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Optimal combination of Histidine, Lysine, Methionine and Leucine affect β-casein synthesis via mTOR signaling pathway in bovine mammary epithelial cells
Assessing the regulatory effect of the optimal ratio of the essential amino acids (EAAs) on milk protein synthesis is vital to AA requirement models for lactation. The study employed response surface methodology (RSM) to determine the optimal ratio of histidine, lysine, methionine and leucine on β-casein expression level in vitro and clarify the effect of four EAAs on β-casein through mechanistic targeting of the rapamycin (mTOR) signaling pathway. A central composite design containing 5 axial points per EAA, and 28 combinations of the four EAAs was performed for our study. The efficiency of RSM and the changes of the mTOR-related signaling proteins were further verified by western-blot. The protein band values from the AA-supplemented cells were related to their AA-deprived controls. The β-casein data from the RSM experimental design were analyzed using a regression model by Design–Expert 8.0.6. The other experimental data were analyzed using the Tukey’s test for post-hoc multiple comparisons of treatment means by SAS. Differences between experimental groups were considered significant at P < 0.05. The results showed that β-casein level was significantly affected by all four EAAs (P<0.01, R2=0.71). The optimal conditions for β-casein expression were as follows: His: Lys: Met: Leu = 5: 6: 1: 7. Only a significant interaction of Leu and Met was observed as to β-casein expression (P<0.01). Further experiments validated that under the optimal mixture of four EAAs, the expression of β-casein was 98% as high as the positive control (i.e., media with all AAs). The phosphorylation of mTOR, Raptor, GβL, S6K1, Rps6 and eEF2 was increased with supplementation of either single EAAs or the optimal combination of EAAs. Finding the best combination of these four EAAs promoted β-casein expression and this appeared to be mediated through activation of the mTORC1 signaling pathway.
Keywords:
Bovine mammary epithelial cells, the optimal ratio of EAAs, mTOR, β-casein