NUTRITIONAL AND HORMONAL REGULATION OF HEPATIC GENES

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The goal of this proposal is to investigate the molecular mechanism of action of thyroid hormone and nutritional factors in modulating specific mRNA levels in the liver. Emphasis will be focused on determining whether regulation occurs at the level of gene transcription and, if so, to developing a system for identifying the DNA elements essential to control. The generation of reduced NADPH necessary for supporting biosynthetic processes in the liver is catalyzed primarily by three enzymes - malic enzyme, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). The hepatic content of these three enzymes is regulated in a similar fashion: elevated in hyperthyroidism or after feeding lipogenic diet and depressed in diabetes, starvation or hyperglucagonemia. Previous work has demonstrated that regulation occurs primarily at a pretranslational level to alter cellular levels of mRNA for each enzyme. Thus, it is conceivable that these 3 NADPH-dependent dehydrogenase represent a coordinately regulated family in rat liver. To further investigate the regulation, specific cDNA clones homologous to mRNA coding for each enzyme will be isolated. These cDNA probes will allow a sensitive and accurate measurement of mRNA levels by molecular hybridization techniques. The effect of thyroid hormone or dietary factors on the rate of nuclear transcription of malic enzyme, G6PGD and G6PGD genes will be tested. Genomic DNA corresponding to each of these enzymes will be isolated from a rat DNA library and compared with respect to gene copy number, number and location of intervening sequences and DNA sequences of 5 feet-flanking regions. Finally, the function of isolated genes will be tested by introduction into an intact cultured cell containing receptor for thyroid hormone and assessing whether mRNA is produced by transcription of these gene in vivo. If so, the ability of thyroid hormone to regulate expression of mRNA from the newly introduced DNA will be examined by varying thyroid hormone levels in the media. Development of such an assay system would allow the definition of the extent and nature of DNA sequences essential for regulation using the techniques of 'in vitro' genetics. This system would provide an excellent opportunity to study the actual mechanism by which the hormonal and nutritional factors can modify gene expression.