过表达磷脂酶D3影响成肌细胞中Akt磷酸化水平

Over-expression of phospholipase D3 inhibits Akt phosphorylation in C2C12 myoblasts

摘要:

磷脂酶D(PLD)催化卵磷脂(Phosphatidylcholine, PC)水解产生胆碱(Choline)和磷脂酸(Phosphatidic acid, PA), 其代谢产物参与调控细胞内许多生理和生化过程。在过表达磷脂酶D3 (PLD3)的成肌细胞(C2C12细胞)中, 研究了PLD3对胰岛素刺激后Akt通路激活的影响。研究结果表明, PLD3过表达细胞的Akt磷酸化水平比对照组低, 并且不受胰岛素浓度变化的调控。虽然PLD3过表达细胞中Akt磷酸化水平随胰岛素刺激时间的延长而有所增加, 但磷酸化总水平比对照组低。磷脂酶D抑制剂丁-1醇能够抑制对照组胰岛素刺激下Akt磷酸化, 却不能抑制PLD3过表达细胞的Akt磷酸化, 并且PLD3过表达细胞Akt磷酸化水平比对照组高6倍。用磷脂酸(PA)做刺激时, 对照组的Akt磷酸化明显增加, 而PLD3过表达细胞株的Akt磷酸化没有显著变化; 用PA和胰岛素同时刺激时, PLD3过表达株和对照组的Akt磷酸化均比PA单独刺激时降低。这说明PLD3的过表达抑制成肌细胞内胰岛素信号的传导。

Abstract:

Phospholipase D (PLD) hydrolyzes phosphocholine into choline and phosphatide acid, and these metabolites play an important role in regulating cell physiology and biochemistry. To study the biological function of phospholipase D3 (PLD3) during the insulin stimulation in C2C12 myoblasts, we constructed PLD3 over-expressed cell lines (C2C12/pPLD3) and investigated the phosphorylation of Akt. The results showed that the level of phosphorylated Akt (P-Akt) was significantly increased in control C2C12 cells when insulin concentration was elevated during cell treatment, whereas the level of P-Akt in C2C12/pPLD3 cells was not changed. When extending the time of insulin treatment, P-Akt level in C2C12/pPLD3 cells was increased around 2 folds, but the total level of P-Akt in C2C12/pPLD3 was still lower than that in control group. 1-Butanol, a PLD inhibitor, could completely block Akt phosphorylation in C2C12 cells that even stimulated by insulin. However, 1-Butanol did not inhibit the Akt phosphorylation in C2C12/pPLD3 cells, but increased the phosphorylation up to 6 folds higher than control cells. The level of Akt phosphorylation in control C2C12 cells was increased significantly when stimulated by phosphatidic acid (PA), while there was no change in C2C12/pPLD3 cells with the similar treatment. When cells simulated by both PA and insulin, P-Akt level in both C2C12/pPLD3 cells and C2C12 cells were down regulated. Our observations indicated that PLD3 over expression may inhibit Akt phosphorylation and further block the transduction of insulin signaling in C2C12 cells.