哺乳动物雷帕霉素靶蛋白信号通路影响精氨酸调控猪肠上皮细胞能量代谢的机制

Regulatory Mechanism of Mammalian Target Protein Rapamycin Signaling Pathway on Regulating Energy Metabolism by Arginine in Porcine Enterocytes

本试验旨在研究哺乳动物雷帕霉素靶蛋白(mTOR)信号通路调控猪肠上皮细胞能量代谢的机制。用含不同浓度精氨酸(Arg)(100、350μmol/L)和雷帕霉素(RAP)(0、10 nmol/L)的DMEM-H培养基培养猪肠上皮细胞72 h后,采用Searhorse XF Analyzers检测细胞线粒体呼吸代谢指标,实时荧光定量PCR检测能量代谢相关酶的mRNA表达量,流式细胞术检测活性氧(ROS)的百分含量以及代谢组学检测代谢物的相对含量。结果表明:1)与仅含100μmol/LArg的培养基相比,添加10 nmol/L RAP至含100或350μmol/L Arg的培养基中后,细胞线粒体呼吸代谢指标基础呼吸、质子渗漏、最大呼吸、储备呼吸率和ATP生成的耗氧率均极显著降低(P<0.01)。培养基中Arg浓度由100μmol/L提高到350μmol/L,除极显著提高ATP生成耗氧率(P<0.01)以外,对细胞线粒体呼吸代谢其他指标无显著影响(P> 0. 05)。2)与仅含100μmol/L Arg的培养基相比,在含100或350μmol/L Arg的培养基中添加10 nmol/L RAP极显著降低了糖酵解过程中相关酶的mRNA相对表达量,包括己糖激酶、丙酮酸脱氢酶、磷酸烯醇式丙酮酸激酶、柠檬酸合酶和异柠檬酸脱氢酶(P<0.01),同时显著降低了脂肪酸代谢过程中脂肪酸合成酶和肉碱棕榈酰转移酶的mRNA相对表达量(P <0. 05)。培养基中Arg浓度由100μmol/L提高到350μmol/L对上述指标没有显著影响(P> 0. 05)。3)与仅含100μmol/LArg的培养基相比,在含100或350μmol/L Arg的培养基中添加10 nmol/L RAP极显著降低了细胞内ROS的百分含量(P<0.01),而不同浓度Arg之间则没有显著变化(P>0.05)。4)与仅含100μmol/L Arg的培养基相比,在含100μmol/L Arg的培养基中添加10 nmol/L RAP显著或极显著降低了细胞提取物中丙氨酸、琥珀酸、柠檬酸、胆碱、肌醇和苏氨酸的相对含量(P<0.05或P<0.01),显著或极显著提高细胞提取物中亮氨酸、β-羟基异丁酸、Arg、赖氨酸、醋酸、糖蛋白和甲酸的相对含量(P<0.05或P<0.01)。综合上述结果可知,抑制mTOR信号通路显著抑制细胞呼吸代谢,即使增加Arg浓度也无法得到缓解,因此mTOR信号通路是Arg调控猪肠上皮细胞能量代谢的关键通路。

The purpose of this study was to investigate the regulatory mechanism of mammalian target protein rapamycin (mTOR) signaling pathway on energy metabolism of porcine intestinal epithelial cells. Intestinal porcine epithelial cells (IPEC-J2) were cultured in Dulbecco ’s modified Eagle’s-high glucose Ham medium (DMEM-H) containing 100 or 350 μmol/L arginine (Arg),0 or 10 nmol/L rapamycin (RAP) for 72 h,and then collected for the determination of mitochondrial respiration using Searhorse XF Analyzers,mRNA expression levels of related enzymes of energy metabolism using real-time fluorescence quantitative PCR (RT-qPCR) and reactive oxygen species (ROS) percentage content using flowcytometry,as well as the relative contents of metabolites using metabonomics. The results showed as follows: 1) compared with added 100μmol/L Arg into the medium,the parameters for mitochondria respiratory metabolism such as basal respiration,proton leak,maximal respiration,spare respiratory capacity,non-mitochondrial respiration and ATP production oxygen consumption rates were significantly reduced after added 10 nmol/L RAP into the medium which containing 100 or 350 μmol/L Arg (P < 0. 01). Elevating the Arg concentration from 100 μmol/L to350 μmol/L in the medium could significant increase the ATP production oxygen consumption rate (P <0.01),but had no significant effects on mitochondrial respiration metabolism parameters (P>0.05). 2) Compared with added 100 μmol/L Arg into the medium,added 10 nmol/L RAP into the medium which containing100 or 350 μmol/L Arg could significantly reduce the mRNA expression levels of related kinases in glycolysis,such as hexokinase,pyruvate dehydrogenase,phosphoenol pyruvate kinase,citrate synthase and isocitrate dehydrogenase (P<0.01),as well as the mRNA expression levels of fatty acid synthase and carnitine palmityltransferase in fatty acid metabolism (P < 0. 01). Elevating the Arg concentration from 100 μmol/L to350 μmol/L had no significant effects on above parameters (P>0.05). 3) Compared with added 100 μmol/L Arg into the medium,added 10 nmol/L RAP into the medium which containing 100 or 350 μmol/L Arg could significantly reduce the percentage content of ROS (P < 0.01),while no significant difference was found between different concentrations of Arg (P>0.05). 4) Compared with added 100 μmol/L Arg into the medium,added 10 nmol/L RAP into the medium which containing 100 μmol/L Arg could significantly decrease the relative contents of alanine,succinate,citrate,choline,myo-inositol and threonine in cell extract (P<0.05 or P<0.01),while significantly increased the relative contents of leucine,β-hydroxy isobutyric acid,arginine,lysine,acetate,glycoprotein and formate (P<0.05 or P<0.01). Taken together,above results suggest that inhibition mTOR signaling pathway significantly blocks the cellular respiration metabolism and can not be alleviated by elevating the Arg concentration. Therefore,mTOR signaling pathway is a key pathway for Arg to regulate energy metabolism in pig enterocytes.