Regulation Mechanism Of Acetate Involved In Milk Fat And Milk Protein Synthesis In The Bovine Mammary Epithelial Cells

This study was conducted to determine the effects of acetate on the synthesis of milk fat and protein and the transcription and translation levels of related genes in bovine mammary epithelial cells (BMECs). Basing on this, we discussed the mechanism and regulatory intersection of acetate involved in milk fat and protein synthesis through supplementing rapamycin and AG490 to inhibit mTOR and JAK2/STAT5 pathway, and to knockdown eIF4E through siRNA method, and to provide theory basis and technology for regulating the milk fat and protein synthesis. This study included four experiments. The purpose of experiment 1 was to examine the effects of different concentrations of acetic acid (0,4,6,8,10 and 12 mmol/L), on cell viability and triacylglycerol (TAG) content, mRNA expression and protein levels of genes relative to milk fat and protein synthesis in BMECs, in order to select the proper concentration of acetate for stimulating the synthesis of milk fat and protein and discuss the regulation mechanism of acetate on milk fat and protein synthesis in the BMECs preliminarily. The experiment 2 was designed on the basis of the experiment 2, adding 6 mmol/L acetate in the BMECs when the mTOR signaling pathway was inhibited with rapamycin, then cell viability and triacylglycerol (TAG) content, mRNA expression and protein levels of genes related to milk fat and protein synthesis were detected to illustrate the regulation mechanism of acetate involved in milk fat and protein synthesis through mTOR pathway. Basing on the experiment 1, the experiment 3 was conducted to examine the cell viability and TAG content, mRNA expression and protein levels of genes related to milk fat and protein synthesis through inhibition of AG490 on JAK2/STAT5 pathway, then the regulation mechanism of acetate involved in milk fat and protein synthesis through JAK2/STAT5 signaling pathway was discussed. The experiment 4 detected cell viability and TAG content, mRNA expression and protein levels of genes related to milk fat and protein synthesis when the gene eIF4E was knocked down, and indicated if eIF4E was the regulatory intersection in the mTOR pathway when acetate involved in milk fat and protein synthesis in the BMECs.The results of this study were showed as follows:① Acetate upregulated TAG synthesis in the BMECs, all concentrations of acetate had significantly stimulation effect on mRNA expression of FASN, ACACA, FABP.3, SREBP1 and PPARG, and 8-12 mmol/L acetate had the better effect; CD36 mRNA abundance was dramatically decreased with the increase of acetate concentration; the protein level of PPARG was increased when supplementing all concentrations of acetate. The mRNA expression of CSN1S1 was significantly down-regulated when the BMECs was treated with acetate, and the value was lower at 8-12 mmol/L. The concentrations of acetate were 6 and 8 mmol/L had stimulatory effect on mRNA expression and protein expression of P-casein, as well as CSN3mRNA expression. The expression of mTOR and genes involved in this pathway was increased with 6 and 8 mmol/L acetate added to the the BMECs. In a word, the synthesis of milk fat and protein in BMECs had better effect when the concentration of acetate was 6 mmol/L.② Cell proliferation was depressed when the mTOR signaling pathway was blocked with rapamycin, the inhibitory effect was more obvious when higher concentration rapamycin was supplemented in the BMECs. The proper concentration of rapamycin was 100nmol/L.③ mTOR pathway was blocked with rapamycin dramatically decreased FASN, ACACA, SCD1, FABP3, SREBP1a nd PPARG mRNA expression and protein expression of PPARG in the BMECs, but TAG content was not influenced. The mRNA expression of CSN1S1, β-casein, CSN3, mTOR, eIF4E,4EBP1and S6K1 was significantly downregulated when the BMECs were treated with rapamycin, and the protein level of P-casein was also decreased. mTOR signaling pathway regulated genes related to milk fat synthesis through the regulation factors SREBP1and PPARG.④ Acetate involved in both milk fat and protein synthesis through mTOR signaling pathway, which is the jointly pathway in controlling milk fat and protein synthesis.⑤ JAK2/STAT5 signaling pathway blocked with AG490 significantly decreased cell proliferation, and the inhibitory effect was more obvious when higher concentration of AG490 was supplemented in the BMECs. The proper concentration of AG490 was 40 umol/L.⑥ The mRNA expression of FASN, ACACA, SCD1, LPL and SREBP1was dramatically downregulated when the JAK2/STAT5 signaling pathway was blocked with 40umol/L AG490, but TAG content and protein expression of PPARG was not influenced. Rapamycin treated significantly decreased mRNA expression of CSN1S1,βP-casein, JAK2 and STAT5, as well as was protein level of β-casein in the BMECs. JAK2/STAT5 signaling pathway regulated genes related to milk fat synthesis through the regulation factors SREBP1.⑦ Acetate involved in both milk fat and protein synthesis through JAK2/STAT5 signaling pathway, which maybe the jointly pathway in controlling milk fat and protein synthesis.⑧ Proliferation of BMECs was inhibited after eIf4E silencing. The mRNA expression of FASN, ACACA, SCD1 and SREBP1 was significantly downregulated after eIf4E inhibition. However, transcription and translation level of PPARG was not affected, as well as eIf4E silencing had no effect on TAG content. eIf4E silencing dramatically decreased CSN1S1, β-casein, CSN3, eIf4E and 4EBP1 mRNA expression, as well as protein level of β-casein.⑨ eIf4E linked milk fat and protein synthesis in the BMECs, and eIf4E as key regulation factor is an intersection in regulating milk fat and protein synthesis. The regulation of acetate on milk fat and protein synthesis through mTOR signaling pathway was closely related to gene eIf4E.Taking all these results into consideration, acetate had significant effect on both milk fat and protein synthesis in the BMECs. mTOR and JAK2/STAT5 signaling pathway regulated milk fat synthesis as well as milk protein synthesis, and it was possible that eIf4E was the intersection of acetate involved in milk fat and protein synthesis in the BMECs.