Effect And Mechanism Of Interaction Of Acetate And Leucine, Acetate And Methionine On Milk Components Synthesis In The Bovine Mammary Epithelial Cells

There is cooperation and competition among milk components precursor(MCP)used for synthesis of milk components in mammary gland.A balance model for maximum synergy maybe exists between MCP.Therefore,it is theoretical and practical significance to study the effect of interaction of milk fat precursor(MFP)and milk protein precursor(MPP)on milk fat,milk protein and lactose.The effect and mechanism of Met and Leu on milk components synthesis in bovine mammary epithelial cells(BMECs)was detected accroding to the gene and protein expression and phosphorylation-level.Based on this,we discussed the effect and mechanism of the interactions between acetic acid and Leu or Met on milk components synthesis,and elucidated their cooperation and competition relationship in regulating milk components synthesis.The mechanism of the interaction between acetic acid and Leu on regulating in milk components synthesis was explained by inhibiting m TOR signaling pathway.It provided theoretical basis and technology for establishing MCP balance model and regulating milk quality by nutritional methods in realistic production.This study contained five experiments.The completed random of single factor was introduced in experiment 1 and 2 to study the effect of Leu(0.45,0.9,1.8,2.7,3.6 and 7.2m M)or Met(0.13,0.26,0.39,0.52,0.65 and 0.78 m M)on the gene and protein expression of genes involved in milk component synthesis.The results showed that Leu improved the gene expression of FASN,ACACA,SREBP1 and protein expression of SREBP1 in dose-dependent,and a better effect was found in 1.8 to 2.7 m M Leu.Leu down-regulated the expression of FABP3,LPL,AGPAT6,GPAM and BTNIA1,and lower expression was detected in 3.6 to 7.2 m M Leu.Leu had positive effect on the gene exoression of milk protein synthesis,and phosphorylation of 4EBP1 and m TOR,showing better results at concentrations of 0.9 to 1.8m M Leu and lower expression at 3.6 to 7.2m M.AMPK had a lower phosphorylation at 0.9 to 7.2 m M Leu.Leu up-regulated the gene expressions of β-4GALT1 and LALBA in dose-dependent,Leu at concentrations of 0.9 to 1.8m M had a better effect,and lower expression at 7.2 m M.It was conclude that at the concentration of 1.8mmol/L,Leu had best effects on the synthesis of milk fat,milk protein and lactose.Met up-regulated the gene expressions of LPL,FASN,ACACA,SREBP1 and PPARG,and protein expression of SREBP1 and PPARG in dose-dependent;and with better outcomes at 0.26 to 0.52 m M,and low up-regulation or suppression at 0.65 to 0.78 m M.Met up-regulated expression of CSN1S1 and CSN3 in dose-dependent,and had better expression at 0.52 m M and down-regulation at 0.78 m M.At concentrations of 0.39 to 0.52 m M,Met had positive effects on expressions of JACK2/STAT5,m TOR pathway,phosphorylation level of S6K1 and m TOR,and ATP,inhibited phosphorylation level of AMPK.Met restrained gene expressions of GLUT1 and LALBA in dose-dependent,with lower levels at 0.26 to 0.52 mmol/L and high lactose content.Met exhibited optimum results of promoting syntheses of milk fat,milk protein,and lactose at 0.52 m M.The experiment 3 included two parts to discuss whether Leu affected the acetic acid regulating milk components synthesis.The first part was a 2 × 6 factorial arrangement with treatments: Leu concentration(0.45 and 1.8 m M)and acetic acid concentration(0,4,6,8,10 and 12 m M)to discuss whether the effect and mechanism of acetic acid on milk components synthesis was Leu concentration.The second part was a 2 × 6 factorial arrangement with treatments: acetic acid concentration(0 and 6 m M)and Leu concentration(0.45,0.9,1.8,2.7,3.6 and 7.2 m M)to discuss whether the regulation of Leu on milk components synthesis was related with acetic acid concentration..The results showed that acetic acid had a positive effect on TAG,gene expression of milk fat sysnthesis,and protein expression of PPARG and SREBP1;as well as gene expression of milk protein and lactose sysnthesis and phosphorylation of m TOR pathway.Acetic acid at 8 to 10 m M had a better effete on these factors.The effect of Leu on milk protein and lactose synthesis was similar with the results of experiment 1,but Leu increased TAG synthesis and expression of AGPAT6,GPAM,LPIN and BTNIA1.Interaction of acetic acid and Leu had a significant effect on milk components synthesis.Leu at 1.8m M had a positive effect on acetic acid up-regulated TAG,expression of genes involved in milk fat synthesis,as well as protein expression of SREBP1 and PPARG.But Leu had a negative effect on acetic acid up-regulated gene expression of FABP3 and LPL.Acetic acid had a positive effect on Leu up-regulated TAG,gene expression of FASN,ACACA,SCD1,AGPAT6,SREBP1 and PPARG,as well as protein expression of SREBP1,and inhibited Leu down-regulating gene expression of FABP3 and LPL.1.8m M Leu had a positive effect on acetic acid up-regulated expression of genes involved in milk protein synthesis,phosphorylation of e IF4 E,S6K1and m TOR,ATP content,as well as down-regulated AMPK.Acetic acid had a positive effect on Leu up-regulatedthe expression of genes involved in milk protein synthesis,phosphorylation m TOR signaling pathway,ATP content,as well as down-regulated phosphorylation of AMPK.Leu at 1.8m M had a positive effect on acetic acid up-regulated gene expression of HKI,HKII and β-4GALT1.Acetic acid had a positive effect on Leu up-regulated lactose synthesis GLUT1,HKII and β-4GALT1.In concluded,the combination of 1.8 m M Leu and 8 to10 m M acetic acid had a better effect on milk fat,milk protein and lactose synthesis.The experiment 4 included two parts to study the effect and mechanism of the interaction of acetic acid and Met on milk fat,milk protein and lactose synthesis.The first part was arranged in a 2(Met concentration;0.13 m M and 0.52 m M)× 6(acetic acid concentration;0,4,6,8,10 and 12 m M)factorial design to discuss whether Met affected the acetic acid regulating milk components synthesis.The second part was arranged in a 2(acetic acid concentration;0 and 6 m M)× 6(Met concentration;0.13,0.26,0.39,0.52,0.65,and 0.78 m M)factorial design to discuss whether acetic acid affected Met regulating milk components synthesis.The results indicated that the effect of acetic acid on milk components was similar with experiment 3,but down-regulated CSN1S1 and 6 m M acetic acid had a better effete on above factors.The effect of Met on milk fat,protein and lactose synthesis was similar with the results of experiment 2.Interaction of acetic acid and Met had a significant effect on milk fat,milk protein and lactose synthesis.Met at 0.52 m M had a positive affect on acetic acid up-regulated TAG,gene expression of FASN,ACACA,AGPAT6,GPAM,as well as protein expression of SREBP1.Acetic acid had a positive effect on Met up-regulated gene expression of LPL,ACACA,SREBP1 and PPARG.Met at 0.52 m M had a positive effect on acetic acid up-regulated the expression of genes related to milk protein synthesis,phosphorylation of m TOR signaling pathway.Acetic acid had a minimum effect on Met effect milk protein synthesis.0.52 m M Met had a negative effect on acetic acid up-regulated gene expression of HKI and HKII.Acetic acid had a positive effect on Met up-regulated lactose synthesis,HKI and β-4GALT1.In concluded,the combination of 0.52 m M Met and 6 m M acetic acid had a better effect on milk fat,milk protein and lactose synthesis.The experiment 5 was conducted based on the results of experiment 3 to inhibit m TOR signaling pathway.The optimal combinations of acetic acid and Leu was selected based on the results of improving milk fat,milk protein and lactose synthesis in experiment 3.This experiment was arranged in a 2(acetic acid concentration;0 vs.8 m M)×2(Leu concentration;0.45 vs.1.8m M)×2(rapamycin concentration;0 vs.100 n M)factorial design.We detected the transcript and translation level of genes involved in milk components synthesis after treating with rapamycin to discuss whether the interaction of acetic acid and Leu regulated milk components synthesis through m TOR signaling pathway.The results indicated that m TOR signaling pathway was blocked by rapamycin decreased the expression of genes involved in milk fat synthesis and protein expression of PPARG and SREBP1,as well as TAG content.The expression of CSN1S1,CSN2,CSN3,gene expression and phosphorylation of m TOR signaling pathway was significantly decreased when rapamycin blocked m TOR signaling pathway.Rapamycin inhibited m TOR signaling pathway resulted in lower lactose content and expression of the related genes.It indicated that m TOR signaling pathway was involved in milk fat,milk protein and lactose synthesis.In summary,acetic acid,Leu and the interaction regulated milk fat,milk protein and lactose synthesis was related with gene and protein expression of milk components synthesis and the phosphorylation of m TOR signaling pathway.The m TOR signaling pathway regulated the milk fat,milk protein and lactose synthesis.The cooperation and competition existed between acetic acid and Leu or Met in milk fat,milk protein and lactose synthesis in BMECs.