Effects Of Increasing The Supplementary Of Met And Branched Amino Acids On Regulation Of Casein Synthesis Pathway In Bovine Mammary Epithelial Cells

Milk protein,one of the compositions in milk,play an important role to human nutrition with the ideal amino acid(AA)profile and concentration.In practice,it has been demonstrated that the addition of rumen-protected Methionine in diet could optimize the amino acid absorption and promte the milk protein synthesis.Moreover,branched amino acids(BCAA)were demonstrated could potentially pomote milk protein synthesis.Furthermore,the bovine mammary gland epithelia cells(BMEC)were confirmed as the important location to milk protein synthesis.But it is still unclear that the mechanism of protein synthesis in BMEC.In sight of these aboved,the objective of this paper was to: 1)evaluate the effect of varying the ratio of Lys:Met(2.9:1)throught increasing methionine supplementary alters mammary cellular metabolites,mTOR signaling,and gene transcription with the technology of GC/MS,RNA sequence,qPCR and western blot;2)evaluate the effect of of increasing the BCAA supplemental(alters mammary cellular metabolites,mTOR signaling,and gene transcription with the technology of GC/MS,RNA sequence,qPCR and western blot.Exp.1 Effects of increasing methionine supplementary on the regulation pathway of casein synthesis in bovine mammary epithelial cellThe bovine mammary gland epithelial cell line,MAC-T cell,was used to cell culture through increasing methionine supplementary to alters the ratio of Lys to Met equal to 2.5:1 and 2.0:1 base on the ratio of Lys:Met=2.9:1 which determined in milk.MAC-T cells were incubated(n = 6replicates/treatment)for 12 h with 3 incremental doses of Met to achieve the following: Lys:Met2.9:1(ideal AA ratio,CON),Lys:Met 2.5:1(LM2.5),and Lys:Met 2.0:1(LM2.0).The ratios of Thr:Phe(1.05:1),Lys:Thr(1.8:1),Lys:His(2.38:1)and Lys:Val(1.23:1)were the same across the 3 treatments.GC/MS,RNA sequence,qPCR and western blot were used to study the effects of increasing the Met supplementral on mammary cellular metabolite,gene transcription,mTOR pathway and the m RNA expression of CSN1S1 and CSN2.1)It was found that the intracellular degradation of Met,Lys,Arg,Phe and BCAA was promoted through the increasing of Met supplemental.Moreover,the utilization efficiency of EAA was decreased.The results of metabolism pathway analysis revealed that the activity of Aminoacyl-t RNA biosynthesis was decreased which could inhibit the activity of mTOR pathway.2)The increasing of Met supplemental increased the m RNA expression of SLC3A2,but decreased the m RNA expression of SLC7A5、SLC36A1、SLC38A2、SLC38A9 and SLC43A1.Compared with LM2.5,LM2.0 further increased the m RNA expression of SLC38A9 and SLC43A1.3)Base on the RNA-seq analysis,LM2.5 resulted the 113 different expression gene with 68 upregulation and 45 down-regulation compared with CON.LM2.0 resulted the 71 different expression gene with 38 up-regulation and 33 down-regulation compared with CON.The activity of p53 signaling pathway、HIF-1 signaling pathway、protein kinase binding、regulation of phosphorylation、positive regulation of peptidyl-serine phosphorylation和PI3K-Akt signaling pathway were significantly effected by increasing the Met supplemental.Of these,the p53 signaling pathway with negeative regulation of mTOR pathway and PI3K-Akt with position regulation of mTOR pathway were activated and inhibited respectively.The the expression of RHEB,S6K1 and EIF4 E of mTOR pathway positive regulator was increased with the Met addition.The activity of mTOR pathway was decreased by increasing the Met supplementary.The phosphorylation ratio of Akt and mTOR was increased but decreased respectively,which reveled that the increasing of Met inhibited the mTOR pathway activity on phosphorylation level.Increasing the Met supplementary inhibited the gene expression of β-casein.The activity of regulation pathway of casein synthesis was inhibited with the increasing the Met supplementary base on the ratio of Lys to Met equal to 2.9 in milk.Exp.2 Effects of increasing BCAA supplementary on the regulation pathway of casein synthesis in bovine mammary epithelial cellBase on the results of experiment 1,the bovine mammary gland epithelial cell line,MAC-T cell,was used to cell culture through increasing BCAA supplementary to alters the ratio of Lys to Ile,Val and Leu equal to 1.29:1,1.12:1 and 0.78:1respectively base on the ratio of Lys to Ile,Val and Leu equal to 1.45:1,1.23:1 and 0.85:1 respectively,which base on the control group in experiment.1)Metabolomics via GC-MS revealed that, compared with CON,LV1.12 resulted the greater EAA concentration of His,Met,Ile and Leu and NEAA concentration of Gln,Glu,Ser,Gly and Asp and the cocnetration of uric,N-Acetylglutamic acid and fructose,but low concentration of glucose-6-p.LI1.29 resulted the greater intracellular concentration of AA(Pro,Leu,Asp,Glu and Gln)and uric acid,N-Acetylglutamic acid and glycolic acid,but decresed the concentration of pyruvic acid,phosphoric acid,glutaric acid,3-p-glycerol.LL0.78 resulted the greater concentration of phosphoric acid,uric acid and succinic acid,but decreasted the glucose-6-p,pyruvic acid and fumaric acid.The pathway of Ala-Asp-Glu,Cys-Met,Glu-Gln,Gly-Ser-Thr and Arg-Pro was signigicantly regulated by the increasing of BCAA supplementary.2)LL0.78 significantly increased the m RNA expression of SLC3A2,SLC7A5,SLC36A1 SLC38A2 and SLC38A9.LV1.12 significantly increased the m RNA expression of SLC3A2 and SLC38A9.LI1.29 significantly increased the m RNA expression of SLC3A2,but decreased the m RNA expression of SLC7A5,SLC38A2 and SLC38A9.3)The results of RNA-seq analysis shown that LV1.12 resulte the 951 different expression gene with 570 up-regulation and 381 down-regulation compared with CON.LL0.78 resulted the 157 different expression gene with 104 up-regulation and 53 down-regulation compared with CON.The increasing of Val promoted the activity of mTOR pathway,but the increasing of Leu inhibited the activity of mTOR pathway.4)On the phosphorylation level,Compared with other treatments,LV1.12 led to greater phosphorylation status of Akt、mTOR、S6K1 and RPS but decreased the EIF2α,which could activate the mTOR pathway.LI1.29 significantly increased the phosphorylation of mTOR,4EBP1 and RPS6 but decreased the phosphorylation of EIF2αand EEF2,which could activate the mTOR pathway.The mTOR pathway activity was decreased by the LL0.78 with the lower phosphorylation of mTOR.The m RNA expression of CSN1S1 was increased by the increasing supplementary of BCAA.LV1.12 increased the m RNA expression of CSN2 but the LI1.29 decreased.The increasing of Val resulted the greater activity of casein synthesis regulation.In coclusion,Met,Ile,Val and Leu are with different regulation of casein synthesis signaling pathway in bovine mammary gland epithelial cells.The increasing supplementary of Met decreased the pathway activity of casein synthesis through down-regulating the activity of mTOR pathway base on the regulation of protein phosphorylation.The difference of pathway activity of casein synthesis was existed with the increasing supplementary of Ile,Val and Leu.The increasing addition of Val enhanced the mTOR pathway activity by the regulation of phosphorylation.Val paly an important role to regulate the pathway activity of casein synthesis compared with Ile and Leu.