The long term objective of the proposed research is to gain an understanding of the molecular mechanisms by which ethanol, alone and in combination with the glucocorticoids and insulin, regulate the Level of mRNA encoding hepatic glucose-6-phosphate dehydrogenase (G6PD). Alcohol consumption causes changes in hepatic lipid metabolism, ultimately resulting in accumulation of fat in liver. The precise etiology of alcoholic fatty liver is unclear although there is a strong suggestion of glucocorticoid involvement. Extensive studies in man and laboratory animals have shown that ethanol stimulates the hypothalamic-pituitary-adrenal axis resulting in glucocorticoid secretion. Our previous work has shown that ethanol, alone and in combination with the glucocorticoids and insulin, regulate the level of G6PD mRNA. Since hepatic G6PD is a lipogenic enzyme and is regulated by ethanol and the glucocorticoids, we are proposing to study the transcriptional and post-transcriptional regulation of G6PD gene expression as a model for ethanol/glucocorticoid modulation of hepatic gene expression. To this end, the specific aims of this proposal are: 1 Assess the effect of ethanol, alone and in combination with the glucocorticoids and insulin, on transcriptional regulation of the G6PD gene. 2 Assess the effect of ethanol, alone and in combination with the glucocorticoids and insulin, on post-transcriptional regulation of G6PD gene expression. Chimeric genes constructed from the G6PD promoter region and a reporter gene will be transfected into cells to assess transcriptional regulation. Post-transcriptional regulation will be studied by analyzing mRNA transcribed from chimeric genes constructed from a reporter gene (pSV2CAT) with the sequences from the 3' region of the G6PD cDNA or genomic clone inserted into the appropriate region of the reporter gene. In each specific aim, the sequences bestowing regulation to the chimeric genes will be specifically identified by deletional and site-directed mutational analysis. Results of this study may illustrate how ethanol and the glucocorticoids interact to regulate expression of a gene that encodes a key hepatic, lipogenic enzyme.
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