The Cellular Regulatory Mechanism Of Negative Feedback Between Autophagy And MTOR Under Starvation

This study explored the regulatory network among mTOR, autophagy and amino acid, based on previous study. The aim of this study is to dissect the mechanisms of the following four questions.1. The regulatory mechanism of extracellular amino acid and amono acid generated through autophagy with mTOR reactivation under starvation.2. The function of amino acid transporters under mTOR reactivation.3. The effect of mTOR reactivation for amino acid transporter expression.4. The contribution of mTOR and autophagy for cell survival under starvation. The main goal of this project is to explore the basic regulatory mechanism among mTOR, autophagy and amino acid, and to provide the novel principles and insights for further research.This project composed of three parts:(1) Construct the stable cell line with autophagy essential gene (Beclin-1) knocked down. Analyze autophagy level and mTOR activity under prolonged starvation by immunofluoresces and western blot. (2) Compare mTOR activity in serum or serum+amino acid starvation, and analyze the influence between mTOR and amino acid transporters. (3) Test the contribution of mTOR and autophagy in cell survival rate under different treatment of starvation.Our data showed that, (1) The level of S6K phosphorylation which is downstream of mTOR decreased very significantly (P<0.01) at serum-starvation 4h. Under prolonged starvation, the level of S6K phosphorylation elevated very significantly (P<0.01), which showed regular and significant fluctuations. At the same time, the number of autophagosomes increased very significantly (P<0.01) at 4h, and decreased significantly (P<0.01) at 14h. It also showed regular and significant fluctuations like mTOR. But the difference was that the peak of autophagy level was earlier than the peak of mTOR activation. We further analyzed the same phenomenon in Beclinl KD cells with the same method and condition. The results showed that mTOR could not be reactivated in this cell line. Compared with serum starvation and DPBS starvation, the level of S6K phosphorylation showed regular and significant fluctuations in serum starvation condition which was different from DPBS. The level of S6K phosphorylation decreased very significantly (P<0.01) at DPBS-starvation 3h but it could not be up-regulated later.2.5mM GPNA and 50mM BCH based experiments was identified to be the proper concentration to inhibit amino acid transporters SLC1A5 and SLC7A5 through the concentration gradient experiments. Compared with NC, the level of S6K phosphorylation was not be up-regulated with GPNA addition. The differences were obvious versus NC (P<0.01). the trend of S6K phosphorylation level with BCH treatment was similar with GPNA. The expression of amino acid transporter SLC7A5 showed a significant increase (P<0.01) at 16h. When mTOR reactivity was inhibited by RAPA or GPNA, the up-regulation of SLC7A5 expression was also limited. The difference was dramatic with control (P< 0.01). But the expression of SLC7A8 was not affected in the same condition. (3) Comparison of normal NRK cells, NRK+ RAPA, BeclinlKD stable cells, BeclinlKD + RAPA on cell viability, the viability were 120%,80%,50% and 40% at 50h. The differences were obvious. Impaired autophagy or mTOR have accelerated cell death. The expression of Bcl2 decreased very significantly (P<0.01) at 3h detected by Western Blot, and the expression of Bcl2 increased very significantly (P<0.01) at 21h. Overexpression Bcl2 in Beclinl KD cells could rescue cell death significantly.Together, our data indicated that in prolonged starvation both the activity of mTOR and autophagy level presented an oscillation pattern that correlated with each other; mTOR reactivation relied on extracellular amino acid. Amino acid transporters activated mTOR by importing amino acid into the cell, while mTOR reactivation also contributed to up-regulate protein level of these transporters; mTOR and autophagy regulated each other through amino acid to maintain cell anabolism and catabolism at proper level to reduce cell death under stress.