Performance of non-invasive tests and histology for the prediction of clinical outcomes in patients with non-alcoholic fatty liver disease: an individual participant data meta-analysis

BackgroundHistologically assessed liver fibrosis stage has prognostic significance in patients with non-alcoholic fatty liver disease (NAFLD) and is accepted as a surrogate endpoint in clinical trials for non-cirrhotic NAFLD. Our aim was to compare the prognostic performance of non-invasive tests with liver histology in patients with NAFLD.MethodsThis was an individual participant data meta-analysis of the prognostic performance of histologically assessed fibrosis stage (F0–4), liver stiffness measured by vibration-controlled transient elastography (LSM-VCTE), fibrosis-4 index (FIB-4), and NAFLD fibrosis score (NFS) in patients with NAFLD. The literature was searched for a previously published systematic review on the diagnostic accuracy of imaging and simple non-invasive tests and updated to Jan 12, 2022 for this study. Studies were identified through PubMed/MEDLINE, EMBASE, and CENTRAL, and authors were contacted for individual participant data, including outcome data, with a minimum of 12 months of follow-up. The primary outcome was a composite endpoint of all-cause mortality, hepatocellular carcinoma, liver transplantation, or cirrhosis complications (ie, ascites, variceal bleeding, hepatic encephalopathy, or progression to a MELD score ≥15). We calculated aggregated survival curves for trichotomised groups and compared them using stratified log-rank tests (histology: F0–2 vs F3 vs F4; LSM: 2·67; NFS: 0·676), calculated areas under the time-dependent receiver operating characteristic curves (tAUC), and performed Cox proportional-hazards regression to adjust for confounding. This study was registered with PROSPERO, CRD42022312226.FindingsOf 65 eligible studies, we included data on 2518 patients with biopsy-proven NAFLD from 25 studies (1126 [44·7%] were female, median age was 54 years [IQR 44–63), and 1161 [46·1%] had type 2 diabetes). After a median follow-up of 57 months [IQR 33–91], the composite endpoint was observed in 145 (5·8%) patients. Stratified log-rank tests showed significant differences between the trichotomised patient groups (p<0·0001 for all comparisons). The tAUC at 5 years were 0·72 (95% CI 0·62–0·81) for histology, 0·76 (0·70–0·83) for LSM-VCTE, 0·74 (0·64–0·82) for FIB-4, and 0·70 (0·63–0·80) for NFS. All index tests were significant predictors of the primary outcome after adjustment for confounders in the Cox regression.InterpretationSimple non-invasive tests performed as well as histologically assessed fibrosis in predicting clinical outcomes in patients with NAFLD and could be considered as alternatives to liver biopsy in some cases

A systems biology approach to the pathogenesis and progression of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) refers to a spectrum of diseases ranging from simple steatosis (isolated hepatic fat accumulation) to steatohepatitis (NASH; hepatic fat accumulation with lipotoxicity, hepatic cell damage and inflammation), eventually progressing to fibrosis, cirrhosis and potentially hepatocellular carcinoma. NAFLD is a common disease highly associated with the Metabolic Syndrome. Hence, it has been rising in prevalence in parallel with the increasing incidence of diabetes and obesity. The transition between the different stages of the disease could be a discriminant for a benign prognosis or a higher mortality risk due to cardiovascular or chronic liver disease. The diagnosis of NAFLD can be based on imaging studies (e.g., ultrasound technology or MRI spectroscopy). However, the specific stage of the disease, the presence of hepatocellular damage, inflammation, and amount of fibrosis in the liver can be detected only by biopsies. Such invasive methods cannot be applied outside specialist practice, present a significant risk of complications and are subject to sampling error; thus, they constitute an imperfect gold standard. To date, there is a lack of 1) tractable non-invasive biomarkers that could aid the diagnosis, risk stratification and monitoring of patients; 2) approved therapies; 3) reliable pre-clinical models of the disease to test drug and biomarker effectiveness/accuracy. A better understanding of NAFLD/NASH pathophysiology in humans will help achieve identifying new targets, improving the management of NAFLD patients. During my PhD, I have integrated and analysed transcriptomics data from rodent models and human NAFLD patients of different stages of the disease to identify specific mechanisms that explain the progression between the different stages of the disease. I have also compared these data to preclinical disease models to rank them against human pathophysiology and point to the most desirable ones. The thesis is organised into six chapters. In addition to the “Introduction” and the “Final Discussion”, the four “Results” chapters address the following topics: 1. Molecular characterisation of rodent models to determine their suitability for preclinical studies using human data as reference. Using an unbiased approach that allows ranking the murine models based on metabolic phenotyping, histology, and transcriptomics (compared to human data), I have ranked multiple preclinical models of NAFLD based on their “proximity to human disease”. My results suggest that rodents fed with diets enriched in refined carbohydrates, saturated lipids, and cholesterol (Western Diets), with/without sugar water (American Lifestyle approach) are the closest models to human NASH and, therefore, the most representative of human pathophysiology. Additionally, some genetic models of obesity (ob/ob, MC4r) augment liver damage induced by these diets, making them valuable tools to achieve more advanced disease stages and/or faster models. 2. Identification of the molecular landscape of NAFLD progression. All previous work describing NAFLD progression has been based on the division of the datasets in artificially defined, discrete disease stages. Here, I implemented a pseudotemporal ordering method that successfully captures the disease trajectory in a continuum. Based on the pathway enrichment and upstream regulator analysis results, I show that my analysis matches the results of the discrete approach supervised by histology, providing additional granularity and better defining the transition among disease stages. An expression-module-based analysis also defined relevant processes representing the molecular signature of the disease during its progression. Using Random Forest models, I identified a list of genes predictive of the disease progression; intriguingly, this list of hits features multiple drivers of NASH well characterised mechanistically plus novel targets, worth future investigation. I expect this detailed molecular profile of NAFLD progression to help understand the mechanisms underpinning NASH progression and improve the stratification of patients and the ranking of pre-clinical models. 3. Comparison of NAFLD progression for patients with and without Type 2 diabetes. Using transcriptomics data of NAFLD patients with and without diabetes, the aim is to answer whether there are differences between the two populations in NAFLD progression. Utilising the pseudotemporal ordering approach mentioned above, I identified specific T2DM-associated biological processes that due to the complex nature of the disease and the limitations of relatively small human datasets, could not be identified with standard bioinformatics approaches. 4. Contribution of MBOAT7 and INSIG1 in NAFLD progression in mice and humans. This chapter provides a proof of concept of how transcriptomic studies can be reverse-translated into mice to study the contribution of the targets I identified in mechanisms of NASH progression. Here I show that the partial genetic ablation of MBOAT7 alters the hepatic transcriptome and the dependence of these effects on the dietary challenge. Additionally, I show that an INSIG1 ablation induces an increased lipid remodelling and cholesterol biosynthesis, acting as a protective mechanism that prevents NASH progression. Overall, my work paves the way for a better understanding of the NAFLD disease progression and defines new approaches to study NASH with system biology and translational approaches, exploiting novel methods that had never been used in NAFLD research.MRC (Medical Research Council) scholarship fundin

An unbiased ranking of murine dietary models based on their proximity to human Metabolic Dysfunction–associated Steatotic Liver Disease (MASLD)

Metabolic dysfunction-Associated Steatotic Liver Disease (MASLD), previously known as non-alcoholic fatty liver disease (NAFLD), encompasses steatosis and steatohepatitis (NASH/MASH), leading to cirrhosis and hepatocellular carcinoma. Preclinical MASH research is mainly performed in rodents; however, the model that best recapitulates human disease is yet to be defined. We conducted a wide-ranging retrospective review (metabolic phenotype, liver histopathology, transcriptome benchmarked against humans) of murine models (mostly male), and ranked them using an unbiased MASLD “Human Proximity Score” (MHPS) to define their metabolic relevance and ability to induce MASH-fibrosis. Here, we show that Western Diets align closely with human MASH; high cholesterol content, extended study duration, and/or genetic manipulation of disease-promoting pathways are required to intensify liver damage and accelerate significant (F2+) fibrosis development. Choline-deficient models rapidly induce MASH-fibrosis while showing relatively poor translatability. Our ranking of commonly used MASLD models, based on their proximity to human MASLD, helps with selection of appropriate in vivo models to accelerate preclinical research.This study has been conducted as part of the Preclinical work package of the LITMUS (Liver Investigation: Testing Marker Utility in Steatohepatitis) project. The LITMUS study is a large multi-centre study aiming to evaluate Non-Alcoholic Fatty Liver Disease biomarkers. The Innovative Medicines Initiative 2 (IMI2) Joint Undertaking under Grant Agreement 777377, funded the LITMUS study. This Joint Undertaking receives support from the European Union's Horizon 2020 research and innovation program and EFPIA. EMBL-EBI Core funding supported EP and IK through funding and computing resources from EMBL-EBI. Funding from the MRC (Medical Research Council) supported IK. M.V. is supported by the University of Bari (Horizon Europe Seed cod. id. S06-miRNASH), the Foundation for Liver Research (Intramural Funding), Associazione Italiana Ricerca sul Cancro (IG2022 Grant n. 27521) and Ministry of University and Research on Next Generation EU Funds [COD: P202222FCC, CUP: H53D23009960001, D.D. MUR 1366 (01-09-2023), Title: “System Biology” approaches in HCV Patients with Residual Hepatic Steatosis after Viral Eradication; Cod PE00000003, CUP: H93C22000630001, DD MUR 1550, Title: “ON Foods - Research and innovation network on food and nutrition Sustainability, Safety and Security – Working ON Foods”; Cod: CN00000041, CUP: H93C22000430007, Title PNRR “National Center for Gene Therapy and Drugs based on RNA Technology”, M4C2-Investment 1.4; Code: CN00000013, CUP: H93C22000450007, Title PNNR: “National Centre for HPC, Big Data and Quantum Computing”). A.V-P. is funded by MRC MDU, MRC Metabolic Diseases Unit (MC_UU_00014/5): Disease Model Core, Biochemistry Assay Lab, Histology Core and British Heart Foundation. F.O. is funded by UK Medical Research Council Program Grants MR/K0019494/1 and MR/R023026/1. C.M.P.R. is supported by Fundação para a Ciência e Tecnologia (PTDC/MED-FAR/3492/2021) and La Caixa Foundation (LCF/PR/HR21/52410028). Q.M.A. is supported by the Newcastle NIHR Biomedical Research Centre. S.L.F. and W.S. are supported by the NIH (NIH R01 DK128289; NCI 5P30CA196521-08 to S.L.F.; NIH R01 DK136016 to W.S.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript

Unraveling the Developmental Roadmap toward Human Brown Adipose Tissue.

Increasing brown adipose tissue (BAT) mass and activation is a therapeutic strategy to treat obesity and complications. Obese and diabetic patients possess low amounts of BAT, so an efficient way to expand their mass is necessary. There is limited knowledge about how human BAT develops, differentiates, and is optimally activated. Accessing human BAT is challenging, given its low volume and anatomical dispersion. These constraints make detailed BAT-related developmental and functional mechanistic studies in humans virtually impossible. We have developed and characterized functionally and molecularly a new chemically defined protocol for the differentiation of human pluripotent stem cells (hPSCs) into brown adipocytes (BAs) that overcomes current limitations. This protocol recapitulates step by step the physiological developmental path of human BAT. The BAs obtained express BA and thermogenic markers, are insulin sensitive, and responsive to β-adrenergic stimuli. This new protocol is scalable, enabling the study of human BAs at early stages of development.ER

Suppression of insulin-induced gene 1 (INSIG1) function promotes hepatic lipid remodelling and restrains NASH progression.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a silent pandemic associated with obesity and the metabolic syndrome, and also increases cardiovascular- and cirrhosis-related morbidity and mortality. A complete understanding of adaptive compensatory metabolic programmes that modulate non-alcoholic steatohepatitis (NASH) progression is lacking. METHODS AND RESULTS: Transcriptomic analysis of liver biopsies in patients with NASH revealed that NASH progression is associated with rewiring of metabolic pathways, including upregulation of de novo lipid/cholesterol synthesis and fatty acid remodelling. The modulation of these metabolic programmes was achieved by activating sterol regulatory element-binding protein (SREBP) transcriptional networks; however, it is still debated whether, in the context of NASH, activation of SREBPs acts as a pathogenic driver of lipotoxicity, or rather promotes the biosynthesis of protective lipids that buffer excessive lipid accumulation, preventing inflammation and fibrosis. To elucidate the pathophysiological role of SCAP/SREBP in NASH and wound-healing response, we used an Insig1 deficient (with hyper-efficient SREBPs) murine model challenged with a NASH-inducing diet. Despite enhanced lipid and cholesterol biosynthesis, Insig1 KO mice had similar systemic metabolism and insulin sensitivity to Het/WT littermates. Moreover, activating SREBPs resulted in remodelling the lipidome, decreased hepatocellular damage, and improved wound-healing responses. CONCLUSIONS: Our study provides actionable knowledge about the pathways and mechanisms involved in NAFLD pathogenesis, which may prove useful for developing new therapeutic strategies. Our results also suggest that the SCAP/SREBP/INSIG1 trio governs transcriptional programmes aimed at protecting the liver from lipotoxic insults in NASH.NIHR Cambridge Biomedical Research Centre EU Horizon2020 (Grant Agreement 634413 – EpoS) Evelyn Trus

Suppression of insulin-induced gene 1 (INSIG1) function promotes hepatic lipid remodelling and restrains NASH progression.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a silent pandemic associated with obesity and the metabolic syndrome, and also increases cardiovascular- and cirrhosis-related morbidity and mortality. A complete understanding of adaptive compensatory metabolic programmes that modulate non-alcoholic steatohepatitis (NASH) progression is lacking. METHODS AND RESULTS: Transcriptomic analysis of liver biopsies in patients with NASH revealed that NASH progression is associated with rewiring of metabolic pathways, including upregulation of de novo lipid/cholesterol synthesis and fatty acid remodelling. The modulation of these metabolic programmes was achieved by activating sterol regulatory element-binding protein (SREBP) transcriptional networks; however, it is still debated whether, in the context of NASH, activation of SREBPs acts as a pathogenic driver of lipotoxicity, or rather promotes the biosynthesis of protective lipids that buffer excessive lipid accumulation, preventing inflammation and fibrosis. To elucidate the pathophysiological role of SCAP/SREBP in NASH and wound-healing response, we used an Insig1 deficient (with hyper-efficient SREBPs) murine model challenged with a NASH-inducing diet. Despite enhanced lipid and cholesterol biosynthesis, Insig1 KO mice had similar systemic metabolism and insulin sensitivity to Het/WT littermates. Moreover, activating SREBPs resulted in remodelling the lipidome, decreased hepatocellular damage, and improved wound-healing responses. CONCLUSIONS: Our study provides actionable knowledge about the pathways and mechanisms involved in NAFLD pathogenesis, which may prove useful for developing new therapeutic strategies. Our results also suggest that the SCAP/SREBP/INSIG1 trio governs transcriptional programmes aimed at protecting the liver from lipotoxic insults in NASH.NIHR Cambridge Biomedical Research Centre EU Horizon2020 (Grant Agreement 634413 – EpoS) Evelyn Trus

Oxygen is a critical regulator of cellular metabolism and function in cell culture

SUMMARY Cell culture, the workhorse of biomedical research, is generally considered to be hyperoxic. However, oxygen consumption by cells is underappreciated. High cellular respiration rates can rapidly deplete oxygen, resulting in local hypoxia. Increasing pericellular oxygen levels rewired the metabolism of multiple post-mitotic cell-lines, both in monolayer and organoid culture. Under standard conditions, cultured adipocytes are hypoxic and highly glycolytic. Increased oxygen availability diverted glucose flux toward mitochondria and increased lipogenesis from glucose-derived carbon. These metabolic changes were coupled to thousands of gene expression changes, and rendered adipocytes more sensitive to insulin and lipolytic stimuli. Importantly, pathway analyses revealed increasing oxygen tension made in vitro adipocytes more similar to in vivo adipose tissue. hPSC-derived hepatocytes and cardiac organoids were also functionally enhanced by increased oxygen. Our findings suggest that oxygen is limiting in many standard cell culture systems, and highlight how controlling oxygen availability can improve translatability of cell models

Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.These studies were supported by the Wellcome-MRC Institute of Metabolic Science (IMS) Metabolic Research Laboratories, Imaging Core (Wellcome Trust Major Award (208363/Z/17/Z)), and the MRC MDU Mouse Biochemistry Laboratory (MC_UU_00014/5). RNAseq was performed by the IMS Genomics and transcriptomics core facility and supported by the UK MRC Metabolic Disease Unit (MRC_MC_UU_00014/5) and a Wellcome Trust Major Award (208363/Z/17/Z). S.V. was supported by BHF (RG/18/7/33636). O.J.C. was supported by a Wellcome Trust PhD studentship. I.K. was supported by a Medical Research Council (MRC) PhD studentship. C.P. was supported by a BBSRC project grant (BB/W005905/1). A.J.M was supported by BBSRC [BB/F016581/1] and BHF [FS/17/61/33473]. D.C.G. is funded by a Sir Henry Dale Fellowship from the Wellcome Trust/Royal Society (210481). J.A.N was supported by a Wellcome Senior Clinical Research Fellowship (215477/Z/19/Z). J.E.H. was supported by a Snow Medical Fellowship. The L.V. lab is funded by the ERC advanced grant New-Chol and the core support grant from the Wellcome Trust and Medical Research Council (MRC) of the Wellcome–Medical Research Council Cambridge Stem Cell Institute. For K.H.F.-W. the work was supported in part by DOD-W81XWH-19-1-0213. C.F. and M.Y. were supported by the MRC Core award (MRC_MC_UU_12022/6). A.V-P. was supported by BHF (RG/18/7/33636) and MRC (MC_UU_12012/2). D.J.F. was supported by a Medical Research Council Career Development Award (MR/S007091/1) and a Wellcome Institution Strategic Support Fund award (204845/Z/16/Z)

Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.

Acknowledgements: These studies were supported by the Wellcome-MRC Institute of Metabolic Science (IMS) Metabolic Research Laboratories, Imaging Core (Wellcome Trust Major Award (208363/Z/17/Z)), and the MRC MDU Mouse Biochemistry Laboratory (MC_UU_00014/5). RNAseq was performed by the IMS Genomics and transcriptomics core facility and supported by the UK MRC Metabolic Disease Unit (MRC_MC_UU_00014/5) and a Wellcome Trust Major Award (208363/Z/17/Z). S.V. was supported by BHF (RG/18/7/33636). O.J.C. was supported by a Wellcome Trust PhD studentship. I.K. was supported by a Medical Research Council (MRC) PhD studentship. C.P. was supported by a BBSRC project grant (BB/W005905/1). A.J.M was supported by BBSRC [BB/F016581/1] and BHF [FS/17/61/33473]. D.C.G. is funded by a Sir Henry Dale Fellowship from the Wellcome Trust/Royal Society (210481). J.A.N was supported by a Wellcome Senior Clinical Research Fellowship (215477/Z/19/Z). J.E.H. was supported by a Snow Medical Fellowship. The L.V. lab is funded by the ERC advanced grant New-Chol and the core support grant from the Wellcome Trust and Medical Research Council (MRC) of the Wellcome–Medical Research Council Cambridge Stem Cell Institute. For K.H.F.-W., the work was supported in part by DOD-W81XWH-19-1-0213. C.F. and M.Y. were supported by the MRC Core award (MRC_MC_UU_12022/6). A.V-P. was supported by BHF (RG/18/7/33636) and MRC (MC_UU_12012/2). D.J.F. was supported by a Medical Research Council Career Development Award (MR/S007091/1) and a Wellcome Institution Strategic Support Fund award (204845/Z/16/Z).Funder: cambridgeuniversity | Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge (Wellcome - MRC Cambridge Stem Cell Institute); doi: http://dx.doi.org/10.13039/501100021773The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.These studies were supported by the Wellcome-MRC Institute of Metabolic Science (IMS) Metabolic Research Laboratories, Imaging Core (Wellcome Trust Major Award (208363/Z/17/Z)), and the MRC MDU Mouse Biochemistry Laboratory (MC_UU_00014/5). RNAseq was performed by the IMS Genomics and transcriptomics core facility and supported by the UK MRC Metabolic Disease Unit (MRC_MC_UU_00014/5) and a Wellcome Trust Major Award (208363/Z/17/Z). S.V. was supported by BHF (RG/18/7/33636). O.J.C. was supported by a Wellcome Trust PhD studentship. I.K. was supported by a Medical Research Council (MRC) PhD studentship. C.P. was supported by a BBSRC project grant (BB/W005905/1). A.J.M was supported by BBSRC [BB/F016581/1] and BHF [FS/17/61/33473]. D.C.G. is funded by a Sir Henry Dale Fellowship from the Wellcome Trust/Royal Society (210481). J.A.N was supported by a Wellcome Senior Clinical Research Fellowship (215477/Z/19/Z). J.E.H. was supported by a Snow Medical Fellowship. The L.V. lab is funded by the ERC advanced grant New-Chol and the core support grant from the Wellcome Trust and Medical Research Council (MRC) of the Wellcome–Medical Research Council Cambridge Stem Cell Institute. For K.H.F.-W. the work was supported in part by DOD-W81XWH-19-1-0213. C.F. and M.Y. were supported by the MRC Core award (MRC_MC_UU_12022/6). A.V-P. was supported by BHF (RG/18/7/33636) and MRC (MC_UU_12012/2). D.J.F. was supported by a Medical Research Council Career Development Award (MR/S007091/1) and a Wellcome Institution Strategic Support Fund award (204845/Z/16/Z)

An unbiased ranking of murine dietary models based on their proximity to human metabolic dysfunction-associated steatotic liver disease (MASLD).

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease, encompasses steatosis and metabolic dysfunction-associated steatohepatitis (MASH), leading to cirrhosis and hepatocellular carcinoma. Preclinical MASLD research is mainly performed in rodents; however, the model that best recapitulates human disease is yet to be defined. We conducted a wide-ranging retrospective review (metabolic phenotype, liver histopathology, transcriptome benchmarked against humans) of murine models (mostly male) and ranked them using an unbiased MASLD 'human proximity score' to define their metabolic relevance and ability to induce MASH-fibrosis. Here, we show that Western diets align closely with human MASH; high cholesterol content, extended study duration and/or genetic manipulation of disease-promoting pathways are required to intensify liver damage and accelerate significant (F2+) fibrosis development. Choline-deficient models rapidly induce MASH-fibrosis while showing relatively poor translatability. Our ranking of commonly used MASLD models, based on their proximity to human MASLD, helps with the selection of appropriate in vivo models to accelerate preclinical research.This study has been conducted as part of the Preclinical work package of the LITMUS (Liver Investigation: Testing Marker Utility in Steatohepatitis) project. The LITMUS study is a large multi-centre study aiming to evaluate Non-Alcoholic Fatty Liver Disease biomarkers. The Innovative Medicines Initiative 2 (IMI2) Joint Undertaking under Grant Agreement 777377, funded the LITMUS study. This Joint Undertaking receives support from the European Union's Horizon 2020 research and innovation program and EFPIA. EMBL-EBI Core funding supported EP and IK through funding and computing resources from EMBL-EBI. Funding from the MRC (Medical Research Council) supported IK. M.V. is supported by the University of Bari (Horizon Europe Seed cod. id. S06-miRNASH), the Foundation for Liver Research (Intramural Funding), Associazione Italiana Ricerca sul Cancro (IG2022 Grant n. 27521) and Ministry of University and Research on Next Generation EU Funds [COD: P202222FCC, CUP: H53D23009960001, D.D. MUR 1366 (01-09-2023), Title: “System Biology” approaches in HCV Patients with Residual Hepatic Steatosis after Viral Eradication; Cod PE00000003, CUP: H93C22000630001, DD MUR 1550, Title: “ON Foods - Research and innovation network on food and nutrition Sustainability, Safety and Security – Working ON Foods”; Cod: CN00000041, CUP: H93C22000430007, Title PNRR “National Center for Gene Therapy and Drugs based on RNA Technology”, M4C2-Investment 1.4; Code: CN00000013, CUP: H93C22000450007, Title PNNR: “National Centre for HPC, Big Data and Quantum Computing”). A.V-P. is funded by MRC MDU, MRC Metabolic Diseases Unit (MC_UU_00014/5): Disease Model Core, Biochemistry Assay Lab, Histology Core and British Heart Foundation. F.O. is funded by UK Medical Research Council Program Grants MR/K0019494/1 and MR/R023026/1. C.M.P.R. is supported by Fundação para a Ciência e Tecnologia (PTDC/MED-FAR/3492/2021) and La Caixa Foundation (LCF/PR/HR21/52410028). Q.M.A. is supported by the Newcastle NIHR Biomedical Research Centre. S.L.F. and W.S. are supported by the NIH (NIH R01 DK128289; NCI 5P30CA196521-08 to S.L.F.; NIH R01 DK136016 to W.S.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript