The overall objective of the proposed research is to gain an understanding of the mechanism by which ethanol, alone and in combination with the glucocorticoids, regulate the level of mRNA encoding hepatic glucose 6-phosphate dehydrogenase (G6PD). The accumulation of fat in liver is observed after either acute or chronic ethanol consumption and may lead ultimately to the development of more severe liver damage. The precise etiology of alcoholic fatty liver is uncertain although there is a strong suggestion of glucocorticoid involvement. Ethanol stimulates the hypothalamic-pituitary-adrenal axis resulting in glucocorticoid secretion. In humans, excess ethanol consumption can cause pseudo-Cushing's syndrome. In laboratory animals, adrenalectomy blocks the development of alcoholic fatty liver. Our preliminary results have established that ethanol, alone and in combination with the glucocorticoids, increase the cellular content of mRNA encoding G6PD as measured either by in vitro translation or by hybridization with G6PD cDNA probes. Since G6PD can be considered a lipogenic enzyme in adult liver and is regulated by ethanol and the glucocorticoids, we are proposing to study transcriptional and post-transcriptional regulation of mRNA encoding G6PD as a model for ethanol-glucocorticoid modulation of hepatic gene expression. To this end, the specific aims of this proposal are: 1. Construct and characterize full length cDNA probes to G6PD mRNA. 2. Assess the effect of ethanol, alone and in combination with the glucocorticoids, on the production of G6PD mRNA in hepatocytes. Following development and characterization of the full length G6PD cDNA probes, we will utilize these tools to assess the regulation of production of G6PD mRNA. Primary cultures of adult rat liver parenchymal cells in a chemically defined medium will be incubated with combinations of ethanol and the glucocorticoids. Experiments will be carried out to ascertain whether ethanol and the glucocorticoids affect G6PD mRNA transcription, processing or turnover. Results of this study may illustrate how ethanol and the glucocorticoids interact to regulate the expression of a key hepatic, lipogenic enzyme.
