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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.

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.Liver-specific and hepatocyte-specific Vmp1 knockout mice as well as Vmp1 knock-in mice were generated by crossing Vmp1Hepatocyte-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.Our results provide novel insights on the role of VMP1 in regulating hepatic phospholipid synthesis and lipoprotein secretion in the pathogenesis of NAFLD/NASH.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.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of preterm infants characterized by intestinal inflammation and intestinal epithelial cells (IEC) death. Evidence suggests that scattered crypt IEC apoptosis is a critical pathogenic event that subsequently leads to excessive villus necrosis in human NEC and rodent pups with experimental NEC. Necroptosis, programmed necrosis, is a proinflammatory cell death process that has been linked to several intestinal diseases. However, the critical role of necroptosis and its upstream pathways in the pathogenesis of NEC remains largely unexplored. Here, we aimed to study the linkage between scattered crypt IEC apoptosis and necroptosis during NEC development using a mouse genetic engineering approach. First, a unique triple-transgenic mouse model, 3xTg-iEAP (inducible Enterocyte specific Apoptosis Phenotype), was developed to induce IECs-specific over-expression of FasL using LoxP-Villin-Cre-mediated recombination strategy in combination with the rtTA-based regulatory system. Second, we created 3xTg-iEAPIfng-/- and 3xTg-iEAPRip3-/- mice by breeding 3xTg-iEAP with either Ifnγ knockout (KO) mice or Rip3 KO mice. The three days old neonatal pups of 3xTg-iEAP, 3xTg-iEAPIfng-/-, 3xTg-iEAPRip3-/- and littermate controls were challenged with a single dose doxycycline (Dox; 20 μg/gm, subcutaneously). Their growth was monitored on daily basis. The intestinal tissue samples were collected at different time points (8 h, 24 h, 48 h, and 72 h) and subjected to histological and qRT-PCR analysis. We confirmed that 3xTg-iEAP, 3xTg-iEAPIfng-/-, and 3xTg-iEAPRip3-/- mice developed normal, viable, and fertile, and indistinguishable from littermate controls without exposure to Dox. Treatment with Dox resulted in scattered IEC apoptosis in crypts of 3xTg-iEAP, 3xTg-iEAPIfng-/-, and 3xTg-iEAPRip3-/- mice within 8 h. Compared to littermate controls, we observed NEC-like damage with profound intestinal villus epithelial necrosis at 48 h after Dox treatment in 3xTg-iEAP mice that mimics histological features of human NEC. qRT-PCR analysis showed that the development of villus necrosis was associated with the increased expression of pro-inflammatory mediator genes and necroptosis-associated genes including Ifnγ and Rip3 in 3xTg-iEAP mice during the phase of NEC development. Interestingly, compared to 3xTg-iEAP mice, 3xTg-iEAPIfng-/- and 3xTg-iEAPRip3-/- mice displayed a lack of NEC-like incidence at 48h after Dox treatment, suggesting the crucial role of IFN-γ and RIP3 in scattered IEC apoptosis-induced NEC in our model. Taken together, the inhibition of necroptosis in IECs prevents intestinal injury during NEC. Our findings may advance knowledge in preventing or treating this devastating disorder in humans. Grants from NIH and the U.S. Department of Veterans Affairs support this work. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Receptor-interacting protein kinase 3 (Ripk3) is one of the critical mediators of inflammatory cytokine-stimulated signaling. Here we show that Ripk3 signaling selectively regulates both the number and the function of hematopoietic stem cells (HSCs) during stress conditions. Ripk3 signaling is not required for normal homeostatic hematopoiesis. However, in response to serial transplantation, inactivation of Ripk3 signaling prevents stress-induced HSC exhaustion and functional HSC attenuation, while in response to fractionated low doses of ionizing radiation (IR), inactivation of Ripk3 signaling accelerates leukemia/lymphoma development. In both situations, Ripk3 signaling is primarily stimulated by tumor necrosis factor-α. Activated Ripk3 signaling promotes the elimination of HSCs during serial transplantation and pre-leukemia stem cells (pre-LSCs) during fractionated IR by inducing Mlkl-dependent necroptosis. Activated Ripk3 signaling also attenuates HSC functioning and represses a pre-LSC-to-LSC transformation by promoting Mlkl-independent senescence. Furthermore, we demonstrate that Ripk3 signaling induces senescence in HSCs and pre-LSCs by attenuating ISR-mediated mitochondrial quality control.

bFGF treatment increases autophagy regulated secreted factors

Supplementary Figures from Oncogenic transformation confers a selective susceptibility to the combined suppression of the proteasome and autophagy

Includes legends for all supplementary figures. S1: CAFs exhibit increased LC3 puncta per cell compared to NF. S2: CAF autophagy inhibition significantly decreases HNSCC in vitro progression. S3: IL-6 and IL-8 induce fibroblast autophagy. S4: bFGF treatment increases autophagy regulated secreted factors. S5: Autophagy inhibition reduces HNSCC in combination therapy, and does not affect CAF proliferation.

Autophagy inhibition reduces HNSCC in combination therapy, and does not affect CAF proliferation

Examples of Electron Microscopy Autophagosomes

The proteasome and the autophagy systems are two evolutionarily conserved mechanisms for degrading intracellular materials. They are functionally coupled and suppression of the proteasome promotes autophagy. Although suppression of the proteasome leads to cell death, suppression of autophagy can be either prodeath or prosurvival. To understand the underlining mechanism of this dichotomy and its potential clinical implications, we treated various transformed and nontransformed human cells with proteasome inhibitors. We found that whether autophagy served a prosurvival role in this scenario was contingent on the cellular oncogenic status. Thus, autophagy suppression enhanced apoptosis induced by proteasome inhibitors in transformed cells, but not in nontransformed cells. Oncogenic transformation enhanced the ability of cells to initiate autophagy in response to stress, reflecting a stronger dependence of transformed cells on autophagy for survival. Indeed, a combined use of bortezomib, the only Food and Drug Administration–approved proteasome inhibitor for clinical use, and chloroquine, which inhibits autophagy by disturbing lysosomal functions, suppressed tumor growth more significantly than either agent alone in a xenograft model. These findings indicate that suppression of both intracellular degradation systems could constitute a novel strategy for enhanced cancer control in a tumor-specific way. [Mol Cancer Ther 2009;8(7):2036–45]

Supplementary Fig. from A coordinated action of Bax, PUMA, and p53 promotes MG132-induced mitochondria activation and apoptosis in colon cancer cells

Despite therapeutic advancements, there has been little change in the survival of patients with head and neck squamous cell carcinoma (HNSCC). Recent results suggest that cancer-associated fibroblasts (CAF) drive progression of this disease. Here, we report that autophagy is upregulated in HNSCC-associated CAFs, where it is responsible for key pathogenic contributions in this disease. Autophagy is fundamentally involved in cell degradation, but there is emerging evidence that suggests it is also important for cellular secretion. Thus, we hypothesized that autophagy-dependent secretion of tumor-promoting factors by HNSCC-associated CAFs may explain their role in malignant development. In support of this hypothesis, we observed a reduction in CAF-facilitated HNSCC progression after blocking CAF autophagy. Studies of cell growth media conditioned after autophagy blockade revealed levels of secreted IL6, IL8, and other cytokines were modulated by autophagy. Notably, when HNSCC cells were cocultured with normal fibroblasts, they upregulated autophagy through IL6, IL8, and basic fibroblast growth factor. In a mouse xenograft model of HNSCC, pharmacologic inhibition of Vps34, a key mediator of autophagy, enhanced the antitumor efficacy of cisplatin. Our results establish an oncogenic function for secretory autophagy in HNSCC stromal cells that promotes malignant progression. Cancer Res; 77(23); 6679–91. ©2017 AACR.

CAFs exhibit increased LC3 puncta per cell compared to NF.

IL-6 and IL-8 induce fibroblast autophagy

Targeting the ubiquitin-proteasome degradation pathway has become a promising approach for cancer therapy. Previous studies have shown that proteasome inhibition leads to apoptosis in various cancer cells. The mechanism by which apoptosis occurs are not fully understood and can be cell type and/or inhibitor specific. In this study, we investigated the mechanism of mitochondrial activation by proteasome inhibitors in colon cancer cells. We found that Bax activation and mitochondria translocation were required for apoptosis induced by multiple proteasome inhibitors. In contrast, reactive oxygen species did not seem to be induced by MG132 or bortezomib and antioxidants had no effects on MG132-induced apoptosis. In contrast, treatment with MG132 or bortezomib induced a significant accumulation of p53 and PUMA. Genetic deletion of either p53 or PUMA led to a marked suppression of apoptosis induced by these inhibitors, accompanied with reduced Bax activation and cytochrome c release. Consistently, inhibition of translation by cycloheximide could also effectively abolish the accumulation of p53 and PUMA and suppress MG132-induced Bax activation and apoptosis. These findings thus strongly indicate the critical involvement of p53-, PUMA-, and Bax-mediated mitochondrial activation in proteasome inhibitor–induced apoptosis in colon cancer cells. [Mol Cancer Ther 2007;6(3):1062–9]

CAF autophagy inhibition significantly decreases HNSCC in vitro progression.

Supplementary Figures 1-5, Table 1 from Following Cytochrome c Release, Autophagy Is Inhibited during Chemotherapy-Induced Apoptosis by Caspase 8–Mediated Cleavage of Beclin 1

Supplementary Methods from Following Cytochrome c Release, Autophagy Is Inhibited during Chemotherapy-Induced Apoptosis by Caspase 8–Mediated Cleavage of Beclin 1

Equilibration of phospholipids between the two monolayers of the lipid bilayer of cellular membranes is mediated by scramblases acting as phospholipid shuttling proteins that are critical for cellular function, particularly during inter-organelle contact. Recent work has identified several protein scramblases, including TMEM41B, VMP1 and ATG9 that are critical in autophagy. More recently, ATG9, TMEM41B, and VMP1 have also been discovered to be important regulators of cellular lipid homeostasis. In vivo mouse models involving ablation of TMEM41B in liver have shown that knockout of these proteins can lead to rapid development of non-alcoholic steatohepatitis (NASH) and systemic dyslipidemia, though this has not been explored yet with ATG9. The resulting phenotype is likely due to the combined effects of a severe lipid secretion defect caused by stalled neutral lipids export from the endoplasmic reticulum (ER) membrane bilayer coupled with increased lipogenesis. Here we briefly discuss recent exciting findings on the topic of scramblases in autophagy, their relevance to human non-alcoholic fatty liver disease (NAFLD)/NASH, as well as future directions in this research.

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. We found that the numbers of both total and larger tumors increased significantly in DEN-treated mice fed with ethanol diet than in mice fed with 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. To our surprise, no differences were found 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. Our findings indicate that chronic ethanol feeding promotes DEN-initiated liver tumor development, which tends to be attenuated by genetic deletion of hepatic TFEB.

Alcohol-related liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are the primary causes of chronic liver disease in western countries. Liver transplantation is currently one of the most efficient approaches to save patients with liver failure, which is often associated with hepatic ischemia-reperfusion (IR) injury. IR injury is exacerbated by hepatic steatosis, yet the mechanism remains elusive. Necroptosis is a form of regulated cell death mediated by receptor-interacting protein kinase 1 (RIP1), RIP3 and mixed lineage kinase domain-like (MLKL) protein, which has been implicated in the pathogenesis of ALD and NAFLD. Though necroptosis plays an important role in IR injury of high fat diet - induced steatotic livers, the role of necroptosis in IR injury of ethanol - induced steototic livers has not been investigated. In the present study, we used chronic plus binge alcohol (Gao-binge) feeding followed by IR surgery to investigate IR liver injury with ethanol-associated steatosis. We found that the levels of key necroptotic proteins MLKL and RIP3 increased in alcohol-fed mouse livers. Moreover, we observed increased liver injury after IR in control diet-fed mice, which was further exacerbated by alcohol feeding based on serum alanine aminotransferase (ALT) levels and TUNEL staining of necrotic cells. Hepatic neutrophil infiltration also increased in alcohol-fed mice after IR surgery. However, deletion of

VMP1 is an ER membrane protein with phospholipid scramblase activity that has a critical role in regulating phagophore expansion and autophagosome closure. VMP1 also regulates lipid droplet formation and lipoprotein secretion in cultured cells and zebrafish. In a recent study, we showed that mice with hepatic deletion of Vmp1 have impaired very-low-density lipoprotein (VLDL) secretion and develop nonalcoholic steatohepatitis (NASH) even when fed with regular chow diet. Mechanistically, deletion of Vmp1 leads to decreased hepatic phosphatidylcholine (PC) and phosphatidylethanolamine (PE) levels as well as altered PC and PE acyl chain compositions resulting in the accumulation of neutral lipid structures in the ER phospholipid bilayer and decreased pre-VLDL assembly. These studies provide novel mechanistic insights into the non-autophagic functions of VMP1 in regulating lipoprotein secretion.

BACKGROUND & AIMS: Primary liver tumors contain distinct subtypes. A subset of iCCAs can arise from cell fate reprogramming of mature hepatocytes in mouse models. However, the underpinning of cell fate plasticity during hepatocarcinogenesis is still poorly understood, hampering therapeutic development to treat HCC and iCCA. As YAP activation induces liver tumor formation and cell fate plasticity, we investigated the role of Sox9, a transcription factor downstream of Yap activation and expressed in biliary epithelial cells (BECs), in Yap-induced cell fate plasticity during hepatocarcinogenesis. METHODS: To evaluate the function of Sox9 in YAP-induced hepatocarcinogenesis in vivo, we performed inducible hepatocyte-specific YAP activation with simultaneous Sox9 removal in several mouse genetic models. Cell fate reprogramming was determined by lineage tracing and immunohistochemistry. The molecular mechanism underlying Yap and Sox9 function in hepatocyte plasticity was investigated by transcription and transcriptomic analyses of mouse and human liver tumors. RESULTS: Sox9, a marker of liver progenitor cells (LPCs) and BECs, is differentially required in YAP-induced stepwise hepatocyte programming. While Sox9 has limited function in hepatocyte dedifferentiation to LPCs, it is required for BEC differentiation from LPCs. YAP activation in Sox9-deficient hepatocytes resulted in more aggressive HCC with enhanced Yap activity at the expense of iCCA-like tumors. Furthermore, we showed that 20% of primary human liver tumors were associated with a YAP activation signature, and tumor plasticity is highly correlated with YAP activation and SOX9 expression. CONCLUSION: Our data demonstrated that Yap-Sox9 signaling determines hepatocyte plasticity and tumor heterogeneity in hepatocarcinogenesis in both mouse and human liver tumors. We identified Sox9 as a critical transcription factor required for Yap-induced hepatocyte cell fate reprogramming during hepatocarcinogenesis.

BACKGROUND AND AIMS: Human TM6SF2 variant rs58542926 is associated with nonalcoholic fatty liver disease (NAFLD) and hepatocellular cancer (HCC). However, conflicting reports in germline Tm6sf2 knockout mice suggest no change or decreased VLDL secretion and either unchanged or increased hepatic steatosis, with no increased fibrosis. We generated liver specific Tm6Sf2 knockout mice (Tm6 LKO) to study VLDL secretion and the impact on development and progression of NAFLD. APPROACH AND RESULTS: Two independent lines of Tm6 LKO mice exhibited spontaneous hepatic steatosis. Targeted lipidomic analyses showed increased triglyceride (TG) species whose distribution and abundance phenocopied findings in mice with liver specific deletion of microsomal triglyceride transfer protein. VLDL TG secretion was reduced, with small, underlipidated particles and unchanged or increased APOB. Liver-specific adeno-associated viral (AAV8-TBG) rescue using either wild type (WT) or mutant E167K-Tm6 reduced hepatic steatosis and improved VLDL secretion. Tm6 LKO mice fed a high milk-fat diet for 3 weeks exhibited increased steatosis and fibrosis and those phenotypes were further exacerbated when mice were fed fibrogenic, high fat/fructose diets for 20 weeks. In two models of HCC, either neonatal mice injected with streptozotocin (NASH/STAM) and high fat fed or with diethylnitrosamine (DEN) injection plus fibrogenic diet feeding, Tm6 LKO mice exhibited increased steatosis, greater tumor burden and increased tumor area versus Tm6 flox controls. Additionally, DEN-injected and fibrogenic diet fed Tm6 LKO mice administered WT Tm6 or E167K-mutant Tm6 AAV8 revealed significant tumor attenuation, with tumor burden inversely correlated with Tm6 protein levels. CONCLUSIONS: Liver-specific Tm6sf2 deletion impairs VLDL secretion, promoting hepatic steatosis, fibrosis and accelerated development of HCC, which was mitigated with AAV8- mediated rescue.

Obesity is the major risk factor for nonalcoholic fatty liver disease (NAFLD), for which effective cures are lacking. Despite the notion that obesity is associated with aberrant levels and action of pituitary hormones that are essential for maintaining hepatic metabolic and inflammatory states, the intrinsic pituitary endocrine abnormalities and their systemic consequences are incompletely defined. By characterizing the impact of diet-induced obesity (DIO) on the pituitary whole tissue and single cell transcriptome, we demonstrated that obesity disrupts pituitary endoplasmic reticulum (ER) homeostasis by suppressing the inositol-requiring enzyme-α (IRE1α)-mediated adaptive unfolded protein response (UPR). We further showed that defective pituitary UPR by IRE1α-deletion in the anterior pituitary strikingly augmented obesity-associated systemic metabolic abnormalities, particularly the NAFLD-associated pathologies. Conversely, enhancing the adaptive UPR in the anterior pituitary, by genetic gain-of-function of spliced X-box binding protein 1 (sXBP1), ameliorated the systemic and hepatic metabolic defects observed in mice with pituitary IRE1α deletion. Intriguingly, disruption of the UPR in the pituitary resulted in impaired hepatic UPR, which was in part due to a defective thyroid hormone receptor (THR)-mediated activation of hepatic Xbp1. In contrast, activation of the hepatic THR signaling improved obesity-associated glucose intolerance and attenuated the impaired hepatic ER homeostasis in anterior pituitary-IRE1α deficient mice. Together, our study provides the first insight into disruption of endocrine signaling-mediated inter-organ UPR communication drives obesity-associated hepatic pathologies. Unraveling these connections might uncover new therapeutic targets for NAFLD and other obesity-associated diseases.

The 4th Chinese American Liver Society (CALS)/Society of Chinese Bioscientists in America (SCBA) Hepatology Division Annual Symposium was held virtually on October 29–30, 2021. The goal of the CALS Symposium was to present and discuss the recent research data on the pathogeneses and therapeutic targets of liver diseases among the CALS members, trainees and invited speakers. Here we briefly introduce the history of the CALS/SCBA Hepatology Division and highlight the presentations that focus on the current progresses on basic and translational research in liver metabolism, bile acid biology, alcohol-related liver disease, drug-induced liver injury, cholestatic liver injury, non-alcoholic fatty liver disease/non-alcoholic steatohepatitis and liver cancer.

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.

Background and Aims TRIM21 is a ubiquitin E3 ligase that is implicated in numerous biological processes including immune response, cell metabolism, redox homeostasis, and cancer development. We recently reported that TRIM21 can negatively regulate the p62-Keap1-Nrf2 antioxidant pathway by ubiquitylating p62 and prevents its oligomerization and protein sequestration function. As redox homeostasis plays a pivotal role in many cancers including liver cancer, we sought to determine the role of TRIM21 in hepatocarcinogenesis. Methods We examined the correlation between TRIM21 expression and the disease using publicly available data sets and 49 cases of HCC clinical samples. We used TRIM21 genetic knockout mice to determine how TRIM21 ablation impact HCC induced by the carcinogen DEN plus phenobarbital (PB). We explored the mechanism that loss of TRIM21 protects cells from DEN-induced oxidative damage and cell death. Results There is a positive correlation between TRIM21 expression and HCC. Consistently, TRIM21-knockout mice are resistant to DEN-induced hepatocarcinogenesis. This is accompanied by decreased cell death and tissue damage upon DEN treatment, hence reduced hepatic tissue repair response and compensatory proliferation. Cells deficient in TRIM21 display enhanced p62 sequestration of Keap1 and are protected from DEN-induced ROS induction and cell death. Reconstitution of wild-type but not the E3 ligase-dead and the p62 binding-deficient mutant TRIM21 impedes the protection from DEN-induced oxidative damage and cell death in TRIM21-deficient cells. Conclusions Increased TRIM21 expression is associated with human HCC. Genetic ablation of TRIM21 leads to protection against oxidative hepatic damage and decreased hepatocarcinogenesis, suggesting TRIM21 as a preventive and therapeutic target.

Glutamine synthetase (GS) catalyzes de novo synthesis of glutamine that facilitates cancer cell growth. In the liver, GS functions next to the urea cycle to remove ammonia waste. As a dysregulated urea cycle is implicated in cancer development, the impact of GS's ammonia clearance function has not been explored in cancer. Here, we show that oncogenic activation of β-catenin (encoded by CTNNB1) led to a decreased urea cycle and elevated ammonia waste burden. While β-catenin induced the expression of GS, which is thought to be cancer promoting, surprisingly, genetic ablation of hepatic GS accelerated the onset of liver tumors in several mouse models that involved β-catenin activation. Mechanistically, GS ablation exacerbated hyperammonemia and facilitated the production of glutamate-derived nonessential amino acids, which subsequently stimulated mechanistic target of rapamycin complex 1 (mTORC1). Pharmacological and genetic inhibition of mTORC1 and glutamic transaminases suppressed tumorigenesis facilitated by GS ablation. While patients with hepatocellular carcinoma, especially those with CTNNB1 mutations, have an overall defective urea cycle and increased expression of GS, there exists a subset of patients with low GS expression that is associated with mTORC1 hyperactivation. Therefore, GS-mediated ammonia clearance serves as a tumor-suppressing mechanism in livers that harbor β-catenin activation mutations and a compromised urea cycle.

Autophagy plays an important role in maintaining cellular homeostasis. Defects in autophagy have been linked to various human diseases, such as cancer, neurodegenerative diseases, and cardiovascular diseases. Therefore, it is useful to develop an assay that can measure the functions of autophagy and also be used to identify autophagy modulators by screening a large number of compounds. This chapter describes a cell-based high content green fluorescent protein (GFP)-LC3 assay using mouse embryonic fibroblasts (MEF) stably expressing GFP-LC3.

Exercise is critical for improving metabolic health and putatively maintains or enhances mitochondrial quality control in metabolic tissues. Although previous work has shown that exercise elicits hepatic mitochondrial biogenesis, it is unknown if acute exercise activates hepatic mitophagy, the selective degradation of damaged or low-functioning mitochondria. We tested if an acute bout of treadmill running increased hepatic mitophagic flux both right after and 2-h postexercise in 15- to 24-wk-old C57BL/6J female mice. Acute exercise did not significantly increase markers of autophagic flux, however, mitophagic flux was activated 2-h post-treadmill running as measured by accumulation of both LC3-II and p62 in isolated mitochondria in the presence of leupeptin, an inhibitor of autophagosome degradation. Furthermore, mitochondrial-associated ubiquitin, which recruits the autophagy receptor protein p62, was also significantly increased at 2 h. Further examination via Western blot and proteomics analysis revealed that acute exercise elicits a time-dependent, dynamic activation of mitophagy pathways. Moreover, the results suggest that exercise-induced hepatic mitophagy is likely mediated by both polyubiquitination and receptor-mediated signaling pathways. Overall, we provide evidence that acute exercise activates hepatic mitophagic flux while also revealing specific receptor-mediated proteins by which exercise maintains mitochondrial quality control in the liver. NEW & NOTEWORTHY This study provides evidence that acute exercise activates hepatic mitophagic flux and mitochondrial polyubiquitination while additionally revealing specific receptor-mediated proteins by which exercise maintains mitochondrial quality control in the liver.

Autophagy is a highly conserved catabolic process that degrades cytosolic proteins and organelles via formation of autophagosomes that fuse with lysosomes to form autolysosomes, whereby autophagic cargos are degraded. Numerous studies have demonstrated that autophagy plays a critical role in the regulation of liver physiology and homeostasis, and impaired autophagy leads to the pathogenesis of various liver diseases such as viral hepatitis, alcohol associated liver diseases (AALD), non-alcoholic fatty liver diseases (NAFLD), and liver cancer. Recent evidence indicates that autophagy may play a dual role in liver cancer: inhibiting early tumor initiation while promoting progression and malignancy of already formed liver tumors. In this review, we summarized the progress of current understanding of how hepatic viral infection, alcohol consumption and diet-induced fatty liver diseases impair hepatic autophagy. We also discussed how impaired autophagy promotes liver tumorigenesis, and paradoxically how autophagy is required to promote the malignancy and progression of liver cancer. Understanding the molecular mechanisms underlying how autophagy differentially affects liver cancer development and progression may help to design better therapeutic strategies for prevention and treatment of liver cancer.

Rationale: Kupffer cells (KCs) play a crucial role in liver immune homeostasis through interacting with other immune cells and liver sinusoidal endothelial cells (LSECs). However, how KCs exactly interact with these cells for maintaining the homeostasis still require the further investigation. CXCL10 is a chemokine that has been implicated in chemoattraction of monocytes, T cells, NK cells, and dendritic cells, and promotion of T cell adhesion to endothelial cells. Although CXCL10 is also known to participate in the pathogenesis of hepatic inflammation, the degree to which it is functionally involved in the crosstalk between immune cells and regulation of immune response is still unclear. Methods: To dynamically investigate the function of KCs, we used our recently developed rapid cell ablation model, intermedilysin (ILY)/human CD59 (hCD59)-mediated cell ablation tool, to selectively ablate KC pool under normal condition or concanavalin A (Con A)- induced hepatitis. At certain time points after KCs ablation, we performed flow cytometry to monitor the amount of hepatic infiltrating immune cells. mRNA array was used to detect the change of hepatic cytokines and chemokines levels. Cytokines and chemokines in the serum were further measured by LEGENDplexTM mouse proinflammatory chemokine panel and inflammation panel. Evans blue staining and transmission electron microscopy were used to investigate the interaction between KCs and LSECs in steady condition. CXCL10 neutralizing antibody and CXCL10 deficient mouse were used to study the role of CXCL10 in immune cell migration and pathogenesis of Con A-induced hepatitis. Results: At steady state, elimination of KCs results in a reduction of hepatic infiltrating monocytes, T, B, and NK cells and a list of cytokines and chemokines at transcriptional level. In the meantime, the depletion of KCs resulted in increased sinusoidal vascular permeability. In the pathological condition, the KCs elimination rescues Con A-induced acute hepatitis through suppressing proinflammatory immune responses by down-regulation of hepatitis-associated cytokines/chemokines in serum such as CXCL10, and recruitment of infiltrating immune cells (monocytes, T, B, and NK cells). We further documented that deficiency or blockade of CXCL10 attenuated the development of Con A-induced hepatitis associated with reduction of the infiltrating monocytes, especially inflammatory Ly6Chi monocytes. Conclusions: This study supports the notion that KCs actively interact with immune cells and LSECs for maintaining immune response and liver homeostasis. Our data indicate that the interplay between KCs and infiltrated monocytes via CXCL10 contribute to Con A-induced hepatitis.

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.

Background & Aims Alcohol abuse is the major cause of experimental and human pancreatitis but the molecular mechanisms remain largely unknown. We investigated the role of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in the pathogenesis of alcoholic pancreatitis. Methods Using a chronic plus acute alcohol binge (referred to as Gao-binge) mouse model, we analyzed pancreas injury, autophagic flux, zymogen granule removal, TFEB nuclear translocation and lysosomal biogenesis in GFP-LC3 transgenic mice, acinar cell-specific Atg5 knockout (KO) and TFEB KO mice as well as their matched wild type mice. Results We found that Gao-binge alcohol induced typical features of pancreatitis in mice with increased serum amylase and lipase activities, pancreatic edema, infiltration of inflammatory cells, accumulation of zymogen granules (ZGs) and expression of inflammatory cytokines. While Gao-binge alcohol increased the number of autophagosomes, it also concurrently inhibited TFEB nuclear translocation and TFEB-mediated lysosomal biogenesis resulting in insufficient autophagy. Acinar cell-specific Atg5 KO and acinar cell-specific TFEB KO mice developed severe inflammatory and fibrotic pancreatitis in both Gao-binge alcohol and control diet-fed mice. In contrast, TFEB overexpression inhibited alcohol-induced pancreatic edema, accumulation of zymogen granules and serum amylase and lipase activities. In line with our findings in mice, decreased LAMP1 and TFEB nuclear staining were also observed in human alcoholic pancreatitis tissues. Conclusions our results indicate that TFEB plays a critical role in maintaining pancreatic acinar cell homeostasis. Impairment of TFEB-mediated lysosomal biogenesis by alcohol may lead to insufficient autophagy and promote alcohol-induced pancreatitis.

SQSTM1/p62 (sequestosome 1) is a macroautophagy/autophagy receptor protein that is degraded by selective autophagy. Intracellular accumulation of SQSTM1 activates multiple cell survival signaling pathways including NFΚB/NF-κB (nuclear factor kappa B), MTOR (mechanistic target of rapamycin kinase) and NFE2L2/Nrf2 (nuclear factor, erythroid derived 2, like 2). Both SQSTM1 and NFE2L2 have been considered as oncogenic, and increased accumulation of SQSTM1 and NFE2L2 activation have been frequently observed in various cancers including hepatocellular carcinoma. In a recent study, we found that deletion of Sqstm1 improved hepatic metabolic reprogramming and cell repopulation resulting in the attenuation of liver injury in mice with liver-specific deletion of Atg5 and Tsc1 that have defective hepatic autophagy and persistent MTOR complex 1 (MTORC1) activation. To our surprise, hepatocytic deletion of Sqstm1 promotes liver tumorigenesis in liver-specific atg5 and tsc1 double-knockout mice. Overall, these findings reveal a complex interplay among autophagy, SQSTM1 and MTORC1 and their differential roles either as oncogenic or tumor suppressor in liver tumorigenesis depending on the disease stage and context.

Acetaminophen (APAP) overdose can induce liver injury and is the most frequent cause of acute liver failure in the United States. We investigated the role of p62/SQSTM1 (referred to as p62) in APAP-induced liver injury (AILI) in mice. We found that the hepatic protein levels of p62 dramatically increased at 24 h after APAP treatment, which was inversely correlated with the hepatic levels of APAP-adducts. APAP also activated mTOR at 24 h, which is associated with increased cell proliferation. In contrast, p62 knockout (KO) mice showed increased hepatic levels of APAP-adducts detected by a specific antibody using Western blot analysis but decreased mTOR activation and cell proliferation with aggravated liver injury at 24 h after APAP treatment. Surprisingly, p62 KO mice recovered from AILI whereas the wild-type mice still sustained liver injury at 48 h. We found increased number of infiltrated macrophages in p62 KO mice that were accompanied with decreased hepatic von Willebrand factor (VWF) and platelet aggregation, which are associated with increased cell proliferation and improved liver injury at 48 h after APAP treatment. Our data indicate that p62 inhibits the late injury phase of AILI by increasing autophagic selective removal of APAP-adducts and mitochondria but impairs the recovery phase of AILI likely by enhancing hepatic blood coagulation., Graphical abstract p62 inhibits the late injury phase of acetaminophen-induced liver injury (AILI) by increasing autophagic selective removal of acetaminophen-adducts and mitochondria but impairs the recovery phase of AILI likely by enhancing hepatic blood coagulation.Image 1

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 of how autophagy impacts mTORC1 activation-mediated liver metabolic changes and tumorigenesis. 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/Sqstm1 F/F (p62) and whole body Nrf2 KO mice to generate L-Atg5/Tsc1/p62 and L-Atg5/Tsc1, Nrf2 triple KO (TKO) mice. These mice were housed for various time points 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 of L-Tsc1 KO mice 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 was also found in a subset of human hepatocellular carcinoma. 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 with increased mTOR activation. Using multiple genetic engineered mice that are defective of hepatic autophagy with persistent mTOR activation, we dissected the complex interplay among mTOR, autophagy, p62 and Nrf2 on liver cell repopulation, metabolic reprogramming and redox homeostasis in liver tumorigenesis. Our results uncovered an unexpected novel tumor suppressor function of p62/Sqstm1 by regulating liver cell repopulation, ductular reaction and metabolic reprogramming in liver tumorigenesis.

Fatty liver is a highly heterogenous condition driven by various pathogenic factors in addition to the severity of steatosis. Protein insufficiency has been causally linked to fatty liver with incompletely defined mechanisms. Here we report that fatty liver is a sulfur amino acid insufficient state that promotes metabolic inflexibility via limiting coenzyme A availability. We demonstrate that the nutrient-sensing transcriptional factor EB synergistically stimulates lysosome proteolysis and methionine adenosyltransferase to increase cysteine pool that drives the production of coenzyme A and glutathione, which support metabolic adaptation and antioxidant defense during increased lipid influx. Intriguingly, mice consuming an isocaloric protein-deficient Western diet exhibit selective hepatic cysteine, coenzyme A and glutathione deficiency and acylcarnitine accumulation, which are reversed by cystine supplementation without normalizing dietary protein intake. These findings support a pathogenic link of dysregulated sulfur amino acid metabolism to metabolic inflexibility that underlies both overnutrition and protein malnutrition-associated fatty liver development.