PCPE-1, a brown adipose tissue-derived cytokine, promotes obesity-induced liver fibrosis.

Metabolic dysfunction-associated steatohepatitis (MASH, previously termed non-alcoholic steatohepatitis (NASH)), is a major complication of obesity that promotes fatty liver disease. MASH is characterized by progressive tissue fibrosis and sterile liver inflammation that can lead to liver cirrhosis, cancer, and death. The molecular mechanisms of fibrosis in MASH and its systemic control remain poorly understood. Here, we identified the secreted-type pro-fibrotic protein, procollagen C-endopeptidase enhancer-1 (PCPE-1), as a brown adipose tissue (BAT)-derived adipokine that promotes liver fibrosis in a murine obesity-induced MASH model. BAT-specific or systemic PCPE-1 depletion in mice ameliorated liver fibrosis, whereas, PCPE-1 gain of function in BAT enhanced hepatic fibrosis. High-calorie diet-induced ER stress increased PCPE-1 production in BAT through the activation of IRE-1/JNK/c-Fos/c-Jun signaling. Circulating PCPE-1 levels are increased in the plasma of MASH patients, suggesting a therapeutic possibility. In sum, our results uncover PCPE-1 as a novel systemic control factor of liver fibrosis.

Portal Fibrosis and the Ductular Reaction: Pathophysiological Role in the Progression of Liver Disease and Translational Opportunities.

A consistent feature of chronic liver diseases and the hallmark of pathologic repair is the so-called ductular reaction. This is a histological abnormality characterized by an expansion of dysmorphic cholangiocytes inside and around portal spaces infiltrated by inflammatory, mesenchymal, and vascular cells. The ductular reaction is a highly regulated response based on the reactivation of morphogenetic signaling mechanisms and a complex crosstalk among a multitude of cell types. The nature and mechanism of these exchanges determine the difference between healthy regenerative liver repair and pathological repair. An orchestrated signaling among cell types directs mesenchymal cells to deposit a specific extracellular matrix with distinct physical and biochemical properties defined as portal fibrosis. Progression of fibrosis leads to vast architectural and vascular changes known as liver cirrhosis. The signals regulating the ecology of this microenvironment are just beginning to be addressed. Contrary to the tumor microenvironment, immune modulation inside this "benign" microenvironment is scarcely known. One of the reasons is that both the ductular reaction and portal fibrosis have been primarily considered a manifestation of cholestatic liver disease, whereas this phenomenon is also present, albeit with distinctive features, in all chronic human liver diseases. Novel human-derived cellular models and progress in "omics" technologies are increasing our knowledge at a fast pace. Most importantly, this knowledge is on the edge of generating new diagnostic and therapeutic advances. Here, we will critically review the latest advances, in terms of mechanisms, pathophysiology, and treatment prospects. In addition, we will delineate future avenues of research including innovative translational opportunities.

The "Domino effect" in MASLD: The inflammatory cascade of steatohepatitis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is an increasingly common complication of obesity, affecting over a quarter of the global adult population. A key event in the pathophysiology of MASLD is the development of metabolic-associated steatohepatitis (MASH), which greatly increases the chances of developing cirrhosis and hepatocellular carcinoma. The underlying cause of MASH is multifactorial, but accumulating evidence indicates that the inflammatory process in the hepatic microenvironment typically follows a pattern that can be roughly divided into three stages: (1) Detection of hepatocyte stress by tissue-resident immune cells including γδ T cells and CD4-CD8- double-negative T cells, followed by their secretion of pro-inflammatory mediators, most notably IL-17A. (2) Recruitment of pro-inflammatory cells, mostly of the myeloid lineage, and initiation of inflammation through secretion of effector-type cytokines such as TNF, TGF-ß, and IL-1ß. (3) Escalation of the inflammatory response by recruitment of lymphocytes including Th17, CD8 T, and B cells leading to chronic inflammation, hepatic stellate cell activation, and fibrosis. Here we will discuss these three stages and how they are consecutively linked like falling domino tiles to the pathophysiology of MASH. Moreover, we will highlight the clinical potential of inflammation as a biomarker and therapeutic target for the treatment of MASLD.

YAP activation in liver macrophages via depletion of MST1/MST2 enhances liver inflammation and fibrosis in MASLD.

Macrophages have been recognized as pivotal players in the progression of MASLD/MASH. However, the molecular mechanisms underlying their multifaceted functions in the disease remain to be further clarified. In the current study, we developed a new mouse model with YAP activation in macrophages to delineate the effect and mechanism of YAP signaling in the pathogenesis of MASLD/MASH. Genetically modified mice, featuring specific depletion of both Mst1 and Mst2 in macrophages/monocytes, were generated and exposed to a high-fat diet for 12 weeks to induce MASLD. Following this period, livers were collected for histopathological examination, and liver non-parenchymal cells were isolated and subjected to various analyses, including single-cell RNA-sequencing, immunofluorescence and immunoblotting and qRT-PCR to investigate the impact of YAP signaling on the progression of MASLD. Our data revealed that Mst1/2 depletion in liver macrophages enhanced liver inflammation and fibrosis in MASLD. Using single-cell RNA-sequencing, we showed that YAP activation via Mst1/2 depletion upregulated the expressions of both pro-inflammatory genes and genes associated with resolution/tissue repair. We observed that YAP activation increases Kupffer cell populations (i.e., Kupffer-2 and Kupffer-3) which are importantly implicated in the pathogenesis of MASLD/MASH. Our data indicate that YAP activation via Mst1/2 deletion enhances both the pro-inflammatory and tissue repairing functions of Kupffer-1 and -2 cells at least in part through C1q. These YAP-regulatory mechanisms control the plasticity of liver macrophages in the context of MASLD/MASH. Our findings provide important evidence supporting the critical regulatory role of YAP signaling in liver macrophage plasticity and the progression of MASLD. Therefore, targeting the Hippo-YAP pathway may present a promising therapeutic strategy for the treatment of MASH.

Spatial lipidomics reveals zone-specific hepatic lipid alteration and remodeling in metabolic dysfunction-associated steatohepatitis.

Alteration in lipid metabolism plays a pivotal role in developing metabolic dysfunction-associated steatohepatitis (MASH). However, our understanding of alteration in lipid metabolism across liver zonation in MASH remains limited. Within this study, we investigated MASH-associated zone-specific lipid metabolism in a diet and chemical-induced MASH mouse model. Spatial lipidomics using mass spectrometry imaging in a MASH mouse model revealed 130 lipids from various classes altered across liver zonation and exhibited zone-specific lipid signatures in MASH. Triacylglycerols, diacylglycerols, sphingolipids and ceramides showed distinct zone-specific changes and re-distribution from pericentral to periportal localization in MASH. Saturated and monounsaturated fatty acids (FA) were the primary FA composition of increased lipids in MASH, while polyunsaturated FAs were the major FA composition of decreased lipids. We observed elevated fibrosis in the periportal region, which could be the result of observed metabolic alteration across zonation. Our study provides valuable insights into zone-specific hepatic lipid metabolism and demonstrates the significance of spatial lipidomics in understanding liver lipid metabolism. Identifying unique lipid distribution patterns may offer valuable insights into the pathophysiology of MASH and facilitate the discovery of diagnostic markers associated with liver zonation.

EASL-EASD-EASO Clinical Practice Guidelines on the management of metabolic dysfunction-associated steatotic liver disease (MASLD): Executive Summary.

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously termed non-alcoholic fatty liver disease (NAFLD), is defined as steatotic liver disease (SLD) in the presence of one or more cardiometabolic risk factor(s) and the absence of harmful alcohol intake. The spectrum of MASLD includes steatosis, metabolic dysfunction-associated steatohepatitis (MASH, previously NASH), fibrosis, cirrhosis and MASH-related hepatocellular carcinoma (HCC). This joint EASL-EASD-EASO guideline provides an update on definitions, prevention, screening, diagnosis and treatment for MASLD. Case-finding strategies for MASLD with liver fibrosis, using non-invasive tests, should be applied in individuals with cardiometabolic risk factors, abnormal liver enzymes and/or radiological signs of hepatic steatosis, particularly in the presence of type 2 diabetes or obesity with additional metabolic risk factor(s). A stepwise approach using blood-based scores (such as the fibrosis-4 index [FIB-4]) and, sequentially, imaging techniques (such as transient elastography) is suitable to rule-out/in advanced fibrosis, which is predictive of liver-related outcomes. In adults with MASLD, lifestyle modification-including weight loss, dietary changes, physical exercise and discouraging alcohol consumption-as well as optimal management of comorbidities-including use of incretin-based therapies (e.g. semaglutide, tirzepatide) for type 2 diabetes or obesity, if indicated-is advised. Bariatric surgery is also an option in individuals with MASLD and obesity. If locally approved and dependent on the label, adults with non-cirrhotic MASH and significant liver fibrosis (stage ≥2) should be considered for a MASH-targeted treatment with resmetirom, which demonstrated histological effectiveness on steatohepatitis and fibrosis with an acceptable safety and tolerability profile. No MASH-targeted pharmacotherapy can currently be recommended for the cirrhotic stage. Management of MASH-related cirrhosis includes adaptations of metabolic drugs, nutritional counselling, surveillance for portal hypertension and HCC, as well as liver transplantation in decompensated cirrhosis.

Molecular pathogenesis of metabolic dysfunction-associated steatotic liver disease, steatohepatitis, hepatic fibrosis and liver cirrhosis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by intense deposition of fat globules in the hepatic parenchyma that could potentially progress to liver cirrhosis and hepatocellular carcinoma. Here, we evaluated a rat model to study the molecular pathogenesis of the spectrum of MASLD and to screen therapeutic agents. SHRSP5/Dmcr rats were fed a high-fat and cholesterol (HFC) diet for a period of 12 weeks and evaluated for the development of steatosis (MASLD), steatohepatitis, fibrosis and cirrhosis. A group of animals were sacrificed at the end of the 4th, 6th, 8th and 12th weeks from the beginning of the experiment, along with the control rats that received normal diet. Blood and liver samples were collected for biochemical and histopathological evaluations. Immunohistochemical staining was performed for α-SMA and Collagen Type I. Histopathological examinations demonstrated steatosis at the 4th week, steatohepatitis with progressive fibrosis at the 6th week, advanced fibrosis with bridging at the 8th week and cirrhosis at the 12th week. Biochemical markers and staining for α-SMA and Collagen Type I demonstrated the progression of steatosis to steatohepatitis, hepatic fibrosis and liver cirrhosis in a stepwise manner. Control animals fed a normal diet did not show any biochemical or histopathological alterations. The results of the present study clearly demonstrated that the HFC diet-induced model of steatosis, steatohepatitis, hepatic fibrosis and cirrhosis is a feasible, quick and appropriate animal model to study the molecular pathogenesis of the spectrum of MASLD and to screen potent therapeutic agents.

Identification of crosstalk genes relating to ECM-receptor interaction genes in MASH and DN using bioinformatics and machine learning.

This study aimed to identify genes shared by metabolic dysfunction-associated fatty liver disease (MASH) and diabetic nephropathy (DN) and the effect of extracellular matrix (ECM) receptor interaction genes on them. Datasets with MASH and DN were downloaded from the Gene Expression Omnibus (GEO) database. Pearson's coefficients assessed the correlation between ECM-receptor interaction genes and cross talk genes. The coexpression network of co-expression pairs (CP) genes was integrated with its protein-protein interaction (PPI) network, and machine learning was employed to identify essential disease-representing genes. Finally, immuno-penetration analysis was performed on the MASH and DN gene datasets using the CIBERSORT algorithm to evaluate the plausibility of these genes in diseases. We found 19 key CP genes. Fos proto-oncogene (FOS), belonging to the IL-17 signalling pathway, showed greater centrality PPI network; Hyaluronan Mediated Motility Receptor (HMMR), belonging to ECM-receptor interaction genes, showed most critical in the co-expression network map of 19 CP genes; Forkhead Box C1 (FOXC1), like FOS, showed a high ability to predict disease in XGBoost analysis. Further immune infiltration showed a clear positive correlation between FOS/FOXC1 and mast cells that secrete IL-17 during inflammation. Combining the results of previous studies, we suggest a FOS/FOXC1/HMMR regulatory axis in MASH and DN may be associated with mast cells in the acting IL-17 signalling pathway. Extracellular HMMR may regulate the IL-17 pathway represented by FOS through the Mitogen-Activated Protein Kinase 1 (ERK) or PI3K-Akt-mTOR pathway. HMMR may serve as a signalling carrier between MASH and DN and could be targeted for therapeutic development.

Perilipins: A family of five fat-droplet storing proteins that play a significant role in fat homeostasis.

Lipid droplets are organelles with unique spherical structures. They consist of a hydrophobic neutral lipid core that varies depending on the cell type and tissue. These droplets are surrounded by phospholipid monolayers, along with heterogeneous proteins responsible for neutral lipid synthesis and metabolism. Additionally, there are specialized lipid droplet-associated surface proteins. Recent evidence suggests that proteins from the perilipin family (PLIN) are associated with the surface of lipid droplets and are involved in their formation. These proteins have specific roles in hepatic lipid droplet metabolism, such as protecting the lipid droplets from lipase action and maintaining a balance between lipid storage and utilization in specific cells. Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by the accumulation of lipid droplets in more than 5% of the hepatocytes. This accumulation can progress into metabolic dysfunction-associated steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. The accumulation of hepatic lipid droplets in the liver is associated with the progression of MASLD and other diseases such as sarcopenic obesity. Therefore, it is crucial to understand the role of perilipins in this accumulation, as these proteins are key targets for developing novel therapeutic strategies. This comprehensive review aims to summarize the structure and characteristics of PLIN proteins, as well as their pathogenic role in the development of hepatic steatosis and fatty liver diseases.

Interleukin-21 receptor signaling promotes metabolic dysfunction-associated steatohepatitis-driven hepatocellular carcinoma by inducing immunosuppressive IgA+ B cells.

BACKGROUND: Dysregulation of immune surveillance is tightly linked to the development of metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC); however, its underlying mechanisms remain unclear. Herein, we aimed to determine the role of interleukin-21 receptor (IL-21R) in MASH-driven HCC. METHODS: The clinical significance of IL-21R was assessed in human HCC specimens using immunohistochemistry staining. Furthermore, the expression of IL-21R in mice was assessed in the STAM model. Thereafter, two different MASH-driven HCC mouse models were applied between IL-21R-deficient mice and wild type controls to explore the role of IL-21R in MASH-driven HCC. To further elucidate the potential mechanisms by which IL-21R affected MASH-driven HCC, whole transcriptome sequencing, flow cytometry and adoptive lymphocyte transfer were performed. Finally, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescent staining, chromatin immunoprecipitation assay and western blotting were conducted to explore the mechanism by which IL-21R induced IgA+ B cells. RESULTS: HCC patients with high IL-21R expression exhibited poor relapse-free survival, advanced TNM stage and severe steatosis. Additionally, IL-21R was demonstrated to be upregulated in mouse liver tumors. Particularly, ablation of IL-21R impeded MASH-driven hepatocarcinogenesis with dramatically reduction of lipid accumulation. Moreover, cytotoxic CD8+ T lymphocyte activation was enhanced in the absence of IL-21R due to the reduction of immunosuppressive IgA+ B cells. Mechanistically, the IL-21R-STAT1-c-Jun/c-Fos regulatory axis was activated in MASH-driven HCC and thus promoted the transcription of Igha, resulting in the induction of IgA+ B cells. CONCLUSIONS: IL-21R plays a cancer-promoting role by inducing IgA+ B cells in MASH-driven hepatocarcinogenesis. Targeting IL-21R signaling represents a potential therapeutic strategy for cancer therapy.

Liver biopsy evaluation in MASH drug development: Think thrice, act wise.

During recent decades, the metabolic dysfunction-associated steatohepatitis (MASH) field has witnessed several paradigm shifts, including the recognition of liver fibrosis as the main predictor of major adverse liver outcomes. Throughout this evolution, liver histology has been recognised as one of the main hurdles in MASH drug development due to its invasive nature, associated cost, and high inter- and intra-reader variability. Collective experience demonstrates the importance of consistency in the central reading process, where consensus methods have emerged as appropriate ways to mitigate against well-known challenges. Using crystalized knowledge in the field, stakeholders should collectively work towards the next paradigm shift, where non-invasive biomarkers will be considered surrogate endpoints for accelerated approval. In this review, we provide an overview of the evolution of the regulatory histology endpoints and the liver biopsy reading process, within the MASH trial landscape, over recent decades; we then review the biggest challenges associated with liver biopsy endpoints. Finally, we discuss and provide recommendations on the best practices for liver biopsy evaluation in MASH drug development.

Increases and decreases in liver stiffness measurement are independently associated with the risk of liver-related events in NAFLD.

BACKGROUND & AIMS: The clinical significance of change in liver stiffness measurement (LSM) by vibration-controlled transient elastography (VCTE) in patients with non-alcoholic fatty liver disease (NAFLD) is not well-understood. We prospectively defined rates of progression to and regression from LSM-defined compensated advanced chronic liver disease (cACLD) and their associations with liver-related events (LREs). METHODS: Participants in the NASH Clinical Research Network-led NAFLD Database 2 and 3 studies were included. Progression to cACLD was defined as reaching LSM ≥10 kPa in participants with LSM <10 kPa on initial VCTE; regression from cACLD was defined as reaching LSM <10 kPa in participants with baseline LSM ≥10 kPa. LREs were defined as liver-related death, liver transplant, hepatocellular carcinoma, MELD >15, development of varices, or hepatic decompensation. Univariate and multivariable interval-censored Cox regression analyses were used to compare the cumulative LRE probability by LSM progression and regression status. RESULTS: In 1,403 participants, 89 LREs developed over a mean follow-up of 4.4 years, with an annual incidence rate for LREs of 1.5 (95% CI 1.2-1.8). In participants at risk, progression to LSM ≥10 or ≥15 kPa occurred in 29% and 17%, respectively, whereas regression to LSM <10 or <15 kPa occurred in 44% and 49%, respectively. Progressors to cACLD (≥10 kPa) experienced a higher cumulative LRE rate vs. non-progressors (16% vs. 4%, adjusted hazard ratio 4.0; 95% (1.8-8.9); p <0.01). Regressors from cACLD (to LSM <10 kPa) experienced a lower LRE rate than non-regressors (7% vs. 32%, adjusted hazard ratio 0.25; 95% CI 0.10-0.61; p <0.01). CONCLUSIONS: Change in LSM over time is independently and bi-directionally associated with risk of LRE and is a non-invasive surrogate for clinical outcomes in patients with NAFLD. IMPACT AND IMPLICATIONS: The prognostic value of change in LSM in patients with NAFLD is not well understood. In this large prospective study of patients with NAFLD and serial vibration-controlled transient elastography exams, baseline and dynamic changes in LSM were associated with the risk of developing liver-related events. LSM is a useful non-invasive surrogate of clinical outcomes in patients with NAFLD.

FGF21 agonists: An emerging therapeutic for metabolic dysfunction-associated steatohepatitis and beyond.

The worldwide epidemics of obesity, hypertriglyceridemia, dyslipidaemia, type 2 diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD)/metabolic dysfunction-associated steatohepatitis (MASH) represent a major economic burden on healthcare systems. Patients with at-risk MASH, defined as MASH with moderate or significant fibrosis, are at higher risk of comorbidity/mortality, with a significant risk of cardiovascular diseases and/or major adverse liver outcomes. Despite a high unmet medical need, there is only one drug approved for MASH. Several drug candidates have reached the phase III development stage and could lead to several potential conditional drug approvals in the coming years. Within the armamentarium of future treatment options, FGF21 analogues hold an interesting position thanks to their pleiotropic effects in addition to their significant effect on both MASH resolution and fibrosis improvement. In this review, we summarise preclinical and clinical data from FGF21 analogues for MASH and explore additional potential therapeutic indications.

Organoid-guided precision hepatology for metabolic liver disease.

Metabolic dysfunction-associated steatotic liver disease affects millions of people worldwide. Progress towards a definitive cure has been incremental and treatment is currently limited to lifestyle modification. Hepatocyte-specific lipid accumulation is the main trigger of lipotoxic events, driving inflammation and fibrosis. The underlying pathology is extraordinarily heterogenous, and the manifestations of steatohepatitis are markedly influenced by metabolic communications across non-hepatic organs. Synthetic human tissue models have emerged as powerful platforms to better capture the mechanistic diversity in disease progression, while preserving person-specific genetic traits. In this review, we will outline current research efforts focused on integrating multiple synthetic tissue models of key metabolic organs, with an emphasis on organoid-based systems. By combining functional genomics and population-scale en masse profiling methodologies, human tissues derived from patients can provide insights into personalised genetic, transcriptional, biochemical, and metabolic states. These collective efforts will advance our understanding of steatohepatitis and guide the development of rational solutions for mechanism-directed diagnostic and therapeutic investigation.

Global survey of stigma among physicians and patients with nonalcoholic fatty liver disease.

BACKGROUND & AIMS: Patients with fatty liver disease may experience stigma from the disease or comorbidities. In this cross-sectional study, we aimed to understand stigma among patients with nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) and healthcare providers. METHODS: Members of the Global NASH Council created two surveys about experiences/attitudes toward NAFLD and related diagnostic terms: a 68-item patient and a 41-item provider survey. RESULTS: Surveys were completed by 1,976 patients with NAFLD across 23 countries (51% Middle East/North Africa [MENA], 19% Europe, 17% USA, 8% Southeast Asia, 5% South Asia) and 825 healthcare providers (67% gastroenterologists/hepatologists) across 25 countries (39% MENA, 28% Southeast Asia, 22% USA, 6% South Asia, 3% Europe). Of all patients, 48% ever disclosed having NAFLD/NASH to family/friends; the most commonly used term was "fatty liver" (88% at least sometimes); "metabolic disease" or "MAFLD" were rarely used (never by >84%). Regarding various perceptions of diagnostic terms by patients, there were no substantial differences between "NAFLD", "fatty liver disease (FLD)", "NASH", or "MAFLD". The most popular response was being neither comfortable nor uncomfortable with either term (56%-71%), with slightly greater discomfort with "FLD" among the US and South Asian patients (47-52% uncomfortable). Although 26% of patients reported stigma related to overweight/obesity, only 8% reported a history of stigmatization or discrimination due to NAFLD. Among providers, 38% believed that the term "fatty" was stigmatizing, while 34% believed that "nonalcoholic" was stigmatizing, more commonly in MENA (43%); 42% providers (gastroenterologists/hepatologists 45% vs. 37% other specialties, p = 0.03) believed that the name change to metabolic dysfunction-associated steatotic liver disease (or MASLD) might reduce stigma. Regarding the new nomenclature, the percentage of providers reporting "steatotic liver disease" as stigmatizing was low (14%). CONCLUSIONS: The perception of NAFLD stigma varies among patients, providers, geographic locations and sub-specialties. IMPACT AND IMPLICATIONS: Over the past decades, efforts have been made to change the nomenclature of nonalcoholic fatty liver disease (NAFLD) to better align with its underlying pathogenetic pathways and remove any potential stigma associated with the name. Given the paucity of data related to stigma in NAFLD, we undertook this global comprehensive survey to assess stigma in NAFLD among patients and providers from around the world. We found there is a disconnect between physicians and patients related to stigma and related nomenclature. With this knowledge, educational programs can be developed to better target stigma in NAFLD among all stakeholders and to provide a better opportunity for the new nomenclature to address the issues of stigma.

Histological improvements following energy restriction and exercise: The role of insulin resistance in resolution of MASH.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatohepatitis (MASH) is one of the most common liver diseases worldwide and is characterized by multi-tissue insulin resistance. The effects of a 10-month energy restriction and exercise intervention on liver histology, anthropometrics, plasma biochemistries, and insulin sensitivity were compared to standard of care (control) to understand mechanisms that support liver health improvements. METHODS: Following medical diagnosis of MASH, individuals were randomized to treatment (n = 16) or control (n = 8). Liver fat (magnetic resonance spectroscopy), 18-hour plasma biochemical measurements, and isotopically labeled hyperinsulinemic-euglycemic clamps were completed pre- and post-intervention. Body composition and cardiorespiratory fitness (VO2peak) were also measured mid-intervention. Those in the treatment group were counseled to reduce energy intake and completed supervised, high-intensity interval training (3x/week) for 10 months. Controls continued physician-directed care. RESULTS: Treatment induced significant (p <0.05) reductions in body weight, fat mass, and liver injury, while VO2peak (p <0.05) and non-esterified fatty acid suppression (p = 0.06) were improved. Both groups exhibited reductions in total energy intake, hemoglobin A1c, hepatic insulin resistance, and liver fat (p <0.05). Compared to control, treatment induced a two-fold increase in peripheral insulin sensitivity which was significantly related to higher VO2peak and resolution of liver disease. CONCLUSIONS: Exercise and energy restriction elicited significant and clinically meaningful treatment effects on liver health, potentially driven by a redistribution of excess nutrients to skeletal muscle, thereby reducing hepatic nutrient toxicity. Clinical guidelines should emphasize the addition of aerobic exercise in lifestyle treatments for the greatest histologic benefit in individuals with advanced MASH. IMPACT AND IMPLICATIONS: The mechanisms that underpin histologic improvement in individuals with metabolic dysfunction-associated steatohepatitis (MASH) are not well understood. This study evaluated the relationship between liver and metabolic health, testing how changes in one may affect the other. We investigated the effects of energy restriction and exercise on the association between multi-tissue insulin sensitivity and histologic improvements in participants with biopsy-proven MASH. For the first time, these results show that an improvement in peripheral (but not hepatic) insulin sensitivity and systemic markers of muscle function (i.e. cardiorespiratory fitness) were strongly related to resolution of liver disease. Extrahepatic disposal of substrates and improved fitness levels supported histologic improvement, confirming the addition of exercise as crucial to lifestyle interventions in MASH. CLINICAL TRIAL NUMBER: NCT03151798.

Induction of steatosis in primary human hepatocytes recapitulates key pathophysiological aspects of metabolic dysfunction-associated steatotic liver disease.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common cause of chronic liver disease. Owing to limited available treatment options, novel pre-clinical models for target selection and drug validation are warranted. We have established and extensively characterized a primary human steatotic hepatocyte in vitro model system that could guide the development of treatment strategies for MASLD. METHODS: Cryopreserved primary human hepatocytes from five donors varying in sex and ethnicity were cultured with free fatty acids in a 3D collagen sandwich for 7 days and the development of MASLD was followed by assessing classical hepatocellular functions. As proof of concept, the effects of the drug firsocostat (GS-0976) on in vitro MASLD phenotypes were evaluated. RESULTS: Incubation with free fatty acids induced steatosis, insulin resistance, mitochondrial dysfunction, inflammation, and alterations in prominent human gene signatures similar to patients with MASLD, indicating the recapitulation of human MASLD in this system. The application of firsocostat rescued clinically observed fatty liver disease pathologies, highlighting the ability of the in vitro system to test the efficacy and potentially characterize the mode of action of drug candidates. CONCLUSIONS: Altogether, our human MASLD in vitro model system could guide the development and validation of novel targets and drugs for the treatment of MASLD. IMPACT AND IMPLICATIONS: Due to low drug efficacy and high toxicity, clinical treatment options for metabolic dysfunction-associated steatotic liver disease (MASLD) are currently limited. To facilitate earlier stop-go decisions in drug development, we have established a primary human steatotic hepatocyte in vitro model. As the model recapitulates clinically relevant MASLD characteristics at high phenotypic resolution, it can serve as a pre-screening platform and guide target identification and validation in MASLD therapy.

ACMSD inhibition corrects fibrosis, inflammation, and DNA damage in MASLD/MASH.

BACKGROUND & AIMS: Recent findings reveal the importance of tryptophan-initiated de novo nicotinamide adenine dinucleotide (NAD+) synthesis in the liver, a process previously considered secondary to biosynthesis from nicotinamide. The enzyme α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), primarily expressed in liver and kidney, acts as a modulator of de novo NAD+ synthesis. Boosting NAD+ levels has previously demonstrated remarkable metabolic benefits in mouse models. In this study, we aimed to investigate the therapeutic implications of ACMSD inhibition in the treatment of metabolic dysfunction-associated steatotic liver disease/steatohepatitis (MASLD/MASH). METHODS: In vitro experiments were conducted in primary rodent hepatocytes, Huh7 human liver carcinoma cells and iPSC-derived human liver organoids (HLOs). C57BL/6J male mice were fed a western-style diet and housed at thermoneutrality to recapitulate key aspects of MASLD/MASH. Pharmacological ACMSD inhibition was given therapeutically, following disease onset. Steatohepatitis HLO models were used to assess the DNA damage responses by ACMSD inhibition in human contexts. RESULTS: Inhibiting ACMSD with a novel specific pharmacological inhibitor promotes de novo NAD+ synthesis and reduces DNA damage ex vivo, in vivo, and in HLO models. In mouse models of MASLD/MASH, de novo NAD+ biosynthesis is suppressed, and transcriptomic DNA damage signatures correlate with disease severity; in humans, Mendelian randomization-based genetic analysis suggests a notable impact of genomic stress on liver disease susceptibility. Therapeutic inhibition of ACMSD in mice increases liver NAD+ and reverses MASLD/MASH, mitigating fibrosis, inflammation, and DNA damage, as were observed in HLO models of steatohepatitis. CONCLUSIONS: Our findings highlight the benefits of ACMSD inhibition to enhance hepatic NAD+ levels and enable genomic protection, underscoring its therapeutic potential in MASLD/MASH. IMPACT AND IMPLICATIONS: Enhancing NAD+ levels has shown remarkable health benefits in mouse models of MASLD/MASH, yet liver-specific NAD+ boosting strategies remain underexplored. Here, we present a novel pharmacological approach to enhance liver NAD+de novo synthesis by inhibiting ACMSD, an enzyme highly expressed in the liver. Inhibiting ACMSD increases NAD+ levels, enhances mitochondrial respiration, and maintains genomic stability in hepatocytes ex vivo and in vivo. These molecular benefits prevent disease progression in both mouse and human liver organoid models of steatohepatitis. Our preclinical study identifies ACMSD as a promising target for MASLD/MASH management and lays the groundwork for developing ACMSD inhibitors as a clinical treatment.

Prospective head-to-head comparison of non-invasive scores for diagnosis of fibrotic MASH in patients with type 2 diabetes.

BACKGROUND & AIMS: Non-invasive scores have been proposed to identify patients with fibrotic, metabolic dysfunction-associated steatohepatitis (MASH), who are at the highest risk of progression to complications of cirrhosis and may benefit from pharmacologic treatments. However, data in patients with type 2 diabetes (T2DM) are lacking. The aim of this multicenter prospective study was to perform a head-to-head comparison of FAST (FibroScan-aspartate aminotransferase [AST]), MAST (MRI-AST), MEFIB (magnetic resonance elastography [MRE] plus FIB-4), and FNI (fibrotic NASH index) for detecting fibrotic MASH in patients with T2DM. METHODS: A total of 330 outpatients with T2DM and biopsy-proven metabolic dysfunction-associated steatotic liver disease (MASLD) from the QUID-NASH study (NCT03634098), who underwent FibroScan, MRI-proton density fat fraction and MRE at the time of liver biopsy were studied. The main outcome was fibrotic MASH, defined as NAS ≥4 (with at least one point for each parameter) and fibrosis stage ≥2 (centrally reviewed). RESULTS: All data for score comparisons were available for 245 patients (median age 59 years, 65% male, median BMI 31 kg/m2; fibrotic MASH in 39%). FAST and MAST had similar accuracy (AUROCs 0.81 vs. 0.79, p = 0.41) but outperformed FNI (0.74; p = 0.01) and MEFIB (0.68; p <0.0001). When using original cut-offs, MAST outperformed FAST, MEFIB and FNI when comparing the percentage of correctly classified patients, in whom liver biopsy would be avoided (69% vs. 48%, 46%, 39%, respectively; p <0.001). When using cut-offs specific to our population, FAST outperformed FNI and MAST (56% vs. 40%, and 38%, respectively; p <0.001). CONCLUSION: Our findings show that FAST, MAST, MEFIB and FNI are accurate non-invasive tools to identify patients with T2DM and fibrotic MASH in secondary/tertiary diabetes clinics. Cut-offs adapted to the T2DM population should be considered. IMPACT AND IMPLICATIONS: Among patients with type 2 diabetes (T2DM), identifying those with metabolic dysfunction-associated steatohepatitis and significant fibrosis, who are the most at risk of developing clinical liver-related outcomes and who may benefit from pharmacologic treatments, is an unmet need. In this prospective multicenter study, we compared four non-invasive scores, three based on imaging (MRI or ultrasound technologies) and one on laboratory blood tests, for this purpose, using original and study-specific cut-offs. Our findings show that FAST, MAST, MEFIB and FNI are accurate non-invasive tools to identify patients with T2DM and fibrotic MASH in secondary/tertiary diabetes clinics. Cut-offs adapted to the T2DM population should be considered. TRIAL REGISTRATION NUMBER: NCT03634098.

NIS2+TM as a screening tool to optimize patient selection in metabolic dysfunction-associated steatohepatitis clinical trials.

BACKGROUND & AIMS: Strategies to reduce liver biopsy (LB) screen failures through better patient selection are needed for clinical trials. Standard fibrosis biomarkers were not derived to detect "at-risk" metabolic dysfunction-associated steatohepatitis (MASH; MASH with metabolic dysfunction-associated steatotic liver disease score ≥4 and fibrosis stage ≥2). We compared the performance of screening pathways that incorporate NIS2+™, an optimized version of the blood-based NIS4® technology designed to identify at-risk MASH, with those incorporating fibrosis (FIB)-4 within the RESOLVE-IT clinical trial (NCT02704403), aiming for optimized selection of patients for LB. METHODS: A retrospective simulation analysis was conducted in the RESOLVE-IT screening pathway (RSP) cohort. LB failure rate (LBFR), number of patients needed to screen, and overall cost estimations of different pathways were calculated for a range of NIS2+™ and FIB-4 cut-offs and compared with those of the RSP, which relied on investigators' local practices. An analysis of potential recruitment bias based on histology, sex, age, or comorbidities was performed. RESULTS: The analysis cohort included 1,929 patients, 765 (40%) with at-risk MASH. The NIS2+™ pathway resulted in a significantly lower LBFR (39%) compared with the FIB-4 pathway (58%) or the RSP (60%) when using cost-optimized cut-offs (NIS2+™, 0.53; FIB-4, 0.58). For every 1,000 inclusions, NIS2+™ significantly reduced unnecessary LBs (632 vs. 1,522; -58%) and screening costs (US$12.7 million vs. US$15.0 million) vs. the RSP, while the number of patients needed to screen increased moderately (3,220 to 4,033). NIS2+™ alone is better than FIB-4 alone or combined with FIB-4. CONCLUSIONS: This analysis demonstrated that patient selection for LB using NIS2+™ significantly reduced unnecessary biopsies and screening costs, which could greatly improve the feasibility of MASH clinical trials. IMPACT AND IMPLICATIONS: Simple and accurate non-invasive strategies to optimize the selection of patients who should be referred for liver biopsy for inclusion in MASH clinical trials is critical to reduce the high liver biopsy failure rates. While the use of the Fibrosis-4 index alone did not lead to a significant improvement of the screening process, selecting patients using NIS2+™, a recently developed optimization of the NIS4® technology for the detection of at-risk MASH, showed improved performance by simultaneously reducing liver biopsy failure rates and the overall cost of the trial, while maintaining the number of patients needed to screen at a manageable level and not generating any bias in included patients' characteristics. This makes NIS2+™ an accurate and reliable screening tool that could improve the recruitment of patients in future MASH clinical trials, and would lead to increased patient comfort and security, ensuring timely and cost-efficient trial completion.