The long-term objective of this work is to understand the molecular mechanism by which dietary polyunsaturated fats inhibit gene expression. Americans are encouraged to consume less fat and a higher polyunsaturated to saturated fat ratio. This recommended dietary goal is a preventive health measure because of the correlation between fat intake, serum lipids and the risk of heart disease. Little is known about the intracellular events resulting from changing the type and quantity of fat in the diet. Polyunsaturated fatty acids have a myriad of actions within the organism including the transcriptional regulation of a number of genes. However, the mechanisms by which fatty acids alter gene expression remain to be determined. Glucose-6-phosphate dehydrogenase (G6PD) is inhibited both by dietary polyunsaturated fats and by polyunsaturated fatty acids in hepatocytes in culture. Consequently, G6PD will be used as our model gene. The research program described in this application will determine if the inhibition of G6PD by polyunsaturated fatty acids occurs at a pre- or post-transcriptional step and then to determine the molecular basis by which this inhibition occurs.
In Specific Aim 1, the genomic DNA probes will be characterized and a cDNA generated for the analysis of transcriptional activity and mRNA accumulation. Only probes which are free of repetitive elements and unique with respect of their occurrence within the genome will be used.
In Specific Aim 2, the relative importance of transcriptiona1 versus post-transcriptional processes on the regulation of G6PD by dietary fat will be determined. Mice fed a high glucose, low fat diet will be compared to mice fed a high glucose diet supplemented with safflower oil. Transcriptional activity will be measured using the nuclear run-on assay and compared with changes in the accumulation of G6PD mRNA and activity of the enzyme.
In Specific Aim 3, the molecular level at which fatty acids inhibit G6PD expression in hepatocytes will be determined. Transcriptional activity, mRNA abundance and enzyme activity will be compared in primary cultures of mouse hepatocyte+/- linoleate and arachidonate.
In Specific Aim 4, the molecular basis for the inhibition of G6PD expression by fatty acids will be determined. If regulation is primarily transcriptional, the cis-acting DNA element in the G6PD 5'- flanking DNA will be identified using DNase I hypersensitivity assays, functional transfection analyses of deletion mutants and in vitro DNA binding assays. If regulation is post-transcriptional, the precise step will be localized and the cis-acting element within the mRNA sequences will be identified using functional transfection analyses of G6PD RNA sequences linked to a reporter gene. In both cases the putative response element will be characterized for its ability to confer fatty acid regulation on a heterologous promoter or gene and will be defined by sequence specific mutations.
