Effect Of Interaction Of Lysine And Acetate On Expression Of Genes Involved In Milk Protein,Fat And Lactose Synthesis In The Bovine Mammary Epithelial Cells

The objective of this study was to determine the effect and mechanism of Lys on expression of genes involved in milk fat,milk protein and lactose synthesis in bovine mammary epithelial cells(BMECs).Based on this,we discussed the effect and mechanism of the interactions between acetic acid and Lys on milk component synthesis,and elucidated their cooperation and competition relationship in regulating milk component synthesis,and made a preliminary exploration of its mechanism in transcript and translation level.It provided a theoretical basis for regulating milk quality by milk components precursor.The research included three experiments:Experiment 1 was conducted to study the effect of Lys(0.5(basal medium,control),1.0,2.0,4.0,8.0 and 16.0 mM)on expression of genes involved in milk protein,milk fat and lactose synthesis in BMECs.The completed random of single factor was introduced in this experiment.The results showed that the concentration of Lys significantly or tend to significantly improved the content of ATP,cell proliferation,and gene's expression of CSNIS1,C.SN3,mTOR,eIF4E,AMPKal and phosphorylation of mTOR,S6K1 and decreased AMPK phosphorylation in dose-dependent;those parameters showed better results at concentrations of 1.0 to 2.0 mM Lys,but lower expression of CSN1S1,CSN3,STAT5 and mTOR signal pathway at 16.0 mM Lys,lower gene expression of 4EBP1 from 1.0 to 16.0 mM Lys.The results showed that Lys improved significantly or tend to significantly improved the content of TAG and genes expression of FABP3,LPL,FASN,AGPAT6 and GPAM in dose-dependent;and those parameters showed better results at concentrations of 2.0 to 4.0 mM Lys,but the high concentration of Lys down-regulated genes and protein expression of SREBP1,and had lower genes expression of PPARG,FASN,SCD1,AGPAT6 and GPAM.The results also showed that Lys improved the content of lactose and genes expression of GLUT1,HK1 and HKII in positive dose-dependent;and the better results of the synthesis of lactose was detected at concentrations of 2.0 to 8.0 mM Lys,but lower gene expression of HKII at concentrations of 1.0 to 2.0 mM and down-regulation phosphorylation of glucose and the synthesis of lactose at concentrations of 16.0 mM.The optimal Lys concentrations for milk fat,milk protein and lactose synthesis was 2.0 mM.Experiment 2 was to discuss whether the effect of acetic acid on expression of genes involved in milk protein.milk fat and lactose synthesis was related with Lys concentration in BMECs.It was a 2 × 6 factorial arrangement with treatments:Lys concentration(0.5 and 2.0 mM)and acetic acid concentration(0.4.0,6.0,8.0,10.0 and 12.0 mM)to discuss the interaction effect between Lys and acetic acid on milk fat,milk protein and lactose and its suitable combination.The results showed that acetic acid improved genes and protein expression and phosphorylation of milk fat,milk protein and lactose,but high concentration of acetic acid could down-regulate this effect,and the better results was detected at concentrations of 6.0 mM.The effect of Lys on milk fat,milk protein and lactose synthesis was similar with the results of experiment 1.But Lys down-regulated the gene expression of HKI in this experiment.Lys at 2.0 mM had a positive effect on acetic acid up-regulated genes expression of STAT5,elF4E and phosphorylation of,TOR signal pathway,and relieved the acetic acid down-regulated gene expression of CSN1S1:Lys at 2.0 mM had a positive effect on acetic acid up-regulated expression of genes involved in uptake and transport for LCFA,lipid droplet,as well as genes and protein expression of PPARG and SREBP1.Lys at 2.0 mM had a positive effect on acetic acid up-regulated gene expression of ?-4GALT1,but had a negative effect on acetic acid up-regulated gene expression of HKI.Interaction of acetic acid and Lys had a significant effect on synthesis of milk fat,milk protein and lactose.In concluded,the combination of 2.0 mM Lys and 6.0 mM acetic acid had a better effect on milk fat,milk protein and lactose synthesis.Experiment 3 was to discuss whether the effect of Lys on expression of genes involved in milk protein,milk fat and lactose synthesis was related with acetic acid concentration.Experiment 3 was a 2×6 factorial arrangement with treatments:Acetic acid concentration(0 and 6.0 mM)and Lys concentration(0.5,1.0,2.0,4.0.8.0 and 16.0 mM)to deeply discuss the interaction effect of Lys and acetic acid on milk fat,milk protein and lactose synthesis and the suitable combination.The effect of acetic acid on milk fat,milk protein and lactose synthesis was similar with the results of experiment 2.The effect of Lys on milk fat,milk protein and lactose synthesis was similar with the results of experiment 1 and 2.Acetic acid at 6.0 mM had a positive effect on Lys up-regulated the content of ATP,cell proliferation,genes expression of CSN2,CSN3,JAK2,and mTOR signal pathway and its relevant genes expression and phosphorylation level,but acetic acid had a negative effect on low concentration of Lys up-regulated gene expression of CSNIS1.Acetic acid could relieve the down-regulated effect of high concentration of Lys on gene expression of CSN1S1.Acetic acid at 6.0 mM had a positive effect on Lys up-regulated expression of genes involved in milk fat synthesis,as well as genes and protein expression of PPARG and SREBP1 but acetic acid had a negative effect on Lys up-regulated genes expression of LPL and SCD1,acetic acid could relieve the down-regulated the expression of AGPAT6.Acetic acid had a positive effect on Lys up-regulated expression of genes involved in lactose synthesis and might relieve Lys down-regulated the expression of HKII.In conclusion,the combination of 6.0 mM acetic acid and 1.0 to 2.0 mM Lys had a better effect on milk fat,milk protein and lactose synthesis.In summary,the interaction effect of Lys and acetic acid significantly regulated expression of genes involved in milk protein,milk fat and lactose synthesis.The JAK2/STAT5 and mTOR signaling pathway might be involved in the milk fat,milk protein and lactose synthesis.There was cooperation and competition between Lys and acetic acid in milk fat,milk protein and lactose synthesis in BMECs,the combination of 2.0 mM Lys and 6.0 mM acetic acid had a better effect on milk fat,milk protein and lactose synthesis.