Role and mechanisms of autophagy in acetaminophen-induced liver injury

Abstract

Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in the USA and many other countries. Although the metabolism and pathogenesis of APAP has been extensively investigated for decades, the mechanisms by which APAP induces liver injury are incompletely known, which hampers the development of effective therapeutic approaches to tackle this important clinical problem. Autophagy is a highly conserved intracellular degradation pathway, which aims at recycling cellular components and damaged organelles in response to adverse environmental conditions and stresses as a survival mechanism. There is accumulating evidence indicating that autophagy is activated in response to APAP overdose in specific liver zone areas, and pharmacological activation of autophagy protects against APAP-induced liver injury. Increasing evidence also suggests that hepatic autophagy is impaired in nonalcoholic fatty livers (NAFLD), and NAFLD patients are more susceptible to APAP-induced liver injury. Here, we summarized the current progress on the role and mechanisms of autophagy in protecting against APAP-induced liver injury.

Keywords: acetaminophen; acetaminophen protein adducts; autophagy; liver injury; mitophagy.

Figure 1. A simplified scheme for the regulation of autophagy in mammalian cells

When nutrients are sufficient, mTORC1 is activated and phosphorylates ULK1/2 and inhibits its activity. In contrast, when cells suffer from insufficient energy, AMPK is activated and phosphorylates ULK1/2 to activate ULK1/2. The active ULK1/2 phosphorylates Beclin-1, leading to VPS34 activation and phagophore formation. ULK1/2 functions in a complex with FIP200, Atg13 and Atg101, whereas VPS34 function requires a regulatory subunit, p150 and Beclin-1. Nascent LC3 is first processed by a protease, Atg4, to expose its C terminus glycine to form LC3-I form. LC3-I is then further activated by E1-like enzyme, Atg7, then transferred to an E2-like conjugating enzyme, Atg3. Finally, LC3 conjugates with PE to form a membrane associated LC3-II form, with the Atg12-Atg5-Atg16L1 complex participating as an E3-like ligase. Atg9-mediated cycling systems containing the core protein Atg9, Atg2 and WIPI1/2 contributes to the elongation of the phagophore by delivering membrane from donor sources. The closure of an elongated phagophore marks the formation of a mature autophagosome, which eventually fuses with a lysosome, leading to cargo degradation. GTPase Rab7 and some components of the SNARE family proteins, such as VAMP7, VAMP8, VAMP9 and STX17, and lysosomal-associated membrane protein 1/2 are implicated in autophagosome fusion with lysosomes.

Figure 2. A proposed model for the mechanisms underlying the protection of autophagy against acetaminophen hepatotoxicity

APAP is first metabolized by cytochrome P450 enzymes (mainly via Cyp2E1) to generate the reactive metabolite NAPQI, which depletes hepatic GSH and binds to cellular and mitochondrial proteins to form APAP-Adducts (APAP-AD) resulting in a mitochondrial oxidant stress. The increasing oxidative stress and mitochondrial protein adducts trigger mitochondrial damage. Damaged mitochondria can lead to necrotic cell death, inflammation and subsequent liver injury. p62, an autophagy receptor protein, is recruited to APAP-AD, which may facilitate APAP-AD transition to the detergent insoluble form and allow their recognition and sequestration by autophagosomes and eventual removal by autolysosomes. Damaged mitochondria could be removed through PINK1-Parkin mediated selective mitophagy. Damaged mitochondria can stabilize mitochondrial PINK1 that recruits Parkin to mitochondria to trigger the ubiquitination of mitochondrial proteins. The mitochondrial ubiquitination-p62-LC3 complex promotes the recognition and removal of damaged mitochondria by mitophagy although the role of p62 in the APAP context has not been determined. In addition to PINK1/Parkin pathway, several other mitochondrial outer membrane proteins (FUNDC1, Bnip3 and Nix) may directly interact with LC3 through their LC3 interacting region to mediate mitophagy independent of PINK1 and Parkin, but their roles in APAP-induced mitophagy have not been studied. Autophagy can suppress inflammation and inflammasome activation by removing damage mitochondrial and mitochondrial DNA. Torin 1 or rapamycin can target the ULK1 complex to induce autophagy to protect against APAP-induced liver injury via removal of APAP-adducts and damaged mitochondria. In contrast, 3-MA inhibits VPS34 complex to inhibit the upstream autophagosome formation whereas Leupeptin or CQ impairs autolysosome/lysosome functions leading to the exacerbation of APAP-induced necrosis and liver injury, respectively.