Transcription profiling of mouse 3-5 months old males wild-type (129S1/SvImJ) and PPARalpha-null (129S4/SvJae) to investigate PPARalpha-dependent regulation of hepatic lipid metabolism - 4

PPARα 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 PPPARalpha. 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 PPARα-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=4 per group). Liver total RNA from biological replicates was hybridized onto Affymetrix mouse genome 430 2.0 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（peroxisome proliferator-activated receptor alpha，PPARα）是一类配体依赖性转录因子，参与营养代谢与炎症反应的调控。尽管目前对PPARα在肝脂代谢中的功能已有较为深入的认知，但诸多依赖PPARα的信号通路与靶基因仍有待发掘。为系统梳理参与脂代谢的PPARα调控基因，并探索新型候选PPARα靶基因，研究人员通过Affymetrix基因芯片（Affymetrix GeneChips）分析了多项动物实验中经PPARα激活或敲除的肝脏样本，最终鉴定出大量与脂代谢相关的新型PPARα调控基因。从该基因集合中，筛选出8个基因开展后续研究，以解析其在小鼠肝脏以及小鼠、大鼠、人原代肝细胞中的PPARα依赖性调控模式，涉及的基因包括：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）。通过虚拟筛选（in silico screening）策略，研究人员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均参与肝脏甘油三酯水解过程，研究人员在肝脂水平升高的条件下对这三类基因的调控机制展开了深入探究。在喂食高脂饲料的野生型小鼠中，经PPARα激动剂Wy14643处理后，肝脏脂质含量显著下降，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PPARα发挥抗脂肪变性作用的重要潜在机制。本研究证实了转录谱分析在发掘肝脏中新型PPARα调控基因与通路方面的强大效能。实验总体设计：选取3~5月龄的纯种雄性野生型（129S1/SvImJ）与PPARα敲除型（129S4/SvJae）小鼠作为实验对象。实验总体设计：将野生型与PPARα敲除型小鼠分别喂食添加了合成PPARα配体Wy14643（0.1% w/w）的饲料，或普通常规饲料（对照组），持续处理5天（每组n=4）。将各生物学重复样本的肝脏总RNA与Affymetrix小鼠基因组430 2.0基因芯片（Affymetrix mouse genome 430 2.0 GeneChip arrays）进行杂交。实验总体设计：按照ENZO实验流程对5 μg总RNA进行标记与片段化，并严格遵循Affymetrix官方实验流程完成杂交操作。