Autophagy can selectively remove damaged organelles, including mitochondria, and, in turn, protect against mitochondria‐damage–induced cell death. Acetaminophen (APAP) overdose can cause liver injury ...in animals and humans by inducing mitochondria damage and subsequent necrosis in hepatocytes. Although many detrimental mechanisms have been reported to be responsible for APAP‐induced hepatotoxicity, it is not known whether APAP can modulate autophagy to regulate hepatotoxicity in hepatocytes. To test the hypothesis that autophagy may play a critical protective role against APAP‐induced hepatotoxicity, primary cultured mouse hepatocytes and green fluorescent protein/light chain 3 transgenic mice were treated with APAP. By using a series of morphological and biochemical autophagic flux assays, we found that APAP induced autophagy both in the in vivo mouse liver and in primary cultured hepatocytes. We also found that APAP treatment might suppress mammalian target of rapamycin in hepatocytes and that APAP‐induced autophagy was suppressed by N‐acetylcysteine, suggesting APAP mitochondrial protein binding and the subsequent production of reactive oxygen species may play an important role in APAP‐induced autophagy. Pharmacological inhibition of autophagy by 3‐methyladenine or chloroquine further exacerbated APAP‐induced hepatotoxicity. In contrast, induction of autophagy by rapamycin inhibited APAP‐induced hepatotoxicity. Conclusion: APAP overdose induces autophagy, which attenuates APAP‐induced liver cell death by removing damaged mitochondria. (HEPATOLOGY 2012)
Mitochondria are cellular energy powerhouses that play important roles in maintaining cell survival, cell death and cellular metabolic homeostasis. Timely removal of damaged mitochondria via ...autophagy (mitophagy) is thus critical for cellular homeostasis and function. Mitochondria are reticular organelles that have high plasticity for their dynamic structures and constantly undergo fission and fusion as well as movement through the cytoskeleton. In this review, we discuss the most recent progress on the molecular mechanisms and roles of mitochondrial fission/fusion and mitochondrial motility in mitophagy. We also discuss multiple pathways leading to the quality control of mitochondria in addition to the traditional mitophagy pathway under different conditions.
Acetaminophen (APAP) overdose is one of the leading causes of hepatotoxicity and acute liver failure in the United States. Accumulating evidence suggests that hepatocyte necrosis plays a critical ...role in APAP‐induced liver injury (AILI). However, the mechanisms of APAP‐induced necrosis and liver injury are not fully understood. In this study, we found that p53 up‐regulated modulator of apoptosis (PUMA), a B‐cell lymphoma‐2 (Bcl‐2) homology domain 3 (BH3)‐only Bcl‐2 family member, was markedly induced by APAP in mouse livers and in isolated human and mouse hepatocytes. PUMA deficiency suppressed APAP‐induced mitochondrial dysfunction and release of cell death factors from mitochondria, and protected against APAP‐induced hepatocyte necrosis and liver injury in mice. PUMA induction by APAP was p53 independent, and required receptor‐interacting protein kinase 1 (RIP1) and c‐Jun N‐terminal kinase (JNK) by transcriptional activation. Furthermore, a small‐molecule PUMA inhibitor, administered after APAP treatment, mitigated APAP‐induced hepatocyte necrosis and liver injury. Conclusion: Our results demonstrate that RIP1/JNK‐dependent PUMA induction mediates AILI by promoting hepatocyte mitochondrial dysfunction and necrosis, and suggest that PUMA inhibition is useful for alleviating acute hepatotoxicity attributed to APAP overdose.
Acetaminophen (APAP) overdose is a major cause of hepatotoxicity and acute liver failure in the U.S., but the pathophysiology is incompletely understood. Despite evidence for apoptotic signaling, ...hepatic cell death after APAP is generally considered necrotic in mice and in humans. Recent findings suggest that the receptor interacting protein kinase 3 (RIP3) acts as a switch from apoptosis to necrosis (programmed necrosis). Thus, the aim of the current investigation was to determine if RIP3 is involved in APAP‐induced liver cell death. APAP (200‐300 mg/kg) caused glutathione depletion and protein adduct formation, oxidant stress, mitochondrial release of apoptosis inducing factor, and nuclear DNA fragmentation resulting in centrilobular necrosis in C57Bl/6J mice. Inhibiting RIP3 protein induction with antisense morpholinos in wild‐type animals or using RIP3‐deficient mice had no effect on protein adduct formation but attenuated all other parameters, including necrotic cell death, at 6 hours after APAP. In addition, cultured hepatocytes from RIP3‐deficient mice showed reduced injury compared to wild‐type cells after 24 hours. Interestingly, APAP‐induced mitochondrial translocation of dynamin‐related protein 1 (Drp1), the initiator of mitochondrial fission, was inhibited by reduced RIP3 protein expression and the Drp1 inhibitor MDIVI reduced APAP‐induced cell death at 24 hours. All of these protective effects were lost after 24 hours in vivo or 48 hours in vitro. Conclusion: RIP3 is an early mediator of APAP hepatotoxicity, involving modulation of mitochondrial dysfunction and oxidant stress. Controlling RIP3 expression could be a promising new approach to reduce APAP‐induced liver injury, but requires complementary strategies to control mitochondrial dysfunction for long‐term protection. (Hepatology 2013; 58:2099–2108)
Bile acid synthesis plays a key role in regulating whole body cholesterol homeostasis. Transcriptional factor EB (TFEB) is a nutrient and stress-sensing transcriptional factor that promotes lysosomal ...biogenesis. Here we report a role of TFEB in regulating hepatic bile acid synthesis. We show that TFEB induces cholesterol 7α-hydroxylase (CYP7A1) in human hepatocytes and mouse livers and prevents hepatic cholesterol accumulation and hypercholesterolemia in Western diet-fed mice. Furthermore, we find that cholesterol-induced lysosomal stress feed-forward activates TFEB via promoting TFEB nuclear translocation, while bile acid-induced fibroblast growth factor 19 (FGF19), acting via mTOR/ERK signaling and TFEB phosphorylation, feedback inhibits TFEB nuclear translocation in hepatocytes. Consistently, blocking intestinal bile acid uptake by an apical sodium-bile acid transporter (ASBT) inhibitor decreases ileal FGF15, enhances hepatic TFEB nuclear localization and improves cholesterol homeostasis in Western diet-fed mice. This study has identified a TFEB-mediated gut-liver signaling axis that regulates hepatic cholesterol and bile acid homeostasis.
Autophagy is a lysosomal degradation process that degrades long-lived cellular proteins and damaged organelles as a critical cell survival mechanism in response to stress. We recently reported that ...acute ethanol induces autophagy, which then reduces ethanol-induced liver injury. However, the mechanisms by which ethanol induces autophagy are not known. In the present study, ethanol treatment significantly increased both mRNA and protein levels of various essential autophagy-related genes in primary cultured mouse hepatocytes and in mouse liver. Both nuclear translocation of FoxO3a and expression of FoxO3a target genes were increased in ethanol-treated primary hepatocytes and mouse liver. Overexpression of a dominant negative form of FoxO3a inhibited ethanol-induced autophagy-related gene expression and enhanced ethanol-induced cell death in primary hepatocytes, which suggests that FoxO3a is a key factor in regulating ethanol-induced autophagy and cell survival. Resveratrol, a well-known SIRT1 agonist, further enhanced ethanol-induced expression of autophagy-related genes, likely via increased deacetylation of FoxO3a. Moreover, acute ethanol–treated Foxo3a −/− mice exhibited decreased autophagy-related gene expression, but enhanced steatosis and liver injury, compared with wild-type mice. FoxO3a thus plays a critical role in ethanol-induced autophagy in mouse liver. Modulating the FoxO3a autophagy pathway may offer novel therapeutic approaches for treating alcoholic liver pathogenesis.
Defects in lysosome function and autophagy contribute to the pathogenesis of alcoholic liver disease. We investigated the mechanisms by which alcohol consumption affects these processes by evaluating ...the functions of transcription factor EB (TFEB), which regulates lysosomal biogenesis.
We performed studies with GFP-LC3 mice, mice with liver-specific deletion of TFEB, mice with disruption of the transcription factor E3 gene (TFE3-knockout mice), mice with disruption of the Tefb and Tfe3 genes (TFEB and TFE3 double-knockout mice), and Tfebflox/flox albumin cre-negative mice (controls). TFEB was overexpressed from adenoviral vectors or knocked down with small interfering RNAs in mouse livers. Mice were placed on diets of regular ethanol feeding plus an acute binge to induce liver damage (ethanol diet); some mice also were given injections of torin-1, an inhibitor of the kinase activity of the mechanistic target of rapamycin (mTOR). Liver tissues were collected and analyzed by immunohistochemistry, immunoblots, and quantitative real-time polymerase chain reaction to monitor lysosome biogenesis. We analyzed levels of TFEB in liver tissues from patients with alcoholic hepatitis and from healthy donors (controls) by immunohistochemistry.
Liver tissues from mice on the ethanol diet had lower levels of total and nuclear TFEB compared with control mice, and hepatocytes had decreased lysosome biogenesis and autophagy. Hepatocytes from mice on the ethanol diet had increased translocation of mTOR into lysosomes, resulting in increased mTOR activation. Administration of torin-1 increased liver levels of TFEB and decreased steatosis and liver injury induced by ethanol. Mice that overexpressed TFEB in the liver developed less severe ethanol-induced liver injury and had increased lysosomal biogenesis and mitochondrial bioenergetics compared with mice carrying a control vector. Mice with knockdown of TFEB and TFEB-TFE3 double-knockout mice developed more severe liver injury in response to the ethanol diet than control mice. Liver tissues from patients with alcohol-induced hepatitis had lower nuclear levels of TFEB than control tissues.
We found that ethanol feeding plus an acute binge decreased hepatic expression of TFEB, which is required for lysosomal biogenesis and autophagy. Strategies to block mTOR activity or increase levels of TFEB might be developed to protect the liver from ethanol-induced damage.
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Increased megamitochondria formation and impaired mitophagy in hepatocytes have been linked to the pathogenesis of alcohol-associated liver disease (ALD). This study aims to determine the mechanisms ...by which alcohol consumption increases megamitochondria formation in the pathogenesis of ALD.
Human alcoholic hepatitis (AH) liver samples were used for electron microscopy, histology, and biochemical analysis. Liver-specific dynamin-related protein 1 (DRP1; gene name DNM1L, an essential gene regulating mitochondria fission ) knockout (L-DRP1 KO) mice and wild-type mice were subjected to chronic plus binge alcohol feeding. Both human AH and alcohol-fed mice had decreased hepatic DRP1 with increased accumulation of hepatic megamitochondria. Mechanistic studies revealed that alcohol feeding decreased DRP1 by impairing transcription factor EB-mediated induction of DNM1L . L-DRP1 KO mice had increased megamitochondria and decreased mitophagy with increased liver injury and inflammation, which were further exacerbated by alcohol feeding. Seahorse flux and unbiased metabolomics analysis showed alcohol intake increased mitochondria oxygen consumption and hepatic nicotinamide adenine dinucleotide (NAD + ), acylcarnitine, and ketone levels, which were attenuated in L-DRP1 KO mice, suggesting that loss of hepatic DRP1 leads to maladaptation to alcohol-induced metabolic stress. RNA-sequencing and real-time quantitative PCR analysis revealed increased gene expression of the cGAS-stimulator of interferon genes (STING)-interferon pathway in L-DRP1 KO mice regardless of alcohol feeding. Alcohol-fed L-DRP1 KO mice had increased cytosolic mtDNA and mitochondrial dysfunction leading to increased activation of cGAS-STING-interferon signaling pathways and liver injury.
Alcohol consumption decreases hepatic DRP1 resulting in increased megamitochondria and mitochondrial maladaptation that promotes AH by mitochondria-mediated inflammation and cell injury.
Autophagy is a lysosomal degradation pathway that degrades cytoplasmic proteins and organelles. Absence of autophagy in hepatocytes has been linked to promoting liver injury and tumorigenesis; ...however, the mechanisms behind why a lack of autophagy induces these complications are not fully understood. The role of mammalian target of rapamycin (mTOR) in impaired autophagy‐induced liver pathogenesis and tumorigenesis was investigated by using liver‐specific autophagy related 5 knockout (L‐ATG5 KO) mice, L‐ATG5/mTOR, and L‐ATG5/Raptor double knockout (DKO) mice. We found that deletion of mTOR or Raptor in L‐ATG5 KO mice at 2 months of age attenuated hepatomegaly, cell death, and inflammation but not fibrosis. Surprisingly, at 6 months of age, L‐ATG5/mTOR DKO and L‐ATG5/Raptor DKO mice also had increased hepatic inflammation, fibrosis, and liver injury, similar to the L‐ATG5 KO mice. Moreover, more than 50% of L‐ATG5/mTOR DKO and L‐ATG5/Raptor DKO mice already developed spontaneous tumors, but none of the L‐ATG5 KO mice had developed any tumors at 6 months of age. At 9 months of age, all L‐ATG5/mTOR DKO and L‐ATG5/Raptor DKO had developed liver tumors. Mechanistically, L‐ATG5/mTOR DKO and L‐ATG5/Raptor DKO mice had decreased levels of hepatic ubiquitinated proteins and persistent nuclear erythroid 2 p45‐related factor 2 activation but had increased Akt activation compared with L‐ATG5 KO mice. Conclusion: Loss of mTOR signaling attenuates the liver pathogenesis in mice with impaired hepatic autophagy but paradoxically promotes tumorigenesis in mice at a relatively young age. Therefore, the balance of mTOR is critical in regulating the liver pathogenesis and tumorigenesis in mice with impaired hepatic autophagy.
Alcoholic liver disease claims two million lives per year. We previously reported that autophagy protected against alcohol-induced liver injury and steatosis by removing damaged mitochondria. ...However, the mechanisms for removal of these mitochondria are unknown. Parkin is an evolutionarily conserved E3 ligase that is recruited to damaged mitochondria to initiate ubiquitination of mitochondrial outer membrane proteins and subsequent mitochondrial degradation by mitophagy. In addition to its role in mitophagy, Parkin has been shown to have other roles in maintaining mitochondrial function. We investigated whether Parkin protected against alcohol-induced liver injury and steatosis using wild-type (WT) and Parkin knockout (KO) mice treated with alcohol by the acute-binge and Gao-binge (chronic plus acute-binge) models. We found that Parkin protected against liver injury in both alcohol models, likely because of Parkin's role in maintaining a population of healthy mitochondria. Alcohol caused greater mitochondrial damage and oxidative stress in Parkin KO livers compared with WT livers. After alcohol treatment, Parkin KO mice had severely swollen and damaged mitochondria that lacked cristae, which were not seen in WT mice. Furthermore, Parkin KO mice had decreased mitophagy, β-oxidation, mitochondrial respiration, and cytochrome c oxidase activity after acute alcohol treatment compared with WT mice. Interestingly, liver mitochondria seemed able to adapt to alcohol treatment, but Parkin KO mouse liver mitochondria had less capacity to adapt to Gao-binge treatment compared with WT mouse liver mitochondria. Overall, our findings indicate that Parkin is an important mediator of protection against alcohol-induced mitochondrial damage, steatosis, and liver injury.
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