H2O2, the major reactive oxygen species produced during alcohol metabolism induces autophagy without involving mTOR

Background and Aims:

Alcohol-mediated reactive oxygen species (ROS) formation in the liver, mainly H₂O₂, contributes to disease progression and eventually hepatocellular carcinoma development in patients with ALD. Enhancement or activation of autophagy, with the suppression of mTOR signaling, is likely to play an important role in early stages of the alcoholic liver disease (ALD). However, with the progression of the disease, the expression of mTOR increases dramatically leading to the suppression of autophagy. According to recent literature, this is accompanied with significant hepatic lipid accumulation and iron deposition as well as inflammation under persistent alcohol exposure. It is also known, that H₂O₂ is involved in the regulation of autophagy in both acute and chronic ALD models, however, the exact underlying molecular mechanisms are still unclear. Therefore, we investigated in vitro and in vivo by using alcohol mouse model alterations in mTOR signaling as well as downstream effects induced by H₂O₂ and low oxygen tension.

Methods:

Huh7 cells were cultured with the GOX/CAT system, which allows an independent control of hydrogen peroxide as well as oxygen levels, in combination with different doses of ethanol. LC3B, p62, mTOR and autophagy related proteins were analyzed by western blot. Same analyses were performed in liver tissues of C57BL/6 mice treated with acute (alcohol binge) and chronic ethanol (20% ethanol in the drinking water) for 4 weeks (n = 4).

Results:

H₂O₂ significantly increased LC3B activation and this effect could be efficiently blocked by N-acetyl cysteine (NAC), which is a ROS scavenger. Interestingly, even though the LC3B activation was increased by H₂O₂, the m-TOR expression was not suppressed as normally expected. Co-treatment of cells with H₂O₂ and the mTOR inhibitor Rapamycin led to an increased autophagic flux as compared to single treatment. The in vivo experiments showed a combined activation of LC3B and suppressed p62 and AKT levels as well as enhanced p-AMPK expression in the livers of the acute alcohol group. In contrast, mice exposed to chronic alcohol showed blocked autophagic flux with dramatically increased LC3-I and p62 levels.

Conclusion:

Our findings underscore an important role of H₂O₂ in regulating autophagy during acute and chronic alcohol ALD exposure. Further studies will be needed to to identify H₂O₂-induced signaling pathways that regulate autophagy.