Glucose utilization via glycolysis and lipogenesis must be regulated to meet the changing nutritional state of the animals; glycolysis is low and fatty acid synthesis is virtually absent during fasting, and fatty acids and triacylglycerol synthesis for energy storage increase dramatically during feeding. Secretion of insulin, a major anabolic polypeptide hormone, is increased during feeding and is involved in this regulation. In addition, glycolysis and fatty acid synthesis is impaired in diabetic animals and insulin restores the rates to normal levels. Since diabetes is among the most common and serious metabolic diseases, understanding mechanisms of insulin action is of utmost importance. The goal of this research is to determine the molecular mechanisms by which insulin increases transcription of two key enzymes of energy metabolism. Fatty acid synthase (FAS) plays central role in de novo lipogenesis by catalyzing all the reaction in the conversion of acetyl CoA and malonyl CoA to palmitate. Phosphofructokinase-1 (PFK) catalyzes the committed step in glycolysis. We have cloned cDNA and genomic clones for FAS, liver PFK and other insulin-inducible mRNAs and have shown transcriptional stimulation of these genes by insulin. The 5'-flanking regions of FAS and PFK genes contained sequences which confer insulin activation to linked reporter genes when transfected into 3T3-Ll adipocytes. A common mechanism may be involved in the insulin induction of these enzymes. Our goal is to define cis-acting elements and trans- acting factors for the transcriptional activation of these genes by insulin. Using in vitro mutagenesis, essential sequences for insulin regulation will be defined by transfection into adipocytes and by utilizing transgenic mice. The nuclear factor(s) which interact with these sequences will be characterized by mobility shift and DNase footprinting assays. The factor(s) will then be purified and/or cloned to start studying at the molecular level how insulin stimulates transcription of these genes. This will lead us in the future to elucidate a signal transduction pathway of insulin.
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