Transcriptional Regulation of SREBP-1c by Dietary Polyunsaturated Fatty Acids

描述(由申请人提供)：固醇调节元件结合蛋白(SREBP-1c)是主要的转录因子，控制肝脏中一系列致脂基因的表达。在肥胖和高胰岛素血症状态下，SREBP-1c的产生增加，部分原因是转录水平升高。首先，利用肥胖和高胰岛素血症的动物模型，JCR：LA-cp大鼠，通过染色质免疫沉淀分析，确定饮食中补充薄荷油对已知转录因子和辅助激活因子(LXRa、NF-Y、Sp-1、SREBP-1c、SRC-1、CBP/p300)和可能的抑制因子(Kid-1和NrOb2)与SREBP-1c及其靶基因启动子区域的体内相关性的影响。补充鱼油对这些因子丰度的影响将通过实时定量聚合酶链式反应和蛋白质印迹来确定。此外，还将研究新的多不饱和脂肪酸调节机制，如NrOb2、Kid-1转录抑制和胰岛素敏感转录因子/共激活物的磷酸化。其次，为了确定多不饱和脂肪酸抑制胰岛素刺激的SREBP-1c转录的分子机制(S)，我们将对大鼠SREBP-1c启动子中的转录因子结合元件进行突变分析，以剖析介导多不饱和脂肪酸作用的元件。大鼠SREBP-1c启动子驱动的大鼠原代肝细胞荧光素酶分析将用于确定转录活性。针对目标蛋白的siRNA基因敲除将被用于确认启动子突变研究，而候选反式激活子的质粒介导的过表达将被用于在面对PUFAs时“拯救”转录活性。综上所述，目前提出的研究应该为深入了解饮食中多不饱和脂肪酸可以改善SREBP-lc表达升高以及由此导致的肥胖和高胰岛素血症的血脂异常的分子机制提供亟需的见解(S)。相关性：目前的建议试图确定长链多不饱和脂肪酸(PUFA)减少肥胖状态下SREBP-1c表达和脂肪生成的机制(S)，从而改善与肥胖和II型糖尿病相关的血脂异常。

DESCRIPTION (provided by applicant): Sterol regulatory element binding protein (SREBP-1c) is the primary transcription factor that governs expression of a host of lipogenic genes in the liver. In states of obesity and hyperinsulinemia, SREBP-1c production is increased due, in part, to elevated levels of transcription. First, an animal model of obesity and hyperinsulinemia, the JCR:LA-cp rat, will be employed to determine the effect of dietary supplementation with menhaden oil on the in vivo associations of known transcription factors and coactivators (LXRa, NF-Y, Sp-1, SREBP-1c, SRC-1, CBP/p300) and possible repressers (Kid-1 and NrOb2) with the promoter region of SREBP-1c and its target genes by chromatin immunoprecipitation assays. The effect of menhaden oil supplementation on abundance of these factors will be determined by real-time PCR and western blotting. In addition, novel PUFA regulatory mechanisms will be examined such as transcriptional repression by NrOb2, Kid-1, and phosphorylation of insulin-sensitive transcription factors/coactivators. Secondly, to determine the molecular mechanism(s) by which PUFA repress insulin stimulated SREBP-1c transcription, mutational analysis of transcription factor binding elements within the rat SREBP-1c promoter will be performed to dissect the elements which mediate PUFA effects. Rat SREBP-1c promoter driven luciferase assays in rat primary hepatocytes will be used to determine transcriptional activity. Gene knockdown by siRNA directed against target proteins will be used to confirm promoter mutation studies and plasmid mediated overexpression of candidate transactivators will be used to "rescue" transcriptional activity in the face of PUFAs. Taken together, the presently proposed studies should provide much needed insight into the molecular mechanism(s) through which dietary PUFAs can ameliorate elevations in SREBP-lc expression and the resulting dyslipidemia observed in obesity and hyperinsulinemia. Relevance: The current proposal seeks to determine the mechanism(s) by which long chain polyunsaturated fatty acids (PUFAs) decrease expression of SREBP-1c and lipogenesis in the obese state thereby ameliorating dyslipidemia associated with obesity and type II diabetes.