Kokubo Takeshi, Maeda Shyuichi, Tazumi Kyoko, Nozawa Hajime, Miura Yutaka, Kirisako Takayoshi
Research and Development Division, Kirin Company, Limited, Tokyo 164-0001, Japan.
Research Laboratories for Health Science and Food Technologies, Kirin Company, Limited, Yokohama 236-0004, Japan.
Prev Nutr Food Sci. 2015 Dec;20(4):238-45. doi: 10.3746/pnf.2015.20.4.238. Epub 2015 Dec 31.
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.
一种非蛋白质氨基酸，L-鸟氨酸（Orn），已被证明能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是否影响参与蛋白质合成的哺乳动物雷帕霉素靶蛋白（mTOR）复合物1（mTORC1）信号通路。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，随后添加Orn 3小时。将细胞裂解物进行免疫印迹，以评估mTORC1下游靶点（包括p70S6K、S6和4EBP1）的磷酸化情况。为了评估mTORC1对Orn作用的参与情况，在添加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。接下来，我们研究了Orn的作用是否在体内发挥。将Orn口服给予禁食18小时的大鼠，给药后1小时和3小时采集血液和肝脏进行免疫印迹。用Orn处理原代培养细胞3小时可增强p70S6K、S6和4EBP1的磷酸化。此外，雷帕霉素完全阻断了Orn对p70S6K和S6的作用，或部分阻断了对4EBP1的作用。给大鼠口服Orn也显著增强了mTORC1下游靶点在1小时时S6的磷酸化。我们的研究结果表明，Orn有可能诱导大鼠肝脏中mTORC1下游靶点的磷酸化。这可能是由mTORC1活性的增强介导的。
