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

描述（由申请人提供）：甾醇调节元件结合蛋白（SREBP-1c）是控制肝脏中一系列脂肪生成基因表达的主要转录因子。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，通过染色质免疫沉淀法测定肥胖和高胰岛素血症动物模型JCR:LA-cp大鼠饮食中添加鲱鱼油对体内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启动子区及其靶基因的关联的影响。补充鲱鱼油对这些因子丰度的影响将通过实时PCR和western blotting来确定。此外，还将研究新的PUFA调控机制，如NrOb2、Kid-1的转录抑制和胰岛素敏感转录因子/共激活因子的磷酸化。其次，为了确定PUFA抑制胰岛素刺激的SREBP-1c转录的分子机制，将对大鼠SREBP-1c启动子内的转录因子结合元件进行突变分析，以剖析介导PUFA作用的元件。大鼠原代肝细胞中SREBP-1c启动子驱动的荧光素酶测定将用于确定转录活性。针对靶蛋白的siRNA基因敲低将用于确认启动子突变研究，而质粒介导的候选反激活子的过表达将用于在PUFAs面前“拯救”转录活性。综上所述，目前提出的研究应该为膳食PUFAs改善肥胖和高胰岛素血症中SREBP-lc表达升高和由此引起的血脂异常的分子机制提供急需的见解。相关性：目前的建议旨在确定长链多不饱和脂肪酸（PUFAs）在肥胖状态下降低SREBP-1c表达和脂肪生成的机制，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.