A Study On The Signaling Pathway Through Which Leucine Regulates Autophagy

The purpose of this study was to examine whether leucine deprivation induces autophagy or leucine resupplementation blocks autophagy in HEK 293 T cells, and to identify whether mammalian target of rapamycin (mTOR) signaling pathway is dependent or not, and if so, further to clarify whether Rheb, Raptor and GβL that are all regulative proteins upstream of mTOR are necessary.This research includes three parts. (1) Detected autophagic vacuoles by transmission electron microscope and LC3-Ⅱ, p62 abundance by western blot with time-course (Oh, 0.5h, lh,2h,4h) Leucine deprivation in HEK 293T cells. Transfected HEK 293T cells with the LC3-GFP expression plasmid and then detected the distribution of green fluorescence by laser scanning confocal microscope with time-course (0h,0.5h, 1h,2h,4h) leucine deprivation. (2) Detected LC3-Ⅱ, p62 and pS6K1 abundance by western blot after leucine deprivation at two time pionts which autophagy increased the most markedly or after leucine resupplementation at those two time pionts with or without rapamycin. (3)RNA interference of Rheb, Raptor and GβL respectively, then detected abundance of LC3-Ⅱ, p62 and pS6K1 by western blot after leucine deprivation at two time pionts which autophagy increased the most markedly or leucine resupplementation at those two time pionts.Our data show that (1) There was no autophagic vacuoles been watched and green fluorescence of LC3-GFP distributed diffusely in control (Leucine deprivation for Oh). After leucine deprivation for 0.5h, there were some autophagic vacuoles, and diffuse distribution of green fluorescence resulted in a punctate pattern partly. With time-course Leucine deprivation within 4h, amount of autophagic vacuoles increased, and diffuse distribution of green fluorescence resulted in a punctate pattern ultimately, reached the peak at 4h. LC3-Ⅱwas undetected and amount of p62 was the most at Oh, but amount of LC3-Ⅱincreased and p62 decreased both very significantly (P<0.01) after leucine deprivation for 0.5h. Amount of LC3-Ⅱincreased and amount of p62 decreased more and more with time-course Leucine deprivation within 4h. (2) Amount of LC3-Ⅱincreased very significantly (P<0.01) and amount of p62 and pS6K1 both decreased very significantly (P<0.01) after Leucine deprivation for 2h compared to Leucine deprivation for Oh. Amount of LC3-Ⅱand p62 both had no significant (P>0.05) change and amount of pS6K1 decreased very significantly (P<0.01) after leucine resupplementation with Leucine deprivation for 2h compared to Leucine deprivation for Oh. Amount of LC3-Ⅱ decreased very significantly (P<0.01) and p62 and pS6K1 both increased very significantly (P<0.01) after leucine resupplementation with Leucine deprivation for 2h compared to Leucine deprivation for 2h. Amount of LC3-Ⅱincreased very significantly (P<0.01) and amount of p62 and pS6K1 both decreased very significantly (P<0.01) after leucine resupplementation with Leucine deprivation for 2h and rapamycin compared to Leucine deprivation for 0h and compared to leucine resupplementation without rapamycin. Amount of LC3-Ⅱincreased very significantly (P<0.01) and p62 and pS6K1 both had no significant (P>0.05) change after leucine resupplementation with Leucine deprivation for 2h and rapamycin compared to Leucine deprivation for 2h. We get the same results if changed the time of leucine deprivation for 2h to 4h. (3) Amount of LC3-Ⅱincreased very significantly (P<0.01) and amount of p62 and pS6K1 both decreased very significantly (P<0.01) after leucine deprivation or resupplementation, with RNA interference of Rheb or Raptor, compared to Leucine deprivation for 0h. Amount of LC3-Ⅱincreased very significantly (P<0.01) and amount of p62 and pS6K1 both decreased very significantly (P<0.01) after leucine deprivation for 2h with RNA interference of GβL, compared to Leucine deprivation for 0h. Compared to Leucine deprivation for 0h, amount of LC3-Ⅱincreased very significantly (P<0.01) and amount of p62 and pS6K1 both decreased very significantly (P<0.01) after leucine resupplementation with Leucine deprivation for 2h, after RNA interference of GβL. But compared to Leucine deprivation for 2h, amount of LC3-Ⅱdecreased very significantly (P<0.01) and amount of p62 and pS6K1 both increased very significantly (P<0.01) after leucine resupplementation with Leucine deprivation for 2h, after RNA interference of GβL. We get the same results if changed the time of leucine deprivation for 2h to 4h.Together, our results demonstrate that leucine deprivation induces autophagy and this effect will be intensified with the deprivation prolonging within a certain time. Leucine resupplementation blocks autophagy, and this block is mTOR signaling pathway dependent. Rheb and Raptor are both necessary for leucine to regulate autophagy that they are two series-wound bridges for leucine to get to autophagy. GβL is not necessary for leucine to block autophagy.