Regulation Role Of Essential Amino Acid In Synthesis Of The Protein And Fat In Mammary Cell Of Dairy Cow

The objectives of this study were to evaluate the regulation effects of essential amino acid on milk protein and fat synthesis and especially, to explore the role of mTOR in milk protein and fat synthesis pathway. This study contained two parts. In the first part, the effect of essential amino acid ratio on milk protein synthesis and how essential amino acid regulate the milk protein synthesis was investigated, hi the second part, the effect of essential amino acid ratio on milk fat synthesis and how essential amino acid regulate the milk fat synthesis was elucidated. A better understanding of the regulation role of essential amino acid on milk protein and fat synthesis may further lead to beneficial strategies to improve the milk quantity.1. Effect of essential amino acid ratio on milk protein synthesis in MAC-T cells and bovine mammary tissue explants1.1 Effect of essential amino acid ratio on milk protein synthesis in MAC-T cells and bovine mammary tissue explantsAmino acid (AA) not only is building blocks of proteins but also is one of the key molecules that serve as upstream components of the signaling pathways that regulate protein synthesis.In recent years it has become evident that the AA-mediated protein synthesis response within mammary cells is partly regulated through the mTOR pathway. Thus, the specific objective of this study was to investigate how changing the ratio of Lys to Thr, Lys to His, and Lys to Val affected mTOR pathway at transcription and translation level and to test the association of these signaling activities with milk protein synthesis. Target genes plus 3 internal controls were measured using qPCR. And the signaling proteins were tested using Western-blot. Triplicate cultures with the "optimal" AA ratio (OPAA; Lys:Met 2.9:1; Thr:Phe, 1.05:1; Lys:Thr,1.8:1; Lys:His,2.38:1; Lys:Val,1.23:1) plus the mTOR inhibitor rapamycin (OPAARMC, control), OPAA,2.1:1 Lys:Thr (LT2.1),1.3:1 Lys:Thr (LT1.3),3.05:1 Lys:His (LH3.0), and 1.62:1 Lys:Val (LV1.6) were incubated for 12 h. Compared with OPAARMC, the OPAA treatment upregulated SLC1A5, SLC7A5, and RPS6KBJ, and downregulate expression of IRS1, AKT3, TSC2, and EEF1A1, Greater expression of SLC1A5, SLC7A5, SLC2A1, SLC2A8, STAT5B, and RPS6KB1 and lower expression of TSC2 and EEF1A1 were observed in response to LT2.1, LT1.3 and LH3.0 compared with OPAARMC. In addition, treatment with LV1.6 compared with OPAA and control led to greatest upregulation of mTOR. Particularly The OPAA and LT1.3 treatment increased the phosphorylation of mTOR and S6K1. The β-casein expression was significantly increased when mammary tissue explants were treated with OPAA. Overall, our study revealed unique effects of essential AA ratios, and particularly OPAA, on the molecular phenotype associated with milk protein synthesis regulation in mammary cells.1.2 Regulation role of mTOR on milk protein synthesis pathway mediated by essential amino acid in MAC-T cells and bovine mammary tissue explantsThe regulation of mechanism on milk protein synthesis was evaluated in cultured bovine mammary cells and tissues. After incubation with essential amino acid free, essential amino acid and Thr, mammary cells and tissues were collected for determination of mTOR and milk protein expression by qPCR and western blot. The expression of S6K1 and 4EBP1 were also tested. Results showed that the mRNA abundance and phosphorylation of the mTOR were increased when essential amino acids were supplemented. And the expression of β-casein in mammary tissue were improved in the essential amino acids group. Also,essential amino acids enhanced the mRNA and phosphorylation of S6K1 and 4EBP1. Further studies on these these genes function in milk protein synthesis promoted by essential amino acid were conducted by addition of inhibitors for mTOR (rapamycin) and mTOR siRNA. Results showed that inhibition of mTOR decreased the expression of β-casein in both essential amino acid free and essential amino acid group.In addition, the mRNA and phosphorylation of S6K1 and 4EBP1 were reduced when mTOR activity was inhibited. These results suggested that essential amino acid promoted milk protein synthesis through activiting mTOR pathway. During this process, S6K1 and 4EBP1 exerted a directly significant role.2. Effect of essential amino acid ratio on milk fat synthesis in MAC-T cells and bovine mammary tissue explants2.1 Effect of essential amino acid ratio on milk fat synthesis in MAC-T cells and bovine mammary tissue explantsThe objective of this study was to study how changing the ratio of Lys to Thr, Lys to His, and Lys to Val affects the expression of lipogenic target genes in bovine mammary epithelial cell. Triplicate cultures with the "optimal" AA ratio (OPAA; Lys:Met 2.9:1; Thr:Phe,1.05:1; Lys:Thr,1.8:1; Lys:His,2.38:1; Lys:Val,1.23:1) plus rapamycin (OPAARMC, control), OPAA,2.1:1 Lys:Thr (LT2.1),1.3:1 Lys:Thr (LT1.3),3.05:1 Lys:His (LH3.0), and 1.62:1 Lys:Val (LV1.6) were incubated for 12 h. The expression of lipogenic gene networks was evaluated via quantitative PCR of 15 genes plus 3 internal control genes measured using qPCR. Data were log-transformed and statistically analyzed using the GLM of SAS with treatment as a fixed effect and replicate as random effect. The multiple comparisons were corrected using Tukey’s and significance set a P＜0.05. Responses to LT2.1, LT1.3, LH3.0, and LV1.6 relative to the OPAARMC included greater expression of ACSS2, FABP3, ACACA, FASN, SCD, LPIN1, INSIG1, SREBF1, PPARD, and NRlH3. Furthermore, LV1.6 increased expression of ACSL1, DGAT1, and RXRA and reduced PPARG expression. Although no effect of OPAA on expression of PPARG was observed, OPAA increased expression of ACSS2, FABP3, ACACA, FASN, SCD, LPIN1, INSIG1, and SREBF1 compared with OPAARMC. Gene network analysis using Ingenuity Pathway Analysis revealed a potentially important role of EAA ratios in the coordination of milk fat synthesis via PPARG and SREBF1. The upregulation of lipogenic gene networks observed underscore a role of EAA in the regulation of milk fat synthesis during lactation.2.2 Regulation role of mTOR on milk fat synthesis pathway mediated by essential amino acid in MAC-T cells and bovine mammary tissue explantsPreviously study found that esential amino acid can affect the gene network of milk fat synthesis pathway. To elucidate the regulation of mechanism on milk fat synthesis was evaluated in cultured bovine mammary cells and tissues. After incubation with essential amino acid free, essential amino acid and Thr, mammary cells and tissues were collected for determination of SREBP1 and PPARG by qPCR and western blot. The mRAN expression of ACACA, FASN, FABP3 and LPIN1 were also tested. Results showed that the mRNA abundance and protein expression of SREBP1 and PPARG were increased when essential amino acids were supplemented. And the mRNA expression of ACACA, FASN, FABP3, and LPIN1 were improved in the essential amino acids group. Further studies on these these genes function in milk fat synthesis promoted by essential amino acid were conducted by addition of inhibitors for mTOR (rapamycin) and mTOR siRNA. Results showed that inhibition of mTOR decreased the mRNA expression of SREBP1 and PPARG in essential amino acid group.In addition, the mRNA expression of ACACA, FASN, FABP3, and LPIN1 were reduced when mTOR activity was inhibited. These results suggested that essential amino acid promoted the expression of SREBP1 and PPARG through activiting mTOR pathway. During this process, ACACA, FASN, FABP3, and LPIN1 exerted an indirectly significant role, Indicating that SREBP1 might play an important part in mediating this process.In summary, optimal essential amino acid ratio promoted milk protein synthesis significantly in bovine mammary gland and the regulation of mechanism might be via both its direct substrate for protein synthesis and indirect enhancing activating mTOR pathway.In addition, the actived mTOR which was enhanced by optimal essential amino acid ratio also could regulated the fatty acid de novo and triglyceride synthesis through the regulation role of mTOR-SREBP1.This may expand our knowledge of improving milk quantity.