Transcription profiling of mouse wild-type and PPARalpha-null liver treated with the synthetic PPARalpha ligand WY14643

PPARalpha is a ligand-activated transcription factor involved in the regulation of nutrient metabolism and inflammation. Although much is already known about the function of PPARalpha in hepatic lipid metabolism, many PPARalpha-dependent pathways and genes have yet to be discovered. In order to obtain an overview of PPARalpha-regulated genes relevant to lipid metabolism, and to probe for novel candidate PPARalpha target genes, livers from several animal studies in which PPARalpha was activated and/or disabled were analyzed by Affymetrix GeneChips. Numerous novel PPARalpha-regulated genes relevant to lipid metabolism were identified. Out of this set of genes, eight genes were singled out for study of PPARalpha-dependent regulation in mouse liver and in mouse, rat, and human primary hepatocytes, including thioredoxin interacting protein (Txnip), electron-transferring-flavoprotein beta polypeptide (Etfb), electron-transferring-flavoprotein dehydrogenase (Etfdh), phosphatidylcholine transfer protein (Pctp), endothelial lipase (EL, Lipg), adipose triglyceride lipase (Pnpla2), hormone-sensitive lipase (Lipe), and monoglyceride lipase (Mgll). Using an in silico screening approach, one or more PPAR response elements (PPREs) were identified in each of these genes. Since Pnpla2, Lipe, and Mgll contribute to hepatic triglyceride hydrolysis, gene regulation was studied under conditions of elevated hepatic lipids. In wild-type mice fed a high fat diet, the decrease in hepatic lipids following treatment with the PPARalpha agonist Wy14643 was paralleled by significant up-regulation of Pnpla2, Lipe, and Mgll, suggesting that induction of triglyceride hydrolysis may contribute to the anti-steatotic role of PPARalpha. Our study illustrates the power of transcriptional profiling to uncover novel PPARalpha-regulated genes and pathways in liver. Experiment Overall Design: 3-5 months old male pure bred wild-type (129S1/SvImJ) and PPARalpha-null (129S4/SvJae) mice were used. Experiment Overall Design: Wild-type and PPARalpha-null mice were treated with the synthetic PPARalpha ligand WY14643 (0.1% w/w) mixed in the food, or normal food (control) for 5 days (n=5 per group). Liver total RNA of pooled samples (within groups) was hybridized onto Affymetrix MOE430A GeneChip arrays. Experiment Overall Design: Five microgram total RNA was labelled according to the ENZO-protocol, fragmented and hybridized according to Affymetrix's protocols.过氧化物酶体增殖物激活受体α（PPARalpha）是一类配体激活型转录因子，参与营养代谢与炎症反应的调控。尽管学界对PPARalpha在肝脏脂质代谢中的功能已有较为深入的认知，但诸多依赖PPARalpha的信号通路与基因仍有待发掘。为全面解析与脂质代谢相关的PPARalpha调控基因，并探索新型潜在PPARalpha靶基因，研究人员对多项动物实验的肝脏样本开展了分析：实验通过激活或敲除PPARalpha，采用Affymetrix基因芯片（Affymetrix GeneChips）进行检测。研究团队成功鉴定出多个与脂质代谢相关的新型PPARalpha调控基因。 从该基因集合中，研究人员挑选出8个基因，在小鼠肝脏以及小鼠、大鼠、人原代肝细胞中研究其PPARalpha依赖性调控模式，所涉基因包括：硫氧还蛋白相互作用蛋白（thioredoxin interacting protein，Txnip）、电子传递黄素蛋白β多肽（electron-transferring-flavoprotein beta polypeptide，Etfb）、电子传递黄素蛋白脱氢酶（electron-transferring-flavoprotein dehydrogenase，Etfdh）、磷脂酰胆碱转移蛋白（phosphatidylcholine transfer protein，Pctp）、内皮脂肪酶（endothelial lipase，EL, Lipg）、脂肪甘油三酯脂肪酶（adipose triglyceride lipase，Pnpla2）、激素敏感性脂肪酶（hormone-sensitive lipase，Lipe）以及单酰甘油脂肪酶（monoglyceride lipase，Mgll）。 研究人员通过虚拟筛选（in silico screening）策略，在上述每个基因中均鉴定出一个或多个PPAR反应元件（PPAR response elements，PPREs）。 鉴于Pnpla2、Lipe与Mgll参与肝脏甘油三酯水解过程，研究团队在肝脏脂质水平升高的条件下对这些基因的调控机制展开了研究。在喂食高脂饲料的野生型小鼠中，经PPARalpha激动剂Wy14643处理后，肝脏脂质水平显著降低，同时Pnpla2、Lipe与Mgll的表达水平显著上调，这提示诱导甘油三酯水解可能是PPARalpha发挥抗脂肪变性作用的潜在机制之一。 本研究证实了转录谱分析（transcriptional profiling）的应用价值，可用于发掘肝脏中新型的PPARalpha调控基因与通路。 ### 实验总体设计 1. 实验对象：选取3~5月龄的纯种雄性野生型（129S1/SvImJ）与PPARalpha敲除（129S4/SvJae）小鼠。 2. 干预处理：将野生型及PPARalpha敲除小鼠分别喂食添加了合成PPARalpha配体WY14643（0.1% 质量分数）的饲料，或普通饲料（对照组），持续干预5天，每组设置5只生物学重复。 3. 芯片实验：提取各组混合样本的肝脏总RNA，按照ENZO试剂盒说明书对5 μg总RNA进行标记与片段化，随后依照Affymetrix标准流程完成芯片杂交。