Fatty acid synthase (FAS) is a central lipogenic enzyme catalyzing all reactions in the synthesis of palmitate from acetyl CoA and malonyl CoA. FAS transcription drastically increases in liver and adipose tissue when fasted animals are fed a high carbohydrate diet or when diabetic animals are treated with insulin. We previously defined an response sequence at -65 where Upstream Stimulatory Factor (US F) binding brings about insulin-stimulated activation of the FAS promoter in 3T3-Ll adipocytes. We found that Phosphatidylinositol 3-kinase (PI3K)/akt pathway mediates this activation. Using transgenic mice, however, we found that two farther upstream regions are required for FAS promoter activation by feeding/insulin in vivo. One is at -13 1/-278 containing -150 SRE where Sterol Regulatory Element Binding Protein (SREBP) can bind, and the other is at -278/-444 containing -332 E-box where USF can bind. Our goal is to understand transcriptional activation and the signaling pathways for insulin/glucose regulation of the FAS gene. First, we will determine in vivo functions of the USF and SREBP sites by generating transgenic mice carrying the CAT gene driven by the various FAS promoter regions with mutations at the sites, -332 E-box, -150 SRE, and -65 E-box, implicated in the FAS activation by insulin/glucose. Clamping technique will be used to dissect the contribution of insulin and glucose also. Second, by chromatin immunoprecipitation, we will examine in vivo binding of USF, SREBP and other factors. Binding kinetics during feeding and insulin treatment will be determined also. Third, by in vitro binding and transactivation assays, we will examine whether USF and SREBP-1c cooperatively function by direct or indirect interaction via a coactivator such as CBP/p300 in activating FAS promoter. We will also determine changes in histone acetylation that leads to changes in chromatin accessibility of the FAS promoter, Finally, we will examine signaling pathways involved in the FAS promoter activation by insulin/glucose. We will generate mice carrying FAS-CAT transgene in the background of AMP-dependent protein kinase (AMPK) knockout mice to demonstrate the AMPK function in regulation of the FAS promoter in vivo. We will examine potential phosphorylation of USF and SREBP by Akt/PKB and AMPK as well as the functional consequences leading to transcriptional activation of the FAS promoter. Overall, our in vivo approaches will provide physiologically relevant information on cis-trans factors for activation of FAS promoter by insulin/glucose and will help to understand metabolic control of lipogenesis and dysregulation in diabetes and obesity.
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