This proposal is directed toward understanding the transcriptional basis of adipocyte development. The adipocyte plays an important role in the energy balance of vertebrate organisms. Obesity, an excessive adipose mass, is an important risk factor for cardiovasctllar disease, certain cancers and especially, non-insulin dependent diabetes mellitus. An improved understanding of adipose differentiation and adipocytespecific gene expression will offer new opportunities to treat these common disorders. Our recent work has identified a key transcriptional regulator of adipose differentiation, PPARgamma. Ectopic expression of this orphan member of the nuclear hormone receptor family will stimulate adipose differentiation in fibroblastic cells, in a PPAR gamma activator-dependent way. This proposal suggests identifying key domains of PPAR gamma responsible for its adipogenic action, by performing domain swaps with PPAR gamma, an isoform that does not promote adipogenesis. Molecules interacting with this domain that mediate the effects of this factor in differentiation will be cloned by biochemical and genetic techniques. The molecular basis of the synergistic interaction of PPAR gamma and C/EBPa in adipogenesis will be studied by first exatnining protein-protein interactions with molecules synthesized in vitro or in cultured cells. Cooperative interactions at the level of the isolated promoter will be examined through the use of in vitro transcription, employing the aP2 promoter/enhancer. Preliminary data shows that PPAR gamma activation is sufficient to cause withdrawal of cells from the growth cycle. PPAR gamma mutations which disrupt transcriptional activity will be used to determine whether this is likely to occur via transcription control of cell cycle regulatory components, or direct interactions with other growth related proteins such as AP-l factors or cyclin complexes. The role of PPAR gamma in regulating known cyclin dependent kinase inhibitor, as well as the identification of novel molecules with such growth regulatory activity will be investigated. The control of PPAR gamma activity and quantity by certain hormones that promote PPAR gamma-mediated adipogenesis will be studied initially by examing at the effects of insulin, IFG-l and dexamethasone in transient DNA transfections with PPAR gamma and PPAR gamma binding sites linked to a reporter gene. Control of PPAR gamma expression and covalent modification of the PPAR gamma/RXR complex will be subsequently investigated. Finally, and importantly, we will examine the in vivo role of PPAR by creating targeted mutations in the gene in the mouse. Gain of function transgenics will be made overexpressing PPAR gamma in muscle, liver or fat. In all cases, mice will be characterized for degree of adiposity, tissue histology, body composition, hyperlipidemia and blood glucose and insulin. These transgenic mice will also be used to test the hypothesis that PPAR gamma is the physiologically relevant receptor for the anti-diabetic effects of the thiazolidinedione class of drugs.
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