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 4×Phe (0.88 mM) significantly increased (p < 0.05) both the mRNA and protein expression of α-casein (CSN1S1), β-casein (CSN2), and κ-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 4×Phe could increase (p < 0.05) the expression of α-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 (eIF2α), was downregulated by 4×Phe 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, 4×Phe 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 α-casein synthesis by regulating the phosphorylation of 4EBP1 and eIF2α and promoting the formation of the mTOR-centered casein translation initiation complex.
Keywords: amino acid utilization; bovine mammary epithelial cells; mTOR; milk protein synthesis; phenylalanine.