Adiponectin regulates expression of hepatic genes critical for glucose and lipid metabolism

The effects of adiponectin on hepatic glucose and lipid metabolism at transcriptional level are largely unknown. We profiled hepatic gene expression in adiponectin knockout (KO) and wild-type (WT) mice by RNA-Seq. Comparing to WT mice, adiponectin KO mice exhibited decreased mRNA expression of rate-limiting enzymes in several important glucose and lipid metabolic pathways including glycolysis, TCA cycle, fatty-acid activation and synthesis, triglyceride synthesis and cholesterol synthesis. In addition, binding of the transcription factor Hnf4a to DNAs encoding several key metabolic enzymes was reduced in KO mice, suggesting that adiponectin might regulate hepatic gene expression via Hnf4a. Phenotypically, adiponectin KO mice possessed smaller epididymal fat pads and showed reduced body weights comparing to WT mice. When fed a high fat diet, adiponectin KO mice showed significantly reduced lipid accumulation in the livers. These lipogenic defects are consistent with the downregulation of lipogenic genes in the KO mice. Mice were fasted overnight before euthanization. Liver tissues from WT or adiponectin KO male mice (n = 9-10) at 12 weeks of age were harvested and subjected to total RNA extraction using an RNeasy Plus Mini Kit (Qiagen, Valencia, CA). Total RNA from nine to ten mice of the same strain was pooled together as one biological sample. The mRNA sequencing samples were prepared using the Illumina sample preparation protocol (RS-930-1001, Illumina, Inc. San Diego, CA). The cDNA fragments of 200-250 bp were purified on an agarose gel and then enriched by PCR with Phusion polymerase. The cDNA libraries were sequenced by an Illumina Genome analyzer II at the Whitehead Genome Technology Core.脂联素（adiponectin）在转录水平对肝脏糖脂代谢的影响迄今仍未被充分阐明。本研究通过RNA测序（RNA-Seq）技术，对脂联素基因敲除（KO）小鼠与野生型（WT）小鼠的肝脏基因表达谱进行了分析。与野生型小鼠相比，脂联素敲除小鼠体内多条关键糖脂代谢通路中的限速酶mRNA表达水平均出现下调，这些通路包括糖酵解、三羧酸（TCA）循环、脂肪酸活化与合成、甘油三酯合成以及胆固醇合成通路。此外，在脂联素敲除小鼠中，转录因子Hnf4a与数种关键代谢酶编码DNA的结合能力显著下降，这提示脂联素可能通过Hnf4a调控肝脏基因的表达。 表型层面上，脂联素敲除小鼠的附睾脂肪垫体积更小，且体重较野生型小鼠更低。在饲喂高脂饲料后，脂联素敲除小鼠肝脏内的脂质蓄积量显著降低。上述生脂通路的缺陷与脂联素敲除小鼠中脂生成相关基因的下调表达相一致。 所有小鼠在安乐处死前均经过一夜禁食。本研究采集了12周龄的雄性野生型与脂联素敲除小鼠的肝脏组织（每组n=9~10），并使用RNeasy Plus Mini试剂盒（Qiagen，美国加利福尼亚州巴伦西亚）提取总RNA。将同组的9~10只小鼠的总RNA合并为一个生物学样本。mRNA测序文库的构建参照Illumina官方样本制备方案（产品编号RS-930-1001，Illumina公司，美国加利福尼亚州圣地亚哥）进行。通过琼脂糖凝胶纯化得到200~250 bp的cDNA片段，随后使用Phusion高保真DNA聚合酶通过PCR完成文库富集。最终构建的cDNA文库在怀特黑德基因组技术中心使用Illumina Genome Analyzer II测序平台完成测序。