The Effect of Valine on the Synthesis of α-Casein in MAC-T Cells and the Expression and Phosphorylation of Genes Related to the mTOR Signaling Pathway

This study utilized MAC-T cells cultured in vitro as a model to investigate the effects of varying concentrations of valine on α-casein synthesis and its underlying regulatory mechanisms. In this experiment, MAC-T cells were subjected to a 12 h starvation period, followed by the addition of valine in a range of concentrations (a total of seven concentrations: 0.000, 1.596, 3.192, 6.384, 12.768, 25.536, and 51.072 mM, as well as in 10% Fetal Bovine Serum). The suitable range of valine concentrations was determined using enzyme-linked immunosorbent assays (ELISAs). Real-time fluorescent quantitative PCR (RT-qPCR) and Western blot analyses were employed to evaluate the expression levels and phosphorylation states of the casein alpha s1 gene (<i>CSN1S1</i>), casein alpha s2 gene (<i>CSN1S2</i>) and mTOR signaling pathway-related genes. The functionality of the mTOR signaling pathway was further validated through rapamycin (100.000 nM) inhibition experiments. Results indicated that 1× Val (6.384 mM), 2× Val (12.768 mM), 4× Val (25.536 mM), and 8× Val (51.072 mM) significantly enhanced α-casein synthesis (<i>p</i> < 0.01). Within this concentration range, valine significantly upregulated the expression of <i>CSN1S1</i>, <i>CSN1S2</i>, and mTOR signaling pathway-related genes including the RagA gene (<i>RRAGA</i>), RagB gene (<i>RRAGB</i>), RagC gene (<i>RRAGC</i>), RagD gene (<i>RRAGD</i>), <i>mTOR</i>, <i>raptor gene</i> (<i>RPTOR</i>), and <i>4EBP1 gene</i> (<i>EIF4EBP1</i>), <i>eukaryotic initiation factor 4E</i> (<i>EIF4E</i>), and S6 Kinase 1 (<i>S6K1</i>) (<i>p</i> < 0.01). Notably, the expression of the eukaryotic elongation factor 2 (<i>EEF2</i>) gene peaked at 1× Val (6.384 mM), while the expression of other genes reached their maximum at 4× Val (25.536 mM). Additionally, valine significantly increased the phosphorylation levels of mTOR, S6K1, 4E-binding protein-1 (4EBP1), ribosomal <i>protein S6</i> (RPS6), and eEF2 (<i>p</i> < 0.01), with the highest phosphorylation levels of mTOR, S6K1, and RPS6 observed at 4× Val (25.536 mM). Rapamycin treatment significantly inhibited mTOR phosphorylation and α-casein synthesis (<i>p</i> < 0.01); however, the addition of 4× Val (25.536 mM) partially mitigated this inhibitory effect. In conclusion, valine promotes α-casein synthesis by activating the mTOR signaling pathway, with an optimal concentration of 4× Val (25.536 mM).