Obesity is one of the most common metabolic disorders in humans and it is an independent risk factor for non-insulin-dependent diabetes mellitus (NIDDM). In fact, obesity and diabetes are major causes of morbidity and mortality in the U.S., reaching epidemic proportions. It is clear, therefore, that an understanding of the relationship between excess adipose tissue and the development of insulin resistance and diabetes is critical. The long-term objectives of this proposal are to understand the molecular mechanisms that regulate the differentiation of preadipocytes into insulin-responsive adipocytes. Our recent studies have contributed significantly to the elucidation of a network of transcription factors that regulate adipogenesis. At the center of this network are two principal transcription factors, PPARgamma and C/EBPalpha, which function both independently and together to orchestrate the expression of a plethora of genes associated with development of the mature adipocyte. Induction of PPARgamma and C/EBPalpha gene expression appears to be regulated in part by C/EBPbeta and C/EBPdelta. As is the case with most nuclear hormone receptors, the transcriptional activity of PPARgamma also requires its interaction with appropriate ligands. In this regard, recent studies have shown that C/EBPalpha and possibly C/EBPbeta may stimulate processes that lead to the production of endogenous ligands during adipogenesis. Furthermore, other investigations have demonstrated a role for C/EBPalpha along with PPARgamma in regulating the insulin-dependent glucose transport system. We propose, therefore, that domains within C/EBPbeta and C/EBPalpha have separate and discrete activities in promoting the transcription of genes involved in various aspects of the differentiation process. The first two specific aims, therefore, are designed to test this hypothesis.
In Aim 1, we will identify domains within C/EBPbeta and C/EBPalpha responsible for regulating PPARgamma ligand production and insulin sensitivity. Our strategy will be to conditionally express a series of mutants of C/EBPbeta and C/EBPalpha in fibroblasts and preadipocytes, which correspond to various transactivation domains of the proteins. Analysis of the functional properties of the resulting adipocytes will give insight into the activity of each of these domains.
In Aim 2, we will identify cofactors that interact with these domains to facilitate the expression of genes responsible for these functions. The studies in Aim 3 are designed to test the hypothesis that members of the winged-helix forkhead family of transcription factors (FOXC2 and FOXO3a) also participate in regulating C/EBPalpha gene expression and insulin action during adipogenesis. The studies in Aim 4 are designed to test the hypothesis that insulin downregulates C/EBPalpha expression in mature adipocytes by suppressing the activity of the FOX proteins through a phosphorylation-associated retention of FOXO in the cytoplasm. To test this hypothesis, we will conditionally express FOXO3a and a corresponding set of mutants in 3T3-L1 preadipocytes and determine whether they counteract the suppressive effects of insulin on C/EBP alpha transcription.
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