The effects of valine, lysine and threonine and their optimal combinations on the synthesis of α-casein by MAC-T cells.

This study aims to enhance milk production and quality in dairy cows. Using in vitro cultured MAC-T cells as a model, it seeks to investigate the effects of valine, lysine, and threonine, as well as their optimal combinations, on the synthesis of α-casein by MAC-T cells. Following a 12-h serum starvation period, MAC-T cells were supplemented with varying concentrations of each amino acid individually. The appropriate concentration ranges and optimal levels for valine, lysine, and threonine were determined using ELISA. Response surface methodology was employed to identify the optimal combination of the three amino acids. The resulting α-casein synthesis in the combined treatment group (MIX group) was then compared with that achieved at the individual optimal concentrations and validated by ELISA. Furthermore, mRNA expression levels of the α-casein-encoding gene and key components of the mTOR signaling pathway were analyzed by RT-qPCR, while protein phosphorylation levels were assessed via Western blot. To confirm the functional involvement of the mTOR pathway, a rapamycin-based inhibition assay was conducted. The maximal stimulation of α-casein synthesis in MAC-T cells was observed at valine, lysine, and threonine concentrations of 4 × Val (25.528 mmol/L), 1 × Lys (7.364 mmol/L), and 0.5 × Thr (1.473 mmol/L), respectively. The optimal amino acid combination (MIX) was determined to be valine:lysine:threonine = 36.114:9.027:4.602 mmol/L. α-Casein synthesis in the MIX group was significantly higher than in any individual amino acid supplementation group (p < 0.01). Supplementation with the MIX medium markedly upregulated the relative mRNA expression of α-casein encoding genes (CSN1S1 and CSN1S2) and key components of the mTOR signaling pathway (ragA-D, mTOR, MLST8, RPTOR, EIF4EBP1, EIF4E, S6K1, EEF2, and RPS6), as well as enhanced the phosphorylation levels of mTOR pathway-related proteins (mTOR, S6K1, 4EBP1, RPS6, and eEF2) (p < 0.01). Treatment with rapamycin significantly suppressed the mRNA expression of these genes, reduced protein phosphorylation, and inhibited α-casein synthesis (p < 0.01); however, co-supplementation with the optimal amino acid combination partially alleviated this suppression, indicating a protective regulatory role of the MIX formulation. The optimal combination of valine, lysine, and threonine was determined to be 36.114:9.027:4.602 mmol/L, corresponding to an approximate molar ratio of 8:2:1. This specific ratio significantly promotes α-casein synthesis in MAC-T cells through activation of the mTOR signaling pathway.