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Background and Aims: We investigated whether empagliflozin reduces hepatic steatosis in patients with metabolic dysfunction-associated steatotic liver disease without diabetes mellitus., Approach and Results: This was an investigator-initiated, double-blind, randomized, placebo-controlled trial recruiting adult subjects from the community. Eligible subjects without diabetes mellitus (fasting plasma glucose < 7 mmol/L and HbA1c < 6.5%) who had magnetic resonance imaging-proton density fat fraction (MRI-PDFF) ≥ 5% were randomly allocated to receive empagliflozin 10 mg daily or placebo (1:1 ratio) for 52 weeks (end of treatment, EOT). MRI-PDFF was conducted at baseline and EOT. The primary outcome was the difference in change of MRI-PDFF between the 2 groups at EOT. Secondary outcomes were hepatic steatosis resolution (MRI-PDFF < 5%), alanine aminotransferase drop ≥ 17 U/L, MRI-PDFF decline ≥ 30%, a combination of both, and changes of anthropometric and laboratory parameters at EOT. All outcomes were based on intention-to-treat analysis. Of 98 recruited subjects (median age: 55.7 y [IQR:49.5-63.4]; male:54 [55.1%]), 97 (empagliflozin:49, placebo:48; median MRI-PDFF:9.7% vs 9.0%) had MRI-PDFF repeated at EOT. The Empagliflozin group had a greater reduction in median MRI-PDFF compared to the placebo group (-2.49% vs. -1.43%; p = 0.025), with a nonsignificant trend of resolution of hepatic steatosis (44.9% vs. 28.6%; p = 0.094). There was no significant difference in alanine aminotransferase drop ≥ 17 U/L (16.3% vs. 12.2%; p = 0.564), MRI-PDFF drop ≥ 30% (49.0% vs. 40.8%; p = 0.417), and composite outcome (8.2% vs. 8.2%; p = 1.000). Empagliflozin group had a greater drop in body weight (-2.7 vs. -0.2 kg), waist circumference (-2.0 vs. 0 cm), fasting glucose (-0.3 vs. 0 mmol/L), and ferritin (-126 vs. -22 pmol/L) (all p < 0.05)., Conclusions: Empagliflozin for 52 weeks reduces hepatic fat content in subjects with nondiabetic metabolic dysfunction-associated steatotic liver disease. (ClinicalTrials.gov Identifier: NCT04642261)., (Copyright © 2024 American Association for the Study of Liver Diseases.)

Background and Aims: Artificial intelligence-powered digital pathology offers the potential to quantify histological findings in a reproducible way. This analysis compares the evaluation of histological features of NASH between pathologists and a machine-learning (ML) pathology model., Approach and Results: This post hoc analysis included data from a subset of patients (n=251) with biopsy-confirmed NASH and fibrosis stage F1-F3 from a 72-week randomized placebo-controlled trial of once-daily subcutaneous semaglutide 0.1, 0.2, or 0.4 mg (NCT02970942). Biopsies at baseline and week 72 were read by 2 pathologists. Digitized biopsy slides were evaluated by PathAI's NASH ML models to quantify changes in fibrosis, steatosis, inflammation, and hepatocyte ballooning using categorical assessments and continuous scores. Pathologist and ML-derived categorical assessments detected a significantly greater percentage of patients achieving the primary endpoint of NASH resolution without worsening of fibrosis with semaglutide 0.4 mg versus placebo (pathologist 58.5% vs. 22.0%, p < 0.0001; ML 36.9% vs. 11.9%; p =0.0015). Both methods detected a higher but nonsignificant percentage of patients on semaglutide 0.4 mg versus placebo achieving the secondary endpoint of liver fibrosis improvement without NASH worsening. ML continuous scores detected significant treatment-induced responses in histological features, including a quantitative reduction in fibrosis with semaglutide 0.4 mg versus placebo ( p =0.0099) that could not be detected using pathologist or ML categorical assessment., Conclusions: ML categorical assessments reproduced pathologists' results of histological improvement with semaglutide for steatosis and disease activity. ML-based continuous scores demonstrated an antifibrotic effect not measured by conventional histopathology., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background and Aims: DILI accounts for more than half of acute liver failure cases in the United States and is a major health care issue for the public worldwide. As investigative toxicology is playing an evolving role in the pharmaceutical industry, mechanistic insights into drug hepatotoxicity can facilitate drug development and clinical medication., Methods: By integrating multisource datasets including gene expression profiles of rat livers from open TG-GATE database and DrugMatrix, drug labels from FDA Liver Toxicity Knowledge Base, and clinical reports from LiverTox, and with the employment of bioinformatic and computational tools, this study developed an approach to characterize and predict DILI based on the molecular understanding of the processes (toxicity pathways)., Results: A panel of 11 pathways widely covering biological processes and stress responses was established using a training set of six positive and one negative DILI drugs from open TG-GATEs. An entropy weight method-based model was developed to weight responsive genes within a pathway, and an interpretable machine-learning (ML) model XGBoot-SHAP was trained to rank the importance of pathways to the panel activity. The panel activity was proven to differentiate between injured and noninjured sample points and characterize DILI manifestation using six training drugs. Next, the model was tested using an additional 89 drugs (61 positives + 28 negatives), and a precision of 86% and higher can be achieved., Conclusions: This study provides a novel approach to mechanisms-driven prediction modeling, as well as big data integration for insights into pharmacology and other human biology areas., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background and Aim: Drug-induced liver injury occurs frequently and can be life threatening. Although drug-induced liver injury is mainly caused by the direct drug cytotoxicity, increasing evidence suggests that the interplay between hepatocytes and immune cells can define this pathogenic process. Here, we interrogate the role of the pattern recognition scavenger receptor A (SRA) for regulating hepatic inflammation and drug-induced liver injury., Approach and Results: Using acetaminophen (APAP) or halothane-induced liver injury models, we showed that SRA loss renders mice highly susceptible to drug hepatotoxicity, indicated by the increased mortality and liver pathology. Mechanistic studies revealed that APAP-induced liver injury exaggerated in the absence of SRA was associated with the decreased anti-inflammatory and prosurvival cytokine IL-10 concomitant with excessive hepatic inflammation. The similar correlation between SRA and IL-10 expression was also seen in human following APAP uptake. Bone marrow reconstitution and liposomal clodronate depletion studies established that the hepatoprotective activity of SRA mostly resized in the immune sentinel KCs. Furthermore, SRA-facilitated IL-10 production by KCs in response to injured hepatocytes mitigated activation of the Jun N-terminal kinase-mediated signaling pathway in hepatocytes. In addition, supplemental use of IL-10 with N -acetylcysteine, only approved treatment of APAP overdose, conferred mice improved protection from APAP-induced liver injury., Conclusion: We identify a novel hepatocyte-extrinsic pathway governed by the immune receptor SRA that maintains liver homeostasis upon drug insult. Giving that drug (ie, APAP) overdose is the leading cause of acute liver failure, targeting this hepatoprotective SRA-IL-10 axis may provide new opportunities to optimize the current management of drug-induced liver injury., (Copyright © 2023 American Association for the Study of Liver Diseases.)

Background and Aims: Aberrant activation of fatty acid synthase (FASN) is a major metabolic event during the development of HCC. We evaluated the therapeutic efficacy of TVB3664, a FASN inhibitor, either alone or in combination, for HCC treatment., Approach and Results: The therapeutic efficacy and the molecular pathways targeted by TVB3664, either alone or with tyrosine kinase inhibitors or the checkpoint inhibitor anti-programmed death ligand 1 antibody, were assessed in human HCC cell lines and multiple oncogene-driven HCC mouse models. RNA sequencing was performed to elucidate the effects of TVB3664 on global gene expression and tumor metabolism. TVB3664 significantly ameliorated the fatty liver phenotype in the aged mice and AKT-induced hepatic steatosis. TVB3664 monotherapy showed moderate efficacy in NASH-related murine HCCs, induced by loss of phosphatase and tensin homolog and MET proto-oncogene, receptor tyrosine kinase (c-MET) overexpression. TVB3664, in combination with cabozantinib, triggered tumor regression in this murine model but did not improve the responsiveness to immunotherapy. Global gene expression revealed that TVB3664 predominantly modulated metabolic processes, whereas TVB3664 synergized with cabozantinib to down-regulate multiple cancer-related pathways, especially the AKT/mammalian target of rapamycin pathway and cell proliferation genes. TVB3664 also improved the therapeutic efficacy of sorafenib and cabozantinib in the FASN-dependent c-MYC-driven HCC model. However, TVB3664 had no efficacy nor synergistic effects in FASN-independent murine HCC models., Conclusions: This preclinical study suggests the limited efficacy of targeting FASN as monotherapy for HCC treatment. However, FASN inhibitors could be combined with other drugs for improved effectiveness. These combination therapies could be developed based on the driver oncogenes, supporting precision medicine approaches for HCC treatment., (© 2022 American Association for the Study of Liver Diseases.)

Background and Aims: Globally, NAFLD is one of the most common liver disorders, with an estimated prevalence rate of more than 30% in men and 15% in women and an even higher prevalence in people with type 2 diabetes mellitus. Optimal pharmacologic therapeutic approaches for NAFLD are an urgent necessity., Approach and Results: In this study, we showed that compared with healthy controls, hepatic ACSL4 levels in patients with NAFLD were found to be elevated. Suppression of ACSL4 expression promoted mitochondrial respiration, thereby enhancing the capacity of hepatocytes to mediate β-oxidation of fatty acids and to minimize lipid accumulation by up-regulating peroxisome proliferator-activated receptor coactivator-1 alpha. Moreover, we found that abemaciclib is a potent and selective ACSL4 inhibitor, and low dose of abemaciclib significantly ameliorated most of the NAFLD symptoms in multiple NAFLD mice models., Conclusions: Therefore, inhibition of ACSL4 is a potential alternative therapeutic approach for NAFLD., (© 2021 by the American Association for the Study of Liver Diseases.)

Hazard identification regarding adverse effects on the liver is a critical step in safety evaluations of drugs and other chemicals. Current testing paradigms for hepatotoxicity rely heavily on preclinical studies in animals and human data (epidemiology and clinical trials). Mechanistic understanding of the molecular and cellular pathways that may cause or exacerbate hepatotoxicity is well advanced and holds promise for identification of hepatotoxicants. One of the challenges in translating mechanistic evidence into robust decisions about potential hepatotoxicity is the lack of a systematic approach to integrate these data to help identify liver toxicity hazards. Recently, marked improvements were achieved in the practice of hazard identification of carcinogens, female and male reproductive toxicants, and endocrine disrupting chemicals using the key characteristics approach. Here, we describe the methods by which key characteristics of human hepatotoxicants were identified and provide examples for how they could be used to systematically identify, organize, and use mechanistic data when identifying hepatotoxicants., (© 2021 by the American Association for the Study of Liver Diseases.)

Background and Aims: NASH is becoming a leading cause of liver cirrhosis and HCC. Salidroside (p-hydroxyphenethyl-β-D-glucoside; SAL) has various biological and pharmacological activities, including anti-inflammatory, -oxidant, and -cancer activities. However, the therapeutic effect and underlying molecular mechanism of SAL in NASH remain to be further clarified., Methods and Results: In this study, we found that SAL alleviated lipid accumulation and inflammatory response in primary hepatocytes after palmitic acid/oleic acid (PO) stimulation. In addition, SAL effectively prevented high-fat/high-cholesterol (HFHC)-diet-induced NASH progression by regulating glucose metabolism dysregulation, insulin resistance, lipid accumulation, inflammation, and fibrosis. Mechanistically, integrated RNA-sequencing and bioinformatic analysis showed that SAL promoted AMPK-signaling pathway activation in vitro and in vivo, and this finding was further verified by determining the phosphorylation levels of AMPK. Furthermore, the protective effects of SAL on lipid accumulation and inflammation in hepatocytes and livers induced by PO or HFHC stimulation were blocked by AMPK interruption., Conclusions: Our studies demonstrate that SAL protects against metabolic-stress-induced NASH progression through activation of AMPK signaling, indicating that SAL could be a potential drug component for NASH therapy., (© 2021 by the American Association for the Study of Liver Diseases.)

Background and Aims: Hepatocellular dedifferentiation is emerging as an important determinant in liver disease progression. Preservation of mature hepatocyte identity relies on a set of key genes, predominantly the transcription factor hepatocyte nuclear factor 4α (HNF4α) but also splicing factors like SLU7. How these factors interact and become dysregulated and the impact of their impairment in driving liver disease are not fully understood., Approach and Results: Expression of SLU7 and that of the adult and oncofetal isoforms of HNF4α, driven by its promoter 1 (P1) and P2, respectively, was studied in diseased human and mouse livers. Hepatic function and damage response were analyzed in wild-type and Slu7-haploinsufficient/heterozygous (Slu7 +/- ) mice undergoing chronic (CCl 4 ) and acute (acetaminophen) injury. SLU7 expression was restored in CCl 4 -injured mice using SLU7-expressing adeno-associated viruses (AAV-SLU7). The hepatocellular SLU7 interactome was characterized by mass spectrometry. Reduced SLU7 expression in human and mouse diseased livers correlated with a switch in HNF4α P1 to P2 usage. This response was reproduced in Slu7 +/- mice, which displayed increased sensitivity to chronic and acute liver injury, enhanced oxidative stress, and marked impairment of hepatic functions. AAV-SLU7 infection prevented liver injury and hepatocellular dedifferentiation. Mechanistically we demonstrate a unique role for SLU7 in the preservation of HNF4α1 protein stability through its capacity to protect the liver against oxidative stress. SLU7 is herein identified as a key component of the stress granule proteome, an essential part of the cell's antioxidant machinery., Conclusions: Our results place SLU7 at the highest level of hepatocellular identity control, identifying SLU7 as a link between stress-protective mechanisms and liver differentiation. These findings emphasize the importance of the preservation of hepatic functions in the protection from liver injury., (© 2021 The Authors. HEPATOLOGY published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Background and Aims: Nonalcoholic fatty liver disease, especially nonalcoholic steatohepatitis (NASH), has become a major cause of liver transplantation and liver-associated death. NASH is the hepatic manifestation of metabolic syndrome and is characterized by hepatic steatosis, inflammation, hepatocellular injury, and different degrees of fibrosis. However, there is no US Food and Drug Administration-approved medication to treat this devastating disease. Therapeutic activators of the AMP-activated protein kinase (AMPK) have been proposed as a potential treatment for metabolic diseases such as NASH. Cordycepin, a natural product isolated from the traditional Chinese medicine Cordyceps militaris, has recently emerged as a promising drug candidate for metabolic diseases., Approach and Results: We evaluated the effects of cordycepin on lipid storage in hepatocytes, inflammation, and fibrosis development in mice with NASH. Cordycepin attenuated lipid accumulation, inflammation, and lipotoxicity in hepatocytes subjected to metabolic stress. In addition, cordycepin treatment significantly and dose-dependently decreased the elevated levels of serum aminotransferases in mice with diet-induced NASH. Furthermore, cordycepin treatment significantly reduced hepatic triglyceride accumulation, inflammatory cell infiltration, and hepatic fibrosis in mice. In vitro and in vivo mechanistic studies revealed that a key mechanism linking the protective effects of cordycepin were AMPK phosphorylation-dependent, as indicated by the finding that treatment with the AMPK inhibitor Compound C abrogated cordycepin-induced hepatoprotection in hepatocytes and mice with NASH., Conclusion: Cordycepin exerts significant protective effects against hepatic steatosis, inflammation, liver injury, and fibrosis in mice under metabolic stress through activation of the AMPK signaling pathway. Cordycepin might be an AMPK activator that can be used for the treatment of NASH., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Background and Aims: The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates an array of cytoprotective genes, yet studies in transgenic mice have led to conflicting reports on its role in liver regeneration. We aimed to test the hypothesis that pharmacological activation of Nrf2 would enhance liver regeneration., Approach and Results: Wild-type and Nrf2 null mice were administered bardoxolone methyl (CDDO-Me), a potent activator of Nrf2 that has entered clinical development, and then subjected to two-thirds partial hepatectomy. Using translational noninvasive imaging techniques, CDDO-Me was shown to enhance the rate of restoration of liver volume (MRI) and improve liver function (multispectral optoacoustic imaging of indocyanine green clearance) in wild-type, but not Nrf2 null, mice following partial hepatectomy. Using immunofluorescence imaging and whole transcriptome analysis, these effects were found to be associated with an increase in hepatocyte hypertrophy and proliferation, the suppression of immune and inflammatory signals, and metabolic adaptation in the remnant liver tissue. Similar processes were modulated following exposure of primary human hepatocytes to CDDO-Me, highlighting the potential relevance of our findings to patients., Conclusions: Our results indicate that pharmacological activation of Nrf2 is a promising strategy for enhancing functional liver regeneration. Such an approach could therefore aid the recovery of patients undergoing liver surgery and support the treatment of acute and chronic liver disease., (© 2021 The Authors. HEPATOLOGY published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Background and Aims: Real-world data for treatment effectiveness and renal outcomes in chronic hepatitis B (CHB) patients who were switched to the new and safer prodrug tenofovir alafenamide (TAF) from tenofovir disoproxil fumarate (TDF) are limited. Therefore, we aimed to evaluate treatment and renal outcomes of this population., Approach and Results: We analyzed 834 patients with CHB previously treated with TDF for ≥12 months who were switched to TAF in routine practice at 13 US and Asian centers for changes in viral (HBV DNA < 20 IU/mL), biochemical (alanine aminotransferase [ALT] < 35/25 U/L for male/female), and complete (viral+biochemical) responses, as well as estimated glomerular filtration rate (eGFR; milliliters per minute per 1.73 square meters) up to 96 weeks after switch. Viral suppression (P < 0.001) and ALT normalization (P = 0.003) rates increased significantly after switch, with a trend for increasing complete response (P trend = 0.004), while the eGFR trend (P trend  > 0.44) or mean eGFR (P > 0.83, adjusted for age, sex, baseline eGFR, and diabetes, hypertension, or cirrhosis by generalized linear modeling) remained stable. However, among those with baseline eGFR < 90 (chronic kidney disease [CKD] stage ≥2), mean eGFR decreased significantly while on TDF (P = 0.029) but not after TAF switch (P = 0.90). By week 96, 21% (55/267) of patients with CKD stage 2 at switch improved to stage 1 and 35% (30/85) of CKD stage 3-5 patients improved to stage 2 and 1.2% (1/85) to stage 1., Conclusions: Overall, we observed continued improvement in virologic response, ALT normalization, and no significant changes in eGFR following switch to TAF from TDF., (© 2021 by the American Association for the Study of Liver Diseases.)

Background and Aims: Mutational signature analyses are an effective tool in identifying cancer etiology. Humans are frequently exposed to pyrrolizidine alkaloids (PAs), the most common carcinogenic phytotoxins widely distributed in herbal remedies and foods. However, due to the lack of human epidemiological data, PAs are classified as group II hepatocarcinogens by the World Health Organization. This study identified a PA mutational signature as the biomarker to investigate the association of PA exposure with human liver cancer., Approach and Results: Pyrrole-protein adducts (PPAs), the PA exposure biomarker, were measured and found in 32% of surgically resected specimens from 34 patients with liver cancer in Hong Kong. Next, we delineated the mode of mutagenic and tumorigenic actions of retrorsine, a representative PA, in mice and human hepatocytes (HepaRG). Retrorsine induced DNA adduction, DNA damage, and activation of tumorigenic hepatic progenitor cells, which initiated hepatocarcinogenesis. PA mutational signature, as the unique molecular fingerprint of PA-induced mutation, was derived from exome mutations in retrorsine-exposed mice and HepaRG cells. Notably, PA mutational signature was validated in genomes of patients with PPA-positive liver cancer but not patients with PPA-negative liver cancer, confirming the specificity of this biomarker in revealing PA-associated liver cancers. Furthermore, we examined the established PA mutational signature in 1,513 liver cancer genomes and found that PA-associated liver cancers were potentially prevalent in Asia (Mainland China [48%], Hong Kong [44%], Japan [22%], South Korea [6%], Southeast Asia [25%]) but minor in Western countries (North America [3%] and Europe [5%])., Conclusions: This study provides a clinical indication of PA-associated liver cancer. We discovered an unexpectedly extensive implication of PA exposure in patients with liver cancer, laying the scientific basis for precautionary approaches and prevention of PA-associated human liver cancers., (© 2021 by the American Association for the Study of Liver Diseases.)

Background and Aims: HBV-specific T-cell receptor (HBV-TCR) engineered T cells have the potential for treating HCC relapses after liver transplantation, but their efficacy can be hampered by the concomitant immunosuppressive treatment required to prevent graft rejection. Our aim is to molecularly engineer TCR-T cells that could retain their polyfunctionality in such patients while minimizing the associated risk of organ rejection., Approach and Results: We first analyzed how immunosuppressive drugs can interfere with the in vivo function of TCR-T cells in liver transplanted patients with HBV-HCC recurrence receiving HBV-TCR T cells and in vitro in the presence of clinically relevant concentrations of immunosuppressive tacrolimus (TAC) and mycophenolate mofetil (MMF). Immunosuppressive Drug Resistant Armored TCR-T cells of desired specificity (HBV or Epstein-Barr virus) were then engineered by concomitantly electroporating mRNA encoding specific TCRs and mutated variants of calcineurin B (CnB) and inosine-5'-monophosphate dehydrogenase (IMPDH), and their function was assessed through intracellular cytokine staining and cytotoxicity assays in the presence of TAC and MMF. Liver transplanted HBV-HCC patients receiving different immunosuppressant drugs exhibited varying levels of activated (CD39 + Ki67 + ) peripheral blood mononuclear cells after HBV-TCR T-cell infusions that positively correlate with clinical efficacy. In vitro experiments with TAC and MMF showed a potent inhibition of TCR-T cell polyfunctionality. This inhibition can be effectively negated by the transient overexpression of mutated variants of CnB and IMPDH. Importantly, the resistance only lasted for 3-5 days, after which sensitivity was restored., Conclusions: We engineered TCR-T cells of desired specificities that transiently escape the immunosuppressive effects of TAC and MMF. This finding has important clinical applications for the treatment of HBV-HCC relapses and other pathologies occurring in organ transplanted patients., (© 2020 by the American Association for the Study of Liver Diseases.)

Background and Aims: Following mild liver injury, pre-existing hepatocytes replicate. However, if hepatocyte proliferation is compromised, such as in chronic liver diseases, biliary epithelial cells (BECs) contribute to hepatocytes through liver progenitor cells (LPCs), thereby restoring hepatic mass and function. Recently, augmenting innate BEC-driven liver regeneration has garnered attention as an alternative to liver transplantation, the only reliable treatment for patients with end-stage liver diseases. Despite this attention, the molecular basis of BEC-driven liver regeneration remains poorly understood., Approach and Results: By performing a chemical screen with the zebrafish hepatocyte ablation model, in which BECs robustly contribute to hepatocytes, we identified farnesoid X receptor (FXR) agonists as inhibitors of BEC-driven liver regeneration. Here we show that FXR activation blocks the process through the FXR-PTEN (phosphatase and tensin homolog)-PI3K (phosphoinositide 3-kinase)-AKT-mTOR (mammalian target of rapamycin) axis. We found that FXR activation blocked LPC-to-hepatocyte differentiation, but not BEC-to-LPC dedifferentiation. FXR activation also suppressed LPC proliferation and increased its death. These defects were rescued by suppressing PTEN activity with its chemical inhibitor and ptena/b mutants, indicating PTEN as a critical downstream mediator of FXR signaling in BEC-driven liver regeneration. Consistent with the role of PTEN in inhibiting the PI3K-AKT-mTOR pathway, FXR activation reduced the expression of pS6, a marker of mTORC1 activation, in LPCs of regenerating livers. Importantly, suppressing PI3K and mTORC1 activities with their chemical inhibitors blocked BEC-driven liver regeneration, as did FXR activation., Conclusions: FXR activation impairs BEC-driven liver regeneration by enhancing PTEN activity; the PI3K-AKT-mTOR pathway controls the regeneration process. Given the clinical trials and use of FXR agonists for multiple liver diseases due to their beneficial effects on steatosis and fibrosis, the detrimental effects of FXR activation on LPCs suggest a rather personalized use of the agonists in the clinic., (© 2020 by the American Association for the Study of Liver Diseases.)

Background and Aims: The liver plays a central role in all metabolic processes in the body. However, precise characterization of liver metabolism is often obscured by its inherent complexity. Phosphorylated metabolites occupy a prominent position in all anabolic and catabolic pathways. Here, we develop a 31 P nuclear magnetic resonance (NMR)-based method to study the liver "phosphorome" through the simultaneous identification and quantification of multiple hydrophilic and hydrophobic phosphorylated metabolites., Approach and Results: We applied this technique to define the metabolic landscape in livers from a mouse model of the rare disease disorder congenital erythropoietic porphyria (CEP) as well as two well-known murine models of nonalcoholic steatohepatitis: one genetic, methionine adenosyltransferase 1A knockout mice, and the other dietary, mice fed a high-fat choline-deficient diet. We report alterations in the concentrations of phosphorylated metabolites that are readouts of the balance between glycolysis, gluconeogenesis, the pentose phosphate pathway, the tricarboxylic acid cycle, and oxidative phosphorylation and of phospholipid metabolism and apoptosis. Moreover, these changes correlate with the main histological features: steatosis, apoptosis, iron deposits, and fibrosis. Strikingly, treatment with the repurposed drug ciclopirox improves the phosphoromic profile of CEP mice, an effect that was mirrored by the normalization of liver histology., Conclusions: In conclusion, these findings indicate that NMR-based phosphoromics may be used to unravel metabolic phenotypes of liver injury and to identify the mechanism of drug action., (© 2021 by the American Association for the Study of Liver Diseases.)

Background and Aims: Advanced fibrosis attributable to NASH is a leading cause of end-stage liver disease., Approach and Results: In this phase 2b trial, 392 patients with bridging fibrosis or compensated cirrhosis (F3-F4) were randomized to receive placebo, selonsertib 18 mg, cilofexor 30 mg, or firsocostat 20 mg, alone or in two-drug combinations, once-daily for 48 weeks. The primary endpoint was a ≥1-stage improvement in fibrosis without worsening of NASH between baseline and 48 weeks based on central pathologist review. Exploratory endpoints included changes in NAFLD Activity Score (NAS), liver histology assessed using a machine learning (ML) approach, liver biochemistry, and noninvasive markers. The majority had cirrhosis (56%) and NAS ≥5 (83%). The primary endpoint was achieved in 11% of placebo-treated patients versus cilofexor/firsocostat (21%; P = 0.17), cilofexor/selonsertib (19%; P = 0.26), firsocostat/selonsertib (15%; P = 0.62), firsocostat (12%; P = 0.94), and cilofexor (12%; P = 0.96). Changes in hepatic collagen by morphometry were not significant, but cilofexor/firsocostat led to a significant decrease in ML NASH CRN fibrosis score (P = 0.040) and a shift in biopsy area from F3-F4 to ≤F2 fibrosis patterns. Compared to placebo, significantly higher proportions of cilofexor/firsocostat patients had a ≥2-point NAS reduction; reductions in steatosis, lobular inflammation, and ballooning; and significant improvements in alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, bile acids, cytokeratin-18, insulin, estimated glomerular filtration rate, ELF score, and liver stiffness by transient elastography (all P ≤ 0.05). Pruritus occurred in 20%-29% of cilofexor versus 15% of placebo-treated patients., Conclusions: In patients with bridging fibrosis and cirrhosis, 48 weeks of cilofexor/firsocostat was well tolerated, led to improvements in NASH activity, and may have an antifibrotic effect. This combination offers potential for fibrosis regression with longer-term therapy in patients with advanced fibrosis attributable to NASH., (© 2020 by the American Association for the Study of Liver Diseases.)

Background and Aims: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of symptomatic biliary cysts. Current surgical and pharmacological approaches are ineffective, and liver transplantation represents the only curative option. Ursodeoxycholic acid (UDCA) and histone deacetylase 6 inhibitors (HDAC6is) have arisen as promising therapeutic strategies, but with partial benefits., Approach and Results: Here, we tested an approach based on the design, synthesis, and validation of a family of UDCA synthetic conjugates with selective HDAC6i capacity (UDCA-HDAC6i). Four UDCA-HDAC6i conjugates presented selective HDAC6i activity, UDCA-HDAC6i #1 being the most promising candidate. UDCA orientation within the UDCA-HDAC6i structure was determinant for HDAC6i activity and selectivity. Treatment of polycystic rats with UDCA-HDAC6i #1 reduced their hepatomegaly and cystogenesis, increased UDCA concentration, and inhibited HDAC6 activity in liver. In cystic cholangiocytes UDCA-HDAC6i #1 restored primary cilium length and exhibited potent antiproliferative activity. UDCA-HDAC6i #1 was actively transported into cells through BA and organic cation transporters., Conclusions: These UDCA-HDAC6i conjugates open a therapeutic avenue for PLDs., (© 2020 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases.)

Background and Aims: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a pivotal role in the establishment and persistence of HBV infection. Understanding the turnover time of preexisting cccDNA pools would be helpful in designing strategies to clear HBV by fully blocking the de novo generation of cccDNA., Approach and Results: In this study, we retrospectively monitored the emergence and reversion of the rtM204I/V mutant, a signature lamivudine resistance (LAM R ) mutation serving as a biomarker of cccDNA turnover in liver biopsies and longitudinal serum samples from two clinical trials. Methodologies were optimized to differentially isolate and sequence HBV virion DNA, cccDNA, and HBV RNA from clinical samples. A strong correlation was observed between LAM R composition of cccDNA with that of serum and intrahepatic HBV RNA in paired liver and serum samples (r = 0.96 and 0.90, respectively), suggesting that serum HBV RNA can serve as a surrogate marker of cccDNA genetic composition when liver biopsies are unavailable. LAM R mutations emerged and increased from undetectable to 40%-90% within 16-28 weeks in serum HBV RNA from telbivudine-treated patients experiencing virological breakthrough. Similarly, in lamivudine-resistant patients who switched to interferon therapy, serum HBV-RNA population bearing 100% LAM R mutations fully reversed back to wild type within 24-48 weeks., Conclusions: The genetic composition dynamics of serum HBV RNA and biopsy cccDNA in treated HBV patients indicates that cccDNA turnover occurs relatively rapidly (several months), offering a possibility of HBV cure with finite therapy through completely blocking cccDNA replenishment., (© 2020 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Background and Aims: This study examined whether enhanced susceptibility of steatotic liver to ischemia-reperfusion (I/R) injury is due to impaired recruitment of bone marrow (BM) progenitors of liver sinusoidal endothelial cells (LSECs, also called sinusoidal endothelial cell progenitor cells [sprocs]) with diminished repair of injured LSECs and whether restoring signaling to recruit BM sprocs reduces I/R injury., Approach and Results: Hepatic vessels were clamped for 1 hour in rats fed a high-fat, high-fructose (HFHF) diet for 5, 10, or 15 weeks. Matrix metalloproteinase 9 (MMP-9) antisense oligonucleotides (ASO) or an MMP inhibitor were used to induce liver-selective MMP-9 inhibition. HFHF rats had mild, moderate, and severe steatosis, respectively, at 5, 10, and 15 weeks. I/R injury was enhanced in HFHF rats; this was accompanied by complete absence of hepatic vascular endothelial growth factor (VEGF)-stromal cell-derived factor 1 (sdf1) signaling, leading to lack of BM sproc recruitment. Liver-selective MMP-9 inhibition to protect against proteolytic cleavage of hepatic VEGF using either MMP-9 ASO or intraportal MMP inhibitor in 5-week and 10-week HFHF rats enhanced hepatic VEGF-sdf1 signaling, increased BM sproc recruitment, and reduced alanine aminotransferase (ALT) by 92% and 77% at 5 weeks and by 80% and 64% at 10 weeks of the HFHF diet, respectively. After I/R injury in 15-week HFHF rats, the MMP inhibitor reduced active MMP-9 expression by 97%, ameliorated histologic evidence of injury, and reduced ALT by 58%, which is comparable to control rats sustaining I/R injury. Rescue therapy with intraportal MMP inhibitor, given after ischemia, in the 5-week HFHF rat reduced ALT by 71% and reduced necrosis., Conclusions: Lack of signaling to recruit BM sprocs that repair injured LSECs renders steatotic liver more susceptible to I/R injury. Liver-selective MMP-9 inhibition enhances VEGF-sdf1 signaling and recruitment of BM sprocs, which markedly protects against I/R injury, even in severely steatotic rats., (© 2020 by the American Association for the Study of Liver Diseases.)

Background and Aims: Alcohol-associated liver disease (ALD) is a common chronic liver disease worldwide with high morbidity and mortality, and no Food and Drug Administration-approved therapies. Fructose (dietary or endogenous), its metabolite uric acid, and aldose reductase (AR, the only endogenous enzyme that produces fructose) are strongly associated with the development of nonalcoholic fatty liver disease. However, the role of AR or its metabolites in ALD remains understudied and was examined using human specimens, cultured cells, and mouse model systems., Approach and Results: We demonstrated in liver specimens from patients with alcoholic hepatitis, the AR up-regulation and elevated AR metabolites (sorbitol, fructose, and uric acid), which correlated significantly with (1) increased lipid peroxidation byproducts and endoplasmic reticulum (ER) stress, (2) decreased protective ER chaperones, and (3) greater cell death and liver injury. Furthermore, we established a causal role for AR in ALD by showing that the genetic deficiency of AR (knockout mice) prevented alcohol-induced increase in harmful AR metabolites, toxic aldehydes, steatosis, ER stress, apoptosis, and liver injury. Finally, we demonstrated the therapeutic potential of pharmacological AR inhibition against alcohol-induced hepatic injury in experimental ALD., Conclusions: Our data demonstrate that hepatic AR up-regulation, and consequent elevation in fructose, sorbitol and/or uric acid, are important factors contributing to alcohol-induced steatosis, ER stress, apoptosis, and liver injury in both experimental and human ALD. Our study provides a strong rationale to evaluate AR as a potential therapeutic target and to test AR inhibitors to ameliorate alcohol-induced liver injury., (© 2020 by the American Association for the Study of Liver Diseases.)

Background and Aims: Acetaminophen (APAP) overdose induces severe liver injury and hepatic failure. While the activation of c-Jun NH 2 -terminal kinase (JNK) has been implicated as a mechanism in APAP-induced liver injury, the hepatic defense system controlled by nuclear factor erythroid 2-related factor 2 (Nrf2) plays a central role in the mitigation of APAP toxicity. However, the link between the two signaling pathways in APAP-induced liver injury (AILI) remains unclear., Approach and Results: In this study, we demonstrated that the activation of JNK in mouse liver following exposure to APAP was correlated with the phosphorylation of Nrf2 and down-regulation of the antioxidant response element (ARE)-driven genes, NAD(P)H:quinone dehydrogenase 1, glutathione S-transferase α3, glutathione S-transferase M1, glutathione S-transferase M5, and aldo-keto reductase 1C. The JNK inhibitor, SP600125, or knockdown of JNK by infection of adenovirus expressing JNK small interfering RNA, ameliorated the APAP induced liver toxicity, and inhibited the phosphorylation of Nrf2 and down-regulation of detoxifying enzymes by stabilizing the transcription factor. Mechanistically, JNK antagonized Nrf2- and ARE-driven gene expression in a Kelch-like ECH-associated protein 1-independent manner. Biochemical analysis revealed that phosphorylated JNK (P-JNK) directly interacted with the Nrf2-ECH homology (Neh) 1 domain of Nrf2 and phosphorylated the serine-aspartate-serine motif 1 (SDS1) region in the Neh6 domain of Nrf2., Conclusions: Mass spectrometric analysis identified serine 335 in the SDS1 region of mNrf2 as the major phosphorylation site for modulation of Nrf2 ubiquitylation by P-JNK. This study demonstrates that Nrf2 is a target of P-JNK in AILI. Our finding may provide a strategy for the treatment of AILI., (© 2020 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases.)

Background and Aims: STAT3, a member of the signal transducer and activator of transcription (STAT) family, is strongly associated with liver injury, inflammation, regeneration, and hepatocellular carcinoma development. However, the signals that regulate STAT3 activity are not completely understood., Approach and Results: Here we characterize CREB/ATF bZIP transcription factor CREBZF as a critical regulator of STAT3 in the hepatocyte to repress liver regeneration. We show that CREBZF deficiency stimulates the expression of the cyclin gene family and enhances liver regeneration after partial hepatectomy. Flow cytometry analysis reveals that CREBZF regulates cell cycle progression during liver regeneration in a hepatocyte-autonomous manner. Similar results were observed in another model of liver regeneration induced by intraperitoneal injection of carbon tetrachloride (CCl 4 ). Mechanistically, CREBZF potently associates with the linker domain of STAT3 and represses its dimerization and transcriptional activity in vivo and in vitro. Importantly, hepatectomy-induced hyperactivation of cyclin D1 and liver regeneration in CREBZF liver-specific knockout mice was reversed by selective STAT3 inhibitor cucurbitacin I. In contrast, adeno-associated virus-mediated overexpression of CREBZF in the liver inhibits the expression of the cyclin gene family and attenuates liver regeneration in CCl 4 -treated mice., Conclusions: These results characterize CREBZF as a coregulator of STAT3 to inhibit regenerative capacity, which may represent an essential cellular signal to maintain liver mass homeostasis. Therapeutic approaches to inhibit CREBZF may benefit the compromised liver during liver transplantation., (© 2019 by the American Association for the Study of Liver Diseases.)

Spermidine (SPD), a naturally occurring polyamine, has been recognized as a caloric restriction mimetic that confers health benefits, presumably by inducing autophagy. Recent studies have reported that oral administration of SPD protects against liver fibrosis and hepatocarcinogenesis through activation of microtubule associated protein 1S (MAP1S)-mediated autophagy. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor that mediates cellular protection by maintaining the cell's redox, metabolic, and proteostatic balance. In this study, we demonstrate that SPD is a noncanonical NRF2 inducer, and that MAP1S is a component of this noncanonical pathway of NRF2 activation. Mechanistically, MAP1S induces NRF2 signaling through two parallel mechanisms, both resulting in NRF2 stabilization: (1) MAP1S competes with Kelch-like ECH-associated protein 1 (KEAP1) for NRF2 binding through an ETGE motif, and (2) MAP1S accelerates p62-dependent degradation of KEAP1 by the autophagy pathway. We further demonstrate that SPD confers liver protection by enhancing NRF2 signaling. The importance of both NRF2 and p62-dependent autophagy in SPD-mediated liver protection was confirmed using a carbon tetrachloride-induced liver fibrosis model in wild-type, Nrf2 -/- , p62 -/- and Nrf2 -/- ;p62 -/- mice, as the protective effect of SPD was significantly reduced in NRF2 or p62 single knockout mice, and completely abolished in the double knockout mice. Conclusion: Our results demonstrate the pivotal role of NRF2 in mediating the health benefit of SPD, particularly in the context of liver pathologies., (© 2019 by the American Association for the Study of Liver Diseases.)

Lysyl oxidase like-2 (LOXL2) plays a central role in fibrogenesis and is elevated in the serum and liver of patients with primary sclerosing cholangitis (PSC). We evaluated the safety and efficacy of simtuzumab, a monoclonal antibody directed against LOXL2, in patients with PSC. Patients with compensated liver disease caused by PSC were randomized 1:1:1 to receive weekly subcutaneous injections of simtuzumab 75 mg, simtuzumab 125 mg, or placebo for 96 weeks. The primary efficacy endpoint was mean change in hepatic collagen content assessed by morphometry between baseline and week 96. Additional endpoints included change in Ishak fibrosis stage and the frequency of PSC-related clinical events. Overall, 234 patients were randomized and started treatment. At week 96, the mean change from baseline in hepatic collagen content was -0.5% for patients receiving simtuzumab 75 mg (P = 0.73 versus placebo), +0.5% for patients receiving simtuzumab 125 mg (P = 0.33 versus placebo), and 0.0 for patients receiving placebo. Compared with placebo, neither dose of simtuzumab led to significant reductions in Ishak fibrosis stage, progression to cirrhosis, or frequency of clinical events. Overall, 80 (34%) patients had fibrosis progression and 47 (20%) experienced PSC-related clinical events. In a multivariate model of baseline factors, PSC-related clinical events were more frequent in patients with advanced fibrosis (hazard ratio [HR], 2.03; 95% confidence interval [CI], 1.02-4.06; P = 0.045), higher alkaline phosphatase (HR per 10 U/L, 1.01; 95% CI, 1.00-1.02; P = 0.015), and higher enhanced liver fibrosis score (HR per unit, 1.26; 95% CI, 0.98-1.61; P = 0.073). Overall, rates of adverse events and laboratory abnormalities were similar between groups. Conclusion: Treatment with the LOXL2 inhibitor simtuzumab for 96 weeks did not provide clinical benefit in patients with PSC., (© 2018 by the American Association for the Study of Liver Diseases.)

The serine protease plasmin degrades extracellular matrix (ECM) components both directly and indirectly through activation of matrix metalloproteinases. Excessive plasmin activity and subsequent ECM degradation cause hepatic sinusoidal fragility and hemorrhage in developing embryos. We report here that excessive plasmin activity in a murine acetaminophen (APAP) overdose model likewise compromises hepatic sinusoidal vascular integrity in adult animals. We found that hepatic plasmin activity is up-regulated significantly at 6 hours after APAP overdose. This plasmin up-regulation precedes both degradation of the ECM component fibronectin around liver vasculature and bleeding from centrilobular sinusoids. Importantly, administration of the pharmacological plasmin inhibitor tranexamic acid or genetic reduction of plasminogen, the circulating zymogen of plasmin, ameliorates APAP-induced hepatic fibronectin degradation and sinusoidal bleeding. Conclusion: These studies demonstrate that reduction of plasmin stabilizes hepatic sinusoidal vascular integrity after APAP overdose. (Hepatology 2018; 00:1-13)., (© 2018 by the American Association for the Study of Liver Diseases.)

Primary sclerosing cholangitis (PSC) patients are at risk of developing cholangiocarcinoma (CCA). We have shown that (1) histamine increases biliary hyperplasia through H1/H2 histamine receptors (HRs) and (2) histamine levels increase and mast cells (MCs) infiltrate during PSC and CCA. We examined the effects of chronic treatment with H1/H2HR antagonists on PSC and CCA. Wild-type and multidrug-resistant knockout (Mdr2 -/- ) mice were treated by osmotic minipumps with saline, mepyramine, or ranitidine (10 mg/kg body weight/day) or a combination of mepyramine/ranitidine for 4 weeks. Liver damage was assessed by hematoxylin and eosin. We evaluated (1) H1/H2HR expression, (2) MC presence, (3) L-histidine decarboxylase/histamine axis, (4) cholangiocyte proliferation/bile duct mass, and (5) fibrosis/hepatic stellate cell activation. Nu/nu mice were implanted with Mz-ChA-1 cells into the hind flanks and treated with saline, mepyramine, or ranitidine. Tumor growth was measured, and (1) H1/H2HR expression, (2) proliferation, (3) MC activation, (4) angiogenesis, and (5) epithelial-mesenchymal transition (EMT) were evaluated. In vitro, human hepatic stellate cells were evaluated for H1HR and H2HR expression. Cultured cholangiocytes and CCA lines were treated with saline, mepyramine, or ranitidine (25 μM) before evaluating proliferation, angiogenesis, EMT, and potential signaling mechanisms. H1/H2HR and MC presence increased in human PSC and CCA. In H1/H2HR antagonist (alone or in combination)-treated Mdr2 -/- mice, liver and biliary damage and fibrosis decreased compared to saline treatment. H1/H2HR antagonists decreased tumor growth, serum histamine, angiogenesis, and EMT. In vitro, H1/H2HR blockers reduced biliary proliferation, and CCA cells had decreased proliferation, angiogenesis, EMT, and migration. Conclusion: Inhibition of H1/H2HR reverses PSC-associated damage and decreases CCA growth, angiogenesis, and EMT; because PSC patients are at risk of developing CCA, using HR blockers may be therapeutic for these diseases. (Hepatology 2018)., (© 2018 by the American Association for the Study of Liver Diseases.)

Hepatic ischemia-reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and acute/chronic rejection as well as a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although glucocorticoid receptor (GR) signaling may enhance cytoprotective programs, clinical use of glucocorticoid is limited because of adverse effects, whereas clinical relevance of GR-facilitated cytoprotection in OLT remains unknown. We aimed to evaluate the significance of hepatic GR in clinical OLT and verify the impact of recombinant human relaxin (rhRLX), which may function as a GR agonist in a tissue/disease-specific manner. Fifty-one OLT patients were recruited under an institutional research board (IRB) protocol. Liver biopsies were collected after cold storage (presurgery) and 2 hours postreperfusion (before abdominal closure), followed by western blotting-assisted hepatic analyses. Forty-three percent of OLTs failed to increase GR perioperatively under surgical stress. Post-/pre-GR ratios at postoperative day 1 correlated negatively with serum aspartate aminotransferase (AST)/cleaved caspase-3 and positively with B-cell lymphoma-extra large (Bcl-xL)/B-cell lymphoma 2 (Bcl-2) levels. In a murine OLT model with extended (18-hour) cold storage, treatment with rhRLX ameliorated ischemia-reperfusion (IR) damage and improved survival while up-regulating hepatocyte GR and Bcl-xL/Bcl-2 expression in OLT. rhRLX-induced GR suppressed hepatocyte high-mobility group box 1 (HMGB1) translocation/release, accompanied by decreased Toll-like receptor 4 (TLR4)/receptor for advanced glycation end products (RAGE), suppressed interleukin 1 beta (IL1β), chemokine (C-C motif) ligand 2 (CCL2), C-X-C motif chemokine (CXCL)10, tumor necrosis factor alpha (TNFα), CXCL1, and CXCL2 levels, and attenuated neutrophil/macrophage accumulation in OLT. Inhibition of GR in hepatocyte culture and in OLT diminished rhRLX-mediated cytoprotection., Conclusion: This translational study underscores the role of rhRLX-GR signaling as a regulator of hepatocellular protection against IR stress in OLT. In the context of a recent phase III clinical trial demonstrating positive outcomes of rhRLX in patients with acute heart failure, studies on rhRLX for the management of IRI in OLT recipients are warranted. (Hepatology 2018;68:258-273)., (© 2018 by the American Association for the Study of Liver Diseases.)

The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nicotinamide phosphoribosyltransferase overexpressing mice were mildly hyperglycemic at baseline and, similar to mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking nicotinamide phosphoribosyltransferase in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR., Conclusion: NAD availability is limiting during liver regeneration, and supplementation with precursors such as NR may be therapeutic in settings of acute liver injury. (Hepatology 2017;65:616-630)., Competing Interests: R.W.D. is an employee and stockholder of Chromadex Inc., which manufactures and distributes NR. The remaining authors declare no conflicts of interest., (© 2016 by the American Association for the Study of Liver Diseases.)

Hepatitis delta virus (HDV) is the most severe form of viral hepatitis. Pegylated interferon alfa (PEG-IFNα) is effective in only 25%-30% of patients and is associated with frequent side effects. The aim of this study was to analyze the clinical long-term outcome of hepatitis delta in relation to different antiviral treatment strategies. We studied 136 anti-HDV-positive patients who were followed for at least 6 months in a retrospective single-center cohort (mean time of follow-up, 5.2 years; range, 0.6-18.8). Liver cirrhosis was already present in 62 patients at first presentation. Twenty-nine percent of patients did not receive any antiviral treatment, 38% were treated with interferon alfa (IFNα)-based therapies, and 33% received nucleos(t)ide analogues (NAs) only. Clinical endpoints defined as hepatic decompensation (ascites, encephalopathy, and variceal bleeding), hepatocellular carcinoma, liver transplantation, and liver-related death developed in 55 patients (40%). Patients who received IFNα-based therapies developed clinical endpoints less frequently than those treated with NA (P = 0.02; HR, 4.0) or untreated patients (P = 0.05; HR, 2.2; 17%, 64%, and 44%), respectively, which was significant in both chi-square and Kaplan-Meier analysis. In addition, considering various clinical and virological parameters, IFNα therapy was independently associated with a more benign clinical long-term outcome in multivariate logistic regression analysis (P = 0.04; odds ratio, 0.25; 95% confidence interval, 0.07-0.9). Loss of HDV RNA during follow-up was more frequent in IFNα-treated patients and strongly linked with a lower likelihood to experience liver-related complications., Conclusion: IFNα-based antiviral therapy of hepatitis delta was independently associated with a lower likelihood for clinical disease progression. Durable undetectability of HDV RNA is a valid surrogate endpoint in the treatment of hepatitis delta. (Hepatology 2017;65:414-425)., (© 2016 by the American Association for the Study of Liver Diseases.)

Glucagon-like peptide 1 (GLP-1) receptor agonists (GLP-1-RAs) act on multiple tissues, in addition to the pancreas. Recent studies suggest that GLP-1-RAs act on liver and adipose tissue to reduce insulin resistance (IR). Thus, we evaluated the acute effects of exenatide (EX) on hepatic (Hep-IR) and adipose (Adipo-IR) insulin resistance and glucose uptake. Fifteen male subjects (age = 56 ± 8 years; body mass index = 29 ± 1 kg/m 2 ; A1c = 5.7 ± 0.1%) were studied on two occasions, with a double-blind subcutaneous injection of EX (5 μg) or placebo (PLC) 30 minutes before a 75-g oral glucose tolerance test (OGTT). During OGTT, we measured hepatic (HGU) and adipose tissue (ATGU) glucose uptake with [ 18 F]2-fluoro-2-deoxy-D-glucose/positron emission tomography, lipolysis (RaGly) with [U- 2 H 5 ]-glycerol, oral glucose absorption (RaO) with [U- 13 C 6 ]-glucose, and hepatic glucose production (EGP) with [6,6- 2 H 2 ]-glucose. Adipo-IR and Hep-IR were calculated as (FFA 0-120min ) × (Ins 0-120min ) and (EGP 0-120min ) × (Ins 0-120min ), respectively. EX reduced RaO, resulting in reduced plasma glucose and insulin concentration from 0 to 120 minutes postglucose ingestion. EX decreased Hep-IR (197 ± 28 to 130 ± 37; P = 0.02) and increased HGU of orally administered glucose (23 ± 4 to 232 ± 89 [μmol/min/L]/[μmol/min/kg]; P = 0.003) despite lower insulin (23 ± 5 vs. 41 ± 5 mU/L; P < 0.02). EX enhanced insulin suppression of RaGly by decreasing Adipo-IR (23 ± 4 to 13 ± 3; P = 0.009). No significant effect of insulin was observed on ATGU (EX = 1.16 ± 0.15 vs. PLC = 1.36 ± 0.13 [μmol/min/L]/[μmol/min/kg])., Conclusion: Acute EX administration (1) improves Hep-IR, decreases EGP, and enhances HGU and (2) reduces Adipo-IR, improves the antilipolytic effect of insulin, and reduces plasma free fatty acid levels during OGTT. (Hepatology 2016;64:2028-2037)., (© 2016 by the American Association for the Study of Liver Diseases.)

Unlabelled: Wilson disease (WD) is a hepatoneurological disorder caused by mutations in the copper-transporter, ATP7B. Copper accumulation in the liver is a hallmark of WD. Current therapy is based on copper chelation, which decreases the manifestations of liver disease, but often worsens neurological symptoms. We demonstrate that in Atp7b(-/-) mice, an animal model of WD, liver function can be significantly improved without copper chelation. Analysis of transcriptional and metabolic changes in samples from WD patients and Atp7b(-/-) mice identified dysregulation of nuclear receptors (NRs), especially the liver X receptor (LXR)/retinoid X receptor heterodimer, as an important event in WD pathogenesis. Treating Atp7b(-/-) mice with the LXR agonist, T0901317, ameliorated disease manifestations despite significant copper overload. Genetic markers of liver fibrosis and inflammatory cytokines were significantly decreased, lipid profiles normalized, and liver function and histology were improved., Conclusions: The results demonstrate the major role of an altered NR function in the pathogenesis of WD and suggest that modulation of NR activity should be explored as a supplementary approach to improving liver function in WD. (Hepatology 2016;63:1828-1841)., (© 2015 by the American Association for the Study of Liver Diseases.)

Unlabelled: Acetaminophen (APAP) overdose is a frequent cause of drug-induced liver injury and the most frequent cause of acute liver failure in the Western world. Previous studies with mouse models have revealed that impairment of mitochondrial respiration is an early event in the pathogenesis, but the exact mechanisms have remained unclear, and therapeutic approaches to specifically target mitochondria have been insufficiently explored. Here, we found that the reactive oxidative metabolite of APAP, N-acetyl-p-benzoquinoneimine (NAPQI), caused the selective inhibition of mitochondrial complex II activity by >90% in both mouse hepatic mitochondria and yeast-derived complexes reconstituted into nanoscale model membranes, as well as the decrease of succinate-driven adenosine triphosphate (ATP) biosynthesis rates. Based on these findings, we hypothesized that methylene blue (MB), a mitochondria-permeant redox-active compound that can act as an alternative electron carrier, protects against APAP-induced hepatocyte injury. We found that MB (<3 µM) readily accepted electrons from NAPQI-altered, succinate-energized complex II and transferred them to cytochrome c, restoring ATP biosynthesis rates. In cultured mouse hepatocytes, MB prevented the mitochondrial permeability transition and loss of intracellular ATP without interfering with APAP bioactivation. In male C57BL/6J mice treated with APAP (450 mg/kg, intraperitoneally [IP]), MB (10 mg/kg, IP, administered 90 minutes post-APAP) protected against hepatotoxicity, whereas mice treated with APAP alone developed massive centrilobular necrosis and increased serum alanine aminotransferase activity. APAP treatment inhibited complex II activity ex vivo, but did not alter the protein expression levels of subunits SdhA or SdhC after 4 hours., Conclusion: MB can effectively protect mice against APAP-induced liver injury by bypassing the NAPQI-altered mitochondrial complex II, thus alleviating the cellular energy crisis. Because MB is a clinically used drug, its potential application after APAP overdose in patients should be further explored., (© 2014 by the American Association for the Study of Liver Diseases.)

Unlabelled: Drug-induced liver injury (DILI) is an important cause of acute liver failure, with limited therapeutic options. During DILI, oncotic necrosis with concomitant release and recognition of intracellular content amplifies liver inflammation and injury. Among these molecules, self-DNA has been widely shown to trigger inflammatory and autoimmune diseases; however, whether DNA released from damaged hepatocytes accumulates into necrotic liver and the impact of its recognition by the immune system remains elusive. Here we show that treatment with two different hepatotoxic compounds (acetaminophen and thioacetamide) caused DNA release into the hepatocyte cytoplasm, which occurred in parallel with cell death in vitro. Administration of these compounds in vivo caused massive DNA deposition within liver necrotic areas, together with an intravascular DNA coating. Using confocal intravital microscopy, we revealed that liver injury due to acetaminophen overdose led to a directional migration of neutrophils to DNA-rich areas, where they exhibit an active patrolling behavior. DNA removal by intravenous DNASE1 injection or ablation of Toll-like receptor 9 (TLR9)-mediated sensing significantly reduced systemic inflammation, liver neutrophil recruitment, and hepatotoxicity. Analysis of liver leukocytes by flow cytometry revealed that emigrated neutrophils up-regulated TLR9 expression during acetaminophen-mediated necrosis, and these cells sensed and reacted to extracellular DNA by activating the TLR9/NF-κB pathway. Likewise, adoptive transfer of wild-type neutrophils to TLR9(-/-) mice reversed the hepatoprotective phenotype otherwise observed in TLR9 absence., Conclusion: Hepatic DNA accumulation is a novel feature of DILI pathogenesis. Blockage of DNA recognition by the innate immune system may constitute a promising therapeutic venue., (© 2014 by the American Association for the Study of Liver Diseases.)

Unlabelled: Coffee may have hepatoprotective effects and higher coffee consumption has been associated inversely with levels of liver enzymatic markers. However, it is unclear whether decaffeinated coffee is also associated with liver enzymes. The study population included 27,793 participants, age 20 or older, in the U.S. National Health and Nutrition Examination Survey (1999-2010). Coffee intake was evaluated by 24-hour dietary recall. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl transaminase (GGT) were measured. We examined the relationship between coffee intake and enzymatic levels using weighted multiple variable logistic (abnormally elevated levels of enzymes) and linear regression (continuous enzymatic levels). Total coffee consumption was inversely associated with abnormal levels of all four liver enzymes and continuous levels of AST, ALP, and GGT. Compared to those reporting no coffee consumption, participants reporting ≥ 3 cups per day had an odds ratio (OR; 95% confidence interval [CI]) of 0.75 (0.63, 0.89), 0.82 (0.68, 0.98), 0.73 (0.55, 0.95), and 0.69 (0.57, 0.83) for abnormal levels of ALT, AST, ALP, and GGT, respectively. Similar inverse associations were found with decaffeinated coffee intake and abnormal levels of ALT (OR (≥ 2 vs 0 cup/d): 0.62 [0.41, 0.94]), AST (0.74 [0.49, 1.11]), and GGT (0.70 [0.49-1.00])., Conclusion: Higher intakes of coffee, regardless of its caffeine content, were associated with lower levels of liver enzymes., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Unlabelled: Understanding hepatitis C virus (HCV) replication has been limited by access to serial samples of liver, the primary site of viral replication. Our understanding of how HCV replicates and develops drug-resistant variants in the liver is limited. We studied 15 patients chronically infected with genotype 1 HCV treated with telaprevir (TVR)/pegylated-interferon alpha/ribavirin. Hepatic fine needle aspiration was performed before treatment and at hour 10, days 4 and 15, and week 8 after initiation of antiviral therapy. We measured viral kinetics, resistance patterns, TVR concentrations, and host transcription profiles. All patients completed all protocol-defined procedures that were generally well tolerated. First-phase HCV decline (baseline/treatment day 4) was significantly slower in liver than in plasma (slope plasma: -0.29; liver, -0.009; P < 0.001), whereas second-phase decline (posttreatment days 4-15) did not differ between the two body compartments (-0.11 and -0.15, respectively; P = 0.1). TVR-resistant variants were detected in plasma, but not in liver (where only wild-type virus was detected). Based upon nonstructural protein 3 sequence analysis, no compartmentalization of viral populations was observed between plasma and liver compartments. Gene expression profiling revealed strong tissue-specific expression signatures. Human intrahepatic TVR concentration, measured for the first time, was lower, compared to plasma, on a gram per milliliter basis. We found moderate heterogeneity between HCV RNA levels from different intrahepatic sites, indicating differences in hepatic microenvironments., Conclusion: These data support an integrated model for HCV replication wherein the host hepatic milieu and innate immunity control the level of viral replication, and the early antiviral response observed in the plasma is predominantly driven by inhibition of hepatic high-level HCV replication sites., (© 2014 by the American Association for the Study of Liver Diseases.)

Unlabelled: Invariant natural killer T (iNKT) cells are a major subset of lymphocytes found in the liver. These cells mediate various functions, including hepatic injury, fibrogenesis, and carcinogenesis. However, the function of iNKT cells in liver regeneration remains unclear. In the present study, partial hepatectomy (PHx) was used to study liver regeneration. α-Galactosylceramide (α-GalCer), a specific ligand for iNKT cells, was used to induce iNKT cell activation. After PHx, two strains of iNKT cell-deficient mice, CD1d(-/-) and Jα281(-/-) mice, showed normal liver regeneration. Injection of α-GalCer before or after PHx, which rapidly stimulated interferon-gamma (IFN-γ) and interleukin (IL)-4 production by iNKT cells, markedly inhibited liver regeneration. In vitro treatment with IFN-γ inhibited hepatocyte proliferation. In agreement with this in vitro finding, genetic disruption of IFN-γ or its downstream signaling molecule signal transducer and activator of transcription (STAT)1 significantly abolished the α-GalCer-mediated inhibition of liver regeneration. In vitro exposure to IL-4 did not affect hepatocyte proliferation, but surprisingly, genetic ablation of IL-4 or its downstream signaling molecule STAT6 partially eliminated the inhibitory effect of α-GalCer on liver regeneration. Further studies revealed that IL-4 contributed to α-GalCer-induced iNKT cell expansion and IFN-γ production, thereby inhibiting liver regeneration., Conclusion: iNKT cells play a minor role in controlling liver regeneration after PHx under healthy conditions. Activation of iNKT cells by α-GalCer induces the production of IFN-γ, which directly inhibits liver regeneration, and IL-4, which indirectly attenuates liver regeneration by stimulating iNKT cell expansion and IFN-γ production., (© 2014 by the American Association for the Study of Liver Diseases. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Unlabelled: Acetaminophen (APAP)-induced acute liver injury (AILI) is a major health problem. Accumulating evidence suggests that the sympathetic nervous system (SNS) regulates neuronal and hematopoietic progenitors. SNS signaling affects hepatic progenitor/oval cells (HPCs) and β-adrenoceptor agonism will expand HPCs to reduce AILI. Dopamine β-hydroxylase-deficient mice (Dbh-/-), lacking catecholamine SNS neurotransmitters, isolated HPCs, and immature ductular 603B cells were initially used to investigate SNS involvement in HPC physiology. Subsequently, control mice were treated with APAP (350 mg/kg) followed by the β-adrenoceptor agonist, isoproterenol (ISO), or the β-adrenoceptor antagonist, propranolol. Mechanistic studies examined effects of non-SNS HPC expansion on AILI, involvement of the canonical Wnt/β-catenin pathway (CWP) in the action of ISO on HPC expansion and comparison of ISO with the current standard of care, N-acetylcysteine (NAC). Dbh-/- mice lacking catecholamines had low HPC numbers, reconstituted by ISO. In vitro, ISO-induced proliferation of 603B cells was CWP dependent. In control mice, AILI raised HPC numbers, further increased by ISO, with attenuation of liver injury. Delayed administration of NAC did not, but delayed ISO did, reverse AILI. Propranolol worsened AILI. AILI activated the CWP, and ISO enhanced Wnt-ligand production. HPCs were the major source of Wnt ligands. Recombinant Wnt3a and ISO-603B-conditioned media, but not ISO alone, protected isolated hepatocytes from death, reversed by DKK1-a Wnt antagonist. Additionally, tumor-associated weak inducer of apoptosis expanded HPCs and protected against AILI. Furthermore, allotransplantation of HPCs from APAP+ISO-treated mice to other APAP-injured mice improved AILI, an effect antagonized by DKK1., Conclusion: SNS catecholamines expand HPCs, which are both targets and sources of Wnt ligands. Hepatoprotection by ISO is mediated by para- and autocrine effects of Wnt signaling. ISO represents novel pharmacotherapy for AILI., (© 2014 by the American Association for the Study of Liver Diseases.)

Unlabelled: Caffeine is one of the world's most consumed drugs. Recently, several studies showed that its consumption is associated with lower risk for nonalcoholic fatty liver disease (NAFLD), an obesity-related condition that recently has become the major cause of liver disease worldwide. Although caffeine is known to stimulate hepatic fat oxidation, its mechanism of action on lipid metabolism is still not clear. Here, we show that caffeine surprisingly is a potent stimulator of hepatic autophagic flux. Using genetic, pharmacological, and metabolomic approaches, we demonstrate that caffeine reduces intrahepatic lipid content and stimulates β-oxidation in hepatic cells and liver by an autophagy-lysosomal pathway. Furthermore, caffeine-induced autophagy involved down-regulation of mammalian target of rapamycin signaling and alteration in hepatic amino acids and sphingolipid levels. In mice fed a high-fat diet, caffeine markedly reduces hepatosteatosis and concomitantly increases autophagy and lipid uptake in lysosomes., Conclusion: These results provide novel insight into caffeine's lipolytic actions through autophagy in mammalian liver and its potential beneficial effects in NAFLD., (© 2014 by the American Association for the Study of Liver Diseases.)

Unlabelled: Sorafenib--a broad kinase inhibitor--is a standard therapy for advanced hepatocellular carcinoma (HCC) and has been shown to exert antifibrotic effects in liver cirrhosis, a precursor of HCC. However, the effects of sorafenib on tumor desmoplasia--and its consequences on treatment resistance--remain unknown. We demonstrate that sorafenib has differential effects on tumor fibrosis versus liver fibrosis in orthotopic models of HCC in mice. Sorafenib intensifies tumor hypoxia, which increases stromal-derived factor 1 alpha (SDF-1α) expression in cancer and stromal cells and, subsequently, myeloid differentiation antigen-positive (Gr-1(+)) myeloid cell infiltration. The SDF-1α/C-X-C receptor type 4 (CXCR4) pathway directly promotes hepatic stellate cell (HSC) differentiation and activation through the mitogen-activated protein kinase pathway. This is consistent with the association between SDF-1α expression with fibrotic septa in cirrhotic liver tissues as well as with desmoplastic regions of human HCC samples. We demonstrate that after treatment with sorafenib, SDF-1α increased the survival of HSCs and their alpha-smooth muscle actin and collagen I expression, thus increasing tumor fibrosis. Finally, we show that Gr-1(+) myeloid cells mediate HSC differentiation and activation in a paracrine manner. CXCR4 inhibition, using AMD3100 in combination with sorafenib treatment, prevents the increase in tumor fibrosis--despite persistently elevated hypoxia--in part by reducing Gr-1(+) myeloid cell infiltration and inhibits HCC growth. Similarly, antibody blockade of Gr-1 reduces tumor fibrosis and inhibits HCC growth when combined with sorafenib treatment., Conclusion: Blocking SDF-1α/CXCR4 or Gr-1(+) myeloid cell infiltration may reduce hypoxia-mediated HCC desmoplasia and increase the efficacy of sorafenib treatment., (© 2014 by the American Association for the Study of Liver Diseases.)