Insulin resistance is a ubiquitous correlate of obesity and plays a crucial role in several important medical conditions associated with obesity: hypertension, hyperlipidemia and especially, non-insulin dependent diabetes mellitus (NIDDM). Recently, we have shown that the expression of TNF-alpha by adipose cells, particularly in the context of obesity, interferes with the tyrosine kinase activity of the insulin- receptor and causes insulin resistance. This competing renewal application suggests continuing our studies to understand the mechanisms of TNF-induced insulin resistance and its role in obesity-diabetes. Our preliminary data has suggested that IRS-1 is phosphorylated on non- tyrosine residues in response to TNF-a, and immunoprecipitated IRS-1 from TNF treated cells is associated with an inhibitory action on the insulin receptor. We will first map phosphorylation sites on the insulin receptor and IRS-I in response to TNF-alpha and the role of these modifications will be tested by in vitro mutagenesis. The ability of TNF-alpha-induced protein kinases to phosphorylate IRS-1 on key residues will be studied using purified recombinant IRS-1 as a target. Whether this modified IRS-1 can act as a direct inhibitor of the insulin receptor or must first associate with another inhibitory molecule from TNF-~ treated cells will be tested. If such an inhibitory molecule is identified, it will be purified and cloned. In addition to these biochemical studies, the genetic function of IRS-1 in TNF mediated inhibition will be investigated using cells from IRS-1 deficient mice. The extracellular components and intracellular regulators responsible for the expression of TNF-alpha from fat cells will be studied. Preliminary data suggests that cultured adipocytes normally express very little TNF-alpha mRNA but can be induced with high levels of lipids, insulin and dexamethasone. Analysis of the requirements for different lipids will be performed, along with studies of the requirement for certain hormones. Once a system with robust TNF-alpha expression is established, the intracellular regulatory components will be investigated. We will determine first whether the induction of TNF-alpha occurs at the transcriptional or post-transcriptional levels. Ultimately, this will lead to a delineation of required cis-acting nucleic acid sequences and the isolation and cloning of key trans-acting factors. Finally, we will create strains of otherwise normal transgenic mice that express TNF-alpha from fat tissue. The promoterienhancer of the aP2 gene will be linked to the TNF-a coding segment and the resulting mice will be compared to control mice in their insulin sensitivity, degree of adiposity and development of diabetes. Of course, it is possible that obesity is necessary to provide other molecules in addition to TN F- alpha in order to get insulin resistance. We will investigate this by inducing obesity in the transgenic strains by feeding high fat diets. Mice will again be studied for changes in glucose homeostasis and insulin sensitivity via glucose tolerance tests and stimulation of insulin receptor tyrosine phosphorylation in various tissues.
