Mitochondrial dynamics, quality control, and mtDNA in alcohol-associated liver disease and liver cancer.

Mitochondria are intracellular organelles responsible for energy production, glucose and lipid metabolism, cell death, cell proliferation, and innate immune response. Mitochondria are highly dynamic organelles that constantly undergo fission, fusion, and intracellular trafficking, as well as degradation and biogenesis. Mitochondrial dysfunction has been implicated in a variety of chronic liver diseases including alcohol-associated liver disease, metabolic dysfunction-associated steatohepatitis, and HCC. In this review, we provide a detailed overview of mitochondrial dynamics, mitophagy, and mitochondrial DNA-mediated innate immune response, and how dysregulation of these mitochondrial processes affects the pathogenesis of alcohol-associated liver disease and HCC. Mitochondrial dynamics and mitochondrial DNA-mediated innate immune response may thereby represent an attractive therapeutic target for ameliorating alcohol-associated liver disease and alcohol-associated HCC.

SQSTM1/p62 and Hepatic Mallory-Denk Body Formation in Alcohol-Associated Liver Disease.

Sequestosome 1 (SQSTM1/p62; hereafter p62) is an autophagy receptor protein for selective autophagy primarily due to its direct interaction with the microtubule light chain 3 protein that specifically localizes on autophagosome membranes. As a result, impaired autophagy leads to the accumulation of p62. p62 is also a common component of many human liver disease-related cellular inclusion bodies, such as Mallory-Denk bodies, intracytoplasmic hyaline bodies, α1-antitrypsin aggregates, as well as p62 bodies and condensates. p62 also acts as an intracellular signaling hub, and it involves multiple signaling pathways, including nuclear factor erythroid 2-related factor 2, NF-κB, and the mechanistic target of rapamycin, which are critical for oxidative stress, inflammation, cell survival, metabolism, and liver tumorigenesis. This review discusses the recent insights of p62 in protein quality control, including the role of p62 in the formation and degradation of p62 stress granules and protein aggregates as well as regulation of multiple signaling pathways in the pathogenesis of alcohol-associated liver disease.

Loss of hepatic DRP1 exacerbates alcoholic hepatitis by inducing megamitochondria and mitochondrial maladaptation.

BACKGROUND AND AIMS: 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. APPROACH AND RESULTS: 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. CONCLUSION: Alcohol consumption decreases hepatic DRP1 resulting in increased megamitochondria and mitochondrial maladaptation that promotes AH by mitochondria-mediated inflammation and cell injury.

Persistent mTORC1 activation due to loss of liver tuberous sclerosis complex 1 promotes liver injury in alcoholic hepatitis.

BACKGROUND AND AIMS: The aim of the study was to investigate the role and mechanisms of tuberous sclerosis complex 1 (TSC1) and mechanistic target of rapamycin complex 1 (mTORC1) in alcohol-associated liver disease. APPROACH AND RESULTS: Liver-specific Tsc1 knockout (L- Tsc1 KO) mice and their matched wild-type mice were subjected to Gao-binge alcohol. Human alcoholic hepatitis (AH) samples were also used for immunohistochemistry staining, western blot, and quantitative real-time PCR (q-PCR) analysis. Human AH and Gao-binge alcohol-fed mice had decreased hepatic TSC1 and increased mTORC1 activation. Gao-binge alcohol markedly increased liver/body weight ratio and serum alanine aminotransferase levels in L- Tsc1 KO mice compared with Gao-binge alcohol-fed wild-type mice. Results from immunohistochemistry staining, western blot, and q-PCR analysis revealed that human AH and Gao-binge alcohol-fed L- Tsc1 KO mouse livers had significantly increased hepatic progenitor cells, macrophages, and neutrophils but decreased HNF4α-positive cells. Gao-binge alcohol-fed L- Tsc1 KO mice also developed severe inflammation and liver fibrosis. Deleting Tsc1 in cholangiocytes but not in hepatocytes promoted cholangiocyte proliferation and aggravated alcohol-induced ductular reactions, fibrosis, inflammation, and liver injury. Pharmacological inhibition of mTORC1 partially reversed hepatomegaly, ductular reaction, fibrosis, inflammatory cell infiltration, and liver injury in alcohol-fed L- Tsc1 KO mice. CONCLUSIONS: Our findings indicate that persistent activation of mTORC1 due to the loss of cholangiocyte TSC1 promotes liver cell repopulation, ductular reaction, inflammation, fibrosis, and liver injury in Gao-binge alcohol-fed L- Tsc1 KO mice, which phenocopy the pathogenesis of human AH.

TRIM21 Ubiquitylates SQSTM1/p62 and Suppresses Protein Sequestration to Regulate Redox Homeostasis.

TRIM21 is a RING finger domain-containing ubiquitin E3 ligase whose expression is elevated in autoimmune disease. While TRIM21 plays an important role in immune activation during pathogen infection, little is known about its inherent cellular function. Here we show that TRIM21 plays an essential role in redox regulation by directly interacting with SQSTM1/p62 and ubiquitylating p62 at lysine 7 (K7) via K63-linkage. As p62 oligomerizes and sequesters client proteins in inclusions, the TRIM21-mediated p62 ubiquitylation abrogates p62 oligomerization and sequestration of proteins including Keap1, a negative regulator of antioxidant response. TRIM21-deficient cells display an enhanced antioxidant response and reduced cell death in response to oxidative stress. Genetic ablation of TRIM21 in mice confers protection from oxidative damages caused by arsenic-induced liver insult and pressure overload heart injury. Therefore, TRIM21 plays an essential role in p62-regulated redox homeostasis and may be a viable target for treating pathological conditions resulting from oxidative damage.

Loss of Hepatic Transcription Factor EB Attenuates Alcohol-Associated Liver Carcinogenesis.

Alcohol is a well-known risk factor for hepatocellular carcinoma. Autophagy plays a dual role in liver cancer, as it suppresses tumor initiation and promotes tumor progression. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy, which is impaired in alcohol-related liver disease. However, the role of TFEB in alcohol-associated liver carcinogenesis is unknown. Liver-specific Tfeb knockout (KO) mice and their matched wild-type (WT) littermates were injected with the carcinogen diethylnitrosamine (DEN), followed by chronic ethanol feeding. The numbers of both total and larger tumors increased significantly in DEN-treated mice fed ethanol diet than in mice fed control diet. Although the number of tumors was not different between WT and L-Tfeb KO mice fed either control or ethanol diet, the number of larger tumors was less in L-Tfeb KO mice than in WT mice. No differences were observed in liver injury, steatosis, inflammation, ductular reaction, fibrosis, and tumor cell proliferation in DEN-treated mice fed ethanol. However, the levels of glypican 3, a marker of malignant hepatocellular carcinoma, markedly decreased in DEN-treated L-Tfeb KO mice fed ethanol in comparison to the WT mice. These findings indicate that chronic ethanol feeding promotes DEN-initiated liver tumor development, which is attenuated by genetic deletion of hepatic TFEB.

Role of mechanistic target of rapamycin in autophagy and alcohol-associated liver disease.

Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase and a cellular sensor for nutrient and energy status, which is critical in regulating cell metabolism and growth by governing the anabolic (protein and lipid synthesis) and catabolic process (autophagy). Alcohol-associated liver disease (ALD) is a major chronic liver disease worldwide that carries a huge financial burden. The spectrum of the pathogenesis of ALD includes steatosis, fibrosis, inflammation, ductular reaction, and eventual hepatocellular carcinoma, which is closely associated with metabolic changes that are regulated by mTOR. In this review, we summarized recent progress of alcohol consumption on the changes of mTORC1 and mTORC2 activity, the potential mechanisms and possible impact of the mTORC1 changes on autophagy in ALD. We also discussed the potential beneficial effects and limitations of targeting mTORC1 against ALD.

Hepatocytic p62 suppresses ductular reaction and tumorigenesis in mouse livers with mTORC1 activation and defective autophagy.

BACKGROUND & AIMS: Either activation of mTORC1 due to loss of Tsc1 (tuberous sclerosis complex 1) or defective hepatic autophagy due to loss of Atg5 leads to spontaneous liver tumorigenesis in mice. The purpose of this study was to investigate the mechanisms by which autophagy contributes to the hepatic metabolic changes and tumorigenesis mediated by mTORC1 activation. METHODS: Atg5 Flox/Flox (Atg5F/F) and Tsc1F/F mice were crossed with albumin-Cre mice to generate liver-specific Atg5 knockout (L-Atg5 KO), L-Tsc1 KO and L-Atg5/Tsc1 double KO (DKO) mice. These mice were crossed with p62/Sqstm1F/F (p62) and whole body Nrf2 KO mice to generate L-Atg5/Tsc1/p62 and L-Atg5/Tsc1-Nrf2 triple KO mice. These mice were housed for various periods up to 12 months, and blood and liver tissues were harvested for biochemical and histological analysis RESULTS: Deletion of Atg5 in L-Tsc1 KO mice inhibited liver tumorigenesis but increased mortality and was accompanied by drastically enhanced hepatic ductular reaction (DR), hepatocyte degeneration and metabolic reprogramming. Deletion of p62 reversed DR, hepatocyte degeneration and metabolic reprogramming as well as the mortality of L-Atg5/Tsc1 DKO mice, but unexpectedly promoted liver tumorigenesis via activation of a group of oncogenic signaling pathways. Nrf2 ablation markedly improved DR with increased hepatocyte population and improved metabolic reprogramming and survival of the L-Atg5/Tsc1 DKO mice without tumor formation. Decreased p62 and increased mTOR activity were also observed in a subset of human hepatocellular carcinomas. CONCLUSIONS: These results reveal previously undescribed functions of hepatic p62 in suppressing tumorigenesis and regulating liver cell repopulation and metabolic reprogramming resulting from persistent mTORC1 activation and defective autophagy. LAY SUMMARY: Metabolic liver disease and viral hepatitis are common chronic liver diseases and risk factors of hepatocellular carcinoma, which are often associated with impaired hepatic autophagy and increased mTOR activation. Using multiple genetically engineered mouse models of defective hepatic autophagy and persistent mTOR activation, we dissected the complex mechanisms behind this observation. Our results uncovered an unexpected novel tumor suppressor function of p62/Sqstm1, which regulated liver cell repopulation, ductular reaction and metabolic reprogramming in liver tumorigenesis.

Lack of VMP1 impairs hepatic lipoprotein secretion and promotes non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Vacuole membrane protein 1 (VMP1) is an endoplasmic reticulum (ER) transmembrane protein that regulates the formation of autophagosomes and lipid droplets. Recent evidence suggests that VMP1 plays a critical role in lipoprotein secretion in zebra fish and cultured cells. However, the pathophysiological roles and mechanisms by which VMP1 regulates lipoprotein secretion and lipid accumulation in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are unknown. METHODS: Liver-specific and hepatocyte-specific Vmp1 knockout mice as well as Vmp1 knock-in mice were generated by crossing Vmp1flox or Vmp1KI mice with albumin-Cre mice or by injecting AAV8-TBG-cre, respectively. Lipid and energy metabolism in these mice were characterized by metabolomic and transcriptome analyses. Mice with hepatic overexpression of VMP1 who were fed a NASH diet were also characterized. RESULTS: Hepatocyte-specific deletion of Vmp1 severely impaired VLDL secretion resulting in massive hepatic steatosis, hepatocyte death, inflammation and fibrosis, which are hallmarks of NASH. Mechanistically, loss of Vmp1 led to decreased hepatic levels of phosphatidylcholine and phosphatidylethanolamine as well as to changes in phospholipid composition. Deletion of Vmp1 in mouse liver also led to the accumulation of neutral lipids in the ER bilayer and impaired mitochondrial beta-oxidation. Overexpression of VMP1 ameliorated steatosis in diet-induced NASH by improving VLDL secretion. Importantly, we also showed that decreased liver VMP1 is associated with NAFLD/NASH in humans. CONCLUSIONS: Our results provide novel insights on the role of VMP1 in regulating hepatic phospholipid synthesis and lipoprotein secretion in the pathogenesis of NAFLD/NASH. LAY SUMMARY: Non-alcoholic fatty liver disease and its more severe form, non-alcoholic steatohepatitis, are associated with a build-up of fat in the liver (steatosis). However, the exact mechanisms that underly steatosis in patients are not completely understood. Herein, the authors identified that the lack of a protein called VMP1 impairs the secretion and metabolism of fats in the liver and could therefore contribute to the development and progression of non-alcoholic fatty liver disease.

Role of Mechanistic Target of Rapamycin and Autophagy in Alcohol-Induced Adipose Atrophy and Liver Injury.

Chronic alcohol consumption induces adipose tissue atrophy. However, the mechanisms for how alcohol induces lipodystrophy and its impact on liver steatosis and injury are not fully elucidated. Autophagy is a highly conserved lysosomal degradation pathway, which regulates cellular homeostasis. Mice with autophagy deficiency in adipose tissue have impaired adipogenesis. However, whether autophagy plays a role in alcohol-induced adipose atrophy and how altered adipocyte autophagy contributes to alcohol-induced liver injury remain unclear. To determine the role of adipose autophagy and mechanistic target of rapamycin (mTOR) in alcohol-induced adipose and liver pathogenesis, we generated adipocyte-specific Atg5 knockout (KO), adipocyte-specific mTOR KO, adipocyte-specific Raptor KO, and adipocyte-specific tuberous sclerosis complex 1 KO mice by crossing floxed mice with Adipoq-Cre. The KO mice and their matched wild-type mice were challenged with chronic-plus-binge alcohol mouse model. Chronic-plus-binge alcohol induced adipose atrophy with increased autophagy and decreased Akt/mTOR signaling in epididymal adipose tissue in wild-type mice. Adipocyte-specific Raptor KO mice experienced exacerbated alcohol-induced steatosis, but neither adipocyte-specific mTOR nor adipocyte-specific tuberous sclerosis complex 1 KO mice exhibited similar detrimental effects. Adipocyte-specific Atg5 KO mice had increased circulating levels of fibroblast growth factor 21 and adiponectin and were resistant to alcohol-induced adipose atrophy and liver injury. In conclusion, autophagy deficiency in adipose tissue leads to reduced sensitivity to alcohol-induced adipose atrophy, which ameliorates alcohol-induced liver injury in mice.

Trehalose activates hepatic transcription factor EB (TFEB) but fails to ameliorate alcohol-impaired TFEB and liver injury in mice.

BACKGROUND: Recent evidence demonstrates that alcohol activates the mechanistic target of rapamycin (mTOR) and impairs hepatic transcription factor EB (TFEB) reducing autophagy and contributing to alcohol-induced liver injury. Trehalose, a disaccharide, activates TFEB and protects against diet-induced nonalcoholic fatty liver disease in mice. The aim of the present study was to investigate whether trehalose would reverse the impairment of TFEB induced by alcohol and protect against alcohol-induced liver injury. METHODS: Male C57BL/6J mice were subjected to chronic-plus-binge (Gao-binge) alcohol feeding with and without trehalose supplementation. Some mice were also administrered Alda-1, an aldehyde dehydrogenase 2 agonist. RESULTS: We found that Alda-1 did not affect Gao-binge alcohol-induced mTOR activation and impaired TFEB in mouse livers. Trehalose increased TFEB nuclear translocation, elevated levels of LC3-II and lysosomal proteins in mouse livers and cultured AML12 cells, confirming the activation of TFEB by trehalose. However, trehalose did not improve the impairment in TFEB induced by Gao-binge alcohol. Both Alda-1 and trehalose failed to protect against Gao-binge alcohol-induced steatosis and liver injury, based on the serum levels of alanine aminotransferase (ALT), histological analysis, and levels of hepatic triglyceride. Interestingly, trehalose increased expression of pro-inflammatory genes in mouse macrophage RAW264.7 cells and slightly increased the infiltration of hepatic neutrophils and inflammatory cytokine gene expression in Gao-binge alcohol-fed mice livers. CONCLUSIONS: Trehalose fails to improve the impaired TFEB induced by Gao-binge alcohol and does not protect against alcohol-induced liver injury.

Gut-restricted apical sodium-dependent bile acid transporter inhibitor attenuates alcohol-induced liver steatosis and injury in mice.

BACKGROUND: Recent studies have shown that human and experimental alcohol-related liver disease (ALD) is robustly associated with dysregulation of bile acid homeostasis, which may in turn modulate disease severity. Pharmacological agents targeting bile acid metabolism and signaling may be potential therapeutics for ALD. METHODS: The potential beneficial effects of a gut-restricted apical sodium-dependent bile acid transporter (ASBT) inhibitor were studied in a chronic-plus-binge ALD mouse model. RESULTS: Blocking intestinal bile acid reabsorption by the gut-restricted ASBT inhibitor GSK2330672 attenuated hepatic steatosis and liver injury in a chronic-plus-binge ALD mouse model. Alcohol feeding is associated with intestinal bile acid accumulation but paradoxically impaired ileal farnesoid × receptor (FXR) function, and repressed hepatic cholesterol 7α-hydrolase (CYP7A1) expression despite decreased hepatic small heterodimer partner (SHP) and ileal fibroblast growth factor 15 (FGF15) expression. ASBT inhibitor treatment decreased intestinal bile acid accumulation and increased hepatic CYP7A1 expression, but further decreased ileal FXR activity. Alcohol feeding induces serum bile acid concentration that strongly correlates with a liver injury marker. However, alcohol-induced serum bile acid elevation is not due to intrahepatic bile acid accumulation but is strongly and positively associated with hepatic multidrug resistance-associated protein 3 (MRP4) and MRP4 induction but poorly associated with sodium-taurocholate cotransporting peptide (NTCP) expression. ASBT inhibitor treatment decreases serum bile acid concentration without affecting hepatocyte basolateral bile acid uptake and efflux transporters. CONCLUSION: ASBT inhibitor treatment corrects alcohol-induced bile acid dysregulation and attenuates liver injury in experimental ALD.

Impaired protein adduct removal following repeat administration of subtoxic doses of acetaminophen enhances liver injury in fed mice.

Acetaminophen (APAP) is a widely used analgesic and is safe at therapeutic doses. However, an overdose of APAP is hepatotoxic and accidental overdoses are increasingly common due to the presence of APAP in several combination medications. Formation of protein adducts (APAP-CYS) is central to APAP-induced liver injury and their removal by autophagy is an essential adaptive response after an acute overdose. Since the typical treatment for conditions such as chronic pain involves multiple doses of APAP over time, this study investigated APAP-induced liver injury after multiple subtoxic doses and examined the role of autophagy in responding to this regimen. Fed male C57BL/6J mice were administered repeated doses (75 mg/kg and 150 mg/kg) of APAP, followed by measurement of adducts within the liver, mitochondria, and in plasma, activation of the MAP kinase JNK, and markers of liver injury. The role of autophagy was investigated by treatment of mice with the autophagy inhibitor, leupeptin. Our data show that multiple treatments at the 150 mg/kg dose of APAP resulted in protein adduct formation in the liver and mitochondria, activation of JNK, and hepatocyte cell death, which was significantly exacerbated by inhibition of autophagy. While repeated dosing with the milder 75 mg/kg dose did not cause mitochondrial protein adduct formation, JNK activation, or liver injury, autophagy inhibition resulted in hepatocyte death even at this lower dose. These data illustrate the importance of adaptive responses such as autophagy in removing protein adducts and preventing liver injury, especially in clinically relevant situations involving repeated dosing with APAP.

Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)-Mixed Lineage Kinase Domain-Like Protein (MLKL)-Mediated Necroptosis Contributes to Ischemia-Reperfusion Injury of Steatotic Livers.

Increased hepatic ischemia-reperfusion (IR) injury in steatotic livers is a major reason for rejecting the use of fatty livers for liver transplantation. Necroptosis is implicated in the pathogenesis of fatty liver diseases. Necroptosis is regulated by three key proteins: receptor-interacting serine/threonine-protein kinase (RIPK)-1, RIPK3, and mixed-lineage kinase domain-like protein (MLKL). Here, we found that marked steatosis of the liver was induced when a Western diet was given in mice; steatosis was associated with the inhibition of hepatic proteasome activities and with increased levels of key necroptosis-related proteins. Mice fed a Western diet had more severe liver injury, as demonstrated by increases in serum alanine aminotransferase and necrotic areas of liver, after IR than did mice fed a control diet. Although hepatic steatosis was not different between Mlkl knockout mice and wild-type mice, Mlkl knockout mice had decreased hepatic neutrophil infiltration and inflammation and were protected from hepatic IR injury, irrespective of diet. Intriguingly, Ripk3 knockout or Ripk3 kinase-dead knock-in mice were protected against IR injury at the late phase but not the early phase, irrespective of diet. Overall, our findings indicate that liver steatosis exacerbates hepatic IR injury via increased MLKL-mediated necroptosis. Targeting MLKL-mediated necroptosis may help to improve outcomes in steatotic liver transplantation.

Dynamin-1-Like Protein Inhibition Drives Megamitochondria Formation as an Adaptive Response in Alcohol-Induced Hepatotoxicity.

Despite the growing global burden of alcoholic liver diseases, therapeutic options are limited, and novel targets are urgently needed. Accumulating evidence suggests that mitochondria adapt in response to ethanol and formation of megamitochondria in the livers of patients is recognized as a hallmark of alcoholic liver diseases. The processes involved in ethanol-induced hepatic mitochondrial changes, the impact on mitochondria-shaping proteins, and the significance of megamitochondria formation remain unknown. In this study, we investigated the mitochondrial and cellular response to alcohol in hepatoma cell line VL-17A. The mitochondrial architecture rapidly changed after 3 or 14 days of ethanol exposure with double-pronged presentation of hyperfragmentation and megamitochondria, and cell growth was inhibited. Dynamin-1-like protein (Drp1) was identified as the main mediator driving these mitochondrial alterations, and its genetic inactivation was determined to foster megamitochondria development, preserving the capacity of the cells to grow despite alcohol toxicity. The role of Drp1 in mediating megamitochondria formation in mice with liver-specific inactivation of Drp1 was further confirmed. Finally, when these mice were fed with ethanol, the presentation of hepatic megamitochondria was exacerbated compared with wild type fed with the same diet. Ethanol-induced toxicity was also reduced. Our study demonstrates that megamitochondria formation is mediated by Drp1, and this phenomenon is a beneficial adaptive response during alcohol-induced hepatotoxicity.

p53 Up-regulated Modulator of Apoptosis Induction Mediates Acetaminophen-Induced Necrosis and Liver Injury in Mice.

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.

Dual Roles of Mammalian Target of Rapamycin in Regulating Liver Injury and Tumorigenesis in Autophagy-Defective Mouse Liver.

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.

Impaired TFEB-Mediated Lysosome Biogenesis and Autophagy Promote Chronic Ethanol-Induced Liver Injury and Steatosis in Mice.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Impaired Fasting-Induced Adaptive Lipid Droplet Biogenesis in Liver-Specific Atg5-Deficient Mouse Liver Is Mediated by Persistent Nuclear Factor-Like 2 Activation.

Lipid droplets (LDs) are intracellular organelles that store neutral lipids as energy reservoir. Recent studies suggest that autophagy is important in maintaining the homeostasis of intracellular LDs by either regulating the biogenesis of LDs, mobilization of fatty acids, or degradation of LDs in cultured cells. Increasing evidence also supports a role of autophagy in regulating glucose and lipid metabolism in vivo in mammals. In response to fasting/starvation, lipids are mobilized from the adipose tissue to the liver, which increases the number of intracellular LDs and stimulates fatty acid oxidation and ketogenesis. However, it is still controversial and unclear how impaired autophagy in hepatocytes affects the biogenesis of LDs in mouse livers. In the present study, it was demonstrated that hepatic autophagy-deficient (L-Atg)5 knockout mice had impaired adaptation to fasting-induced hepatic biogenesis of LDs. The maladaptation to fasting-induced hepatic biogenesis of LDs in L-Atg5 knockout mouse livers was not due to hepatic changes of de novo lipogenesis, secretion of very-low-density lipoprotein or fatty acid ß-oxidation, but it was due to persistent nuclear factor-like 2 activation because biogenesis of LDs restored in L-Atg5/nuclear factor-like 2 double-knockout mice.