Obesity is frequently associated with insulin resistance and constitutes the major risk factor for non insulin-dependent diabetes mellitus (NIDDM). In recent years, studies have indicated that in obesity, the elevated expression of TNFa by adipose cells plays a critical role in the development of insulin resistance. TNFa interferes with insulin action by inhibiting the tyrosine kinase activity of the insulin receptor (IR) both in cultured cells and in whole animals. This inhibition of IR signaling involves a TNFcxinduced serine phosphorylation of insulin receptor substrate 1 (IRS-i). However, the molecular components that are involved in this process, especially concerning the proximal signaling events activated by TNFa are not known. In this application, we propose to address these components by studying the biological functions of TNFa mediated through its two identified receptors (TNFR1 and TNFR2) in relation to TNFa-induced insulin resistance and other aspects of energy metabolism in cultured cells and whole animals. Our previous experiments in obese mice lacking TNFR1 and TNFR2 demonstrated that while insulin resistance is primarily mediated by TNFR1, other alterations in obesity involve both TNF receptors. To start investigating the signaling pathways used by each receptor in TNFa-induced insulin resistance, we will utilize the preadipocyte cell lines derived from all TNF receptor mutant mice. In these cellular systems we will conduct structure-function analysis of each TNF receptor using the inhibition of the JR tyrosine kinase activity and gene expression as the end point assays. TNFa-target genes in adipose tissue will be determined by expression profiling of adipose tissue obtained from the TNF receptor-deficient obese models. Through introduction of a series of deletion mutants of each TNF receptor into these cells by transfection, we will attempt to identify the intracytoplasmic regions of the TNF receptor(s) responsible for mediating these activities. In addition to this systematic characterization, we will also focus more deeply into the JNK pathway since recent studies have provided strong evidence that this pathway is downstream of TNFa in inducing IRS-i serine phosphorylation and subsequent insulin resistance. To address the role of JNK pathway in insulin resistance in vivo, we will metabolically characterize obese JNK-deficient mice and also obese mice following treatment with a pharmaceutical inhibitor of JNK. In parallel studies with JNK-deficient cell lines we will establish an in vitro model to further study the component of this pathway. These studies should provide insights not only into the molecular mechanisms of TNF-induced insulin resistance and metabolic alterations but also may lead to more general pathways involved distal to other signals resulting in insulin resistance.
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