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The liver is a highly dynamic metabolic organ that plays critical roles in plasma protein synthesis, gluconeogenesis and glycogen storage, cholesterol metabolism and bile acid synthesis as well as drug/xenobiotic metabolism and detoxification. Research from the past decades indicate that autophagy, the cellular catabolic process mediated by lysosomes, plays an important role in maintaining cellular and metabolic homeostasis in the liver. Hepatic autophagy fluctuates with hormonal cues and the availability of nutrients that respond to fed and fasting states as well as circadian activities. Dysfunction of autophagy in liver parenchymal and non-parenchymal cells can lead to various liver diseases including non-alcoholic fatty liver diseases, alcohol associated liver disease, drug-induced liver injury, cholestasis, viral hepatitis and hepatocellular carcinoma. Therefore, targeting autophagy may be a potential strategy for treating these various liver diseases. In this review, we will discuss the current progress on the understanding of autophagy in liver physiology. We will also discuss several forms of selective autophagy in the liver and the molecular signaling pathways in regulating autophagy of different cell types and their implications in various liver diseases.

The pathogenesis of pancreatitis has been linked to disruption of organelle homeostasis including macroautophagy/autophagy dysfunction and endoplasmic reticulum (ER) stress. However, the direct impact of aberrant organelle function on pancreatitis initiation and progression is largely unknown. Recently an ER membrane protein, VMP1 (vacuole membrane protein 1), has been reported to play a crucial role in autophagosome formation. Notably, we found that VMP1 is downregulated in both human chronic pancreatitis (CP) and experimental mouse acute pancreatitis (AP). Pancreatic acinar cell-specific vmp1 deletion promotes inflammation, acinar-to-ductal metaplasia, and fibrosis in mice, sharing histological similarities with human CP. Mechanistically, loss of pancreatic VMP1 leads to defective autophagic degradation and ER stress as well as activation of the NFE2L2/Nrf2 pathway. Genetic ablation of NFE2L2 attenuated pancreatitis in VMP1-deficient mice. Our data highlight the importance of VMP1 in modulating an integrated organelle stress response and its functional role in maintaining pancreas homeostasis in the context of CP. Abbreviations: AMY: amylase; ADM: acinar-to-ductal metaplasia; AP: acute pancreatitis; CASP3: caspase 3; CP: chronic pancreatitis; DDIT3/CHOP: DNA damage inducible transcript 3; DKO, double knockout; ER: endoplasmic reticulum; GCLC: glutamate-cysteine ligase catalytic subunit; GCLM: glutamate-cysteine ligase modifier subunit; HSPA5/BIP: heat shock protein family A (Hsp70) member 5; KO: knockout; KRT19/CK19: keratin 19; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MPO: myeloperoxidase; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; ND: normal donor; NQO1: NAD(P)H quinone dehydrogenase 1; PCNA: proliferating cell nuclear antigen; RIPA: radio-immunoprecipitation; SQSTM1/p62: sequestosome 1; SOX9: SRY-box transcription factor 9; TAP: trypsinogen activation peptide; TFEB: transcription factor EB; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; UB: ubiquitin; VMP1: vacuole membrane protein 1; XBP1: X-box binding protein 1; YAP1, Yes1 associated transcriptional regulator; ZG: zymogen granule.

Nonacholic fatty liver disease, or hepatic steatosis, is the most common liver disorder affecting the western world and currently has no pharmacologic cure. Thus, many investigations have focused on alternative strategies to treat or prevent hepatic steatosis. Our laboratory has shown that chronic heat treatment (HT) mitigates glucose intolerance, insulin resistance, and hepatic steatosis in rodent models of obesity. Here, we investigate the direct bioenergetic mechanism(s) surrounding the metabolic effects of HT on hepatic mitochondria. Utilizing mitochondrial proteomics and respiratory function assays, we show that one bout of acute HT (42°C for 20 min) in male C57Bl/6J mice (n = 6/group) triggers a hepatic mitochondrial heat shock response resulting in acute reductions in respiratory capacity, degradation of key mitochondrial enzymes, and induction of mitophagy via mitochondrial ubiquitination. We also show that chronic bouts of HT and recurrent activation of the heat shock response enhances mitochondrial quality and respiratory function via compensatory adaptations in mitochondrial organization, gene expression, and transport even during 4 weeks of high-fat feeding (n = 6/group). Finally, utilizing a liver-specific heat shock protein 72 (HSP72) knockout model, we are the first to show that HSP72, a protein putatively driving the HT metabolic response, does not play a significant role in the hepatic mitochondrial adaptation to acute or chronic HT. However, HSP72 is required for the reductions in blood glucose observed with chronic HT. Our data are the first to suggest that chronic HT (1) improves hepatic mitochondrial respiratory efficiency via mitochondrial remodeling and (2) reduces blood glucose in a hepatic HSP72-dependent manner.

Defective macroautophagy/autophagy and a failure to initiate the adaptive unfolded protein response (UPR) in response to the endoplasmic reticulum (ER) stress contributes to obesity-associated metabolic dysfunction. However, whether and how unresolved ER stress leads to defects in the autophagy pathway and to the progression of obesity-associated hepatic pathologies remains unclear. Obesity suppresses the expression of hepatic spliced XBP1 (X-box binding protein 1; sXBP1), the key transcription factor that promotes the adaptive UPR. Our RNA-seq analysis revealed that sXBP1 regulates genes involved in lysosomal function in the liver under fasting conditions. Chromatin immunoprecipitation (ChIP) analyzes of both primary hepatocytes and whole livers further showed that sXBP1 occupies the −743 to −523 site of the promoter of Tfeb (transcription factor EB), a master regulator of autophagy and lysosome biogenesis. Notably, this occupancy was significantly reduced in livers from patients with steatosis. In mice, hepatic deletion of Xbp1 (xbp1 LKO) suppressed the transcription of Tfeb as well as autophagy, whereas hepatic overexpression of sXbp1 enhanced Tfeb transcription and autophagy. Moreover, overexpression of Tfeb in the xbp1 LKO mouse liver ameliorated glucose intolerance and steatosis in mice with diet-induced obesity (DIO). Conversely, loss of TFEB function impaired the protective role of sXBP1 in hepatic steatosis in mice with DIO. These data indicate that sXBP1-Tfeb signaling has direct functional consequences in the context of obesity. Collectively, our data provide novel insight into how two organelle stress responses are integrated to protect against obesity-associated metabolic dysfunction. Abbreviations: AAV8: adeno-associated virus serotype 8; ACTB: actin, beta; ANOVA: analysis of variance; ATF6: activating transcription factor-6; ATG: autophagy related; BECN1: beclin 1; BMI: body mass index; ChIP: chromatin immunoprecipitation; CLEAR: coordinated lysosomal expression and regulation; Cre: cre recombinase; DIO: diet-induced obesity; EBSS: Earle’s balanced salt solution; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum (ER) to nucleus signaling 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HFD: high-fat diet; h: hours; HSCs: hepatic stellate cells; INS: insulin; L/A: ammonium chloride and leupeptin; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mRNA: messenger RNA; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; RD: regular diet; RFP: red fluorescent protein; SERPINA7/TBG: serpin family A member 7; SQSTM1/p62: sequestome 1; sXbp1 LOE: liver-specific overexpression of spliced Xbp1; TFEB: transcription factor EB; TG: thapsigargin; TN: tunicamycin; UPR: unfolded protein response; wks: weeks; WT: wild type; XBP1: X-box binding protein 1; xbp1 LKO: liver-specific Xbp1 knockout.

Chronic excessive alcohol use is a well-recognized risk factor for pancreatic dysfunction and pancreatitis development. Evidence from in vivo and in vitro studies indicates that the detrimental effects of alcohol on the pancreas are from the direct toxic effects of metabolites and byproducts of ethanol metabolism such as reactive oxygen species. Pancreatic dysfunction and pancreatitis development are now increasingly thought to be multifactorial conditions, where alcohol, genetics, lifestyle, and infectious agents may determine the initiation and course of the disease. In this review, we first highlight the role of nonoxidative ethanol metabolism in the generation and accumulation of fatty acid ethyl esters (FAEEs) that cause multi-organellar dysfunction in the pancreas which ultimately leads to pancreatitis development. Further, we discuss how alcohol-mediated altered autophagy leads to the development of pancreatitis. We also provide insights into how alcohol interactions with other co-morbidities such as smoking or viral infections may negatively affect exocrine and endocrine pancreatic function. Finally, we present potential strategies to ameliorate organellar dysfunction which could attenuate pancreatic dysfunction and pancreatitis severity.

Liver is the major organ that regulates whole body cholesterol metabolism. Disrupted hepatic cholesterol homeostasis contributes to the pathogenesis of nonalcoholic steatohepatitis, dyslipidemia, atherosclerosis, and cardiovascular diseases. Hepatic bile acid synthesis is the major catabolic mechanism for cholesterol elimination from the body. Furthermore, bile acids are signaling molecules that regulate liver metabolism and inflammation. Autophagy is a highly-conserved lysosomal degradation mechanism, which plays an essential role in maintaining cellular integrity and energy homeostasis. In this review, we discuss emerging evidence linking hepatic cholesterol and bile acid metabolism to cellular autophagy activity in hepatocytes and macrophages, and how these interactions may be implicated in the pathogenesis and treatment of fatty liver disease and atherosclerosis.

Alcohol-related liver disease (ALD) and drug-induced liver injury (DILI) are common causes of severe liver disease, and successful treatments are lacking. Autophagy plays a protective role in both ALD and DILI by selectively removing damaged mitochondria (mitophagy), lipid droplets (lipophagy), protein aggregates and adducts in hepatocytes. Autophagy also protects against ALD by degrading interferon regulatory factor 1 (IRF1) and damaged mitochondria in hepatic macrophages. Specifically, we will discuss selective autophagy for removal of damaged mitochondria and lipid droplets in hepatocytes and autophagy-mediated degradation of IRF1 in hepatic macrophages as protective mechanisms against alcohol-induced liver injury and steatosis. In addition, selective autophagy for removal of damaged mitochondria and protein adducts for protection against DILI is discussed in this review. Development of new therapeutics for ALD and DILI is greatly needed, and selective autophagy pathways may provide promising targets. Drug and alcohol effects on autophagy regulation as well as protective mechanisms of autophagy against DILI and ALD are highlighted in this review.

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., TFEB is a transcriptional regulator of lysosomal biogenesis, activated upon starvation or lysosomal stress. Here the authors report that TFEB regulates hepatic bile acid synthesis downstream of FGF19 signaling.

Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in the United States 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.

Vinyl chloride (VC), a common industrial organochlorine and environmental pollutant, has been shown to directly cause hepatic angiosarcoma and toxicant-associated steatohepatitis at high exposure levels. However, the impact of lower concentrations of VC on the progression of underlying liver diseases (e.g., nonalcoholic fatty liver disease [NAFLD]) is unclear. Given the high prevalence of NAFLD in the United States (and worldwide) population, this is an important concern. Recent studies by our group with VC metabolites suggest a potential interaction between VC exposure and underlying liver disease to cause enhanced damage. Here, a novel mouse model determined the effects of VC inhalation at levels below the current Occupational Safety and Health Administration limit (

In 2012, we briefly summarized the mechanisms, pathophysiological roles and methods for analyzing mitophagy. As then, the mitophagy field has continued to grow rapidly, and many new molecular mechanisms regulating mitophagy and molecular tools for monitoring mitophagy have been discovered and developed. Therefore, the purpose of this review is to update information regarding these advances in mitophagy while focusing on basic molecular mechanisms of mitophagy in different organisms and its pathophysiological roles. We also discuss the advantage and limitations of current methods to monitor and quantify mitophagy in cultured cells andin vivomouse tissues.

Liver-specific deletion of autophagy-related genes in mice leads to hepatomegaly, liver injury and spontaneous liver tumorigenesis. Accumulating evidence indicates that p62/SQSTM1-mediated NFE2L2/Nrf2/(nuclear factor, erythroid 2 like 2) activation plays a critical role in promoting liver injury and tumorigenesis in autophagy-defective livers. However, the mechanisms of how persistent NFE2L2 activation induces liver injury and tumorigenesis are unknown. In a recent study, it was found that deletion of Mtor (mechanistic target of rapamycin kinase) or Rptor/Raptor attenuates hepatomegaly and liver injury in young liver-specific atg5 knockout mice but accelerates liver tumorigenesis in old mice likely due to feedback AKT activation. Overall, these findings suggest that both hyper- and hypo-activation of MTOR are detrimental to the liver resulting in the development of liver tumors. A balanced MTOR activity is critical to maintain the normal physiological functions of the liver, and caution should be exercised when treating hepatocellular carcinomas using MTOR inhibitors. Abbreviations: Atg5: autophgy related 5; DKO: double-knockout; HCC: hepatocellular carcinoma; INS: insulin; INSR: insulin receptor; KEAP1: kelch-like ECH-associated protein 1; KO: knockout; MTOR: mechanistic target of rapamycin kinase; NFE2L2: nuclear factor, erythroid 2 like 2; raptor: regulatory associated protein of MTOR, complex 1; SQSTM1: sequestosome 1: tsc1: TSC complex subunit 1.

Obesity is associated with elevated intracellular nitric oxide (NO) production, which promotes nitrosative stress in metabolic tissues such as liver and skeletal muscle, contributing to insulin resistance. The onset of obesity-associated insulin resistance is due, in part, to the compromise of hepatic autophagy, a process that leads to lysosomal degradation of cellular components. However, it is not known how NO bioactivity might impact autophagy in obesity. Here, we establish that S-nitrosoglutathione reductase (GSNOR), a major protein denitrosylase, provides a key regulatory link between inflammation and autophagy, which is disrupted in obesity and diabetes. We demonstrate that obesity promotes S-nitrosylation of lysosomal proteins in the liver, thereby impairing lysosomal enzyme activities. Moreover, in mice and humans, obesity and diabetes are accompanied by decreases in GSNOR activity, engendering nitrosative stress. In mice with a GSNOR deletion, diet-induced obesity increases lysosomal nitrosative stress and impairs autophagy in the liver, leading to hepatic insulin resistance. Conversely, liver-specific overexpression of GSNOR in obese mice markedly enhances lysosomal function and autophagy and, remarkably, improves insulin action and glucose homeostasis. Furthermore, overexpression of S-nitrosylation–resistant variants of lysosomal enzymes enhances autophagy, and pharmacologically and genetically enhancing autophagy improves hepatic insulin sensitivity in GSNOR-deficient hepatocytes. Taken together, our data indicate that obesity-induced protein S-nitrosylation is a key mechanism compromising the hepatic autophagy, contributing to hepatic insulin resistance.

Mitochondrial biogenesis (MB) is an adaptive response to maintain metabolic homeostasis after mitochondrial dysfunction. Induction of MB during APAP hepatotoxicity has not been studied. To investigate this, mice were treated with toxic doses of APAP and euthanized between 0 and 96 h. At early time points, APAP caused both mitochondrial dysfunction and reduction of mitochondrial mass, indicated by reduced activity of electron transport chain (ETC) complexes I and IV and depletion of mitochondrial DNA (mtDNA), respectively. Both ETC activity and mtDNA gradually recovered after 12 h, suggesting that MB occurs at late time points after APAP overdose. Immunofluorescent staining of mitochondria with mitochondrial outer membrane protein Tom20 further demonstrated that MB occurs selectively in hepatocytes surrounding necrotic areas. MB signaling mediators including PPARγ co-activator 1-α (Pgc-1α), nuclear respiratory factor-1 (Nrf-1) and mitochondrial fission protein dynamin-related protein-1 (Drp-1) were induced. Pgc-1α was selectively increased in hepatocytes surrounding necrotic areas. In addition, the time course of MB induction coincides with increased liver regeneration. Post-treatment with the known MB inducer SRT1720 increased Pgc-1α expression and liver regeneration, resulting in protection against late liver injury after APAP overdose. Thus, induction of MB is an important feature during APAP hepatotoxicity and liver regeneration.

Emerging evidence suggests that mitochondrion–endoplasmic reticulum (ER) and mitochondrion–lipid droplet (LD) contact sites are critical in regulating lipid metabolism in cells. It is well established that intracellular organelles communicate with each other continuously through membrane contact sites to maintain organelle function and cellular homeostasis. The accumulation of LDs in hepatocytes is an early indicator of non-alcoholic fatty liver disease (NAFLD) and alcohol-related liver disease (ALD), which may indicate a breakdown in proper inter-organelle communication. In this review, we discuss previous findings in mitochondrion–ER and mitochondrion–LD contact, focusing on their roles in lipid metabolism in hepatocytes. We also present evidence of a unique mitochondrion–LD contact structure in hepatocytes under various physiological and pathological conditions and propose a working hypothesis to speculate about the role of these structures in regulating the functions of mitochondria and LDs and their implications in NAFLD and ALD.

Pyrrolizidine alkaloids (PAs) are a group of compounds found in various plants and some of them are widely consumed in the world as herbal medicines and food supplements. PAs are potent hepatotoxins that cause irreversible liver injury in animals and humans. However, the mechanisms by which PAs induce liver injury are not clear. In the present study, we determined the hepatotoxicity and molecular mechanisms of senecionine, one of the most common toxic PAs, in primary cultured mouse and human hepatocytes as well as in mice. We found that senecionine administration increased serum alanine aminotransferase levels in mice. H&E and TUNEL staining of liver tissues revealed increased hemorrhage and hepatocyte apoptosis in liver zone 2 areas. Mechanistically, senecionine induced loss of mitochondrial membrane potential, release of mitochondrial cytochrome c as well as mitochondrial JNK translocation and activation prior to the increased DNA fragmentation and caspase-3 activation in primary cultured mouse and human hepatocytes. SP600125, a specific JNK inhibitor, and ZVAD-fmk, a general caspase inhibitor, alleviated senecionine-induced apoptosis in primary hepatocytes. Interestingly, senecionine also caused marked mitochondria fragmentation in hepatocytes. Pharmacological inhibition of dynamin-related protein1 (Drp1), a protein that is critical to regulate mitochondrial fission, blocked senecionine-induced mitochondrial fragmentation and mitochondrial release of cytochrome c and apoptosis. More importantly, hepatocyte-specific Drp1 knockout mice were resistant to senecionine-induced liver injury due to decreased mitochondrial damage and apoptosis. In conclusion, our results uncovered a novel mechanism of Drp1-mediated mitochondrial fragmentation in senecionine-induced liver injury. Targeting Drp1-mediated mitochondrial fragmentation and apoptosis may be a potential avenue to prevent and treat hepatotoxicity induced by PAs., Graphical abstract fx1, Highlights • Senecionine induces apoptosis in primary mouse and human hepatocytes as well as in mouse livers. • Senecionine induces mitochondrial Drp1 translocation, mitochondrial fragmentation, loss of mitochondrial membrane potential and release of mitochondrial cytochrome c in hepatocytes. • Pharmacological inhibition or genetic deletion of Drp1 protects against senecionine-induced hepatotoxicity. • Targeting Drp1-mediated mitochondrial fragmentation and apoptosis may be a potential avenue to prevent and treat hepatotoxicity induced by Pyrrolizidine alkaloids.

Key points Low intrinsic aerobic capacity is associated with increased all-cause and liver-related mortality in humans. Low intrinsic aerobic capacity in the low capacity runner (LCR) rat increases susceptibility to acute and chronic high-fat/high-sucrose diet-induced steatosis, without observed increases in liver inflammation. Addition of excess cholesterol to a high-fat/high-sucrose diet produced greater steatosis in LCR and high capacity runner (HCR) rats. However, the LCR rat demonstrated greater susceptibility to increased liver inflammatory and apoptotic markers compared to the HCR rat. The progressive non-alcoholic fatty liver disease observed in the LCR rats following western diet feeding was associated with further declines in liver fatty acid oxidation and mitochondrial respiratory capacity compared to HCR rats. Abstract Low aerobic capacity increases risk for non-alcoholic fatty liver disease and liver-related disease mortality, but mechanisms mediating these effects remain unknown. We recently reported that rats bred for low aerobic capacity (low capacity runner; LCR) displayed susceptibility to high fat diet-induced steatosis in association with reduced hepatic mitochondrial fatty acid oxidation (FAO) and respiratory capacity compared to high aerobic capacity (high capacity runner; HCR) rats. Here we tested the impact of aerobic capacity on susceptibility for progressive liver disease following a 16-week 'western diet' (WD) high in fat (45% kcal), cholesterol (1% w/w) and sucrose (15% kcal). Unlike previously with a diet high in fat and sucrose alone, the inclusion of cholesterol in the WD induced hepatomegaly and steatosis in both HCR and LCR rats, while producing greater cholesterol ester accumulation in LCR compared to HCR rats. Importantly, WD-fed low-fitness LCR rats displayed greater inflammatory cell infiltration, serum alanine transaminase, expression of hepatic inflammatory markers (F4/80, MCP-1, TLR4, TLR2 and IL-1β) and effector caspase (caspase 3 and 7) activation compared to HCR rats. Further, LCR rats had greater WD-induced decreases in complete FAO and mitochondrial respiratory capacity. Intrinsic aerobic capacity had no impact on WD-induced hepatic steatosis; however, rats bred for low aerobic capacity developed greater hepatic inflammation, which was associated with reduced hepatic mitochondrial FAO and respiratory capacity and increased accumulation of cholesterol esters. These results confirm epidemiological reports that aerobic capacity impacts progression of liver disease and suggest that these effects are mediated through alterations in hepatic mitochondrial function.

How different cell death modes and cell survival pathways cross talk remains elusive. We determined the interrelation of apoptosis, necrosis, and autophagy in tumor necrosis factor (TNF)-α/actinomycin D (ActD) and lipopolysaccharide/D-galactosamine (GalN)-induced hepatotoxicity in vitro and in vivo . We found that TNF-α/ActD-induced apoptosis was completely blocked by a general caspase inhibitor ZVAD-fmk at 24 hours but hepatocytes still died by necrosis at 48 hours. Inhibition of caspases also protected mice against lipopolysaccharide/GalN-induced apoptosis and liver injury at the early time point, but this protection was diminished after prolonged treatment by switching apoptosis to necrosis. Inhibition of receptor-interacting protein kinase (RIP)1 by necrostatin 1 partially inhibited TNF-α/ZVAD-induced necrosis in primary hepatocytes. Pharmacologic inhibition of autophagy or genetic deletion of Atg5 in hepatocytes did not protect against TNF-α/ActD/ZVAD-induced necrosis. Moreover, pharmacologic inhibition of RIP1 or genetic deletion of RIP3 failed to protect and even exacerbated liver injury after mice were treated with lipopolysaccharide/GalN and a pan-caspase inhibitor. In conclusion, our results suggest that different cell death mode and cell survival pathways are closely integrated during TNF-α–induced liver injury when both caspases and NF-κB are blocked. Moreover, results from our study also raised concerns about the safety of currently ongoing clinical trials that use caspase inhibitors.

Acetaminophen (APAP)-induced liver injury is an important clinical and toxicological problem. Understanding the mechanisms and modes of cell death are vital for the development of therapeutic interventions. The histological and clinical features of APAP hepatotoxicity including cell and organelle swelling, karyolysis, and extensive cell contents release lead to the characterization of the cell death as oncotic necrosis. However, the more recent identification of detailed signaling mechanisms of mitochondrial dysfunction, the amplification mechanisms of mitochondrial oxidant stress and peroxynitrite formation by a mitogen-activated protein kinase cascade, mechanisms of the mitochondrial permeability transition pore opening and nuclear DNA fragmentation as well as the characterization of the sterile inflammatory response suggested that the mode of cell death is better termed programmed necrosis. Additional features like mitochondrial Bax translocation and cytochrome c release, mobilization of lysosomal iron and the activation of receptor-interacting protein kinases and the inflammasome raised the question whether other emerging modes of cell death such as apoptosis, necroptosis, ferroptosis and pyroptosis could also play a role. The current review summarizes the key mechanisms of APAP-induced liver injury and compares these with key features of the newly described modes of cell death. Based on the preponderance of experimental and clinical evidence, the mode of APAP-induced cell death should be termed programmed necrosis; despite some overlap with other modes of cell death, APAP hepatotoxicity does not fulfill the characteristics of either apoptosis, necroptosis, ferroptosis, pyroptosis or autophagic cell death.

Mitochondria damage plays a critical role in acetaminophen (APAP)-induced necrosis and liver injury. Cells can adapt and protect themselves by removing damaged mitochondria via mitophagy. PINK1-Parkin pathway is one of the major pathways that regulate mitophagy but its role in APAP-induced liver injury is still elusive. We investigated the role of PINK1-Parkin pathway in hepatocyte mitophagy in APAP-induced liver injury in mice. Wild-type (WT), PINK1 knockout (KO), Parkin KO, and PINK1 and Parkin double KO (DKO) mice were treated with APAP for different time points. Liver injury was determined by measuring serum alanine aminotransferase (ALT) activity, H&E staining as well as TUNEL staining of liver tissues. Tandem fluorescent-tagged inner mitochondrial membrane protein Cox8 (Cox8-GFP-mCherry) can be used to monitor mitophagy based on different pH stability of GFP and mCherry fluorescent proteins. We overexpressed Cox8-GFP-mCherry in mouse livers via tail vein injection of an adenovirus Cox8-GFP-mCherry. Mitophagy was assessed by confocal microscopy for Cox8-GFP-mCherry puncta, electron microscopy (EM) analysis for mitophagosomes and western blot analysis for mitochondrial proteins. Parkin KO and PINK1 KO mice improved the survival after treatment with APAP although the serum levels of ALT were not significantly different among PINK1 KO, Parkin KO and WT mice. We only found mild defects of mitophagy in PINK1 KO or Parkin KO mice after APAP, and improved survival in PINK1 KO and Parkin KO mice could be due to other functions of PINK1 and Parkin independent of mitophagy. In contrast, APAP-induced mitophagy was significantly impaired in PINK1-Parkin DKO mice. PINK1-Parkin DKO mice had further elevated serum levels of ALT and increased mortality after APAP administration. In conclusion, our results demonstrated that PINK1-Parkin signaling pathway plays a critical role in APAP-induced mitophagy and liver injury., Graphical abstract Image 1, Highlights • Cox8-GFP-mCherry is a novel molecular probe to monitor mitophagy in cultured hepatocytes and in mouse livers. . • Double deletion of PINK1 and Parking exacerbates acetaminophen-induced liver injury and mortality in mice.

Impaired macroautophagy/autophagy has been implicated in experimental and human pancreatitis. However, the transcriptional control governing the autophagy-lysosomal process in pancreatitis is largely unknown. We investigated the role and mechanisms of TFEB (transcription factor EB), a master regulator of lysosomal biogenesis, in the pathogenesis of experimental pancreatitis. We analyzed autophagic flux, TFEB nuclear translocation, lysosomal biogenesis, inflammation and fibrosis in GFP-LC3 transgenic mice, acinar cell-specific tfeb knockout (KO) and tfeb and tfe3 double-knockout (DKO) mice as well as human pancreatitis samples. We found that cerulein activated MTOR (mechanistic target of rapamycin kinase) and increased the levels of phosphorylated TFEB as well as pancreatic proteasome activities that led to rapid TFEB degradation. As a result, cerulein decreased the number of lysosomes resulting in insufficient autophagy in mouse pancreas. Pharmacological inhibition of MTOR or proteasome partially rescued cerulein-induced TFEB degradation and pancreatic damage. Furthermore, genetic deletion of tfeb specifically in mouse pancreatic acinar cells increased pancreatic edema, necrotic cell death, infiltration of inflammatory cells and fibrosis in pancreas after cerulein treatment. tfeb and tfe3 DKO mice also developed spontaneous pancreatitis with increased pancreatic trypsin activities, edema and infiltration of inflammatory cells. Finally, decreased TFEB nuclear staining was associated with human pancreatitis. In conclusion, our results indicate a critical role of impaired TFEB-mediated lysosomal biogenesis in promoting the pathogenesis of pancreatitis. Abbreviations: AC: acinar cell; AMY: amylase; ATP6V1A: ATPase, H+ transporting, lysosomal V1 subunit A; ATP6V1B2: ATPase, H+ transporting, lysosomal V1 subunit B2; ATP6V1D: ATPase, H+ transporting, lysosomal V1 subunit D; ATP6V1H: ATPase, H+ transporting, lysosomal V1 subunit H; AV: autophagic vacuole; CDE: choline-deficient, ethionine-supplemented; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; EM: electron microscopy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; H & E: hematoxylin and eosin; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MTORC1: mechanistic target of rapamycin kinase complex 1; ND: normal donor; NEU: neutrophil; PPARGC1A/PGC1α: peroxisome proliferator-activated receptor, gamma, coactivator 1 alpha; RIPA: radio-immunoprecipitation; RPS6: ribosomal protein S6; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TM: tamoxifen; WT: wild-type; ZG: zymogen granule.

Studies using genetic mouse models that have defective autophagy have led to the conclusion that macroautophagy/autophagy serves as a tumor suppressor. One of such models is the liver-specific Atg5 or Atg7 knockout mice, and these knockout mice develop spontaneous liver tumors. It has been generally agreed that p62-mediated Nrf2 activation plays a critical role in promoting autophagy deficiency-induced liver injury and liver tumorigenesis. The mechanisms of how persistent Nrf2 activation induces liver injury and tumorigenesis are incompletely known. We discuss the recent progress on the new roles of HMGB1 and Yap in regulating liver injury and tumorigenesis in mice with liver-specific autophagy deficiency.

Alcoholic liver disease (ALD) is one of the major causes of chronic liver disease worldwide. Currently, no successful treatments are available for ALD. The pathogenesis of ALD is characterized as simple steatosis, fibrosis, cirrhosis, alcoholic hepatitis (AH), and eventually hepatocellular carcinoma (HCC). Autophagy is a highly conserved intracellular catabolic process, which aims at recycling cellular components and removing damaged organelles in response to starvation and stresses. Therefore, autophagy is considered as an important cellular adaptive and survival mechanism under various pathophysiological conditions. Recent studies from our lab and others suggest that chronic alcohol consumption may impair autophagy and contribute to the pathogenesis of ALD. In this chapter, we summarize recent progress on the role and mechanisms of autophagy in the development of ALD. Understanding the roles of autophagy in ALD may offer novel therapeutic avenues against ALD by targeting these pathways.

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.

Xenobiotics exposure increases endoplasmic reticulum (ER) proliferation and cytochrome P-450 (CYP) induction to sustain metabolic requirements. Whether autophagy is essential for the removal of excess ER and CYP and whether an autophagy receptor is involved in this process in mammals remains elusive. In this study, we show that autophagy is induced in mouse livers after withdrawal of the hepatic mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP). Although isolated autophagosomes, autolysosomes, and lysosomes from mouse livers after withdrawal of TCPOBOP contained ER proteins, those in control mouse livers did not. Liver-specific Atg5 knockout mice had higher basal hepatic ER content that was further increased and sustained after withdrawal of TCPOBOP compared with wild-type mice. In addition to regulating ER degradation, our results also suggest that autophagy plays a role in regulating the homeostasis of hepatic CYP because blocking autophagy led to increased CYP2B10 accumulation either at the basal level or following TCPOBOP withdrawal. Furthermore, we found that the autophagy receptor protein sequestosome 1 (SQSTM1)/p62 is associated with the ER. After withdrawal of TCPOBOP, p62 knockout mice had increased ER content in the liver compared with wild-type mice. These results suggest that p62 may act as an autophagy receptor for the autophagic removal of excess ER in the mouse liver. Taken together, our results indicate that autophagy is important for the removal of excess ER and hepatic CYP enzymes in mouse livers, a process associated with the autophagy receptor protein p62.

Background & Aims Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in the US and many other countries. Metabolism of APAP results in formation of APAP protein adducts (APAP-AD) in hepatocytes and triggers mitochondrial dysfunction and necrosis. However, the mechanisms for how APAP-AD are removed from hepatocytes remain unknown. Methods Mice or primary hepatocytes were treated with APAP. APAP-AD were determined by immunoblot, immunostaining and high pressure liquid chomatography with electrochemical detection analysis. Results We found that APAP-AD were detected at 1h, peaked at approximately 2h, declined at 6h and almost full removed at 24h post treatment with APAP in mouse livers and in primary mouse hepatocytes. APAP-AD displayed a punctate pattern and were colocalized with GFP-LC3 positive autophagosomes and Lamp1 positive lysosomes in APAP-treated primary hepatocytes. Moreover, isolated autophagosomes and autolysosomes from APAP-treated mouse livers contained APAP-AD, suggesting autophagy may selectively remove APAP-AD. APAP-AD were detected in both detergent soluble and insoluble pools in APAP-treated mouse livers and hepatocytes. More importantly, pharmacological inhibition of autophagy by leupeptin or chloroquine increased whereas induction of autophagy by Torin 1 decreased serum APAP-AD levels in APAP-treated mice, which correlated with alanine aminotransferase levels and liver necrosis. Furthermore, SQSTM1/p62, an autophagy receptor protein, was recruited to APAP-AD. Adenovirus-mediated shRNA knockdown of SQSTM1/p62 led to increased APAP-AD and necrosis in primary hepatocytes. Conclusions Our data indicate that APAP-AD are removed though selective autophagy. Pharmacological induction of autophagy may be a novel promising approach for treating APAP-induced liver injury. Lay summary Acetaminophen overdose can form acetaminophen protein adducts and mitochondria damage in hepatocytes resulting in liver injury. Activation of autophagy-lysosomal degradation pathway can help to remove acetaminophen protein adducts. Pharmacological induction of autophagy may be a novel promising approach for treating APAP-induced liver injury.

Macroautophagy is a major cellular degradation pathway for long-lived proteins and cellular organelles to maintain cellular homeostasis. Reduced autophagy has been implicated in neurodegenerative diseases, metabolic syndrome, and tumorigenesis. In contrast, increased autophagy has been shown to protect against tissue injury and aging. Here we employed a cell-based quantitative high-throughput image screening (qHTS) for autophagy modulators using mouse embryonic fibroblasts (MEFs) that are stably expressing GFP-LC3. The library of pharmacologically active compounds (LOPAC) was used to screen for the autophagy modulators in compounds alone or in combination with the lysosome inhibitor chloroquine (CQ). The GFP-LC3 puncta were then quantified to measure autophagic flux. The primary screening revealed 173 compounds with efficacy more than 40%. These compounds were cherry-picked and re-tested at multiple different concentrations using the same assay. A number of novel autophagy inducers, inhibitors, and modulators with dual-effects on autophagy were identified from the cherry-pick screening. Interestingly, we found a group of compounds that induce autophagy are related to dopamine receptors and are commonly used as clinical psychiatric drugs. Among them, indatraline hydrochloride (IND), a dopamine inhibitor, and chlorpromazine hydrochloride (CPZ) and fluphenazine dihydrochloride (FPZ), two dopamine receptor antagonists, were further evaluated. We found that FPZ-induced autophagy through mTOR inhibition but IND and CPZ induced autophagy in an mTOR-independent manner. Our data suggest that image-based autophagic flux qHTS can efficiently identify autophagy inducers and inhibitors.

Alcoholic liver disease (ALD) develops in approximately 20% of alcoholic patients, with a higher prevalence in females. ALD progression is marked by fatty liver and hepatocyte necrosis, as well as apoptosis, inflammation, regenerating nodules, fibrosis, and cirrhosis.(1) ALD develops via a complex process involving parenchymal and nonparenchymal cells, as well as recruitment of other cell types to the liver in response to damage and inflammation. Hepatocytes are damaged by ethanol, via generation of reactive oxygen species and induction of endoplasmic reticulum stress and mitochondrial dysfunction. Hepatocyte cell death via apoptosis and necrosis are markers of ethanol-induced liver injury. We review the mechanisms by which alcohol injures hepatocytes and the response of hepatic sinusoidal cells to alcohol-induced injury. We also discuss how recent insights into the pathogenesis of ALD will affect the treatment and management of patients.

Alcoholic liver disease is a major health problem in the United States and worldwide without successful treatments. Chronic alcohol consumption can lead to alcoholic liver disease (ALD), which is characterized by steatosis, inflammation, fibrosis, cirrhosis and even liver cancer. Recent studies suggest that alcohol induces both cell death and adaptive cell survival pathways in the liver, and the balance of cell death and cell survival ultimately decides the pathogenesis of ALD. This review summarizes the recent progress on the role and mechanisms of apoptosis, necroptosis and autophagy in the pathogenesis of ALD. Understanding the complex regulation of apoptosis, necrosis and autophagy may help to develop novel therapeutic strategies by targeting all three pathways simultaneously.

3'-Hydroxyacetanilide orN-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated the absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this,we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT) release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adduct formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves the formation of mitochondrial protein adducts and mitochondrial dysfunction.

Alcoholic liver disease and acetaminophen overdose are common causes of severe liver disease and liver failure in the United States for which there is no cure. Therefore, development of new therapeutic strategies for treatment of liver injury caused by acetaminophen and alcohol is needed. We demonstrated that autophagy protects against alcohol and acetaminophen-induced liver injuries by removing damaged mitochondria via mitophagy, which is a selective form of autophagy specific for degradation of damaged mitochondria. Parkin is well-known to be required for mitophagy induction in in vitro models, and we previously showed that the Parkin-mediated mitophagy pathway likely plays a protective role against alcohol and acetaminophen-induced liver injuries. Therefore, pharmacological upregulation of the Parkin-mediated mitophagy pathway in the liver may provide a novel and effective therapeutic option for treatment of acetaminophen and alcohol-induced liver injuries. In this review, we discuss regulation of Parkin-mediated mitophagy and possible therapeutic targets of intervention in this pathway.

Alcoholic liver disease (ALD) is a major health problem worldwide, and alcohol is well-known to cause mitochondrial damage, which exacerbates alcohol-induced liver injury and steatosis. No successful treatments are currently available for treating ALD. Therefore, a better understanding of mechanisms involved in regulation of mitochondrial homeostasis in the liver and how these mechanisms may protect against alcohol-induced liver disease is needed for future development of better therapeutic options for ALD. Mitophagy is a key mechanism for maintaining mitochondrial homeostasis by removing damaged mitochondria, and mitophagy protects against alcohol-induced liver injury. Parkin, an E3 ubiquitin ligase, is well-known to induce mitophagy in in vitro models although Parkin-independent mechanisms for mitophagy induction also exist. In this review, we discuss the roles of Parkin and mitophagy in protection against alcohol-induced liver injury and steatosis. We also discuss Parkin-independent mechanisms for mitophagy induction, which have not yet been evaluated in the liver but may also potentially have a protective role against ALD. In addition to mitophagy, mitochondrial spheroid formation may also provide a novel mechanism of protection against ALD, but the role of mitochondrial spheroids in protection against ALD progression needs to be further explored. Targeting removal of damaged mitochondria by mitophagy or inducing formation of mitochondrial spheroids may be promising therapeutic options for treatment of ALD.

Because of the liver’s function in the body as both a filter for toxins and an innate immune organ, the liver is predisposed to cellular turnover. Under healthy conditions, this occurs through the ...

Background & Aims The lysosome is an acidic organelle that is important for maintaining cellular and metabolic homeostasis in hepatocytes. Lysosomal dysfunction and chronic inflammation coexist, and both contribute to obesity-associated hepatic insulin resistance. However, in the context of obesity, the interplay between inflammatory signals and hepatic lysosomal function remains largely unknown. Inducible nitric oxide synthase (iNOS) is a hallmark for inflammation, and is activated in obesity. The aim of this study is to understand the molecular link between iNOS-mediated lysosomal nitric oxide (NO) production, hepatic lysosomal function, and autophagy in the context of obesity-associated insulin resistance. Methods The role of iNOS in hepatic autophagy, as related to insulin and glucose homeostasis were studied in mice with diet-induced obesity (DIO). The effects and mechanisms of iNOS-mediated lysosomal NO production on lysosomal function and hepatic autophagy were studied in primary hepatocytes as well as in a mouse model of DIO. Results We demonstrate that obesity promotes iNOS localization to the lysosome and decreases levels of lysosomal arginine, resulting in an accumulation of NO in hepatic lysosomes. This lysosomal NO production is attenuated by treatment with a NO scavenger, while co-overexpression of mTOR and a lysosomal arginine transporter (SLC38A9) enhances lysosomal NO production and suppresses autophagy. In addition, we show that deletion of iNOS ameliorates lysosomal nitrosative stress in the livers of DIO mice, promotes lysosomal biogenesis by activating transcription factor EB (TFEB), and enhances lysosomal function and autophagy. Lastly, deletion of iNOS in mice with DIO improves hepatic insulin sensitivity, which is diminished by suppression of TFEB or autophagy related 7 (Atg7). Conclusions Our studies suggest that lysosomal iNOS-mediated NO signaling disrupts hepatic lysosomal function, contributing to obesity-associated defective hepatic autophagy and insulin resistance.

We previously reported that delayed treatment with Mito-tempo (MT), a mitochondria-targeted superoxide dismutase mimetic, protects against the early phase of acetaminophen (APAP) hepatotoxicity by inhibiting peroxynitrite formation. However, whether this protection is sustained to the late phase of toxicity is unknown. To investigate the late protection, C57Bl/6J mice were treated with 300 mg/kg APAP followed by 20 mg/kg MT 1.5 h or 3 h later. We found that both MT treatments protected against the late phase of APAP hepatotoxicity at 12 and 24 h. Surprisingly, MT-treated mice demonstrated a significant increase in apoptotic hepatocytes, while the necrotic phenotype was observed almost exclusively in mice treated with APAP alone. In addition, there was a significant increase in caspase-3 activity and cleavage in the livers of MT-treated mice. Immunostaining for active caspase-3 revealed that the positively stained hepatocytes were exclusively in centrilobular areas. Treatment with the pan-caspase inhibitor ZVD-fmk (10 mg/kg) 2 h post-APAP neutralized this caspase activation and provided additional protection against APAP hepatotoxicity. Treatment with N-acetylcysteine, the current standard of care for APAP poisoning, protected but did not induce this apoptotic phenotype. Mechanistically, MT treatment inhibited APAP-induced RIP3 kinase expression, and RIP3-deficient mice showed caspase activation and apoptotic morphology in hepatocytes analogous to MT treatment. These data suggest that while necrosis is the primary cause of cell death after APAP hepatotoxicity, treatment with the antioxidant MT may switch the mode of cell death to secondary apoptosis in some cells. Modulation of mitochondrial oxidative stress and RIP3 kinase expression play critical roles in this switch.

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 β-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.

Macroautophagy/autophagy is a dynamic process, and newly synthesized autophagosomes need to fuse with lysosomes to complete the full process, which is referred to as autophagic degradation or autophagic flux. Therefore, the proper number and function of lysosomes are critical for accomplishing autophagic flux. In a recent study, we found that chronic ethanol consumption impairs TFEB (transcription factor EB) function, which leads to decreased lysosomal biogenesis resulting in hepatic steatosis and liver injury in mice. Interestingly, using the autophagic flux assay recommended by the autophagy guidelines, we discovered a novel autophagic flux scenario, which we termed insufficient autophagy. Insufficient autophagy is a scenario in which cells have a decreased number of lysosomes resulting in the accumulation of early autophagosomes. Insufficient autophagy is marked by a partially increased autophagic flux, but the process cannot reach its full degradative capacity due to the lack of a sufficient number of lysosomes. Our work demonstrated that pharmacological or genetic activation of TFEB-mediated lysosomal biogenesis enhances autophagic flux coupled with mitochondrial biogenesis in protecting against ethanol-induced liver injury. Overall, these findings not only identified the steps in which chronic ethanol impairs autophagic flux, but also discovered insufficient autophagy as a novel previously unappreciated autophagic flux scenario.

Bile acids are signaling molecules that critically control hepatocellular function. Disrupted bile acid homeostasis may be implicated in the pathogenesis of chronic liver diseases. Glutathione is an important antioxidant that protects the liver against oxidative injury. Various forms of liver disease share the common characteristics of reduced cellular glutathione and elevated oxidative stress. This study reports a potentially novel physiological function of bile acids in regulating hepatic sulfur amino acid and glutathione metabolism. We found that bile acids strongly inhibited the cysteine dioxygenase type-1-mediated (CDO1-mediated) cysteine catabolic pathway via a farnesoid X receptor-dependent mechanism. Attenuating this bile acid repressive effect depleted the free cysteine pool and reduced the glutathione concentration in mouse liver. Upon acetaminophen challenge, cholestyramine-fed mice showed impaired hepatic glutathione regeneration capacity and markedly worsened liver injury, which was fully prevented by N-acetylcysteine administration. These effects were recapitulated in CDO1-overexpressing hepatocytes. Findings from this study support the importance of maintaining bile acid homeostasis under physiological and pathophysiological conditions, as altered hepatic bile acid signaling may negatively impact the antioxidant defense mechanism and sensitivity to oxidative injury. Furthermore, this finding provides a possible explanation for the reported mild hepatotoxicity associated with the clinical use of bile acid sequestrants in human patients.

Macroautophagy/autophagy plays a dual role in cancer depending on the stage of tumorigenesis. Autophagy prevents tumor initiation by suppressing chronic tissue damage, inflammation, accumulation of damaged organelles and genome instability. Autophagy can also sustain tumor metabolism and provide nutrients for tumor growth and survival via nutrient recycling. Moreover, autophagy is required for benign tumors to progress to malignant tumors. Emerging evidence indicates that autophagy or mitophagy can inactivate tumor suppressors such as TP53/TRP53/p53 to promote tumor progression once carcinogenesis has been initiated.

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.

Alcoholic hepatitis (AH) continues to be a disease with high mortality and no efficacious medical treatment. Although severe AH is presented as acute on chronic liver failure, what underlies this transition from chronic alcoholic steatohepatitis (ASH) to AH, is largely unknown. To address this question, unbiased RNA-seq and proteomic analyses were performed on livers of the recently developed AH mouse model which exhibits the shift to AH from chronic ASH upon weekly alcohol binge, and these results are compared with gene expression profiling data from AH patients. This cross-analysis has identified Casp11 (CASP4 in man) as a commonly upregulated gene known to be involved in non-canonical inflammasome pathway. Immunoblotting confirms CASP11/4 activation in AH mice and patients but not in chronic ASH mice and healthy human livers. Gasdermin-D (GSDMD) which induces pyroptosis (lytic cell death caused by bacterial infection) downstream of CASP11/4 activation, is also activated in AH livers in mice and patients. CASP11 deficiency reduces GSDMD activation, bacterial load in the liver, and the severity of AH in the mouse model. Conversely, the deficiency of IL-18, the key anti-microbial cytokine, aggravates hepatic bacterial load, GSDMD activation, and AH. Further, hepatocyte-specific expression of constitutively active GSDMD worsens hepatocellular lytic death and PMN inflammation. These results implicate pyroptosis induced by CASP11/4-GSDMD pathway in the pathogenesis of AH.

Alcohol consumption leads to injury in multiple organs and systems, including the liver, brain, heart, skeletal muscle, pancreas, bone, immune system, and endocrine system. Emerging evidence indicates that alcohol also promotes adipose tissue dysfunction, which may contribute to injury progression in other organs and systems. Autophagy is a lysosomal degradation pathway that has been shown to regulate adipose tissue homeostasis and adipogenesis. Increasing evidence also demonstrates that alcohol consumption affects autophagy in multiple tissues. This review summarizes current knowledge regarding the effect of autophagy on adipose tissue and its potential roles in alcohol-induced adipose tissue atrophy as well as its contribution to alcohol-induced liver injury. Keywords: Autophagy, Alcohol, Adipose tissue, Liver, Alcoholic liver disease

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.