Quantitative analysis of Bckdhaf/f;RIP-Cre and Bckdhaf/f mice eWAT, iWAT and liver transcriptomes

To explore the underlying mechanism for the regulatory role of branched-chain amino acids (BCAA) catabolism in hypothalamic RIP-Cre neurons to metabolic organs under normal chow diet feeding, we established RIP-Cre neurons selective BCAA catabolic enzyme branched-chain ketoacid dehydrogenase E1 subunit alpha(Bckdha) knockout mice, we conducted RNA sequencing on the inguinal adipose tissue (iWAT), epididymal adipose tissues (eWAT), and liverfrom wild type (WT, Bckdhaf/f) and knockout (KO, Bckdhaf/f;RIP-Cre) mice (two groups in total). Three biological replicates were performed for each group. Methods: RNA sequencing (RNA-Seq) and data analysis were performed by Gene Denovo Biotechnology (Guangzhou, China). Results: In comparison with findings in the Bckdhaf/f group, 393 up-regulated and 214 down-regulated differentially expressed genes (DEGs) were identified in eWAT from Bckdhaf/f;RIP-Cre group . Similarly, 41 up-regulated genes and 152 down-regulated DEGs were identified in iWAT from Bckdhaf/f;RIP-Cre compared to the Bckdhaf/f group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that DEGs in eWAT were enriched in the categories including all subclass pathways in carbohydrate metabolism class, insulin resistance, PPAR signaling, oxidative phosphorylation, thermogenesis, carbon metabolism, fatty acid metabolism, biosynthesis of amino acids and degenerative disease pathways. Similarly, DEGs in iWATwere clustered in the pathways, including the TCA cycle, pyruvate metabolism, glucagon signaling, glycolysis and gluconeogenesis, and carbon metabolism. These changes indicated broad disturbance in WAT energy metabolism in Bckdhaf/f;RIP-Cre mice. Liver transcriptomes showed minor perturbances in subclasses in lipid metabolism, advanced glycation end products and their receptor (AGE-RAGE), PPAR, estrogen, prolactin signaling pathways. Notably, all 13 mitochondrial DNA encoded mRNA were all significantly down-regulated in eWAT of Bckdhaf/f;RIP-Cre mice, along with nucleus DNA encoded mitochondrial located mRNA. Conclusions: Our results using RNA-Seq indicated universal defects of mitochondrial gene expression in eWAT and iWAT. Overall design: iWAT, eWAT, and liver mRNA profiles of 12-week old Bckdhaf/f;RIP-Cre and Bckdhaf/f mice

为探究正常饲料喂养条件下，支链氨基酸（branched-chain amino acids, BCAA）分解代谢在下丘脑RIP-Cre神经元对代谢器官的调控作用背后的潜在机制，我们构建了RIP-Cre神经元选择性敲除支链酮酸脱氢酶E1亚基α（branched-chain ketoacid dehydrogenase E1 subunit alpha, Bckdha）的小鼠模型。随后，我们对野生型（wild type, WT, Bckdhaf/f）与敲除型（knockout, KO, Bckdhaf/f;RIP-Cre）小鼠的腹股沟脂肪组织（inguinal adipose tissue, iWAT）、附睾脂肪组织（epididymal adipose tissues, eWAT）以及肝脏进行了RNA测序（RNA sequencing, RNA-Seq），共设置两组样本，每组包含3个生物学重复。方法：RNA测序及数据分析由广州吉诺生物技术有限公司（Gene Denovo Biotechnology, Guangzhou, China）完成。结果：与Bckdhaf/f组相比，Bckdhaf/f;RIP-Cre组小鼠附睾脂肪组织中鉴定出393个上调差异表达基因（differentially expressed genes, DEGs）与214个下调差异表达基因。同理，相较于Bckdhaf/f组，Bckdhaf/f;RIP-Cre组小鼠腹股沟脂肪组织中鉴定出41个上调差异表达基因与152个下调差异表达基因。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析显示，附睾脂肪组织中的差异表达基因显著富集于碳水化合物代谢类别的所有亚类通路、胰岛素抵抗、过氧化物酶体增殖物激活受体（PPAR）信号通路、氧化磷酸化、产热、碳代谢、脂肪酸代谢、氨基酸生物合成以及退行性疾病通路。同样，腹股沟脂肪组织中的差异表达基因富集于三羧酸循环、丙酮酸代谢、胰高血糖素信号通路、糖酵解与糖异生以及碳代谢通路。上述变化提示Bckdhaf/f;RIP-Cre小鼠的白色脂肪组织（WAT）能量代谢存在广泛紊乱。肝脏转录组分析显示，脂代谢、晚期糖基化终末产物及其受体（advanced glycation end products and their receptor, AGE-RAGE）、PPAR、雌激素、催乳素信号通路的部分亚类存在轻微扰动。值得注意的是，Bckdhaf/f;RIP-Cre小鼠附睾脂肪组织中的13个线粒体DNA编码mRNA均显著下调，同时伴随核DNA编码的线粒体定位mRNA表达异常。结论：本研究通过RNA测序结果表明，Bckdhaf/f;RIP-Cre小鼠的附睾与腹股沟脂肪组织存在线粒体基因表达的普遍缺陷。整体实验设计：12周龄Bckdhaf/f;RIP-Cre与Bckdhaf/f小鼠的腹股沟脂肪组织、附睾脂肪组织及肝脏的mRNA表达谱。