The Regulate Function Of MTORC1 And MTORC2 On The Expression Of Key Enzymes Of Amino Acid Metabolism In Human Hepatocytes

The liver is an important metabolic organ of human body,and the substance metabolism in hepatocytes is a complex process,which is regulated by various internal and external factors,especially the signal transduction pathway plays an important role.The level of key enzyme for amino acid metabolism reflect on liver healthy state,such as aspartate aminotransferase(AST),glutamate dehydrogenase(GDH),glutamate decarboxylase(GAD),ornithine decarboxylase(ODC),glutathione thiotransferase(GST),alpha-ketoglutarate dehydrogenase(OGDH),and so on.However,the regulate mechanism of those enzyme remains poorly understood.Beside,as the central coordinator of cell growth and metabolism,the regulation mechanisms of mTORC1 and mTORC2 on key enzyme genens expression in amino acid metabolism in hepatocytes have been poorly understood.In this study,firstly,human normal hepatocytes(HL-7702)and hepatocellular carcinoma(HepG2)cells were used as experimental models to study different expression levels of six key enzyme relative to amino acid metabolism;Then,the function and regulation mechanism of mTORC1 and mTORC2 on key enzyme gene expression in amino acid metabolism were checked in HL-7702 cells;Finally,we compared and ensured the different functions of mTORC1 and mTORC2 in regulate amino acid metabolism key enzyme gene expression.The mRNA level of key enzymes genes were detected by qPCR.The changes of synthesis and secretion of six enzymes were tested by ELISA.The mTOR signaling pathway and transcription factors,such as NF-?B,STAT3,were measured by Western blot.The results showed that:(1)The intracellular content of AST,OGDH and GDH in HL-7702 cells were significantly higher than HepG2(p<0.01),while the intracellular level of GAD,ODC and GST were strikingly lower than HepG2(p<0.01);The extracellular expression of AST,OGDH,GDH,GAD,ODC and GST were decreased in HL-7702 cells;(2)After HL-7702 cells were treated with rapamycin,the phosphorylation level of mTOR,S6 and 4EBP1 were notably inhibited,the mRNA level of AST,OGDH,GDH,GAD,ODC and GST genes were remarkably attenuated(p<0.01),the enzyme content of intracellular and extracellular were also obviously alleviated(p<0.01);(3)After gene silencing of Raptor in HL-7702 cell,compared with the group of control,the phosphorylation level of mTOR,S6 and 4EBP1 were dramaticlly declined,the mRNA level of AST,OGDH,GDH,GAD,ODC and GST genes were remarkably attenuated(p<0.01),the enzyme content of intracellular also were obviously alleviated(p<0.01);(4)After the gene of Rheb overexpressed in HL-7702 cell,compared with the group of control,the phosphorylation level of mTOR,S6 and 4EBP1 were clearly increased,the mRNA level of AST,OGDH,GDH,GAD,ODC and GST genes were remarkably heighten(p<0.01),the enzyme content of intracellular were also obviously increased(p<0.01);(5)The mTORC1 pathway and NF-?B activity were activated by glutamate,while STAT3 activity remained unaffected;the mRNA level of AST gene and content of enzyme were increased by exogenous glutamate treatment(p<0.01),in HL-7702 cell;(6)After treatment with ornithine in HL-7702 cells,the mTORC1 pathway and NF-?B activity were increased,while STAT3 activity remained unaffected,the mRNA level of ODC gene and content of enzyme(p<0.05)were increased;(7)After gene silencing of the gene of Rictor in HL-7702 cell,the protein phosphorylation level of mTOR and AKT of mTORC2 pathway were relieved,the mRNA level of AST,OGDH,GDH,GAD,ODC and GST genes were remarkably attenuated(p<0.01),the enzyme content of intracellular were also obviously alleviated(p<0.01);(8)After overexpressed of mTORC2 key component mSIN1 in HL-7702 cells,the phosphorylation level of mTOR and AKT were apparently heighten,the mRNA level of AST,OGDH,GDH,GAD,ODC and GST genes were remarkably heighten(p<0.01),the enzyme content of intracellular were also obviously increased(p<0.01);(9)By comparing mTORC1 and mTORC2 with the content of key enzyme of amino acid metabolism influence in HL-7702 cell,we considered that the regulate function of mTORC1 was better than mTORC2.Taken together,the synthesis and secretion of amino acid metabolism key enzyme of AST,OGDH,GDH,GAD,ODC and GST were different in human normal hepatocytes HL-7702 and in hepatocelluar carcinoma cells HepG2,which provides basic data for the prediction of malignant transformation of hepatocytes using the changes of these enzyme contents in the future.mTORC1 regulate the gene expression and enzyme secretion of AST,OGDH,GDH,GAD,ODC and GST relative to amino acid metabolism key enzyme;mTORC1 adjust the expression of AST or ODC by regulation NF-?B activity and mediate the signal of glutamate and ornithine.Meanwhile,mTORC2 also participate in above six genes expression that regulating function was weaken than mTORC1.This study indicated that the express regulation model of amino acid metabolism key enzyme gene in liver cells,providing scientific basis for further understanding of amino acid metabolic mechanism in liver and liver disease for clinical targeted therapy.