Cardiovascular disease is the principal cause of death and disability in adults with diabetes. Endothelial dysfunction is a hallmark of diabetic vascular disease and is characterized by decreased bioavailability of nitric oxide (NO). Our long-term goal is to elucidate the mechanism by which insulin resistance reduces NO production in endothelial cells and thereby facilitate the development of therapeutics that can be used to attenuate the impact of diabetes and insulin resistance on cardiovascular disease. The specific hypothesis of this revised research proposal is that IKKbeta a key enzyme in the regulation of the NF-kappaB inflammatory pathway mediates the effect of insulin resistance on NO production. This hypothesis is based on the following experimental observations completed in our laboratory. First, IKKbeta is activated in endothelial cells by free fatty acids (FFA), high glucose, and TNF-alpha all of which have been implicated in the pathogenesis of insulin resistance. Second, activation of IKKbeta is associated with impaired NO production. Third, inhibition of IKKbeta pharmacologically using aspirin or genetically using a dominant negative IKKbeta construct blocks the ability of FFA, TNF-alpha, or glucose to impair NO production. Finally, overexpression of wild-type IKKbeta recapitulates the effect of FFA, glucose, or TNF-alpha to impair NO production. The results of these experiments suggest that IKKbeta activation is both necessary and sufficient to cause impairment of endothelial NO production. These results also indicate that inflammatory pathways play a key role in both nutrient excess and cytokine mediated endothelial insulin resistance. Based on these observations the experimental focus of this proposal is on the role of IKKbeta in mediating impaired NO production. We propose to determine how activation of IKKbeta impairs endothelial NO production in both an endothelial cell culture model and in an in vivo model of insulin resistance. We will next address how mediators of insulin resistance might activate IKKbeta and examine a potential role for innate immunity (Toll Like Receptor) in mediating vascular insulin resistance. The following aims are proposed:
Aim 1. To determine the mechanism by which IKKbeta activation impairs NO production in endothelial cells.
Aim 2. To determine whether impaired endothelial insulin signaling and NO production are associated with IKKbeta activation in vivo in a mouse model of diet-induced obesity and insulin resistance.
Aim 3. To determine whether the Toll Like Receptor pathway is necessary for endothelial activation of IKKbeta during the development of vascular insulin resistance.
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