Regulation Of Methionine On Expression Of LAT1 In Dairy Mammary Epithelial Cells

Milk is an important nutrition food.With the increasing population of the world,the demand for milk is increasingly.Casein includes 8 essential amino acids,which has higher nutritional value.Therefore,improving milk quality and the proportion of milk protein is one of the important tasks in the dairy industry.Milk proteins are expressed in the mammary tissues during lactation.90% of milk proteins are synthesized de novo in mammary epithelial cells.Amino acids are raw materials for de novo protein synthesis.The uptake of amino acids by cells requires specific amino acid transporters.Therefore,the expression and activity of amino acid transporters may limit the supply of nutrients and thus influence the synthesis of milk proteins.L-type amino acid transporter 1(LAT1)is an amino acid transporter that plays a major role in the transport of neutral amino acids.It is thought to be an important transporter for the transport of essential amino acids in the mammary gland of dairy cow.LAT1 binds with 4F2 heavy chain(4F2hc)to form a heterodimeric complex and exerts its physiological functions.Methionine is an essential amino acid that can be transported into cells by LAT1.Many studies have shown that methionine has a promoting effect on the synthesis of milk proteins.In dairy cows,LAT1 expression regulates the synthesis of milk proteins.Whether methionine regulates LAT1 expression,thus affecting mik protein synthesis is not clear.The molecular mechanism underlying this has not been clarified.In this study,dairy cow mammary epithelial cells were used as the experimental model.The effects of different concentrations of methionine on the expression of LAT1,4F2 hc and β-casein was examined by qRT-PCR and western blot.The results showed that the expression of LAT1,4F2 hc,and β-casein in mammary epithelial cells increased as the methionine concentration increased.LAT1,4F2 hc and β-casein were expressed highest at the concentration of 0.6 mM methionine.The effect of methionine on the viability of mammary epithelial cells was examined by MTT assay.The results showed that when the methionine concentration reached 0.6 mM,the cell viability was highest.These results indicate that 0.6 mM methionine has the best promoting effect on the expression of LAT1,4F2 hc and β-casein in cultured bovine mammary epithelial cells.Amino acids can regulate the rate of protein synthesis by causing the mediation and integration of cell signaling cascades.The regulation of amino acid on protein synthesis is mostly mediated by the mammalian target of rapamycin(mTOR)signaling pathway.mTOR is an evolutionarily conserved serine/threonine protein kinase that exists in two complexes,the mammalian target of rapamycin complex 1(mTORC1)and the mammalian target of rapamycin complex 2(mTORC2).mTOR binds to protein raptor in mTORC1 and to protein rictor in mTORC2.A variety of amino acids can activate the mTOR signaling pathway to regulate protein synthesis and body growth and metabolism.To determine whether methionine can regulate the expression of LAT1 in mammary epithelial cells and thus influence the synthesis of milk protein,we detected the effects of methionine on mTOR signaling molecules,LAT1,4F2 hc and β-casein expression by western blot analysis.When the mTORC1 signaling pathway was inhibited by rapamycin or RNA interference,the phosphorylation of mTORC1 signaling molecules were also detected.The results showed that methionine can activate mTORC1 and its downstream molecules p70 ribosomal protein S6 kinase(p70S6K)and Eukaryotic Translation Initiation Factor 4E-Binding Protein 1(4E-BP1).When mTORC1 is inhibited,the phosphorylation levels of p70S6 K and 4E-BP1 were decreased.The expression of LAT1,4F2 hc and β-casein was also reduced.These resultes indicated that methionine can regulate LAT1 expression through the mTORC1 pathway,thus affecting milk protein synthesis.However,the expression of LAT1 was not completely inhibited,indicating that there may be other pathways involved in the regulation of LAT1 expression.In this study,the mTORC2 signaling pathway was inhibited by RNA interference.The results showed that the phosphorylation of AKT was decreased,and the expression levels of LAT1,4F2 hc and β-casein were also significantly reduced.These results revealed that mTORC2 is also involved in the regulation of LAT1 expression by methionine,thus affecting milk protein synthesis.The findings enrich the basic theory of lactation biology and provide experimental support for the adjustment of nutritional components of dairy cows through diets or other ways to improve milk quality.