Study On The Difference And Mechanism Of Leucine Regulation Of MTORC1 Signaling In Different Cell Types

The mammalian target of rapamycin(mTOR)complex 1(mTORC1)responds to multiple reactions within cells and regulates various cellular activities,for example,protein synthesis.In addition to nutritional functions,amino acids(AA)also act as signal molecules to regulate the mTORC1 signaling pathway in animals.In the current research progress,leucine(Leu)is considered to have the strongest regulation ability of mTORC1 among all AA,which single deletion inhibits mTORC1 activity to a degree close to the full AA deletion.This has been extensively verified in a variety of cell lines in mice and humans.However,according to the results of previous studies,in cow mammary epithelial cells,the results we observed are not the same.Leu deletion can only partially inhibit the activity of mTORC1,while other essential AA such as isoleucine(Ile)also have strong regulatory role.We speculate that the ability of Leu to regulate mTORC1 may vary among different cells.In order to directly compare the differences between these cells to verify the speculation,and try to analyze its internal reasons,we conducted the following researches.This experiment proved that there are differences in the response of mTORC1 activity to changes in the concentration of extracellular Leu between different cells.Test 1: Comparison of the difference in the regulation of mTORC1 activity by Leu supplementation in MAC-T and MEFs.The results showed that when Leu was added in a gradient,the phosphorylation ratios of MAC-T mTOR(P = 0.019)and S6K1(P = 0.021)both increased first and then decreased,while MEFs in different gradient Leu changes,mTOR(P =0.592)and S6K1(P = 0.596)activities were not significantly different.Test 2: Comparison of the difference of mTORC1 activity in MAC-T,MEFs and Hela in serum-free AA/Leu deletion.The results showed that in the Hela cell line,compared with the positive control,the deletion of AA and Leu reduced the phosphorylation ratio of RPS6 by81.7% and 82%(P < 0.001),respectively,while the phosphorylation ratio of S6K1 was reduced by 57.1% and 63.1%(P < 0.001),respectively.The results of the two deletion treatments were significantly different from the positive control(P < 0.001),but not significant to each other.The results of the MEFs cell line were similar to Hela.The difference is that in MAC-T cells,compared with the positive control,the phosphorylation ratio of RPS6 in the AA and Leu deletion group decreased by 71.7% and 52.0%(P < 0.001),respectively,and the phosphorylation ratio of S6K1 decreased by 75.1% and 61%,respectively(P < 0.001).There is a significant difference between the two.Test 3: Comparison of mTORC1 activity differences in MAC-T,MEFs,Hela and BMECs in serum-containing AA/Leu/Ile deletion.The results showed that the RPS6 activity of AA/Leu/Ile deletion decreased by 82.6%,76.6%,34.3%(P < 0.001),and the S6K1 activity decreased by 81.6%,71.1%,14.2%,respectively,compared with the control group(P < 0.001).The situation of the MEFs cell line is similar to that of Hela.Compared with the control group,the RPS6 activity of the AA/Leu/Ile deletion decreased by 81.5%,74.1%,and 33.8%(P <0.001),while the activity of S6K1 decreased by 80.4%,70.3%,and 17.7%,respectively(P <0.001).The results of the MAC-T cell line are different from the above two cell lines.Compared with the control group,the activity of RPS6 in the absence of AA/Leu/Ile decreased by 62.5%,46.8%,and 40.5%(P < 0.001),while the activity of S6K1 decreased by 74.2%,31.2%,22.5%respectively(P < 0.001).In BMECs cells,the RPS6 activity of AA/Leu/Ile deletion decreased by 79.3%,66.5%,50.4%(P < 0.001),and S6K1 activity decreased by 78.7%,43.6%,36.9%(P< 0.001),respectively.Test 4: Comparison and analysis of transcriptome results in different cells with serumcontaining AA/Leu/Ile deletion.The MEFs cell line transcriptome results show that the differential genes are mainly enriched in MAPK signaling pathway,Cell cycle,Cellular senescence,p53 signaling pathway,mTOR signaling pathway,etc.The transcriptome results of the Hela cell line showed that the differential genes are mainly enriched in Inositol phosphate metabolism,Phosphatidylinositol signaling system,Cell cycle,Apoptosis-multiple species,Peroxisome and other aspects.The MAC-T cell line transcriptome results show that the differential genes are mainly enriched in MAPK signaling pathway,Protein processing in endoplasmic reticulum,Apoptosis,Ribosome,Cellular senescence,etc.In general,in MEFs and Hela,Leu deletion strongly inhibited the mTORC1 signaling pathway,and the level was similar to AA loss,while Ile deletion had a weaker effect on mTORC1 signaling pathway;while in MAC-T,Leu deletion the inhibitory ability of mTORC1 signaling pathway is significantly weaker than that of AA deletion,which is similar to the effect of Ile deletion.In MEFs and the Hela,AA deletion increased the activity of Akt,but not in MAC-T.In MEFs and the Hela,AA/Leu/Ile deletion reduced the expression of LRS,but not in MAC-T.BMECs and MAC-T had similar trends in each treatment group.The transcriptome results of MEFs,Hela and MAC-T were different among the treatment groups.