Study On The Characteristics And Mechanism Of Leucine Deficiency In MTORC1 Signal Of MAC-T Cells

Leucine(Leu)deficiency inhibits protein synthesis through mTORC1 signaling.In growing animals,Leu deficiency strongly inhibits muscle protein synthesis.In HEK293 and other cells,the inhibition of mTORC1 activity by Leu deletion was similar to that of total amino acid(AA)deletion.In dairy cows and other lactation animals,the inhibition of Leu deficiency on milk protein synthesis is relatively mild,suggesting that the effects of Leu deficiency on protein synthesis and mTORC1 signal may be cell-specific,but there is no direct evidence and the underlying mechanism is not clear.In order to solve this scientific problem,MAC-T cells were used to observe the changes of mTORC1 signal and metabolic adaptation caused by Leu deficiency,the effects of serine(Ser)synthesis and branched chain amino acid(BCAA)catabolism on mTORC1 signal regulated by Leu.The response characteristics and mechanism of mTORC1 signal to Leu deficiency in MAC-T cells were studied.The results are as follows:1.Leu deletion response of mTORC1 signal in MAC-T cells(1)MAC-T can quickly adapt to Leu deficiency.When MAC-T cells lacked Leu,the phosphorylation levels of 4EBP1 and RPS6 proteins decreased rapidly and remained stable after 2 h,and the phosphorylation level of 4EBP1 increased after 10 h of culture,which was consistent with the reactivation of mTORC1 caused by cell protein decomposition in other studies.The level of ATF4,a marker of integrated stress response(ISR),also increased rapidly after the absence of Leu,and remained stable after 2 h.The above results show that MAC-T cells can quickly adapt to the environment of Leu deletion.(2)Leu activated mTORC1 signal in a dose-dependent manner.The phosphorylation level of 4EBP1 and RPS6 increased with the increase of Leu concentration in MAC-T cells,cultured in DMEM/F12 medium with 0%,5%,10%,50%,100% and 300% Leu concentration,indicating that Leu activated mTORC1 signal in a dose-dependent manner.The level of ATF4 protein is basically stable when the concentration of Leu is more than 10%,indicating that ISR occurs only at very low Leu levels.(3)Total AA deletion causes a stronger inhibitory effect on mTORC1 signal of MAC-T cells.Different from those reported in 293 T and muscle cells,the response of mTORC1 signal of MAC-T cells to Leu deletion was much lower than that of total AA deletion.Leu deletion for 4 h decreased 4EBP1 phosphorylation level by 19%,total AA deletion for 4 h decreased by 79%,and glucose deficiency for 4 h decreased by 43%.Furthermore,the responses of mTORC1 signals of MAC-T and 293 T cells to Leu and total AA deletions were compared.Leu and total AA deletion decreased the 4EBP1 phosphorylation level of MAC-T cells by24% and 70%,respectively,while that of 293 T cells decreased by 70% and 86%,respectively.The addition of Leu to the total AA deletion medium had no effect on the 4EBP1 phosphorylation level of MAC-T cells,but in 293 T cells the addition increased significantly after 1 hour treatment.The results showed that the responses of the two kinds of cells to Leu deletion were different.The effects of Leu and total AA deficiency on GCN2-e IF2α signaling pathway in MAC-T cells were compared.Both of them significantly increased the level of e IF2α phosphorylation,but the extent of increase was similar.There was no stronger effect of total AA deficiency on e IF2α phosphorylation.(4)Leu deletion has a stronger activating effect on ISR in MAC-T cells.MAC-T cells were cultured in the medium without total AA,Leu or glucose,respectively,and the changes of ATF4 protein expression were observed.Unexpectedly,the expression of ATF4 was the highest when Leu instead of total AA was deleted,and glucose deficiency had the least stimulating effect on ATF4 expression,indicating that Leu deletion had a stronger stimulating effect on ISR in MAC-T cells.The lack of AA can cause cell ISR and promote proteolysis to maintain the stability of intracellular AA concentration,suggesting that ISR may mediate part of the regulation of mTORC1 signal by Leu.(5)ISR can regulate the mTORC1 signal of MAC-T cells.Treatment of MAC-T cells with ISR inhibitors significantly decreased the expression of ATF4 protein and the level of phosphorylation of 4EBP1 and RPS6 in MAC-T cells with Leu deletion.Knockdown of ATF4 gene with sh ATF4 significantly decreased the level of 4EBP1 phosphorylation during Leu deletion,which confirmed that ISR attenuated the inhibitory effect of Leu deletion on mTORC1 signal in MAC-T cells.(6)There was no significant difference in the effect of total AA and Leu deletion on the total protein synthesis rate of MAC-T cells.Puromycin labeling method was used to determine the effect of Leu or total AA deletion on total protein synthesis in MAC-T cells.Both treatments significantly inhibited total protein synthesis,but the inhibition was similar.In summary,the response of mTORC1 signal of MAC-T cells to Leu deletion was faster,dose-dependent and weaker than that of total AA deletion,which was different from that of293 T cells.It was also found that ISR attenuated the inhibitory effect of Leu deletion on mTORC1 signal of MAC-T cells,and the stimulating effect of Leu deletion on ISR of MAC-T cells was stronger than that of total AA deletion,and the stimulating effect of Leu deletion on GCN2-e IF2 α signal of MAC-T cells was similar to that of total AA deletion.The above results provide an explanation for the weak inhibitory effect of Leu deficiency on milk protein synthesis,but it needs to be confirmed by further studies at the body level.2.Adaptive responses of MAC-T cells to Leu deficiency(1)The deletion of Leu causes the adaptive change of cell survival orientation.Leu deletion increased the proportion of early apoptotic cells,but decreased the proportion of necrotic and late apoptotic cells,and the proportion of normal cells was higher than that of the control group.KEGG analysis showed that the transcriptome changes caused by Leu deletion were significantly enriched in cancer-related pathways,MAPK signaling,cell cycle,cell senescence,aminoacyl-t RNA synthesis and so on.There was a power function relationship between cell proliferation and Leu concentration,and changed sharply at very low Leu concentration.The above results suggest that Leu deletion leads to survival-oriented adaptive changes in MAC-T cells.The percentage of normal cells in the early stage of apoptosis,necrosis and late stage of apoptosis increased,while the proportion of normal cells was lower than that in the control group.The loss of total AA was different.Consistent with this,the increase of mitochondrial membrane permeability caused by total AA deletion was much higher than that of Leu deletion.(2)The deletion of Leu reduces the energy level of MAC-T cells.Leu deletion increased the proportion of ADP/ATP in MAC-T cells and decreased the energy level of cells.Leu deletion significantly decreased succinic acid concentration,fumaric acid concentration tended to decrease,and other substrate concentrations increased or had no significant change.The relative pH value of cells decreased significantly and the degree of pyruvate dehydrogenase phosphorylation increased significantly.These results suggest that Leu deficiency may reduce cell energy level mainly by inhibiting mitochondrial oxidation.(3)Leu deletion increased the acetylation level of MAC-T cells.Leu deletion increased the level of protein acetylation in MAC-T cells and decreased in 293 T cells.Transcriptome analysis showed that Leu deletion increased BCAA catabolism,Leu transport,fatty acid βoxidation,protein acetylation and decreased the expression of genes related to de novo fatty acid synthesis in MAC-T cells,suggesting that Leu deletion leads fatty acid metabolism to βoxidation.In summary,Leu deletion decreased the energy level of MAC-T cells,increased the level of protein acetylation,promoted fat β oxidation,inhibited fatty acid synthesis gene expression,and cell metabolism showed survival-oriented changes.3.Ser synthesis-ISR mediates the differential regulation of mTORC1 signal in MAC-T and 293 T cells by Leu deletion.(1)Leu deletion promotes Ser synthesis in MAC-T cells.The deletion of Leu increased the concentration of most AA including Ser in MAC-T cells.KEGG analysis showed that the changes of MAC-T cell transcriptional group and metabolic group caused by Leu deletion were significantly enriched in the pathway related to Ser synthesis,and the expression of Ser synthase gene was increased.When Leu was deleted,the concentration of Ser in MAC-T cells increased significantly.When drugs were used to inhibit Ser synthetase,and the effect of Leu deletion on the increase of Ser concentration disappeared,indicating that Leu deletion stimulated Ser synthesis.Metabonomic results showed that the concentrations of many nucleotide-related metabolites changed significantly after Leu deletion.Since Ser is the precursor of nucleotides and nucleotides are involved in the regulation of mTORC1 signal,the effect of Ser synthesis on the regulation of mTORC1 signal by Leu was studied.(2)Ser synthesis is cell specific in ISR caused by interfering with Leu deficiency.Leu deletion increased the expression of ATF4 in MAC-T and 293 T cells,resulting in ISR.At the same time,deletion of Ser inhibits MAC-T but does not inhibit ISR caused by deletion of Leu in 293 T cells.The drug inhibited the Ser synthesis of the two kinds of cells,significantly activated ISR in MAC-T cells and inhibited ISR in 293 T cells.Because ATF4 regulates the expression of 4EBP1 protein,consistent with this,drug inhibition of Ser synthesis significantly decreased the expression of 4EBP1 in 293 T cells,while increased in MAC-T cells.(3)Ser synthesis mediates that Leu lacks the regulation of mTORC1 signal.The deletion of Leu and Ser enhanced the inhibitory effect of Leu deletion on mTORC1 signal of MAC-T cells,but had no significant effect on the inhibition of mTORC1 signal of 293 T cells by Leu deletion.Drug inhibition of Ser synthesis significantly inhibited mTORC1 signal of MAC-T cells,but had no significant effect on mTORC1 signal of 293 T cells.Knocking down the Ser dehydrase did not significantly affect the regulation of mTORC1 activity by Leu on MAC-T cells,indicating that the regulation of mTORC1 signal by Leu is mainly mediated by Ser synthesis rather than catabolism.(4)Inhibition of Ser synthesis leads to metabolic reprogramming of MAC-T cells.The drug inhibited Ser synthetase and deleted Ser in MAC-T cells,which significantly increased the concentration of seven kinds of TCA cycle substrate and decreased the concentration ofα-KG.The change of TCA cycle substrates concentrations were much stronger than that of Leu deletion,indicating that severe reprogramming occurred in cell metabolism,suggesting that the change of Ser synthesis pathway is an important pathway for Leu to affect cell metabolism.In summary,the Ser synthesis-ISR axis mediates the differential regulation of Leu deletion on different cell metabolism and mTORC1 signals,which is mainly mediated by the process of Ser synthesis.4.Inhibition of Leu catabolism can reduce mTORC1 signal activity in MAC-T cells.(1)Inhibition of BCAT increased Leu concentration but decreased mTORC1 signal.After inhibiting the activity of BCAA transaminase(BCAT)in MAC-T cells,the intracellular Leu concentration was significantly increased,but the phosphorylation levels of 4EBP1 and RPS6 were decreased.Because three kinds of BCAA share BCAT,the activity of MCCC1 enzyme specific to Leu catabolism pathway was further knocked down,and mTORC1 signal decreased,indicating that inhibition of Leu catabolism suppressed mTORC1 signal.(2)The changes of cell metabolism mediate the mTORC1 signal regulation of Leu catabolism inhibition.Drug inhibition of BCAT significantly changed the concentrations of TCA cycle substrates,the concentration of pyruvate decreased,the concentration of acetyl-CoA increased,the concentrations of AA changed widely,the expression of genes related to fatty acid catabolism was generally up-regulated,and the fatty acid composition of MAC-T cells changed significantly.The experiment further evaluated the mediating effect of cell metabolic changes on the regulation of mTORC1 signal by Leu.The addition of acetyl-CoA significantly reduced the inhibitory effect of drug on the decrease of mTORC1 signal.The above results showed that the changes of cell metabolism can interfere with the regulation of mTORC1 signal by Leu to a great extent,suggesting that the structure of cell metabolism is an important basis for the differential regulation of Leu on different types of cells.In summary,this part of the study confirmed that the cell metabolic structure interferes with the regulation of mTORC1 signal by Leu.Conclusion:(1)the effect of Leu deletion on mTORC1 activity is cell specific;(2)the Ser synthesis-ISR axis mediates the differential regulation of mTORC1 signal in MAC-T and293 T cells by Leu deletion;(3)the changes of cellular metabolic structure play an important role in the regulation of mTORC1 signal activity by Leu.