mRNA profiling reveals divergent roles of PPARa and PPARb/d in regulating mouse liver gene expression (PPARa samples)

Little is known about the role of the transcription factor PPARb/d in liver. Here we set out to better elucidate the function of PPARb/d in liver by comparing the effect of PPARa and PPARb/d deletion using whole genome transcriptional profiling and analysis of plasma and liver metabolites. In fed state, the number of genes altered by PPARa and PPARb/d deletion was similar, whereas in fasted state the effect of PPARa deletion was much more pronounced, consistent with the pattern of gene expression of PPARa and PPARb/d. Minor overlap was found between PPARa- and PPARb/d-dependent gene regulation in liver. Pathways upregulated by PPARb/d deletion were connected to innate immunity. Pathways downregulated by PPARb/d deletion included lipoprotein metabolism and various pathways related to glucose utilization, which correlated with elevated plasma glucose and triglycerides and reduced plasma cholesterol in PPARb/d-/- mice. Downregulated genes that may underlie these metabolic alterations included Pklr, Fbp1, Apoa4, Vldlr, Lipg, and Pcsk9, which may represent novel PPARb/d target genes. In contrast to PPARa-/- mice, no changes in plasma FFA, plasma beta-hydroxybutyrate, liver triglycerides and liver glycogen were observed in PPARb/d-/- mice. Our data indicate a role for PPARb/d in hepatic glucose utilization and lipoprotein metabolism but not in the adaptive response to fasting. Keywords: Analysis of target gene regulation by using microarrays Pure-bred Sv129 PPARa -/- mice and corresponding wildtype mice were used. Male mice (n=4-5 per group) were either fed or fasted for 24 hours. At the end of the experiment, mice were anaesthetized with a mixture of isofluorane (1.5%), nitrous oxide (70%) and oxygen (30%). Blood was collected by orbital puncture, after which the mice were sacrificed by cervical dislocation. Livers were dissected, snap frozen in liquid nitrogen and kept at -80ºC until further analysis. For RNA analyses, tissue from the same part of the liver lobe was used.目前学界对转录因子过氧化物酶体增殖物激活受体δ (PPARb/d) 在肝脏中的作用尚不明晰。本研究旨在通过全基因组转录组分析及血浆与肝脏代谢物检测，对比敲除过氧化物酶体增殖物激活受体α (PPARα) 与PPARb/d所产生的调控差异，以更清晰地阐明PPARb/d在肝脏中的功能。在正常喂食状态下，敲除PPARα与PPARb/d所诱导的差异表达基因数量相近；而在禁食状态下，PPARα敲除的调控效应更为显著，这与PPARα与PPARb/d的内源基因表达模式相符。肝脏中依赖PPARα与依赖PPARb/d的基因调控通路仅存在少量重叠。PPARb/d敲除所上调的通路与先天免疫功能密切相关。PPARb/d敲除所下调的通路涵盖脂蛋白代谢及多条葡萄糖利用相关通路，这与PPARb/d基因敲除（PPARb/d-/-）小鼠的血浆葡萄糖、甘油三酯水平升高，血浆胆固醇水平降低的表型高度相关。可能介导上述代谢表型改变的下调基因包括Pklr、Fbp1、Apoa4、Vldlr、Lipg及Pcsk9，上述基因或为PPARb/d的新型靶基因。与PPARα基因敲除（PPARα-/-）小鼠不同，PPARb/d-/-小鼠的血浆游离脂肪酸（FFA）、β-羟基丁酸、肝脏甘油三酯及肝糖原水平均未出现显著变化。本研究数据表明，PPARb/d参与肝脏葡萄糖利用与脂蛋白代谢调控，但不参与机体对禁食的适应性应答过程。关键词：基于微阵列的靶基因调控分析。本研究使用纯种Sv129背景的PPARα基因敲除（PPARα-/-）小鼠及对应野生型小鼠。每组纳入4-5只雄性小鼠，分为正常喂食组与禁食24小时组。实验结束后，采用异氟烷（1.5%）、一氧化二氮（70%）与氧气（30%）的混合气体对小鼠进行麻醉。通过眶静脉穿刺采集血液，随后采用颈椎脱臼法处死小鼠。摘取肝脏组织，置于液氮中快速冷冻，并保存于-80℃以待后续分析。RNA分析所用组织取自肝叶的同一位置。