Effect Of Leucine On The Proliferation And Amino Acid Transport Of Porcine Trophectoderm Cell

To investigate the effect of leucine on proliferation and the regulation of amino acid transporters through m TOR signaling pathway of porcine trophectoderm cell(p Tr). MTT and flow cytometer were used to detect the proliferation and cell cycle of p Tr after treatment with different leucine concentrations(0, 0.1, 0.05, 1, 5, 10 m M) and time(24 h, 48 h, 72 h). Real-time PCR was used to detect the expression of amino acid transporters, the key molecules in m TOR signaling pathway, cyclin dependent dinase 4 and caspase-8 of p Tr after treatment with different leucine concentrations(0, 1, 10 m M) and time(24 h, 48 h) on m RNA level.The results showed as followed:1. After treatment with different concentrations of Leu for 24 h, the cell number of 0.5 m M, 1 m M and 10 m M groups increased significantly(P<0.05); the cell number of all groups decreased very significantly when treated with Leu for 48 h(P<0.01). After treatment with 1m M and 10 m M Leu for 72 h, the cell number decreased very significantly(P<0.01).2. After treatment with different concentrations of leucine for 24 h and 48 h, compaired with the control group, the cell number at phase G0-G1 incresed and at phase S decreased in a dose- and time-dependent manner. The result of RT-PCR showed that the expression of CDK4 m RNA decreased as the concentration of leucine increasde and after treatment with the 10 m M group for 48 h, the difference reached the level of significant(P<0.05).3. After treatment with different concentrations of leucine for 24 h and 48 h, compared with the control group, there were no significant difference among them of the expression of caspase-8 m RNA(P>0.05).4. After treatment with different concentrations of leucine, the 1 m M group reduced the the expression of 4E-BP1 significantly after 24 h(P<0.05), but with no significant difference when compared to the 10 m M group(P>0.05); the 1 m M and 10 m M groups redcued the expression of 4E-BP1 very significantly after 48 h(P<0.01). The 1 m M group reduced the the expression of e IF4 G significantly after 24 h(P<0.05), but there was no significant difference when treated with 48 h(P>0.05). The 10 m M group increased the expression of m TOR m RNA very signifiantly after 24 h when compared with the control group and the 1 m M group(P<0.01), and there was a significant increasement after 48 h(P<0.05).The expression of S6K1 and PKB m RNA showed no significant difference after treated with Leu for 24 h and 48 h(P>0.05).5. After treatment with different concentrations of leucine, the expression of the animo acid transporters SNAT1?SNAT2?LAT1?4F2hc?r BAT m RNA in all groups decreased compared with the control group. After treated with leucine for 24 h, except the 1 m M group reduced the expression of SNAT1 m RNA very significantly(P<0.01), and lower than the 10 m M group significant(P<0.05), the expression of the animo acid transporters SNAT2?LAT1?4F2hc?r BAT m RNA were lower than the control group and there were no significant difference among them(P>0.05). After treated with leucine for 48 h, 1 m M group reduced reduced the expression of the animo acid transporters LAT1 m RNA significantly(P<0.05), but there were no significant different of the expression of the animo acid transporters SNAT1?SNAT2?4F2hc?r BAT m RNA(P>0.05). 10 m M group reduced the expression of the animo acid transporters SNAT1?SNAT2?LAT1 m RNA very significantly(P<0.01), reduced the expression of the animo acid transporter r BAT m RNA significantly(P<0.05), but there were no significant different of the expression of the animo acid transporter 4F2 hc m RNA significantly(P>0.05).All the currently avaliable data demonstrate that high concentratin of leucine can block cell cycle progression, then suppress the proliferation of p Tr, reduce the expression of amino acid transpoters on m RNA level by reducing the m TOR signaling pathway.