| This study was carried out to investigate:Experiment Ⅰ:This study employed the immortalized bovine mammary epithelial cells(CMEC-H) as a model to clarify the regulatory effects on single supplement of leucine(Leu) on casein synthesis molecular mechanism via mammalian target of rapamycin(m TOR) signaling pathway. We used the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide(MTT) method to evaluate the effect of varying concentrations of Leu on the ratio of relative growth rate of CMEC-H. We used the real-time fluorescence quantitative PCR(q RT-PCR) and western-blotting(WB) investigate the effects of varying concentrations of Leu on m TOR signaling pathway-related proteins and caseins expression in CMEC-H. The Earle’s Balanced Salt Solution(EBSS) was set as control group, and other treatment groups based on t he EBSS were added with different concentrations of Leu. The MTT results showed that after the addition of different concentrations of Leu for 12 and 24 h, CMEC-H proliferation increased remarkably(P<0.01) with Leu in the range of 0.15~5.4 mmol/L. The q RT- PCR results showed that, compared with the negative control, the expression of CSN1S1, CSN1S2, CSN2 and CSN3 genes measured at 6 h with Leu in the range of 0.45~10.8mmol/L were simultaneously up-regulated(P<0.05). The expression of m TOR pathway related genes m TOR, raptor, Gβl, 4EBP1 and e EF2 were highest(P <0.01) with the addition of 1.35 mmol/L Leu, while the expression of S6K1 gene reduced along with the increasing Leu concentrations. The WB results showed that, compared to the control group, the expression of caseins and phosphorylation of m TOR(Ser2481), raptor(Ser792), e IF4E(Ser209), e EF2(Thr56)(P< 0.01) were increased with the Leu concentrations in range of 0.45 to 10.8 mmol/L, while P-S6K1(Thr389) from 1.35 to 10.8 mmol/L, 4EBP1(Thr37) and GβL at 10.8 mmol/L, P-RPS6(Ser235/236) from 5.4 to 10.8 mmol/L were decreased. Whereas αs1-casein expression was only reduced at 9.6 mmol/L, GβL at 0.15 and 9.6 mmol/L and P-RPS6(Ser235/236) from 4.8 to 9.6 mmol/L. Our linear regression model assay suggested that αs1-casein expression was positively correlated with P-m TOR(Ser2481), P-S6K1(Thr389)(P<0.01) and P-e EF2(Thr56)(P<0.01) for the addition of Leu.Experiment Ⅱ: This study employed the CMEC-H as a model to clarify the regulatory effects on single supplement of histidine(His) on casein synthesis molecular mechanism via m TOR signaling pathway. We used the MTT method to evaluate the effect of varying concentrations of His on the relative growth rate of CMEC-H. We used the q RT- PCR andWB investigate the effects of varying concentrations of His on m TOR signaling pathwayrelated proteins and caseins expression in CMEC-H. The EBSS was set as control group, and other treatment groups based on the EBSS were added with different concentrations of His. The MTT results showed that after the addition of different concentrations of Leu for 12 and 24 h, CMEC-H proliferation increased remarkably(P<0.01) with His in the range of 0.15 ~ 9.6 mmol/L. The q RT-PCR results showed that, compared with the negative control, the expression of CSN1S1, CSN1S2, CSN2 and CSN3 genes measured at 6 h with His in the range of 0.15~9.6 mmol/L were simultaneously significantly up- regulated(P <0.05). When adding His to the negative medium, the expression of 4EBP1, e EF2, e IF4 E and rps6 genes increased along with the increasing His concentrations, while the expression of m TOR genes reduced along with the increasing His concentrations; GβL gene acquired its highest expression at 4.8 m mol/L and S6K1 gene at 1.2 mmol/L. The WB results showed that, compared to the control group, under the concentrations from 0.15 t o 9.6 mmol/L, His supplementation increased the expression of αs2- casein, β-casein and κ-casein and P-m TOR(Ser2481), P-raptor(Ser792), P-S6k1(Thr389), P-4EBP1(Thr37), P-e IF4E(Ser209), P-e EF2(Thr56)(P<0.01) phosphorylation in CMEC-H. Whereas αs1-casein expression was only reduced at 9.6 mmol/L, GβL at 0.15 and 9.6 mmol/L and P-RPS6 from 4.8 to 9.6 mmol/L. Our linear regression model assay suggested that the expression of β-casein(P<0.01) and κ-casein(P<0.01) were positively correlated with P-e EF2(Thr56) for the addition of His. Experiment Ш:The ratio of different essential amino acids(EAAs) in the diets of cows is vital to improve milk protein yield. β-casein is one of the important milk protein with high nutritive value. 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 m TOR signaling pathway. A central composite design containing 5 axial points per EAA, and 28 combinations of the four EAAs was used in our study. The results of RSM and the changes of the m TOR-related signaling proteins were further verified by WB. 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 are as follows: His: Lys: Met: Leu = 5: 6: 1: 7. A significant interaction of Leu and Met was observed for β-casein expression(P<0.01). Further experiments validated that the phosphorylation of m TOR(Ser2481), Raptor(Ser792), S6K1(Thr389), RPS6(Ser235/236) and e EF2(Thr56) was increased with supplementation of either single EAAs or an optimal combination of EAAs. However, P-4EBP1(Thr37) wasdecreased with the addition of Lys, Met or Leu alone. The P-e IF2α(Ser51) was decreased when supplementing Met alone. 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). Finding the best combination of these four EAAs promoted β-casein expression and this appeared to be mediated through activation of the m TORC1 signaling pathway. |
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