Phenylalanine Regulates Milk Protein Synthesis via LAT1-mTOR Signaling Pathways in Bovine Mammary Epithelial Cells

Phenylalanine (Phe) is a potentially limiting amino acid for lactating cows. The mechanism by which Phe regulates milk protein synthesis remains unclear. The present study elucidates the mechanisms by which phenylalanine affects milk protein synthesis, amino acid utilization, and related signaling pathways in bovine mammary epithelial cells (BMECs). The BMECs were treated with five concentrations (0, 0.22, 0.44, 0.88, 1.76 mM, and serum free). Rapamycin inhibitors and RNA interference (RNAi) were used to inhibit the phosphorylation of the mammalian target of rapamycin (mTOR) signaling pathway and the expression of relevant amino acid transporters, respectively. The results showed that 4xPhe (0.88 mM) significantly increased (p < 0.05) both the mRNA and protein expression of alpha-casein (CSN1S1), beta-casein (CSN2), and kappa-casein (CSN3), as well as L-type amino acid transporter-1 (LAT1) mRNA expression. Protein expression and modification assays of mTOR-related proteins showed that 4xPhe could increase (p < 0.05) the expression of alpha-casein and eukaryotic initiation factor 4E-binding protein-1 (4EBP1) and tended to increase the expression of ribosomal protein S6 protein kinase (S6K1, p = 0.054). The general control nonderepressible 2 (GCN2) signaling pathway factor, eukaryotic initiation factor 2 (eIF2 alpha), was downregulated by 4xPhe treatment (p < 0.05). The rapamycin inhibition test showed that Phe regulated casein synthesis via the mTOR signaling pathway. RNAi experiments showed that LAT1 mediated the entry of Phe into cells. Moreover, 4xPhe treatment tended to decrease (0.05 < p < 0.10) the consumption of valine, leucine, histidine, tyrosine, cysteine, alanine, asparagine, and serine in the medium. Collectively, phenylalanine enhanced alpha-casein synthesis by regulating the phosphorylation of 4EBP1 and eIF2 alpha and promoting the formation of the mTOR-centered casein translation initiation complex.