injury, leading to increased expression of inducible nitric oxide (iNOS), and release of high levels of nitric oxide (NO). Based on compelling and convincing preliminary data, we hypothesize that iNOS, via release of NO with superoxide, plays an important role in insulin resistance of burn by altered signaling via IR, IRSs, PI 3-K, Akt/PKB and GSK-3. The following Specific Aims will test the above hypothesis in burn/sham-injured rodents in vivo, in cultured cells and in reconstituted in vitro systems: Specific Aim 1 will test the hypothesis that iNOS is required for insulin resistance. Specific Aim 2 will test the hypothesis that the exaggerated production of NO by iNOS decreases tyrosine kinase activity of IR and tyrosyl phosphorylation of IRSs. The molecular mechanism of inactivation of JR and IRSs (S-nitrosylation vs. nitration) will also be identified. Specific Aim 3 will test the hypothesis that the exaggerated production of NO by iNOS alters the kinase activity of Akt/PKB, the further downstream molecule of PI 3-K, independent of IR and IRSs. The molecular mechanisms responsible for inactivation (post-translational modifications) of Ak/IPKB by NO will also be identified. Specific Aim 4 will test the hypothesis that exaggerated production of NO by iNOS increases activity of GSK-3, due to effects related to both decreased AktJPKB activity and direct effects of NO on GSK-3. The direct role of NO on activation of GSK-3 (independent of AktIPKB) will be tested with NO donors and scavengers. The in vivo studies will include the use of burn and sham-injured rats, and iNOS knock out (-/-) and wild type (+/+) mice. Insulin mediated signaling changes, and the post-translational modifications in the signaling molecules enumerated above with and without specific iNOS inhibitor (1400W) will be evaluated. Functional changes, evaluated using 2-deoxyglucose uptake in muscle and adipocyte, will be correlated to signaling changes. Using adipocyte and myocyte cell lines and primary cultures from iNOS -/- and iNOS +/+ mice, the role of iNOS/NO will be evaluated with and without NO donors or scavengers. The role of NO will be confirmed in in vitro reconstitution system containing active signaling molecules. The post-translational modifications (nitration vs. S-nitrosylation) associated with 1NOS/NO will be studied by biochemical, spectrophotometric and immunoblot techniques. Several lines of evidence suggest that protein S-nitrosylationl-denitrosylation and tyrosine nitration/denitration may serve as regulatory components. The involvement of NO in insulin resistance will be assessed in the light of this new concept. The immediate short-term goals of these studies are, therefore, to characterize the molecular and biochemical mechanisms inducing insulin resistance, so that in the long-term, insulin resistance of burn injury in humans can be reversed. The studies together will thus provide significant insights into the pathogenesis of insulin resistance and provide information on novel therapeutic strategies to treat burn, and other stress or inflammation-induced insulin resistance.