Malic enzyme and fatty acid synthase are two enzymes in the pathway for de novo biosynthesis of long-chain fatty acids. The relative synthesis rates of these enzymes are regulated by nutritional and hormonal stimuli. In vivo, feeding stimulates and starvation inhibits the synthesis of these enzymes. In chick- embryo hepatocytes in culture, triiodothyronine (T3) and insulin stimulate, and glucagon (via cyclic AMP) inhibits. We have isolated cloned cDNA sequences for malic enzyme and fatty acid synthase and established that enzyme synthesis rates are regulated by controlling the amounts of malic enzyme and fatty acid synthase mRNAs. One exception is stimulation of translation of fatty acid synthase mRNA by insulin. In vivo and in culture, regulation of the level of malic enzyme mRNA involves a combination of transcription and post-transcriptional controls, with the latter predominant. Regulation of fatty acid synthase mRNA level in vivo is primarily transcriptional. The point of regulation in hepatocytes in culture has not been ascertained. Kinetic and inhibitor experiments suggest that there is an intermediate in the T3-induced stimulation of the accumulation of mRNAs for both enzymes. We speculate that cyclic AMP may regulate the activity of that intermediate. Our objective in the next grant period is to understand the molecular mechanisms by which T3 and glucagon regulate expression of the gene for malic enzyme. We plan to determine which step subsequent to transcription initiation in the pathway for gene expression is regulated by T3, and to determine if a step other than degradation of mature mRNA is regulated by glucagon. Infective retroviral vectors will be used to develop a new method for gene transfer into chick-embryo hepatocytes. Putative regulatory sequences in the malic enzyme gene or transcript (hormone regulatory elements) will be tested functionally by attaching them to reporter genes containing hormone-neutral promoters and expressing the hybrid DNAs in hormone-sensitive hepatocytes. To achieve these objectives also will require completion of the cloning and characterization of the malic enzyme gene, a gene of more than 63 kb. Finally, we plan to initiate the identification, purification, and characterization of regulatory factors which bind to hormone regulatory elements in the malic enzyme gene or transcripts, with the ultimate aim of understanding how such factors interact with specific DNA or RNA, and with other regulatory factors, to achieve multifactorial regulation of gene expression.
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