Effects Of Leucine On Casein Synthesis And Its Regulating Mechanism In Bovine Mammary Epithelial Cell

The study was to utilize bovine mammary epithelial cells as the model toinvestigate effects of leucine at different levels on proliferation and casein-relatedgene expressions in vitro. Moreover, the study established a cell-free model based oncrude lysosomes and cytosol proteins from an immortalized cow mammary epithelialcell line (CMEC-H) in order to provide a new approach to research into effects ofamino acids on milk protein synthesis. The study was composed of three parts:1. Effects of leucine on bovine mammary epithelial cells proliferation wereevaluated by MTT colorimetric assay. Supplementation levels of leucine were0.00mM/L (0group, control group),0.1125mM/L (0.25group),0.225mM/L (0.5group),0.45mM/L (1group),0.9mM/L (2group),1.8mM/L (4group),3.6mM/L (8group),7.2mM/L (16group),14.4mM/L (32group), respectively.Experiments were conducted with24h,48h and72h. All experiments were repeated3times. The results showed that0.45mM/L to3.6mM/L (1group~8group) leucineat48h significantly improved the proliferation of bovine mammary epithelial cells.2. Effects of leucine on casein-related gene expression regulating milk proteinsynthesis in bovine mammary epithelial cells were evaluated by RealTime-PCR(RT-PCR). The cells were subsequently treated with leucine (0.00,0.25,0.5,1,2,4,8,16,32mM/L). After4hours of amino acid starvation, all cells were treated with48hours. All experiments and every treatment were repeated3times.1) Relativeexpression of CSN1S1, CSN1S2, CSN2and CSN3genes were all significantlyincreased （P<0.05）in all experimental groups, and the upper-regulated expressionlevels reached the highest when leucine concentration was at0.9mM/L (2group).2)Relative expression of mTOR, S6K1, JAK2and STAT5genes were all significantly increased （P<0.05）in all experimental groups, and relative expression of4E-BP1was significantly decreased （P<0.05） in all groups. The upper-regulated anddown-regulated expression levels all reached the highest when leucine concentrationwas also at0.9mM/L (2group).3. We established a cell-free model duplicating amino acid regulating milkprotein synthesis, which is based on an immortalized cow mammary epithelial cellline (CMEC-H). This model is mainly composed of crude lysosomes and cytosolproteins from CMEC-H. The results showed that this cell-free model can effectivelyduplicate main aspects of amino acids signaling mTOR program in vitro. The additionof1leucine solely and1essential amino acids led mTOR to be activated,phosphorylated and then moved towards the crude lysosome fractions in the cell-freemodel. However, combined with1leucine and1essential amino acids,20nmrapamycin was not able to completely prevent mTOR from being phosphorylated.Besides, by trypan blue dye exclusion assay, we found passing through gauge-26needle25times was a good cell lysis method of CMEC-H.In conclusion, when leucine concentration was at0.9mM/L (2group), cellproliferation, relative expression of four casein-encoding genes and genes related tomilk protein synthesis (except4E-BP1) reached the highest (significantly increased).The cell-free model constructed can effectively duplicate main features of amino acidssignaling mTOR program in vitro.