Metabolic and transcriptome responses of RNAi-mediated AMPKa knockdown in Tribolium castaneum

Background: The AMP-activated protein kinase (AMPK) is an intracellular fuel sensor for lipid and glucose metabolism. In addition to the short-term regulation of metabolic enzymes by phosphorylation, AMPK may also exert long-term effects on the transcription of downstream genes through the regulation of transcription factors and coactivators. In this study, RNA interference (RNAi) was conducted to investigate the effects of knockdown of TcAMPKa on lipid and carbohydrate metabolism in the red flour beetle, Tribolium castaneum, and the transcriptome profiles of dsTcAMPKa-injected and dsEGFP-injected beetles under normal conditions were compared by RNA-sequencing. Results: RNAi-mediated suppression of TcAMPKa increased whole-body triglyceride (TG) level and the ratio between glucose and trehalose, as was confirmed by in vivo treatment with the AMPK-activating compound, 5-Aminoimidazole-4-carboxamide1-ß-D-ribofuranoside (AICAR). A total of 1184 differentially expressed genes (DEGs) were identified between dsTcAMPKa-injected and dsEGFP-injected beetles. These include genes involved in lipid and carbohydrate metabolism as well as insulin/insulin-like growth factor signaling (IIS). Real-time quantitative polymerase chain reaction analysis confirmed the differential expression of selected genes. Interestingly, metabolism-related transcription factors such as sterol regulatory element-binding protein 1 (SREBP1) and carbohydrate response element-binding protein (ChREBP) were also significantly upregulated in dsTcAMPKa-injected beetles. Conclusions: AMPK plays a critical role in the regulation of beetle metabolism. The findings of DEGs involved in lipid and carbohydrate metabolism provide valuable insight into the role of AMPK signaling in the transcriptional regulation of insect metabolism. Overall design: Total mRNA profiles of 20-day old dsEGFP-injected and dsTcAMPKa-injected larvae were generated by transcriptome sequencing under normal conditions with three replicates.

背景：腺苷酸活化蛋白激酶（AMP-activated protein kinase, AMPK）是参与脂质与葡萄糖代谢的细胞内能量感受器。除了通过磷酸化对代谢酶进行短期调控外，AMPK还可通过调控转录因子及辅激活因子，对下游基因的转录产生长期影响。本研究借助RNA干扰（RNA interference, RNAi）技术，探究敲低TcAMPKa对赤拟谷盗（Tribolium castaneum）脂质与碳水化合物代谢的影响，并通过RNA测序（RNA-sequencing）比较了正常条件下注射dsTcAMPKa与注射dsEGFP的赤拟谷盗的转录组表达谱。
结果：RNAi介导的TcAMPKa敲低提升了赤拟谷盗全身体内甘油三酯（triglyceride, TG）水平以及葡萄糖与海藻糖的比值，该结果经AMPK激活剂5-氨基咪唑-4-甲酰胺-1-β-D-呋喃核糖苷（AICAR）的体内处理实验得到验证。在注射dsTcAMPKa与注射dsEGFP的赤拟谷盗样本中，共鉴定出1184个差异表达基因（differentially expressed genes, DEGs），这些基因涉及脂质与碳水化合物代谢以及胰岛素/胰岛素样生长因子信号通路（insulin/insulin-like growth factor signaling, IIS）。实时定量聚合酶链反应验证了部分筛选基因的差异表达情况。值得注意的是，在注射dsTcAMPKa的赤拟谷盗体内，固醇调节元件结合蛋白1（sterol regulatory element-binding protein 1, SREBP1）与碳水化合物反应元件结合蛋白（carbohydrate response element-binding protein, ChREBP）等代谢相关转录因子的表达量也显著上调。
结论：AMPK在赤拟谷盗的代谢调控中发挥关键作用。本研究中鉴定出的涉及脂质与碳水化合物代谢的差异表达基因，为理解AMPK信号通路在昆虫代谢转录调控中的功能提供了重要参考。
总体设计：在正常培养条件下，对20日龄的注射dsEGFP与注射dsTcAMPKa的赤拟谷盗幼虫进行转录组测序，获取其总mRNA表达谱，每组设置3个生物学重复。