The activities of several enzymes involved in the pathway for the de novo synthesis of long-chain fatty acids are low in starved animals and high in animals fed high-carbohydrate diets. The primary regulatory mechanism is transcriptional. We are using malic enzyme as a model to investigate the molecular basis of this phenomenon. Transitions between the fed and starved states are mimicked quantitatively in chick-embryo hepatocytes incubated in chemically-defined medium. Insulin and triiodothyronine (T3) stimulate transcription of the gene for malic enzyme by 30- to 50-fold; glucagon, cAMP, unesterified fatty acids and isoquinoline sulfonamide protein kinase inhibitors inhibit transcription by 80 to 95% or more. Corticosterone and peroxisome-proliferating agents such as clofibrate have no effects on transcription of malic enzyme by themselves but stimulate the response to T3 in cells incubated in culture for more than 3 days. The cell culture system that we have developed is thus ideally suited for analyzing the molecular mechanisms by which these agents regulate transcription of the malic enzyme gene. In next grant period, we propose to identify and characterize cis-acting response elements for T3, cAMP, hexanoate, isoquinoline sulfonamide protein kinase inhibitors, corticosterone and peroxisome-proliferating agents. We also propose to characterize cis-acting elements that interact with the T3 response element and modify its transcriptional response to T3. In these analyses, we will test promoter function of various parts of the malic enzyme gene with a transient transfection assay. Uptake of DNA into chick-embryo hepatocytes will be facilitated by lipofectin. We have already established that a 5.8 kb fragment of 5'-flanking DNA from the malic enzyme gene confers regulation by T3, cAMP and hexanoate on a linked CAT gene. We also propose to identify and characterize the trans-acting factors that bind to the cis-acting elements and mediate the actions of the hormones and metabolites listed above. DNase I and methylation interference footprinting and gel mobility-shift assays will be used to assess binding of protein factors to cis-acting response elements. Finally, we propose to purify those factors that have not been described previously and to clone and characterize the corresponding cDNAs.
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