ORIGINAL ARTICLE—ALIMENTARY TRACT Indomethacin suppresses LAMP-2 expression and induces lipophagy and lipoapoptosis in rat enterocytes via the ER stress pathway Ken Narabayashi • Yuko Ito • Nabil Eid • Kentaro Maemura • Takuya Inoue • Toshihisa Takeuchi • Yoshinori Otsuki • Kazuhide Higuchi Received: 12 February 2014 / Accepted: 20 August 2014 Ó Springer Japan 2014 Abstract Background Indomethacin enhances small intestinal epi- thelial cell apoptosis, which may account for mucosal ulceration. However, the involvement of autophagy in indomethacin-induced enterocyte damage is unreported. Methods Using light microscopy and electron micros- copy techniques, Western blot analysis, and pharmaco- logical inhibition of autophagy, we investigated the autophagic response of cultured rat enterocytes to indo- methacin treatment (200 lM) at various time points. Fur- thermore, autophagy was examined in enterocytes of rats given indomethacin by gavage (10 mg/kg). Results Our data indicate that indomethacin induced accumulation of cytoplasmic lipid droplets (LDs) in cul- tured enterocytes, which was associated with time-depen- dent autophagic responses. Initially (0–6 h), mediated by endoplasmic reticulum stress and suppression of mamma- lian target of rapamycin, a predominant cytoprotective lipophagy was activated in indomethacin-treated entero- cytes, as evidenced by induction and colocalization of LC3-II with LDs, excessive formation of autophagosomes sequestering LDs (autolipophagosomes; ALPs), and decreased viability of enterocytes on blocking autophagy with 3-methyladenine. On prolonged exposure to indo- methacin (6–24 h), there was a decrease of LAMP-2 expression in enterocytes coupled with accumulation of ALPs and LDs with fewer autolysosomes in addition to an elevation of lipoapoptosis. These time-dependent auto- phagic and apoptotic responses to indomethacin treatment were detected in enterocytes of indomethacin-treated rats, confirming in vitro results. Conclusions The findings of this study describe a novel mechanism of enterocyte damage by indomethacin medi- ated by endoplasmic reticulum stress, accumulation of LDs, and subsequent activation of the early phase of cy- toprotective lipophagy. This is followed by a late phase characterized by reduced expression of lysosomal auto- phagic proteins, accumulation of ALPs, and enhanced lipoapoptosis. Keywords Enterocytes  Indomethacin  LAMP-2  Lipid droplet  Lipophagy  Lipoapoptosis  Endoplasmic reticulum stress  4-Phenylbutyrate  LC3 puncta Abbreviations ALP Autolipophagosome CHOP CCAAT/enhancer-binding protein homologous protein DAB 3,3 0 -Diaminobenzidine ER Endoplasmic reticulum HRP Horseradish peroxidase LAMP-2 Lysosomal-associated membrane protein 2 LC3 Microtubule-associated protein light chain 3 LD Lipid droplet 3-MA 3-Methyladenine mTOR Mammalian target of rapamycin MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide Electronic supplementary material The online version of this article (doi:10.1007/s00535-014-0995-2) contains supplementary material, which is available to authorized users. K. Narabayashi  T. Inoue  T. Takeuchi  K. Higuchi Second Department of Internal Medicine, Osaka Medical College, Daigakumachi, Takatsuki, Osaka 569-8686, Japan Y. Ito  N. Eid  K. Maemura  Y. Otsuki (&) Division of Life Sciences, Department of Anatomy and Cell Biology, Osaka Medical College, 2-7 Daigakumachi, Takatsuki, Osaka 569-8686, Japan e-mail: an1001@art.osaka-med.ac.jp 123 J Gastroenterol DOI 10.1007/s00535-014-0995-2