The Effect of L-Ornithine on the Phosphorylation of mTORC1 Downstream Targets in Rat Liver.
The Effect of L-Ornithine on the Phosphorylation of mTORC1 Downstream Targets in Rat Liver.
复制标题
DOI：
10.3746/pnf.2015.20.4.238
复制
发表时间：
2015-12-01
影响因子：
--
通讯作者：
Kirisako, Takayoshi
中科院分区：
文献类型：
--
作者：
Kokubo, Takeshi;Maeda, Shyuichi;Kirisako, Takayoshi
一种非蛋白质氨基酸，L-鸟氨酸（Orn），已被证明可以刺激肝脏中的尿素循环和组织蛋白质合成。本研究的目的是评估Orn是否影响哺乳动物雷帕霉素靶蛋白复合物1（mTOR 1）通路，该通路参与蛋白质合成。将从Wistar大鼠肝脏分离的原代培养细胞在无氨基酸培养基中孵育，然后加入Orn孵育3 h。对细胞裂解物进行免疫印迹，以评估mTORC 1下游靶标（包括p70 S6 K、S6和4 EBP 1）的磷酸化。为了评估mTORC 1参与Orn的作用，在加入Orn之前用mTOR抑制剂雷帕霉素预处理细胞，并对细胞裂解物进行免疫印迹。我们接下来检查Orn的作用是否在体内发挥。禁食18 h大鼠灌胃给予Orn，于给药后1 h和3 h取血和肝脏进行免疫印迹。Orn处理原代培养细胞3小时可增强p70 S6 K、S6和4 EBP 1的磷酸化。此外，雷帕霉素完全（p70 S6 K和S6）或部分（4 EBP 1）阻断Orn的作用。大鼠口服Orn后，1 h时mTORC 1下游靶点的磷酸化水平显著升高，尤其是在S6。我们的研究结果表明，Orn有可能诱导大鼠肝脏中mTORC 1下游靶点的磷酸化。这可能是由mTORC 1活性增强介导的。
A non-protein amino acid, L-ornithine (Orn), has been shown to stimulate the urea cycle and tissue protein synthesis in the liver. The purpose of the current study was to assess whether Orn affects the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) pathway, which is involved in protein synthesis. Primary cultured cells isolated from Wistar rat liver were incubated in an amino acid-free medium, followed by addition of Orn for 3 h. The cell lysate was subjected to immunoblotting to evaluate the phosphorylation of downstream targets of mTORC1, including p70S6K, S6, and 4EBP1. To assess the involvement of mTORC1 for the effect of Orn, the cells were pretreated with the mTOR inhibitor rapamycin before the addition of Orn and the cell lysate was subjected to immunoblotting. We next examined whether the effects of Orn were exerted in vivo. Orn was orally administered to 18 h food-deprived rats, the blood and the livers were collected at 1 and 3 h after administration for immunoblotting. Orn treatment for primary cultured cells for 3 h enhanced the phosphorylation of p70S6K, S6, and 4EBP1. In addition, rapamycin blocked the effects of Orn completely (p70S6K and S6) or partially (4EBP1). The oral administration of Orn to the rat also augmented the phosphorylation of mTORC1 downstream targets notably in S6 at 1 h. Our findings demonstrate that Orn has the potential to induce the phosphorylation of downstream targets of mTORC1 in the rat liver. This may be mediated by the augmentation of mTORC1 activity.