Mechanistic Insights into Glycine’s Regulation of Milk Protein Synthesis via PI3K-AKT-mTOR Signaling

Amino acids play a dual role in milk protein synthesis, functioning as both metabolic precursors and signaling molecules. This study aimed to elucidate the molecular mechanism by which glycine regulates α-casein production in bovine mammary epithelial cells (MAC-T). Under serum-free conditions, MAC-T cells were exposed to graded concentrations of glycine (1.105, 2.209, 4.418, 8.836, 17.673, and 35.345 mM) for 24 h. α-Casein levels in cell lysates and culture supernatants were quantified by ELISA. Transcriptional activity of casein-encoding genes (CSN1S1, CSN1S2) and PI3K-AKT-mTOR pathway components was assessed by RT-qPCR. Phosphorylation status of pathway proteins was analyzed by Western blot. The functional involvement of the PI3K-AKT-mTOR pathway was validated using the specific inhibitor LY294002. Glycine stimulated α-casein synthesis and secretion in a concentration-dependent manner, with maximal efficacy at 4.418 mM. At this concentration, glycine upregulated CSN1S1, CSN1S2, and PI3K-AKT-mTOR pathway gene expression, and enhanced phosphorylation of the corresponding proteins. Inhibition of PI3K-AKT-mTOR signaling by LY294002 abolished glycine-induced α-casein synthesis, and this effect was reversed by glycine co-treatment. These findings demonstrate that glycine enhances α-casein synthesis through activation of the PI3K-AKT-mTOR pathway.