Effects of the ideal profiles of lysine, methionine, threonine, phenylalanine, histidine, and valine on milk protein synthesis gene network expression in bovine mammary epithelial cells
Amino acids (AA) are essential precursors for milk protein synthesis in mammals. In recent years it has become evident that the AA-mediated protein synthesis response within mammary cells is partly regulated through the mTOR pathway. Thus, AA not only are building blocks of proteins but also are one of the key molecules that serve as upstream components of the signaling pathways that regulate protein synthesis. Although the effects of AA on signaling through mTOR in mammary cells have been explored, little is known about the transcriptional response, particularly regarding the 6 essential AA [lysine (Lys), methionine (Met), threonine (Thr), phenylalanine (Phe), histidine (His), and valine (Val)] for which “ideal” recommendations have been proposed. The specific objective of this study was to investigate how changing the ratio of Lys to Thr, Lys to His, and Lys to Val affected the expression of genes associated with pathways of insulin, mTOR, and Jak2-Stat5 signaling and also glucose and AA transport in MacT cells. Target genes plus 3 internal controls were measured using qPCR. Triplicate cultures with the “optimal” AA ratio (OPAA; Lys:Met 2.9:1; Thr:Phe, 1.05:1; Lys:Thr, 1.8:1; Lys:His, 2.38:1; Lys:Val, 1.23:1) plus the mTOR inhibitor rapamycin (OPAARMC, control) or OPAA, 2.1:1 Lys:Thr (LT2.1), 1.3:1 Lys:Thr (LT1.3), 3.05:1 Lys:His (LH3.0), and 1.62:1 Lys:Val (LV1.6) were incubated for 12 h. Compared with OPAARMC the OPAA treatment upregulated SLC1A5, SLC7A5, and RPS6KB1, and downregulated expression of IRS1, AKT3, TSC2, and EEF1A1. Greater expression of SLC1A5, SLC7A5, SLC2A1, SLC2A8, STAT5B, and RPS6KB1 and lower expression of TSC2 and EEF1A1 were observed in response to LT2.1, LT1.3 and LH3.0 compared with OPAARMC. Treatment with LV1.6 as compared with LT2.1, LT1.3 and LH3.0 had similar effects on expression of SLC1A5, SLC7A5, SLC2A1, SLC2A8, STAT5B, and RPS6KB1. In addition, treatment with LV1.6, LH3.0, and LT2.1 compared with OPAA and control led to greatest upregulation of mTOR. However, only LV1.6 upregulated TSC1 and TSC2 and downregulated EIF4EBP1 relative to OPAA and control. Overall, our study revealed unique effects of essential AA ratios, and particularly Lys:Val, on the molecular phenotype associated with milk protein synthesis regulation in mammary cells.
Keywords: mTOR, nutrigenomics, milk protein synthesis