| Balanced dietary protein and amino acids not only benefit pig growth,development and reproduction,but also improve feed efficiency and reduce environmental pollution.Amino acids may affect protein synthesis by activating mTORC1 signaling.Amino acids can not free to pass through the cell membrane and require the assistance of amino acid transporter.Lysosome is recognized as a key intracellular organelle for mTORC1 activation by amino acids,and SLC38A9 and SLC36A1 on the lysosomal membrane are amino acid transceptors whose primary function is to sense amino acids and initiate mTORC1 signaling pathway through the amino acid sensing complex,rather than to mediate bulk amino acid transport.To determine the effect of reduced dietary protein levels on the expression of amino acid transporters and mTORC1 signaling pathway related genes,and elucidated the mechanism of SLC38A9 and SLC36A1 sensing amino acids and regulating mTORC1 signaling pathway.The main results are as follows:1.The effects of reduced dietary protein levels on amino acid transporters and mTORC1 signaling pathway were measured by QPCR chip.The results indicated that there was no significant difference in the weight of longissimus dorsi and the phosphorylation levels of mTOR and S6K1 between 20% crude protein(CP)diet(normal recommended)and 17% CP diet(3% less than the recommended level)group after 25-day and 45-day feeding.mRNA expression of amino acid transporters SLC38A2,SLC1A7,SLC7A1,SLC7A5,SLC16A10 and SLC3A2 in the 14% CP diet group were significantly(P < 0.05)higher than those in the 17% CP diet or 20% CP diet group,and the weight of longissimus dorsi and the phosphorylation of mTOR and S6K1 decreased in the 14% CP diet group after 25-day feeding.Furthermore,the vitro experimental results further confirmed that the mRNA levels for SLC7A1,SLC7A5,SLC3A2,SLC38A2 and SLC36A1 were increased and the phosphorylation of mTOR and S6K1 was decreased when the concentration of amino acids in C2C12 myoblasts was reduced.All these results indicated that properly reduced protein(3% less than recommended level)diet did not affect the mRNA expression level of amino acid transceptors and longissimus dorsi growth,but 14% CP diet diet and amino acid deficiency induced the high expression of amino acid transporters and the inhibition of the mTORC1 activity,which resulting in restriction on protein synthesis and longissimus dorsi growth.2.Overexpression vectors of SLC38A9 or SLC36A1 were constructed with mouse SLC38A9 or SLC36A1 cDNA cloned into pcDNA3.1,and individual siRNAs were transfected to knockdown SLC38A9 or SLC36A1.The binding of SLC38A9 and SLC36A1 and the interaction of them in expression level and lysosomal membrane localization,and the effects of SLC38A9 and SLC36A1 on lysosomal membrane localization and phosphorylation of mTOR were analysed by QPCR,western blot,immunofluorescence and co-immunoprecipitation.All these results indicated that under normal state,SLC38A9 and SLC36A1 were associated and required to release inactivated mTOR from lysosomal surface to the cytoplasm,and upregulated the activity of mTORC1.In the absence of amino acids,the expressions of SLC38A9 and SLC36A1 were increased,with SLC38A9 losing the binding with SLC36A1,resulting in the dispersion of mTOR from lysosome throughout the cytoplasm and the inhibition of the activity of mTORC1.In the presence of leucine,the expressions of SLC38A9 and SLC36A1 were also enhanced but SLC38A9 was tightly associated with SLC36A1,leading to the recruitment of mTOR from cytoplasm to lysosome and the enhancement of the activity of mTORC1.Additionally,the interaction proteins of SLC38A9 were identified by HA-immunoprecipitations and LC-MS/MS.The interacting proteins of SLC38A9 were analyzed by KEGG pathway to participate in most enriched pathways which were associated with amino acid sensing and protein synthesis,such as mTOR signaling pathway,ribosome,protein processing in endoplasmic reticulum,glutathione metabolism,and AMPK signaling pathway.3.Two AARE of SLC38A9 binding to ATF4 was identified by ChIP-PCR and the AARE at the first intron of SLC38A9 was in the core promoter region of SLC38A9.Overexpression vector of ATF4 was constructed and individual siRNAs were transfected to knockdown ATF4.ATF4 regulated the mRNA level of SLC38A9 measured by QPCR.It indicated that ATF4 might regulate the expression level of SLC38A9 by binding to the AARE of SLC38A9.The effect of amino acid deficiency or leucine on the mRNA levels of ATF4 and SLC38A9 were detected by QPCR.The results indicated that mRNA level of ATF4 was decreased and mRNA level of SLC38A9 was increased in the absence of amino acids,and mRNA levels of ATF4 and SLC38A9 were enhanced by the addition of leucine after amino acid starvation.Overall,amino acid starvation decreased the activity of mTORC1 which resulting in restriction on expression of ATF4 and protein synthesis,but induced the binding of ATF4 to AARE on SLC38A9,which in turn increased SLC38A9 expression.In the presence of leucine,mTORC1 signaling pathway and protein synthesis were activated,ATF4 was activated by mTORC1 and could promote the expression of amino acid transporters,and the expression of SLC38A9 was increased which resulting in activity of mTORC1 signaling pathway. |
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