The long-term objective is to understand the molecular mechanisms underlying insulin-induced transcription of lipogenic genes. Insulin is the principal hormone that controls blood glucose. When circulating insulin is high there is an increase in fatty acid and fat synthesis in adipose tissue and liver; these processes are impaired when insulin is low and insulin restores the rates to normal. Diabetes is among the most common and serious metabolic diseases and understanding insulin action is of utmost importance. The goal of this research is to understand at a molecular level insulin induction of fatty acid synthase (FAS), the central lipogenic enzyme that catalyzes all reactions in the synthesis of palmitate from acetyl CoA and malonyl CoA. We previously found that insulin rapidly and markedly increases FAS gene transcription and recently defined insulin response sequence (IRS) at the proximal promoter region of the FAS gene and found upstream stimulatory factor (USF) binding to this sequence in vitro. The resent studies are to characterize the cis-trans system and signaling pathway for insulin- stimulated FAS transcription with the following specific aims: 1. To examine whether USF is involved in insulin regulation of the FAS insulin response complex. USF as an insulin response mediator will be established by correlating functional and bind activities, using in vitro mutagenesis of the IRS and by expressing dominant-negative USFs. GAL4-USF fusion will also be used to assess the role of USF as an insulin response mediator. 2. To isolate and clone other potential component of the FAS insulin response complex. If USF is proven to be a component of insulin response complex, a novel heterodimerization partner of the USF will be cloned by yeast interaction trap or by in vitro protein-protein interaction. If USF is proven not to be involved in insulin regulation, the putative IRS binding protein will be purified and/or cloned. 3. To explore signaling pathway by examining effects of specific inhibitors and dominant negative forms of signal molecules on insulin activated FAS promoter-luciferase activity. We will also examine whether USF or other protein component undergo phosphorylation- dephosphorylation. 4. To further examine the FAS promoter sequence in vivo. The first 2.1 kb of the 5'-flanking sequence of the FAS gene is sufficient for tissue-specific and hormonally regulated expression in transgenic mice. The sequence for insulin regulation will be further defined by transgenic mice that contain 5'deletion FAS-CAT constructs to demonstrate and confirm its role in vivo and by genomic footprinting by ligation-mediated PCR to demonstrate in vivo protein-DNA interactions.
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