The overall objective of this project is to further our understanding of the mechanisms of carbohydrate regulation of gene expression. During the last several years we have demonstrated that a large number of hepatic genes are regulated by alterations in the amount of carbohydrate presented to the liver cell. The mRNA for one of these genes, mRNA-S14, has proven to be an excellent model for the study of carbohydrate responsive mRNAs. With this model we have recnetly demonstrated that carbohydrate administration rapidly induces this mRNA. Furthermore, preliminary data indicate that the induction of mRNA-S14 is associated with a rise in an intracellular nucleotide. We now propose to utilize HPLC techniques to identify this nucleotide, and demonstrate that this nucleotide is the proximate intracellular mediator of the carbohydrate effect. To acccomplish this goal we shall quantitate the temporal relationship between the synthesis of this nucleotide and the accumulation of mRNA-S14 in livers from rats given various stimuli known to induce mRNA-S14. In addition we propose to identify this nucleotide by mass spectrometry. The identified nucleotide will be added to primary hepatocyte cultures in order to demonstrate that an increase in the intracellular content of the nucleotide leads to enhanced formation of mRNA- S14. Further studies on the mechanism of carbohydrate regulation of mRNA-S14 will be performed by direct measurements of the synthesis rate of this mRNA with (3H)- Uridine and measurements of degradation rates following Actinomycin D addition to hepatocyte cultures. We shall measure the rate of accumulation of the nuclear precursor of mRNA-S14 in order to demonstrate that the carbohydrate effects occurs within the nucleus of the cell. Lastly, in order to establish the nexus between animal models and human disease we shall study carbohydrate regulation of gene expression in man by examining the spectrum of mRNAs present in peripheral mononuclear cells of diabetic patients. The studies outlined in this proposal should provide a greater understanding of the mechanisms by which alterations in carbohydrate status lead to changes in gene expression.
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