Transcription profiling of mouse liver wild type and PPARalpha-nulls -1

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 PPARα in hepatic lipid metabolism, many PPARalpha-dependent pathways and genes have yet to be discovered. In order to obtain an overview of PPARα-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 β 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 PPARα. 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: Fed mice were killed at the end of the dark cycle (wild-type and PPARalpha-null mice; n=5 per group), mice were fasted for 24h before sacrifice (wild-type and PPARalpha-null mice; 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）是一种配体激活的转录因子，参与营养代谢与炎症调控。尽管目前对PPARalpha在肝脏脂质代谢中的功能已有较为深入的认知，但仍有大量PPARalpha依赖的信号通路与基因有待发掘。为系统梳理与脂质代谢相关的PPARalpha调控基因，并探索新型PPARalpha候选靶基因，研究人员采用Affymetrix基因芯片（Affymetrix GeneChips）对多项通过激活和/或敲除PPARalpha构建的动物实验模型的肝脏样本进行了转录组分析。本次研究共鉴定出多个与脂质代谢相关的新型PPARalpha调控基因。从该基因集合中筛选出8个基因，在小鼠肝脏以及小鼠、大鼠和人原代肝细胞中开展其PPARalpha依赖的调控模式研究，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 β 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）。通过计算机虚拟筛选（in silico screening）方法，研究人员在上述每一个基因中均鉴定出了一个或多个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，研究人员在肝脏脂质水平升高的条件下对这些基因的调控机制进行了深入探究。在喂食高脂饲料的野生型小鼠中，经PPARalpha激动剂Wy14643处理后，肝脏脂质含量显著降低，同时Pnpla2、Lipe和Mgll的表达水平明显上调，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重要机制之一。本研究证实了转录组谱分析在发掘肝脏中新型PPARalpha调控基因与信号通路方面的强大效能。实验总体设计：选用3至5月龄的纯系雄性野生型（129S1/SvImJ）与PPARalpha敲除（PPARalpha-null，129S4/SvJae）小鼠作为实验对象。实验总体设计：正常饲喂组小鼠于暗周期结束时处死（野生型与PPARalpha敲除小鼠，每组n=5）；禁食组小鼠在处死前禁食24小时（野生型与PPARalpha敲除小鼠，每组n=5）。将各组内混合样本的肝脏总RNA与Affymetrix MOE430A基因芯片（Affymetrix MOE430A GeneChip arrays）进行杂交。实验总体设计：按照ENZO实验流程对5μg总RNA进行标记与片段化，并依照Affymetrix标准操作流程完成杂交反应。