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Apoptosis of enterocytes and nitration of junctional complex proteins promote alcohol-induced gut leakiness and liver injury

  • Young-Eun Cho
    Affiliations
    Section of Molecular Pharmacology & Toxicology, Laboratory of Membrane Biochemistry, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD 20892, USA
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  • Li-Rong Yu
    Affiliations
    Biomarkers and Alternative Models Branch, Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079, USA
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  • Mohamed A. Abdelmegeed
    Affiliations
    Section of Molecular Pharmacology & Toxicology, Laboratory of Membrane Biochemistry, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD 20892, USA
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  • Seong-Ho Yoo
    Affiliations
    Department of Forensic Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea
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  • Byoung-Joon Song
    Correspondence
    Corresponding author. Address: Laboratory of Membrane Biochemistry, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD 20892, USA. Tel.: +1 301 496 3985.
    Affiliations
    Section of Molecular Pharmacology & Toxicology, Laboratory of Membrane Biochemistry, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD 20892, USA
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Published:February 17, 2018DOI:https://doi.org/10.1016/j.jhep.2018.02.005

      Highlights

      • Binge alcohol exposure caused apoptosis of gut enterocytes in rats and WT mice.
      • Binge alcohol exposure reduced the levels of tight junction and adherent junction proteins.
      • Nitration of the junctional proteins led to ubiquitin-dependent degradation.
      • Apoptosis of enterocytes and reduced junctional proteins led to gut leakiness.
      • Binge alcohol-induced gut leakiness was observed in mice, rats and humans.

      Background & Aims

      Binge alcohol exposure causes gut leakiness, contributing to increased endotoxemia and inflammatory liver injury, although the molecular mechanisms are still elusive. This study was aimed at investigating the roles of apoptosis of enterocytes and nitration followed by degradation of intestinal tight junction (TJ) and adherens junction (AJ) proteins in binge alcohol-induced gut leakiness.

      Methods

      The levels of intestinal (ileum) junctional complex proteins, oxidative stress markers and apoptosis-related proteins in rodents, T84 colonic cells and autopsied human ileums were determined by immunoblot, immunoprecipitation, immunofluorescence, and mass-spectral analyses.

      Results

      Binge alcohol exposure caused apoptosis of gut enterocytes with elevated serum endotoxin and liver injury. The levels of intestinal CYP2E1, iNOS, nitrated proteins and apoptosis-related marker proteins were significantly elevated in binge alcohol-exposed rodents. Differential, quantitative mass-spectral analyses of the TJ-enriched fractions of intestinal epithelial layers revealed that several TJ, AJ and desmosome proteins were decreased in binge alcohol-exposed rats compared to controls. Consistently, the levels of TJ proteins (claudin-1, claudin-4, occludin and zonula occludens-1), AJ proteins (β-catenin and E-cadherin) and desmosome plakoglobin were very low in binge alcohol-exposed rats, wild-type mice, and autopsied human ileums but not in Cyp2e1-null mice. Additionally, pretreatment with specific inhibitors of CYP2E1 and iNOS prevented disorganization and/or degradation of TJ proteins in alcohol-exposed T84 colonic cells. Furthermore, immunoprecipitation followed by immunoblot confirmed that intestinal TJ and AJ proteins were nitrated and degraded via ubiquitin-dependent proteolysis, resulting in their decreased levels.

      Conclusions

      These results demonstrated for the first time the critical roles of CYP2E1, apoptosis of enterocytes, and nitration followed by ubiquitin-dependent proteolytic degradation of the junctional complex proteins, in promoting binge alcohol-induced gut leakiness and endotoxemia, contributing to inflammatory liver disease.

      Lay summary

      Binge alcohol exposure causes gut leakiness, contributing to increased endotoxemia and inflammatory liver injury. Our results demonstrated for the first time the critical roles of apoptosis of enterocytes and nitration followed by ubiquitin-dependent proteolytic degradation of the junctional complex proteins in promoting this gut leakiness and endotoxemia. These results provide insight into the molecular mechanisms of alcohol-induced inflammatory liver disease.

      Graphical abstract

      Keywords

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