Regulatory Mechanism of Mammalian Target Protein Rapamycin Signaling Pathway on Regulating Energy Metabolism by Arginine in Porcine Enterocytes
XIAO Hao1,2, WU Miaomiao2, WANG Li1, TAN Bi2
1. State Key Laboratory of Livestock and Poultry Breeding, Ministry of Agriculture Key Laboratory of Animal Nutrition and Feed Science in South China, Guangdong Public Laboratory of Animal Breeding and Nutrition, Guangdong Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
2. Observation and Experiment Station of Animal Nutrition and Feed Science in South-Central China, Ministry of Agriculture, Hunan Provincial Engineering Research Center for Healthy Livestock and Poultry Production, Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China
Abstract：
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 flow cytometry, as well as the relative contents of metabolites using metabonomics. The results showed as follows:1) compared with added 100 μmol/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 to 350 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 containing 100 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 to 350 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.
. Regulatory Mechanism of Mammalian Target Protein Rapamycin Signaling Pathway on Regulating Energy Metabolism by Arginine in Porcine Enterocytes[J]. Chinese Journal of Animal Nutrition, 2019,V31(3): 1266-1277.
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2019, Vol. 31 
