JHEP Reports

Background and aimsAlcohol disturbs hepatic lipid synthesis and transport, but the role of lipid dysfunction in the severity of alcohol-related liver disease (ALD) is unclear. We therefore characterised the liver and plasma lipidome in a biopsy-controlled cohort of patients with early ALD. MethodsWe performed ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry for lipidomics of the liver and plasma from 315 patients, and of plasma from 51 healthy controls matched for age, gender and BMI. We correlated lipid levels with histological fibrosis, inflammation and steatosis, after correction for multiple testing and adjustment for age, gender, statin use, BMI, HbA1c, HOMA-IR, and ongoing drinking. Moreover, we investigated the mechanism of dysregulated sphingolipid metabolism by whole-blood transcriptomics and qPCR sequencing of miRNA. ResultsWe detected 198 lipids in the liver and 236 lipids in the circulation from 18 lipid classes. Nearly all ceramides, sphingomyelins and lyso-phosphocholines in plasma decreased as fibrosis progressed. This was paralleled by a comparable decrease in the liver. Circulating and liver sphingomyelins were also inversely associated with hepatic inflammation. The lipidomic signature of healthy controls was only comparable to ALD patients with no fibrosis. Three circulating miRNA, highly involved in sphingomyelin metabolism, were dysregulated together with the mRNA expression of enzymes in the sphingomyelin degradation pathway. Mendelian randomization in Finnish and UK population biobanks externally validated our findings, suggesting a causal relationship between genetic disposition to ALD and low sphingolipid levels. ConclusionsLiver fibrosis severity in alcohol-related liver disease is characterized by selective lipid depletion in blood and liver, indicating profound effects of progressive disease on the bioactive sphingolipids, already from early stages of fibrosis. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/21260429v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@ad651org.highwire.dtl.DTLVardef@17c7c95org.highwire.dtl.DTLVardef@1d742f7org.highwire.dtl.DTLVardef@16da292_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThe lipidome in the liver and circulation in alcohol-related liver disease (ALD) C_LIO_LISphingolipid, phospholipid and triacylglycerol levels were lowered in fibrosis C_LIO_LIExpression and regulation of genes in the sphingolipid pathway were affected C_LIO_LIALD has a causal link to lowered sphingomyelin levels in blood C_LI Lay summaryAlcohol causes a dysfunctional fat metabolism in the liver. In this study, we detected 198 different types of lipids in the liver and 236 in the blood stream of patients with different severity of alcohol-related liver disease. We found that patients with more severe scarring (fibrosis) of the liver, and more severe liver inflammation, had lower levels of sphingolipids both in the circulation and the liver. Sphingolipids regulate cell survival and inflammation, so they may be involved in the mechanism of progressive alcohol-related liver disease.

Background and aimsAlcohol-associated hepatitis (AH) is the most life-threatening form of alcohol-associated liver disease (ALD). AH is characterized by severe inflammation attributed to increased levels of ethanol, microbes or microbial components, and damage-associated molecular pattern (DAMP) molecules in the liver. HSPB1 (Heat Shock Protein Family B (Small) Member 1; also known as Hsp25/27) is a DAMP that is rapidly increased in and released from cells experiencing stress, including hepatocytes. The goal of this study was to define the role of HSPB1 in AH pathophysiology. MethodsSerum HSPB1 was measured in a retrospective study of 184 heathy controls (HC), heavy alcohol consumers (HA), patients with alcohol-associated cirrhosis (AC), and patients with AH recruited from major hospital centers. HSPB1 was also retrospectively evaluated in liver tissue from 10 HC and AH patients and an existing liver RNA-seq dataset. Finally, HSPB1 was investigated in a murine Lieber-DeCarli diet model of early ALD as well as cellular models of ethanol stress in hepatocytes and hepatocyte-macrophage communication during ethanol stress. ResultsCirculating HSPB1 was significantly increased in AH patients and levels positively correlated with disease-severity scores. Likewise, HSPB1 was increased in the liver of patients with severe AH and in the liver of ethanol-fed mice. In vitro, ethanol-stressed hepatocytes released HSPB1, which then triggered TNF-mediated inflammation in macrophages. Anti-HSPB1 antibody prevented TNF release from macrophages exposed to media conditioned by ethanol-stressed hepatocytes. ConclusionsOur findings support investigation of HSPB1 as both a biomarker and therapeutic target in ALD. Furthermore, this work demonstrates that anti-HSPB1 antibody is a rational approach to targeting HSPB1 with the potential to block inflammation and protect hepatocytes, without inactivating host defense. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/24313193v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@141816eorg.highwire.dtl.DTLVardef@1985d66org.highwire.dtl.DTLVardef@1c0ed4org.highwire.dtl.DTLVardef@118ca9_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIHSPB1 is significantly increased in serum and liver of patients with alcohol-associated hepatitis. C_LIO_LIEthanol consumption leads to early increases in HSPB1 in the mouse liver. C_LIO_LIHepatocytes subjected to ethanol stress release HSPB1 into the extracellular environment where it activates TNF-mediated inflammation in macrophages. C_LIO_LIAnti-HSPB1 antibody blocks hepatocyte-triggered TNF in a model of hepatocyte-macrophage communication during ethanol stress. C_LI

BackgroundAlcohol overuse disrupts liver function and alters microbial gut communities, with alcohol-related liver disease (ALD) accounting for half of all liver-related deaths worldwide. Bile acids (BAs) regulate liver and gut function, but their metabolism becomes disrupted in ALD. While it is known that gut microbes transform primary to secondary BAs, which are subsequently reabsorbed via the enterohepatic circulation, BA metabolism during ALD progression remains poorly understood. MethodsWe investigated BA co-metabolism in a cross-sectional cohort of individuals with ALD (n=462) and healthy controls (n=148). We validated key findings in two independent ALD cohorts (n=34 and n=52). We integrated BA concentrations, measured by targeted mass spectrometry in feces and plasma, with liver proteomics, and gut microbiome profiles derived from metagenomic and metatranscriptomic sequencing of fecal samples. ResultsAdvanced fibrosis was associated with decreased hepatic BA synthesis and impaired BA transport. Despite this, disease progression corresponded with increased levels of primary and secondary BAs in plasma and feces. The abundance of microbial secondary BA dehydroxylation and epimerization pathways in the gut microbiome changed with disease severity. Genes encoding early steps in the multi-step dehydroxylation pathway increased, whereas those involved in later steps were depleted, indicating a community-level microbial imbalance. In ALD, we identified Eggerthella lenta as a key mediator of BA dehydroxylation, while Mediterraneibacter torques and Bacteroides thetaiotaomicron facilitated most of the BA epimerization as a detoxification mechanism. ConclusionFibrotic ALD is characterized by disrupted primary BA synthesis and transport, leading to BA accumulation in the gut and blood circulation. Altered microbial secondary BA metabolism reflects a compensatory mechanism that becomes impaired at advanced fibrosis stages. Our findings highlight the gut-liver axis as an important factor influencing ALD progression. Impact and ImplicationsO_LIWith the progression of alcohol-related liver disease (ALD), levels of bile acids (BA) in serum and feces increase, but BA production and transport are impaired in the liver. C_LIO_LISecondary microbial BA metabolism, particularly epimerization and dehydroxylation, increases in ALD. However, a key enzyme, baiN, is depleted, illustrating a microbial community-level metabolic dysbiosis. C_LIO_LIThe main contributing microbial species were, among others, Mediterraneibacter torques, Bacteroides thetaiotaomicron, and Eggerthella lenta, which could serve as potential targets for future microbial-targeted interventions. C_LI Lay summaryAlcohol-related liver disease (ALD) from long-term alcohol overuse affects how the liver and gut interact, especially in handling bile acids (BAs), which are molecules produced by the liver and transformed by gut bacteria. Our study found that in people with ALD, the liver produces fewer BAs, but BAs accumulate in the gut and blood because their transport is impaired. We also observed that bacterial transformations of these BA change as the disease progresses, most likely due to an imbalance in the gut microbiome. For the first time, we identify specific bacterial species that strongly influence BA levels in ALD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/25332046v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@19966f0org.highwire.dtl.DTLVardef@352be6org.highwire.dtl.DTLVardef@d5146dorg.highwire.dtl.DTLVardef@1304022_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 0:C_FLOATNO Graphical Abstract C_FIG

Alterations in cellular metabolism represent an important response to proliferative signals in both normal and transformed tissues. The benign proliferative process of liver regeneration after partial hepatectomy offers insight into homeostatic mechanisms to control liver mass, which are disrupted in liver disease induced by viral factors, alcohol, or associated with obesity. Moreover, successful targeting of cancer depends on the identification of genes and pathways that are selectively activated in the transformed state. Here, we present a differential transcriptomic and metabolomic analysis of benign proliferative and transformed liver, including associated plasma metabolite and lipid species. Using partial hepatectomy-induced liver regeneration and diethylnitrosamine (DEN) induced carcinogenesis, we identify and analyze alterations specific to multiple regenerative and transformed tissue states. Transcriptomics and LC/MS based metabolite profiling reveal fatty acid import and storage are specifically rewired during liver regeneration in a time dependent manner, a phenomenon not observed in liver tumors. In contrast, liver tumors exhibit preferential activation of numerous metabolic pathways, including glycolysis, serine biosynthesis, and polyamine metabolism. Alterations in serine metabolism occur at the earliest detectable stages in tumorigenesis and promote survival upon serine restriction. These data demonstrate that transformation-induced alterations in metabolism are distinct from those observed in normal regenerative cell division, which may be used to identify transformation-specific liabilities.

BackgroundRecently, the new nomenclature metabolic dysfunction-associated steatohepatitis (MASH) was proposed to supersede non-alcoholic steatohepatitis (NASH). To optimize the management of these patients, it is crucial to comprehend the similarities and differences between individuals with NASH and MASH. MethodsWe analyzed data from 13,846 participants in the third National Health and Nutrition Examination Surveys, along with their linked mortality through 2019. NASH and MASH were defined based on respective criteria. Survey-weight adjusted multivariable Cox proportional model was used to examine mortality. ResultsThe overall prevalence of steatohepatitis, NASH and MASH was 5.7% (n=788), 4.1% (n=564) and 5.5% (n=763), respectively. Most individuals with NASH (96.8%) could be categorized as MASH, but only 69.7% individuals with MASH qualified as NASH. During a median follow-up of 27 years, individuals with MASH exhibited a 53% higher risk of all-cause mortality (adjusted hazard ratio [aHR] 1.53, 95% CI 1.24-1.89). But individuals with NASH didnt show an association with all-cause mortality after adjustment for metabolic risk factors (aHR 1.14, 95% CI 0.91-1.44). Notably, individuals who met the criteria for MASH but not NASH (NASH(-)/MASH(+)) had a higher risk of all-cause mortality (aHR 2.47, 95% CI 1.71-3.57) compared to those with NASH(+)/MASH(+) (aHR 1.22, 95% CI 0.97-1.55). Moreover, advanced fibrosis was only associated with an increased risk of all-cause mortality in individuals with MASH, not NASH. ConclusionsMASH, rather than NASH, was associated with an elevated risk of all-cause mortality after adjusting for metabolic risk factors. Well-designed prospective studies are needed to assess and validate our findings.

Background & AimsAlterations in the glycosylation of blood proteins affect protein functionality and have been linked to various diseases. Metabolic dysfunction- associated steatotic liver disease (MASLD) is a silent disease, of which progression to advanced disease stages including metabolic dysfunction-associated steatohepatitis (MASH), fibrosis and cirrhosis often goes unnoticed. As current non- invasive diagnostic tests lack specificity, the purpose of this work was to study total blood protein N-glycosylation in individuals with MASLD and various degrees of fibrosis as compared to healthy controls. MethodsIn two independent cross-sectional cohort studies, blood N-glycosylation analysis was performed by mass spectrometry on released glycans of overall 132 MASLD patients and 99 age- and sex-matched healthy controls. Relationships between glycosylation traits and the disease spectrum of MASLD including fibrotic MASLD were investigated in comparison to healthy controls. Furthermore, publicly available transcriptomics datasets were used to explore glycosyltransferase expression in patients with MASLD. ResultsGlobally lower 2,3-sialylation distinguished MASLD from healthy controls (OR [CI]=0.36; [0.18-0.67]; p-value=0.019, and 0.11 [0.04-0.24]; p-value<0.000001), as well as non-fibrotic MASLD from its fibrotic counterparts (OR: 0.13 [0.06-0.26]; p- value<0.0001), but showed no association with steatohepatitis activity. Hepatic ST3GAL6, a sialyltransferase responsible for N-glycan 2,3-sialylation, negatively associated with fibrosis progression, similar to the observed glycomic signature. Both glycomic and transcriptomic signatures were replicated in independent cohorts. ConclusionsFibrotic MASLD is characterized by a global decrease of blood protein 2,3-sialylation and according decrease in hepatic 2,3-sialyltransferase expression, associating with disease progression. These findings suggest alterations in the N- glycan biosynthetic pathway and are potentially useful in the early diagnosis of fibrosis in MASLD. Lay SummaryNon-invasive markers of fibrotic MASLD perform suboptimal. This research identified that changes in blood protein glycosylation coincide with fibrosis development, offering blood-based markers that could potentially replace a liver biopsy. What You Need to KnowO_ST_ABSBACKGROUND AND CONTEXTC_ST_ABSThe majority of the plasma glycoproteins is synthesized in the liver and changes to their glycosylation are known to affect their function and to associate with liver disorders. NEW FINDINGSMASLD patients exhibit lower 2,3-sialylation on the complete range of their blood protein N-glycans, which coincides with the histological appearance of fibrosis, mediated likely via downregulation of hepatic ST3GAL6. LIMITATIONSWhile the findings of this study has could have implications for diagnosing fibrotic MASLD, the identified glycomic signature needs to be confirmed in a larger, ideally prospective patient cohort. CLINICAL RESEARCH RELEVANCEBy identifying specific signatures in the blood protein N-glycome, this research offers potential non-invasive markers for early diagnosis and monitoring of fibrosis in MASLD. Non-invasive diagnosis could potentially lessen the need for liver biopsies, and allow for timely intervention and improved disease management, ultimately leading to improvement of patient outcomes and the reduction of liver-related morbidity and mortality. BASIC RESEARCH RELEVANCEThe observed glycomic and transcriptomic signatures offer molecular-level insights into fibrosis development in MASLD. This paves the way for future research at the intersection of glycoscience and hepatology, that will offer deeper insights into the pathophysiology of this liver disease.

Introduction and ObjectivesMetabolic dysfunction-associated steatotic liver disease (MASLD) affects about one-quarter of adults worldwide, and liver fibrosis is its strongest predictor of liver-related morbidity and mortality. Using combined in-silico screening and clinical validation, we aimed to identify circulating biomarkers associated with fibrosis progression. Fibulin-3 was identified, and its diagnostic performance was evaluated in biopsy-proven MASLD cohorts. Materials and MethodsThe GSE125251 RNA-seq dataset was reanalyzed to compare liver transcriptomes from MASLD subjects with minimal (F0-F1) versus moderate to advanced fibrosis (F2/F3-F4). Differentially expressed genes (DEGs) were filtered to retain plasma-secreted, protein-coding candidates. Top-ranked genes were evaluated in liver biopsies from a morbidly obese cohort (n = 65) stratified by fibrosis stage, and their plasma levels were measured via ELISA in an independent bariatric cohort (n = 225). ResultsAmong 106 DEGs, 22 encoded plasma-circulating proteins. Six top candidates (EFEMP1, LTBP2, LUM, DPT, CHI3L1, CCL20) were prioritized. EFEMP1 (Fibulin-3) showed the strongest association with fibrosis, with significantly higher hepatic mRNA and protein expression in F2/F3-F4 versus F0-F1 (p < 0.005). Plasma Fibulin-3 levels correlated with fibrosis stage ({rho} = 0.40, p < 0.0001), increasing from 9.4 ng/mL in F0-F1 to 21.7 ng/mL in F2/F3-F4. Its diagnostic performance for F [&ge;] 2 (AUROC = 0.78) exceeded that of APRI, FIB-4, NFS, and HSI. A combined index including Fibulin-3, HSI, platelets, and GGT increased the AUROC to 0.87 (CI: 0.79-0.92). ConclusionsPlasma Fibulin-3 is notably higher in individuals with advanced MASLD and represents a promising non-invasive biomarker for liver fibrosis stratification in metabolically unhealthy obese populations.

Background and ObjectivesBariatric surgery is a highly effective obesity treatment, yet it may predispose individuals to alcohol-related liver injury. While altered ethanol metabolism following procedures like Roux-en-Y gastric bypass (RYGB) is well described, the long-term hepatic consequences, particularly the risk of portal hypertension in patients who develop alcohol-related hepatitis (AH,) remain poorly defined. MethodsUsing the TriNetX US Collaborative Network, we identified adult patients diagnosed with AH or alcohol-related cirrhosis. We compared outcomes between patients with a history of RYGB or sleeve gastrectomy (SG) who subsequently developed AH (Bariatric+AH group) and those with AH and no history of bariatric surgery (AH-only group). Propensity score matching was performed on over 44 demographic, clinical, and laboratory variables. Cox proportional hazards models and Kaplan-Meier survival curves were used to estimate the risk of clinically significant portal hypertension (PH) events, liver transplantation, and all-cause mortality at three-, five-, and seven-year follow-ups. ResultsAfter matching, 772 patients were included in each cohort. At 7 years post-index event, the Bariatric + AH group exhibited a significantly higher risk of PH-related complications compared to the AH-only group (HR 1.519; 95% CI, 1.15-2.005; p = 0.003). No significant differences were observed in liver transplantation (HR 1.412; 95% CI, 0.850-2.346; p = 0.181) or all-cause mortality (HR 1.085; 95% CI, 0.904-1.303; p = 0.381). These findings were consistent across all follow-up intervals. ConclusionBariatric surgery is associated with an increased long-term risk of portal hypertension in patients who develop alcohol-related hepatitis despite similar mortality and transplantation rates. These findings underscore the need for targeted postoperative counseling, liver-focused surveillance strategies, and integration of hepatologic risk assessment into metabolic surgery care pathways.

Background & AimsHepatitis B virus (HBV) drives hepatocellular carcinoma in part through the activity of its X protein (HBx), yet the mechanisms by which HBx alters hepatocyte function remain incompletely understood. Progress has been limited by the lack of relevant human models that support controlled HBx expression in mature hepatocytes. Here, we use an improved hepatocyte-like organoid (HLO) platform that supports enhanced hepatocyte maturation to investigate HBx function in a differentiated hepatocyte context. MethodsAdult stem cell-derived HLOs were differentiated using an optimized protocol to generate hepatocyte-like cells with enhanced maturation and transcriptional similarity to primary liver tissue. HBx function was interrogated using both cognate promoter-driven expression and doxycycline-inducible systems across multiple donor-derived organoid lines. Transcriptomic, pathway, and single-cell imaging analyses were performed to assess the impact of HBx expression on hepatocytes. ResultsHBx expression consistently suppressed apoptosis-associated transcripts and reduced expression of core hepatocyte identity genes, including CYP3A4. Pathway analysis revealed downregulation of liver-specific functions, including metabolism, detoxification, complement, and coagulation. At the single-cell level, higher HBx expression was associated with reduced caspase 3/7 activation following apoptotic challenge and decreased hepatocyte marker expression. Functionally, HBx expression increased resistance to apoptosis and enhanced the ability of differentiated hepatocyte-like cells to revert to a proliferative, less differentiated state. ConclusionsHBx expression in differentiated human liver organoids reduces apoptosis and impairs hepatocyte identity, consistently across donors and expression systems. These findings support a model in which HBx promotes a survival-permissive less differentiated state that may contribute to early HBV-driven tumorigenesis. This HLO platform provides a relevant system to dissect HBV-host interactions and reveals a mechanism by which HBV may prime the liver for malignant transformation. Impact and implicationsUnderstanding how HBV promotes hepatocellular carcinoma remains a critical challenge, partly due to the lack of physiologically relevant human derived model systems to study HBx function. Using a differentiated adult human liver organoid system, we show that HBx simultaneously suppresses apoptosis and disrupts hepatocyte identity, providing a mechanistic framework for how HBV may prime hepatocytes for malignant transformation. These findings are particularly relevant for researchers studying HBV pathogenesis and liver cancer, as well as for clinicians aiming to better understand early disease progression. While further validation in more complex multicellular systems is needed, this platform can support the identification of HBx-targeted therapeutic strategies and guide the development of improved adult human derived models for virus-host interaction studies.

Background and AimsThe newly proposed nomenclature for steatotic liver diseases (SLD) aims to reduce the stigma associated with "non-alcoholic fatty liver disease" (NAFLD), increase awareness, and provide a framework for delineating pathogenic pathways. Approach and ResultsWe projected the new nomenclatures diagnostic scheme onto National Health and Nutrition Examination Survey (NHANES) data and determined SLD prevalence, fibrosis risk factors, subtypes, and consistency with previous classifications. Steatosis grade and fibrosis stage were estimated from vibration controlled transient elastography (VCTE). At a threshold of 240 dB/m, 62.1% [95% confidence interval (CI), 59.8-64.3%] of adults ([&ge;] 20 years) and 30.5% (95% CI, 27.1-34.0%) of adolescents (12-19 years) had SLD. By American Gastroenterological Association criteria, 19.3 million (95% CI, 15.8-22.8) adults with SLD qualify for hepatology referral. Over 98% of adults but only 85% of adolescents with NAFLD met criteria for definite MASLD. Significant fibrosis ([&ge;] 8.6 kPa) occurred in 13.5 million (95% CI, 10.9-16.2) adults with MASLD; risk factors varied by race and ethnicity. Significant fibrosis occurred in over 1.5 million adults without any identified LD and was associated with lead (Pb) exposure, odds ratio = 3.89 (95% CI, 2.00-7.56). ConclusionsThe overarching term, SLD, changes the diagnostic algorithm and creates an umbrella classification that highlights the extraordinary prevalence of liver steatosis. The more precise nomenclature establishes a valuable patient-centric platform for research and clinical care, clarifying risk groups and risk factors, including adolescents with NAFLD but without definite MASLD and adults without SLD in whom toxic exposures may increase fibrosis risk.

BackgroundDihydrosphingolipids are lipid molecules biosynthetically related to ceramides. An increase in ceramides is associated with enhanced fat storage in the liver and inhibition of their synthesis is reported to prevent the appearance of steatosis in animal models. However, the precise association of dihydrosphingolipids with non-alcoholic fatty liver disease (NAFLD) is yet to be established. We employed a diet-induced NAFLD mouse model to study the association between this class of compounds and disease progression. MethodsMice were fed a high-fat diet enriched in cholesterol and supplemented with glucose and fructose up to 40 weeks. A mouse subgroup was treated with carbon tetrachloride to accelerate fibrosis development. Animals were sacrificed at different time-points to reproduce the full spectrum of histological damage found in human disease, including steatosis (NAFL) and steatohepatitis (NASH) with and without significant fibrosis. Blood and liver tissue samples were obtained from patients (n=195) whose NAFLD severity was assessed histologically. Lipidomic analysis was performed using liquid chromatography-tandem mass spectrometry. ResultsTriglyceride, cholesterol ester and dihydrosphingolipid levels were increased in the liver of model mice in association with the degree of steatosis. Dihydroceramide concentrations increased with the histological severity of the disease in liver samples of mice (0.024 {+/-} 0.003 vs 0.049 {+/-} 0.005, non-NAFLD vs NASH-fibrosis, p<0.0001) and patients (0.105 {+/-} 0.011 vs 0.165 {+/-} 0.021, p=0.0221). Several dihydroceramide and dihydrosphingomyelin species were increased in plasma of NAFLD patients and correlated with accumulation of liver triglycerides. ConclusionsDihydrosphingolipids accumulate in the liver in response to increased free fatty acid overload and are correlated with progressive histological damage in NAFLD. The increase in dihydrosphingolipids is related to upregulation of hepatic expression of enzymes involved in de novo synthesis of ceramides. HIGHLIGHTSO_LINeutral lipids and dihydrosphingolipids accumulate in liver in correlation with the histological severity of NAFLD in both mice and humans. C_LIO_LIThe ceramide pathway is stimulated to alleviate the free fatty acid excess in liver of NAFLD models. C_LIO_LIAppearance of significant fibrosis is associated with reduced concentrations of neutral lipids but not dihydrosphingolipids in a mouse model of NAFLD. C_LI

Background and AimsChronic hepatitis C virus (HCV) infection can lead to cirrhosis, development of hepatocellular carcinoma (HCC) and several extrahepatic manifestations. A sustained virological response (SVR) is achieved with direct-acting antivirals (DAA) in over 95% of the patients, but sequelae do not improve in all patients, suggesting permanent biological alterations induced by HCV infection. Therefore, we investigated the influence of chronic HCV infection, viral elimination and cirrhosis on inflammatory immune mediators. Approach and ResultsIn 102 chronic HCV patients, 46 with and 56 without cirrhosis, 92 soluble immune mediators (SIM) were measured in plasma samples at therapy start, end of treatment and long-term follow-up (median 96 weeks). 39 HBsAg positive persons with HBeAg negative infection served as controls. At baseline, 42 SIM were altered in chronic HCV patients (adj.p <0.05). Notably, patients with cirrhosis displayed a higher frequency and severity of alterations. At long-term follow-up, the SIM profile of the non-cirrhotic patients recovered to the level of the control group, while 41 SIM remained altered in cirrhotic patients. 33 of these SIM correlated with elastography, among them SIM linked to carcinogenesis as e.g. HGF, IL8 and IL6 (KEGG Pathways hsa05202, hsa05200). ConclusionsHCV-related changes in the inflammatory milieu can persist even after HCV elimination, specifically in cirrhotic patients. These changes are closely associated with liver damage and carcinogenesis. Our findings underscore the need for HCV elimination before extensive liver injury occurs and suggest further investigation of the relationship between persistent inflammatory milieu changes and long-term sequelae after HCV elimination. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/23297828v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@3a885borg.highwire.dtl.DTLVardef@ce3eb1org.highwire.dtl.DTLVardef@7601f6org.highwire.dtl.DTLVardef@17ad655_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background & AimsThe metabolic interplay between obesity and myopenia (MP) in chronic liver disease (CLD) remains poorly understood. We investigated whether progressive liver dysfunction mediates an obesity-MASLD (metabolic dysfunction{square}associated steatotic liver disease)-MP cascade and assessed the prognostic impact of MP and myopenic obesity (MO) in advanced CLD (ACLD). MethodsWe analyzed 859 CLD patients cross{square}sectionally and 169 obese patients longitudinally (median 38{square}months), using multimodal MRI to measure liver stiffness (LS), proton{square}density fat fraction (PDFF), and body composition. Temporal relationships between changes ({Delta}) in adiposity, muscle mass, and liver injury markers were assessed. Prognosis in ACLD (n=328) was evaluated using Cox regression. ResultsMP and MO were present in 29% and 8% of patients, respectively. In the longitudinal cohort, MO prevalence increased significantly from 15% to 23% (P{square}<{square}0.01). In fibrosis stages F0-2, {Delta}visceral adipose tissue significantly correlated with {Delta}PDFF, {Delta}ALT, and {Delta}LS (all P{square}<{square}0.01), whereas {Delta}muscle mass decreased, likely from weight loss. In F3-4, {Delta}ALBI score and {Delta}PDFF (hepatic fat "burning{square}out") independently correlated with {Delta}muscle mass (both P{square}<{square}0.01). In ACLD, MP--but not obesity itself--was an independent predictor of liver{square}related death (HR{square}2.27, 95%{square}CI{square}1.08-4.78, P{square}={square}0.025). ConclusionsOur findings suggest an obesity-MASLD-MP cascade driven by a liver-centered metabolic paradox: preserved hepatic function promotes harmful fat accumulation, whereas hepatic dysfunction leads to fat depletion (energy deficiency) and muscle loss. Recognition of this dynamic loop highlights the need for stage-specific strategies: fat reduction initially, followed by aggressive muscle preservation and energy repletion in ACLD.

Background & AimsAlcohol-associated liver disease (ALD) is a major cause of liver disease worldwide with scarce therapeutic options. Animal models poorly recapitulate advanced ALD precluding the development of new treatments. Organoids have emerged as a powerful human-based preclinical tool. However, current patient-derived liver organoids fail to recapitulate the epithelial heterogeneity and its generation requires liver surgical resections, thus limiting personalized disease modeling. Here, we report the development of organoids from liver needle biopsies (b-Orgs) from patients with ALD. Methodsb-Orgs were generated from tru-cut biopsies from patients at early (n=28) and advanced (n=34) stages of ALD. b-Orgs were characterized by immunofluorescence, bulk and single cell RNA-sequencing and compared to parental tissues. b-Orgs were used to model ALD progression, identify pathogenic drivers, induce alcohol-associated hepatitis (AH) and evaluate response to prednisolone. ResultsPhenotypic and functional analysis of b-Orgs showed hepatocyte- enriched features. Single-cell RNA-sequencing revealed a heterogeneous cell composition comprising hepatocyte, biliary and progenitor populations, mirroring the epithelial landscape found in patients with advanced ALD. Moreover, b-Orgs preserved disease-stage features and allowed to identify the association of ELF3 with cell plasticity and disease progression. Finally, stimulation of b-Orgs with drivers of ALD induced pathophysiological features of alcohol-associated hepatitis, including ROS production, lipid accumulation, inflammation and decreased cell proliferation, which were mitigated in response to prednisolone. Conclusions Overall, we provide a human-based model that recapitulates epithelial complexity and patient specific features, allowing to identify drivers of cell plasticity and expanding organoid-based liver disease modeling for personalized medicine. Impact and implications Here, we describe the generation of biopsy-derived organoids (b-Orgs) from patients with liver disease. b-Orgs reproduce the liver epithelial cell composition found in patients liver tissue and are efficiently generated from different stages of the disease, providing a platform for patient- tailored disease modeling and drug testing.

Background and aims. The first direct-acting antivirals (DAAs) to treat the viral hepatitis C (HCV) became available in 2011. Despite numerous clinical studies of patient outcomes after treatment, few have evaluated changes in the liver microenvironment. Despite achieving sustained virologic response (SVR), patients may still experience adverse outcomes like cirrhosis and hepatocellular carcinoma. By comparing gene and protein expression in liver biopsies collected before and after treatment, we sought to determine whether specific signatures correlated with disease progression and adverse clinical outcomes. Methods. Biopsies were collected from 22 patients before and after DAA treatment. We measured [~]770 genes and used multispectral imaging with custom machine learning algorithms to analyze phenotypes of intrahepatic macrophages (CD68, CD14, CD16, MAC387, CD163) and T cells (CD3, CD4, CD8, CD45, FoxP3). Results. Before DAA treatment, patients showed two distinct gene expression patterns: one with high pro-inflammatory and antiviral gene expression and another with weaker expression. Patients with adverse outcomes exhibited significantly (p<0.05) more inflammatory activity and had more advanced fibrosis stages in their baseline biopsies than those with liver disease resolution. Patients who achieved SVR had significantly decreased liver enzymes, reduced inflammatory scores, and restored type 1 interferon pathways similar to controls. However, after DAA treatment, patients with persistently high gene expression (67%, pre-hot) still had significantly worse outcomes (p<0.049) despite achieving SVR. A persistent lymphocytic infiltrate was observed in a subset of these patients (76.5%). After therapy, anti-inflammatory macrophages (CD16+, CD16+CD163+, CD16+CD68+) increased, and T cell heterogeneity was more pronounced, showing a predominance of helper and memory T cells (CD3+CD45RO+, CD4+CD45RO+, CD3+CD4+CD45RO+). Conclusions. Patients who have more inflamed livers and more advanced fibrosis before DAA treatment should be closely followed for the development of adverse outcomes, even after achieving SVR. We can enhance patient risk stratification by integrating gene and protein expression profiles with clinical data. This could identify those who may benefit from more intensive monitoring or alternative therapeutic approaches, inspiring a new era of personalized patient care. Lay SummaryDirect-acting antiviral (DAA) therapy has dramatically improved the treatment of chronic HCV, making it curable for most people. This study determined gene and protein expression differences in the liver before and after treatment of HCV. These results will lead to a deeper understanding of the changes in the hepatic immune microenvironment with and without the virus present in the liver in hopes of improving patient surveillance, prognosis, and outcome in the future.

Background & Aims: Per- and polyfluoroalkyl substances (PFAS) are persistent endocrine-disrupting chemicals associated with metabolic dysfunction, including metabolic dysfunction-associated steatotic liver disease (MASLD). While PFAS perturb lipid and bile acid (BA) metabolism in a sex-specific manner, the underlying mechanisms remain unclear. We tested whether steroid hormones mediate PFAS-associated metabolic alterations. Methods: In 104 patients with biopsy-characterized MASLD, we performed sex-stratified analyses applied liquid chromatography coupled to mass spectrometry (LC-MS) for chemical analysis, integrating circulating steroids, PFAS exposure, hepatic lipidomics and BA profiles. Results: Steroid hormones were associated with MASLD severity in a sexually-dimorphic manner. Dihydrotestosterone showed consistent inverse associations with steatosis, fibrosis, necroinflammation and insulin resistance, particularly in females. PFAS exposure was associated with altered steroid profiles, predominantly indicating suppressed steroidogenesis in females. These PFAS-associated hormonal changes were linked to downstream alterations in hepatic lipids and BAs. Mediation analysis supported indirect effects of PFAS on metabolic pathways via steroids, including testosterone/epi-testosterone-mediated effects on ether phospholipids and estradiol-mediated effects on lithocholic acid. Females exhibited stronger PFAS-steroid-BA associations, whereas males showed weaker, lipid-centric effects. Conclusions: PFAS exposure is associated with sex-specific disruption of steroid hormone pathways that may link environmental exposure to lipid and BA dysregulation in MASLD. These findings identify steroid hormones as potential key mediators of PFAS-associated metabolic dysfunction and highlight sex as a critical determinant in environmental liver disease.

Bile acids (BAs) are liver-synthesized steroids that facilitate lipid digestion and regulate diverse metabolic pathways. Because of their cytotoxicity, excessive hepatic BA accumulation has been implicated in the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD). Although circulating BAs are frequently elevated in MASLD, it is unclear whether similar alterations occur within the liver. Moreover, the strong overlap between MASLD and metabolic syndrome complicates efforts to distinguish BA changes driven by liver disease from those arising due to broader metabolic dysfunction. Here we show in a series of human studies that the BA dysmetabolism in MASLD originates from insulin resistance rather than hepatic steatosis. We found that circulating BA concentrations are twofold higher in patients with MASLD than in healthy controls, despite similar intrahepatic levels. Causal inference using a MASLD genetic risk score indicated that this elevation is not attributable to hepatic steatosis. Instead, circulating BAs, but not intrahepatic BAs, associate with glycemia and hepatic insulin sensitivity. We found reduced expression of the BA uptake transporter NTCP in insulin-resistant individuals, implicating impaired hepatic BA clearance. During hepatic vein catheterization, insulin acutely lowered conjugated BA concentrations in hepatic venous blood, consistent with diminished splanchnic BA spillover. Physiology-based simulations of impaired hepatic BA clearance recapitulated the human phenotype. Taken together, our findings argue against a major role for BAs in MASLD pathogenesis but reveal a previously unrecognized link between circulating BA dynamics and hepatic insulin action.

Background & aimsGenome-wide association studies in adults have identified variants in HSD17B13 and MARC1 as protective against NAFLD. It is not known if they are similarly protective in children and, more generally, whether the peri-portal inflammation of pediatric NAFLD and lobular inflammation seen in adults share common genetic influences. Therefore, we aimed to: establish if these variants are associated with NAFLD in children, and to investigate the function of these variants in hepatic metabolism using metabolomics. Methods960 children (590 with NAFLD, 394 with liver histology) were genotyped for rs72613567T>TA in HSD17B13, rs2642438G>A in MARC1. Genotype-histology associations were tested using ordinal regression. Untargeted hepatic proteomics and plasma lipidomics were performed in a subset of samples. In silico tools were used to model the effect of rs2642438G>A (p.Ala165Thr) on MARC1. Resultsrs72613567T>TA in HSD17B13 was associated with lower odds of NAFLD diagnosis (OR 0.7 (95%CI 0.6-0.9) and lower grade of portal inflammation (P<0.001) whilst rs2642438G>A in MARC1 was associated with lower grade of hepatic steatosis (P=0.02). Proteomics found reduced expression of HSD17B13 in carriers of the protective allele, whereas MARC1 levels were not affected by genotype. Both variants showed downregulation of hepatic fibrotic pathways, upregulation of retinol metabolism and perturbation of phospholipid species. Modelling suggests that p.Ala165Thr would disrupt the stability and metal-binding of MARC1. ConclusionsThere are shared genetic mechanisms between pediatric and adult NAFLD, despite their differences in histology. MARC1 and HSD17B13 are involved in phospholipid metabolism and suppress fibrosis in NAFLD.

Background and AimsThe incidence of hepatocellular carcinoma (HCC) is increasingly driven by metabolic risk factors, including obesity and iron overload. Although high-fat and high-iron diets independently promote hepatocarcinogenesis, their shared and distinct molecular effects remain unclear. We sought to define how dietary fat and iron differentially shape HCC development. ApproachMale C3HeB/FeJ mice were exposed to long-term high-fat (HFD), high-iron (HID), or combined (HFD+HID) diets for 16.5 months. Tumor burden, hepatic iron distribution, mTOR signaling, oxidized phospholipid (OxPL) accumulation, and transcriptomic alterations across normal, adjacent non-tumor, and tumor liver tissues were analyzed using biochemical, histological, and RNA sequencing approaches. ResultsAll diets induced HCC with comparable tumor burden. HID increased iron levels in non-tumor liver tissue but resulted in relative iron depletion within tumors, indicating tumor-specific iron utilization. Tumors from all diet groups showed robust mTOR activation and increased OxPL accumulation, with stronger oxidative stress signatures in HFD and HFD+HID tumors. Transcriptomic analyses revealed conserved oncogenic programs alongside diet-specific signatures, with HFD exerting a dominant effect on metabolic reprogramming and gene dysregulation, whereas HID preferentially enhanced immune and inflammatory signaling. Progressive, monotonic changes in gene expression were observed across disease stages. Cross-species analyses linked diet-induced mouse tumors to immunologically "hot" human HCC subtypes. ConclusionsDietary fat and iron promote HCC through overlapping yet distinct molecular pathways, highlighting metabolic and immune mechanisms as key targets in diet-associated liver cancer.

Understanding the genetic and immune heterogeneity in hepatocellular carcinoma (HCC) is crucial as treatments advance towards personalisation. This study characterises the genetic and immune heterogeneity within a population of 56 tumours from 28 patients, 10 of whom have multifocal disease, obtained from patients undergoing surgery at the Leeds Teaching Hospital NHS Trust. These samples represent the most common aetiological groups found in the UK: alcohol-related liver disease, non-alcoholic fatty liver disease and hepatitis C. Whole exome sequencing was performed on DNA from these tumour alongside matched background liver samples to determine tumour-specific variants. Tissue sections were stained for both proliferation and immune cell markers. Tumours across aetiological groups showed significant levels of variation, with high inter-individual variation. Analyses of multifocal tumours revealed significant discordance in genetic and immune cell profiles, both between multi-centric primary and metastatic tumours. This work emphasises the genetic and immuneheterogeneity in HCC across HCC subtypes, between and within individuals, highlighting mechansisms for therapeutic resistance and the need for personalised medicine.