Combination of histidine, lysine, methionine, and leucine promotes β-casein synthesis via the mechanistic target of rapamycin signaling pathway in bovine mammary epithelial cells

The ratio of different AA in the diets of cows is vital to improve milk protein yield. beta-Casein is one of the important milk proteins with high nutritive value. However, the suitable ratio of essential amino acids (EAA) for the expression of beta-casein in the immortalized bovine mammary epithelial cell line is not fully characterized. This study employed response surface methodology to determine the optimal ratio of His, Lys, Met, and Leu on beta-casein expression level in vitro and clarified the effect of the 4 EAA on beta-casein via the mechanistic target of rapamycin (mTOR) signaling pathway. A central composite design containing 5 axial points per EAA and 28 combinations of the 4 EAA was used in our study. The results of response surface methodology and the changes of the mTOR-related signaling proteins were determined by western blot. The results showed that beta-casein level was significantly affected by all 4 EAA (R-2 = 0.71). The optimum conditions for beta-casein expression are found to be 5.47 mM of His, 7.48 mM of Lys, 1.17 mM of Met, and 8.21 mM of Leu (His:Lys:Met:Leu = 5:6:1:7) in the designed scope of concentration. The interaction of Leu and Met significantly affected beta-casein expression (P < 0.01). The phosphorylation of mTOR (Ser(2481)), regulatory associated protein of target of rapamycin (Ser(792)), ribosomal protein S6 kinase 1 (Thr(389)), ribosomal protein S6 (Ser(235/236)), and eukaryotic elongation factor 2 (Thr(56)) was increased with the supplementation of either single EAA or an optimal combination of EAA. However, the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (Thr(37)) was decreased with the addition of Lys, Met, or Leu alone. Furthermore, the phosphorylation (P) of eIF2 alpha (Ser(51)) was decreased when Met was supplemented alone. Under the optimal mixture of 4 EAA, the phosphorylation of mechanistic target of rapamycin complex 1 signaling proteins was significantly greater than the single EAA supplementations and the expression of beta-casein was 98% as high as the positive control (i.e., medium with all AA). A similar trend was found with P-ribosomal protein S6 kinase 1 and P-ribosomal protein S6. In conclusion, the extracellular concentrations of His, Lys, Met, and Leu at a ratio of 5:6:1:7 maximized beta-casein expression in the immortalized bovine mammary epithelial cell line may occur via activation of the mechanistic target of rapamycin complex 1 signaling pathway.