Yang Min, Zhang Xinyu, Ding Yu, Yang Liang, Ren Wanping, Gao Yu, Yao Kangyu, Zhou Yuxin, Shao Wei
Xinjiang Key Laboratory of Meat and Milk Production Herbivore Nutrition, College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China.
Int J Mol Sci. 2025 Mar 29;26(7):3179. doi: 10.3390/ijms26073179.
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 (), casein alpha s2 gene () 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 ( < 0.01). Within this concentration range, valine significantly upregulated the expression of , , and mTOR signaling pathway-related genes including the RagA gene (), RagB gene (), RagC gene (), RagD gene (), , (), and (), (), and S6 Kinase 1 () ( < 0.01). Notably, the expression of the eukaryotic elongation factor 2 () 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 (RPS6), and eEF2 ( < 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 ( < 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).
本研究利用体外培养的MAC-T细胞作为模型，探讨不同浓度缬氨酸对α-酪蛋白合成的影响及其潜在调控机制。在本实验中，MAC-T细胞先经历12小时饥饿期，随后添加一系列浓度的缬氨酸（共七个浓度：0.000、1.596、3.192、6.384、12.768、25.536和51.072 mM，以及添加10%胎牛血清）。使用酶联免疫吸附测定（ELISA）确定缬氨酸浓度的合适范围。采用实时荧光定量PCR（RT-qPCR）和蛋白质免疫印迹分析来评估酪蛋白αs1基因（）、酪蛋白αs2基因（）和mTOR信号通路相关基因的表达水平和磷酸化状态。通过雷帕霉素（100.000 nM）抑制实验进一步验证mTOR信号通路的功能。结果表明，1×缬氨酸（6.384 mM）、2×缬氨酸（12.768 mM）、4×缬氨酸（25.536 mM）和8×缬氨酸（51.072 mM）显著增强α-酪蛋白合成（<0.01）。在此浓度范围内，缬氨酸显著上调了、、以及mTOR信号通路相关基因的表达，这些基因包括RagA基因（）、RagB基因（）、RagC基因（）、RagD基因（）、、（）和（）、（）以及S6激酶1（）（<0.01）。值得注意的是，真核生物延伸因子2（）基因的表达在1×缬氨酸（6.384 mM）时达到峰值，而其他基因的表达在4×缬氨酸（25.536 mM）时达到最大值。此外，缬氨酸显著增加了mTOR、S6K1、4E结合蛋白1（4EBP1）、核糖体（RPS6）和eEF2的磷酸化水平（<0.01），mTOR、S6K1和RPS6的磷酸化水平在4×缬氨酸（25.536 mM）时最高。雷帕霉素处理显著抑制mTOR磷酸化和α-酪蛋白合成（<0.01）；然而，添加4×缬氨酸（25.536 mM）部分减轻了这种抑制作用。总之，缬氨酸通过激活mTOR信号通路促进α-酪蛋白合成，最佳浓度为4×缬氨酸（25.536 mM）。
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