title: Inhibition of mTOR improves the impairment of acidification in autophagic vesicles caused by hepatic steatosis.
abstract: Recent investigations revealed that dysfunction of autophagy involved in the progression of chronic liver diseases such as alcoholic and nonalcoholic steatohepatitis and hepatocellular neoplasia. Previously, it was reported that hepatic steatosis disturbs autophagic proteolysis via suppression of both autophagic induction and lysosomal function. Here, we demonstrate that autophagic acidification was altered by a decrease in lysosomal proton pump vacuolar-ATPase (V-ATPase) in steatohepatitis. The number of autophagic vesicles was increased in hepatocytes from obese KKAy mice as compared to control. Similarly, autophagic membrane protein LC3-II and lysosomal protein LAMP-2 expression were enhanced in KKAy mice liver. Nevertheless, both phospho-mTOR and p62 expression were augmented in KKAy mice liver. More than 70% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, the percentage of acidic autolysosomes was decreased in hepatocytes from KKAy mice significantly (40.1 ± 3.48%). Both protein and RNA level of V-ATPase subunits ATP6v1a, ATP6v1b, ATP6v1d in isolated lysosomes were suppressed in KKAy mice as compared to control. Interestingly, incubation with mTOR inhibitor rapamycin increased in the rate of LTR-positive autolysosomes in hepatocytes from KKAy mice and suppressed p62 accumulation in the liver from KKAy mice which correlated to an increase in the V-ATPase subunits expression. These results indicate that down-regulation of V-ATPase due to hepatic steatosis causes autophagic dysfunction via disruption of lysosomal and autophagic acidification. Moreover, activation of mTOR plays a pivotal role on dysregulation of lysosomal and autophagic acidification by modulation of V-ATPase expression and could therefore be a useful therapeutic target to ameliorate dysfunction of autophagy in NAFLD.
authors.affiliation: Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan.
authors.forename: Eisuke
authors.initials: E
authors.lastname: Nakadera
authors.affiliation: Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan. Electronic address: syamashi@juntendo.ac.jp.
authors.forename: Shunhei
authors.initials: S
authors.lastname: Yamashina
authors.affiliation: Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan.
authors.forename: Kousuke
authors.initials: K
authors.lastname: Izumi
authors.affiliation: Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Inami
authors.affiliation: Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan.
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Sato
authors.affiliation: Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Fukushima
authors.affiliation: Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan.
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Kon
authors.affiliation: Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Ikejima
authors.affiliation: Division of Proteomics and Biomolecular Science, Juntendo University, School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ueno
authors.affiliation: Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan.
authors.forename: Sumio
authors.initials: S
authors.lastname: Watanabe
mesh_terms: D000818:Animals; D001343:Autophagy; D002478:Cells, Cultured; D022162:Cytoplasmic Vesicles / Q000737:chemistry* / Q000378:metabolism* / Q000473:pathology; D005234:Fatty Liver / Q000378:metabolism* / Q000473:pathology; D022781:Hepatocytes / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology; D006863:Hydrogen-Ion Concentration; D050356:Lipid Metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
chemical_list: C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases; D025262:Vacuolar Proton-Translocating ATPases
keywords: Autophagy; Lysosome; Rapamycin; Vacuolar-ATPase