Kokubo, Takeshi
(Research and Development Division, Kirin Company, Limited)
,
Maeda, Shyuichi
(Research Laboratories for Health Science and Food Technologies, Kirin Company, Limited)
,
Tazumi, Kyoko
(Research Laboratories for Health Science and Food Technologies, Kirin Company, Limited)
,
Nozawa, Hajime
(Research Laboratories for Key Technologies, Kirin Company, Limited)
,
Miura, Yutaka
(Research Laboratories for Health Science and Food Technologies, Kirin Company, Limited)
,
Kirisako, Takayoshi
(Research Laboratories for Key Technologies, Kirin Company, Limited)
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 synt...
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.
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.
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문제 정의
Considering the fact that L-citrulline is synthesized endogenously mainly from Orn with carbamoyl phosphate, and constitutes one of the significance reactions in the urea cycle, the stimulatory effect of Orn on protein synthesis is predicted to be involved in the mTORC1 pathway. Therefore, the aim of our study was to investigate whether Orn is able to promote liver protein synthesis through the stimulation of mTORC1 pathway in vitro, and also to compare mTORC1 activation by oral administration of Orn to food-deprived rats.
제안 방법
1). Further studies using specific inhibitors of proposed factors that regulate mTOR activation such as MAPK or PI3K would extend our understanding of the contribution of Orn to the mTORC1 downstream targets.
To evaluate whether Orn stimulates the mTORC1 pathway in vivo as well as in vitro, the phosphorylation of mTOR and its downstream targets in the rat liver was examined. Blood and liver were collected from rats at 1 and 3 h after Orn administration via oral gavage.
대상 데이터
After 3 h incubation, the cells were collected for immunoblotting. All experiments were performed in triplicate.
Male Wistar rats (Japan SLC, Shizuoka, Japan) were purchased at 7 weeks of age and allowed to acclimate to their new surroundings for at least 1 week. Rats ate laboratory rat chow, AIN-93G (Clea Japan, Tokyo, Japan), and drank tap water ad libitum.
이론/모형
Protein concentrations of the samples were determined by the Lowry assay using bovine serum albumin as a standard (Bio-Rad Laboratories Inc., Hercules, CA, USA).
성능/효과
In conclusion, this is the first study to report the effects of Orn on the mTORC1 downstream targets in the rat liver. The results demonstrate that Orn augments the phosphorylation of representative downstream targets of mTORC1, including p70S6K, S6, and 4EBP1, in amino acid-free conditions in rat primary hepatoma cell and rat liver. Based on the mTOR inhibitor experiments, these effects are likely mediated by the mTOR signaling pathway.
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