Experiments are proposed which examine the molecular mechanisms which control lipogenic enzyme synthesis during the differentiation of mammalian adipocytes. The control of lipogenic enzyme development in adipocytes is higly relevant to human obesity, diabetes and cancer of the adipocyte (liposarcoma), the most common sarcoma in humans. The cell types used are cloned preadipocyte lines derived from mouse 3T3 cells used previously to examine the control of lipogenic enzyme syntheiss, predominantly at the protein level. The initial step is the isolation of cDNA clones containing sequences for important and abundant lipogenic enzymes such as glycerophosphate dehydrogenase, malic enzyme and fatty acid synthetase from an adipocyte cDNA library. This has been done first by comparing hybridization of recombinant colonies to cDNA synthesized from preadipocyte or adipocyte mRNA. The identity of particular inserted sequences will be determined by hybridization-selection from adipocyte mRNA and translation in vitro. The regulation of mRNA content for lipogenic enzymes during differentiation is to be examined by measurement of transcription and turnover rates with cloned cDNA probes. Of particular interest is a comparison of a new gene product expressed in the adipocyte (glycerophosphate dehydrogenase) with one of several enzymes quantitatively modulated during differentiation. Also to be examined are mechanisms whereby insulin and cyclic AMP agents, key hormonal influences in vivo, control the levels of enzymes involved in lipid metabolism. The relationship between methylation of genes for lipogenic enzymes, their expression and cellular commitment to the preadipocyte phenotype will be studied with methylation-sensitive restriction enzymes. Whether changes in DNA emthylation correlate with the developmental programming of these genes or with their actual expression will be determined by comparing methylation patterns in cell lines which differ in susceptibility to adipose differentiation. Methylation will also be examined in ells committed to adipocyte differentiation by exposure to 5-azacytidine. The reversibility of differentiation will be determined by replating adipocytes which have had lipid accumulation blocked and retain adhesiveness to the substratum. Subsequent enzyme expression will be monitored at protein, RNA and DNA levels.
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