苯丙氨酸对C2C12细胞葡萄糖摄取能力的影响及与mTOR-p70S6K通路的关系探讨

目的 观察苯丙氨酸对小鼠成肌细胞系C2C12葡萄糖摄取能力的影响，以及刺激后细胞内哺乳动物雷帕霉素靶蛋白（mTOR）-p70S6K信号通路分子的变化。方法 体外培养C2C12细胞，分化诱导为多核的肌管细胞后进行饥饿处理，然后分别用不同浓度苯丙氨酸（1.25、2.5、5、10、20 mmol/L）处理细胞，以KRB平衡液作为对照。通过检测2-NBDG评价细胞葡萄糖摄取能力。用Western blot方法检测细胞内mTOR、p70S6K、胰岛素受体底物（IRS）-1、蛋白激酶B(Akt)的表达情况。结果 与KRB平衡液对照相比，5 mmol/L亮氨酸使C2C12细胞的葡萄糖摄取能力下降了约30%（P=0.001），5 mmol/L苯丙氨酸使C2C12细胞摄取葡萄糖的能力上升了约40%（P＜0.01），且苯丙氨酸的这种促进作用随浓度增大有逐渐增强的趋势。苯丙氨酸对细胞内mTOR Ser2448磷酸化水平无明显影响。除1.25 mmol/L外，其它浓度苯丙氨酸均能抑制p70S6K Thr389的表达。亮氨酸使IRS-1 Ser636/639磷酸化水平表达增高（P＜0.01），除1.25 mmol/L外，其它浓度的苯丙氨酸均有抑制IRS-1 Ser636/639表达的趋势，但与对照相比差异无统计学意义（P=0.381）。高浓度短时间的苯丙氨酸刺激对C2C12细胞内的Akt Ser473表达无明显影响。结论 苯丙氨酸可能通过减少p70S6K的磷酸化，继而减少p70S6K对IRS-1的刺激作用而使细胞葡萄糖摄取能力增强。但苯丙氨酸对mTOR-p70S6K通路的抑制作用不通过Akt的激活。

Effects of Phenylalanine on Glucose Uptake in Mouse Myoblast Cell Line C2C12 and Its Relationship withmTOR-p70S6K Pathway

Objective To investigate the influence of phenylalanine(Phe) on glucose uptake in mouse myoblast cell line C2C12 and to explore its relationship with mTOR-p70S6K pathway．Methods C2C12 cells were cultured to promote formation of multinucleated myotubes in vitro.The cells were deprived and incubated with Phe at different concentrations （1.25, 2.5, 5, 10, 20 mmol/L）．Krebs-Ringer buffer (KRB) was used as control．The 2-NBDG was used to measure glucose uptake of C2C12．The expression of mTOR， p70S6K， IRS-1， and Akt protein were evaluated by Western blot．Results Compared with KBP treatment， glucose uptake of the cells incubated with 5mmol/L leucine (Leu) was decreased by 30% (P=0.001)，while a 40% increase was detected in the cells incubated with 5 mmol/L Phe (P<0.01)．The promotion of glucose uptake was Phe concentration-dependent．Phe stimulation had no effect on the phosphorylation of mTOR at Ser2448. Phosphorylation of p70S6K at Thr389 was inhibited in the cells incubated with Phe at concentration higher than 1.25 mmol/L，but the difference was not significant (P=0.815)． Leu stimulated but Phe over 1.25 mmol/L inhibited phosphorylation of IRS-1 at Ser636/639，although the difference was not significant (P=0.381)．Neither Leu nor Phe affected the expression of phospho-Akt (Ser473) significantly．Conclusion Phenylalanine inhibits phosphorylation of IRS-1 at Ser636/639 possibly through inhibiting the activation of p70S6K．The effect of Phe on mTOR-p70S6K pathway is Akt-independent．