NIH Research Festival
Background: Elevated endotoxemia via leaky gut is a leading cause of multi-organ damage and death in rodents and humans. However, the underlying mechanisms of leaky gut and liver disease caused by alcohol or other substances such as fructose (contained in many soft drinks) and western-style high fat diets are poorly understood. We hypothesized that ethanol-inducible cytochrome P450-2E1 (CYP2E1)-mediated oxidative stress and decreased gut tight junction (TJ) proteins with increased apoptosis of enterocytes play causal roles in binge alcohol- or fructose-mediated gut leakiness, systemic endotoxemia and inflammatory liver injury. Methods: The levels of oxidative stress markers, ileum junctional complex proteins, and apoptosis-related proteins in rodents, T84 colon cells and autopsied people who died from heavy alcohol intoxication and their respective controls were determined by immunoblot, immunoprecipitation, immunofluorescence, confocal imaging and mass-spectral analyses. Results: Binge alcohol or fructose exposure increased oxidative stress marker proteins and caused apoptosis of gut enterocytes with elevated serum endotoxin and liver inflammation. Differential mass-spectral analyses of the purified TJ-enriched fractions of gut epithelial layers showed that several TJ, adherent junction (AJ) and desmosome proteins were markedly decreased in alcohol-exposed rats. Consistently, the levels of intestinal TJ proteins (e.g., claudin-1, claudin-4, occludin, and ZO-1), AJ proteins (e.g., β-catenin and E-cadherin) and desmosome plakoglobin were very low in alcohol- or fructose-exposed rats, wild-type mice, and autopsied alcoholics compared to those of the respective controls and the alcohol-exposed Cyp2e1-null mice. Pretreatment with specific inhibitors of CYP2E1 and iNOS prevented disorganization and degradation of TJ proteins in alcohol- or fructose-exposed T84 colon cells. Immunoprecipitation followed by immunoblot confirmed that gut TJ and AJ proteins were nitrated and degraded via ubiquitin-dependent proteolysis, resulting in their decreased levels in alcohol- or fructose-exposed rats. Conclusion: These mechanistic results for the first time demonstrate the critical roles of CYP2E1 and nitration of gut TJ/AJ proteins in binge alcohol- or fructose-induced gut leakiness and endotoxemia, contributing to liver inflammation or fibrosis. These results can also explain the molecular mechanisms of gut leakiness with decreased TJ proteins in many other disease states.
Scientific Focus Area: Molecular Pharmacology
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