The liver is the primary organ dealing with ingested carbohydrates in mammals and, as such, t is responsible for converting excess dietary carbohydrates to triglycerides. Increased intake of carbohydrate leads t increased hepatic lipogenesis and this process is regulated in part by changes in the production of enzymes involved in the formation of triglycerides. Little is currently known regarding the mechanisms by which nutritional factors control gene expression in mammalian systems and the long range goal of this research is to explore this pathway. For this purpose, we are focusing on the expression of two hepatic genes - pyruvate kinase and the S14 gene. The rates of transcription of both of these genes respond rapidly and markedly to carbohydrate feeding. These changes can be mimicked in cultured primary hepatocytes which allows us to identify DNA sequences critical for the regulation of gene expression by carbohydrate. For both S14 and pyruvate kinase, DNA sequences from the 5'-flanking region were shown to be capable of conferring glucose- activation to a linked reporter gene. We hypothesize that a common carbohydrate-responsive transcription factor is binding to sites in the 5'-flanking region of these genes. Or goal is to identify and characterize the cis-acting DNA sequences and trans-acting factor(s) responsible for controlling this process. This will be accomplished by (1) using mutagenesis techniques, essential DNA sequences for the carbohydrate response of the pyruvate kinase and S14 genes will be defined in transfected hepatocytes and transgenic mice; (2) identifying the hepatic carbohydrate-responsive factor(s) which interacts with these DNA sequences; and (3) purifying and cloning of the carbohydrate- responsive factor from liver. This clone will provide a critical tool for further studies to explore the signalling pathway by which the hepatocyte can 'sense' increased glycolysis and respond by changing specific gene expression. In modern man with the ready availability of simple sugars in the diet, this pathway and its control could be a factor leading to excess conversion to triglycerides and problems associated with obesity. The expression of the S14 gene is also regulated in response to thyroid hormone. The two stimuli control S14 gene expression in a synergistic manner. Recent evidence from the laboratory suggests an interaction between the T3 receptor and components of the carbohydrate pathway. Using CDNA clones to the T3 receptor obtained in the previous funding period, we will test various models for the interaction of the receptor and the carbohydrate-responsive factor. These studies should provide insight into the mechanism by which two distinct stimuli can act to coordinately control gene expression.
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