Adipocytes, long recognized for their importance in energy storage in mammals, are now understood to be critically important in several aspects of energy homeostasis. The current epidemic in obesity and type 2 diabetes underscores the need for understanding these disorders in mechanistic detail, including the development of the fat cell lineage. The core of the transcriptional program is now understood to involve the nuclear receptor PPARgamma in cross-regulation with several members of the C/EBP family. However, several major questions remain.
Aim1 will investigate the nature of the coactivator complex that docks on PPARgamma. Since much of the adipogenic action of this receptor is contained at the N-terminus, we will attempt to purify complexes that interact with this region, specifically those that are modulated by the inhibitory phosphorylation of this region by MAP kinase.
The second Aim will investigate the nature of the natural ligand for PPARy that activates this receptor during the process of fat cell differentiation. We will utilize an unbiased affinity purification of ligands from differentiating cells, combined with several advanced methods of mass spectrometry. The biological significance of a ligand(s) identified will be subject to several critical tests of biological significance. Our last Aim investigates the molecular basis of preadipocyte determination, a process about which little is known. First, we will utilize global transcriptional profiling with various murine 3T3 cell lines that differ greatly in their propensities to differentiate. In a second approach, we will utilize expression libraries from preadipocytes prepared in retroviral libraries, to attempt to convert differentiation-incompetent fibroblasts into determined preadipocytes. With both approaches, our goals will be to create a set of molecular markers with which preadipocyte determination can be characterized and studied in vitro and in vivo. Putative regulatory molecules will be studied by gain and loss of function experiments in cells and mice, including the use of siRNAs and the creation of knock-out animals.
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