The long range goals of this project are to improve our understanding of how the adipocyte regulates lipogenesis and lipolysis through the control of specific gene expression and the corresponding protein products. By improving our understanding of these processes, we may ultimately improve our ability to provide therapy for obesity obesity-linked diabetes and correlative cardiovascular disorders. In the previous funding period, we have isolated and characterized 3 genes which are transcriptionally activated during mouse adipocyte differentiation: glycerophosphate dehydrogenase, adipocyte P2 and adipsin. The DNA sequences necessary to activate these genes will be probed first by a transient transfection-expression assay. After demonstrating proper cell type specificity of expression, as has been done with the adipocyte P2 gene, these DNA segments will be further dissected to determine (1) which sequence elements play a role in activation of its own promoter, (2) whether it can direct transcription from other promoters, and (3) whether it can function in an enhancer-like fashion. Greatest attention will be focused on genetic elements which appear to represent common steps in the pathway of activation of different fat-specific genes. Also under study will be the sequence requirements to get proper responses to 2 hormones key in lipogenesis: cyclic AMP and tumor necrosis factor (cachectin). Experiments will be carried out to isolate and characterize those nuclear factors which bind to and may regulate adipocyte-specific promoters/enhancers/hormone-response elements. Included here will be the FSE2 binding protein which appears to be developmentally regulated and binds in sequence-specific fashion to an element found in at least 2 genes participating in adipocyte differentiation. The ultimate goal will be an in vitro reconstruction of a cell type-specific transcription pattern, using isolated DNA templates. In addition to transcriptional regulation, we will analyze the catalytic and physiological function of adipsin, the serine protease homologue which is produced and secreted by fat cells. This will be done by large scale expression of the cloned cDNA in a baculovirus vector and subsequent study of the proteolytic activity of this enzyme toward an variety of extracellular substrates of potential importance in adipose physiology. Biological activity will also be examined by neutralizing adipsin activity with monospecific antibodies and following subsequent effects on adipocyte differentiation and lipogenesis.
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