The long term objectives of our program are to determine the physiologic, biochemical, and molecular mechanisms responsible for the nutritional and hormonal regulation of hepatic gene expression. The studies in this application will delineate the mechanisms that regulate the transcription and processing of a hepatic mRNA, mRNA-S14. We have taken advantage of the rapid response of this mRNA to dietary carbohydrate to show that the transcriptional response reflects the intracellular metabolism of sugars and not a response to insulin. Therefore, this MRNA is an excellent model to study the effects of nutritional signals on the regulation of gene expression. Further, the induction by feeding a sucrose containing diet or the administration of thyroid hormone is similar to that of the hepatic lipogenic enzymes. Therefore, this mRNA is also an excellent model to study the response of the lipogenic enzyme mRNAs. The dietary response is due both to enhanced transcription of the gene and increased efficiency of processing of pre-mRNA to mature mRNA. We can reproduce the transcriptional regulation of mRNA-S14 in primary hepatocyte cultures by altering the concentration of glucose presented to the hepatocyte. To define the mechanisms responsible for the dietary regulation of this hepatic gene, we propose to introduce a series of deletion mutations in the 5'flanking DNA of the S14 gene. The mutant constructs will be transfected into primary hepatocyte cultures and the reporter luciferase activity measured as an indicator of S14 gene transcription. These studies will define the specific DNA sequences responsible for the regulation of gene transcription by dietary carbohydrate. To explore the mechanism of the synergistic interaction between thyroid hormone and dietary carbohydrates we will create constructs containing a thyroid response element and a carbohydrate response element of the S14 gene and test the ability of these constructs to show a synergistic response to thyroid hormone and carbohydrates. These studies will define the minimum DNA sequences that lead to interaction between thyroid hormone and nutritional signals. We will also define new hepatic transcription factors that are responsible for the carbohydrate regulation of mRNA-S14. The time course and magnitude of response of these proteins to dietary and hormonal stimuli will be measured. Lastly, we will use in vitro splicing reactions to demonstrate that dietary carbohydrate enhances the efficiency of pre-mRNA-S14 processing. The studies outlined in this proposal should further our understanding of the molecular and physiologic basis of dietary regulation of hepatic gene expression. Since altered glucose metabolism leads to changes in gene expression in many tissues, these studies have major implications for the pathophysiologic mechanisms that lead to complications associated with diabetes mellitus.
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