Molecular Mechanism Of SNAT2 Mediated Amino Acid Regulation On Milk Synthesis In Bovine Mammary Epithelial Cells

Amino acids are the most important organic small molecules in life.It has been found that amino acids can be used as raw materials for milk protein synthesis to promote the synthesis of milk proteins in cells,not only that,amino acids can also act as signaling molecules to activate important regulatory enzymes in the cell,such as phosphatidylinositol 3-kinas?PI3K?,the mammalian target of rapamycin?mTOR?,regulating the synthesis of lactoprotein and milk fat in cells^[1,2,3,4].In recent years,it has been reported that the regulation of cellular physiological function by amino acids as signaling molecules may be mediated by amino acid transporters,but its molecular mechanism is not clear and thorough.And the regulation of amino acid between milk fat and amino acid transporter is much less understood.Amino acid transporter is a lipid soluble carrier protein located on the plasma membrane.It can actively absorb and transport the free amino acids outside the cells into the cells or transfer the amino acids from the cytoplasm to the cells^[5,6,7].The purpose of this study is to explore the function of the sodium-dependent neuteral amino acid transporter 2?SNAT2/SLC38A2?in the synthesis of milk protein and milk fat in bovine mammary epithelial cells?BEMCs?cultured in vitro,and the molecular mechanism that affects the modulating of amino acids on milk synthesis.This study will provide experimental evidence for further study of lactation regulation and metabolic synthesis in dairy cows.The use of cow mammary tissue cultured cells were obtained and cultured BEMCs,the expression of cytokeratin 18?CK18?in BEMCs was detected by immunofluorescence staining to identify cell types and purity.,the?-casein was detected by Western blotting to verify the cell type and lactation ability.;the synthesis ability and secretion ability of the milk protein and milk fat were expressed in the content of the triglyceride in the serum of the cells and?-casein,and the lactation model of BEMCs was established.;addition of amino acid methionine,lysine and leucine?Met,Lys,Leu?with a final concentration of 0.6 mmol/L were treated with BMECs for 24 hours,by real-time quantitative fluorescence PCR?qRT-PCR?,Western blotting technique,and triglyceride kit,the expression quantity of the?-casein gene and SNAT2 and the secretion of triglyceride in BEMC culture medium were detected.The N-flag-SNAT2 eukaryotic expression vector and SNAT2 siRNA were transferred into cells respectively,and the SNAT2 gene overexpression and knockdown experiment were carried out.By Western blotting and triglyceride kits,the protein expression quantity of SNAT2,mTOR/p-mTOR,Akt/p-Akt,?-casein and the content of triglyceride in BEMCs culture were detected respectively..After 24 hours of treatment of BEMCs with different concentrations of Met,the total protein was collected,and using Western blotting,the best effect concentration of Met on SNAT2 was selected.To transferr the SNAT2si-RNA into the BEMCs treated with Met,and the negative control?NC?was Met+negative RNA.The total protein of cells was collected 24 hours later,and the contents of SNAT2,mTOR/p-mTOR and Akt/p-Akt in the protein samples were detected by Western blotting.To transferr N-flag-SNAT2 true nuclear expression vector into the BEMCs treated with Wortmannin of 0.4mmol/L,and the negative control?NC?was empty gsensor and Wortmannin+empty gsensor.the total protein of cells was collected 24 hours later,and the contents of SNAT2,mTOR/p-mTOR and Akt/p-Aktthe in protein samples were detected by Western blotting.The experimental results show that the obtained BEMCs was tested by immunofluorescence and CK18 in it was positive,.and the?-caein was detected by western blotting;it is found that the 3kinds of amino acids?Met,Lys,Leu?were significantly increased?P<0.05?BMEC secretion of milk protein and milk fat,and activated mTOR signaling pathway,Met and Lys can aslo significantly up-regulate the SNAT2 gene expression.;SNAT2 can positively regulate synthesis of milk protein and milk fat in BMEC,and activated PI3K-Akt and mTOR signaling pathway;after knockdown of SNAT2,the function of methionine in promoting the synthesis of BEMC milk protein were reduced significantly?p<0.05?and the mTOR and PI3K-Akt signaling pathway were inhibited;Compared with the control group,overexpression of SNAT2,adding Wortmannin or blocking the PI3K-Akt pathway would inhibit the mTOR and PI3K-Akt pathway,and make the overexpression effect of SNAT2 disappeared.These results suggest that the activity of mTOR pathway and the expression of SNAT2 are positively regulated by PI3K-Akt pathway.To sum up,we can draw the following conclusions:SNAT2 up-regulates the synthesis of milk protein and milk fat in the BEMCs;SNAT2 regulates the milk synthesis through the PI3K-mTOR pathway;SNAT2 can mediate the amino acids activate the PI3K-mTOR signal pathway.This study provides a theoretical and experimental basis for revealing the mechanism of SNAT2 regulating milk synthesis and amino acid regulation of milk synthesis signaling pathway.