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in vivo MASH (NASH) model, Liver on a Chip, MASH Biomarkers
Translational Research service through Humanized Disease Models

Overview: in vivo MASH (NASH) model


Metabolic Dysfunction Associated Steatohepatitis (MASH) is one of the most prevalent chronic liver diseases, which is closely associated with obesity, insulin resistance and dyslipidemia. These important features therefore need to be reflected in a preclinical model as well. Based on 25 years of research on translational metabolic disease models, TNO has developed the Ldlr-/-.Leiden MASH mouse model that accurately mimics the etiology and pathology of MASH and fibrosis in humans. By using a high-fat diet, with a macronutrient composition comparable to human diets (e.g., without added cholesterol), the Ldlr-/-.Leiden MASH mouse model develops obesity, insulin resistance, adipose tissue inflammation, increased gut permeability with altered microbiota composition, and MASH with bridging fibrosis [F3 stage].

Features:the Ldlr-/-.Leiden MASH mouse model


  Translational disease induction and clinical phenotype
  Severe obesity (with adipose tissue inflammation)
  Insulin resistance
  Hyperlipidemia (humanized lipoprotein profiles), high triglycerides, high (V)LDL, low HDL)
  Translational histology: steatosis, inflammatory aggregates and bridging fibrosis (up to F3)
  Atherosclerosis development
  Translational underlying pathways, verified on transcriptomics, proteomics and metabolomics level
  Extensive validation with multiple interventions (including the FDA approved drug)
  Metabolic overload-induced complications in multiple organs.

Time lines・Readout Paramters ; The Ldlr-/-.Leiden MASH mouse model


The Ldlr-/-.Leiden MASH model has been validated with many different interventions

Intervention Target Publication
Caspase-1 inhibitor Caspase 1 Morrison, Int J Obes 2016
Cenicriviroc CCR2 & CCR5 Poster at EASL, 2024
(olive oil polyphenols)
Oxidative stress Luque-Sierra et al., Mol Nutr Food Res, 2018
Alvarez-Amor et al., Nature Sci Rep. 2021
Rosi-, Pioglitazone PPAR-γ Mulder et al., Nat Sci Rep 2016 & in-house
Lanifibranor PPARs In preparation
EPA, DHA from krill oil PPARs, oxylipins, resolvins Gart et al., Nutrients 2021
Obeticholic acid FXR Salic et al., PLoS One 2019
Volixibat IBAT Mulder et al., Nat Sci Rep 2016 & in-house
Butyric acid i.a. TGF-β fibrosis sign. Arnoldussen et al., Int J Obes 2017
Gart et al., Biomedicines 2021
Propionic acid TCA cycle Gart et al., FASEB J 2020
Semaglutide GLP-1 Inia et al., Int J of Mol Sci 2023
DGAT2 inhibitor DAGT2 In preparation
L-carnitine and nicotinamide riboside β-oxidation, mitochondria Salic and Gart et al., Int J Mol Sc 2019
Casein hydrolysate i.a. lipid modulators Schoemaker et al., PLoS One 2017
Branched chain amino acids Mitochondria, TCA cycle Gart et al., FASEB J. 2022
van den Hoek et al., Metabolism 2021
Milk-fat globule membrane Metabolism Arnoldussen et al., Int J Obes 2021
Natural cholesterol lowering compounds Neutrophils Morrison et al., Front Endocrinol 2021
Resmetirom THR-β Poster at EASL, 2024
Translation to Human Translational Value Head to head with 1099 patients

Download; latest publication list (pdf)

● New paper in Nature comm (May 29, 2024); Development of a novel non-invasive biomarker panel for hepatic fibrosis in MASLD

TNO presented the following four posters related to MASH (NASH) research at EASL congress in Milan, Italy (June 5th to 8th, 2024).

  ● Resmetirom protects against diet-induced MASLD and reduces atherogenic risk factors in obese Ldlr-/-.Leiden mice.

  ● Metabolic flux analysis using a 14C microtracer approach combined with accelerator mass spectrometry analysis
- a proof of concept study on de novo lipogenesis.

  ● Development of a novel non-invasive biomarker panel for hepatic fibrosis in MASLD.

  ● Treatment with the CCR2/CCR5 antagonist Cenicriviroc does not affect MASH and fibrosis development in Ldlr-/-.Leiden mice, translational to clinical phase 3 trial results

Recent poster at AASLD 2022:  The bispecific anti FGFR1/KLB agonist antibody bFKB1 attenuates non alcoholic steatohepatitis and atherosclerosis in Ldlr -/-.Leiden mice.
Poster-1 at AASLD 2023:  Temporal dynamics of metabolic dysfunctions in liver, adipose tissue and the gut during diet-induced NASH development in Ldlr-/-.Leiden mice.
Poster-2 at AASLD 2023:  Clinical translatability of Ldlr-/-.Leiden mouse model for NASH

Flux Analysis using 14C and AMS; De Novo Lipogenesis, Muscle Protein Turnover, etc

Metabolic flux measurements play an important role in advancing our understanding of (patho)physiology, disease mechanisms and the development of new therapeutics. By incorporating stable or radioactive isotopes into specific molecules (tracers), the distribution and fate of the isotope can be followed ? thereby providing insight into the movement and metabolic transformation of biomolecules. An isotope that is frequently used for such analyses is 13C, which is analyzed by isotope ratio mass spectrometry. The high natural abundance of 13C (~1%) requires the use of large amounts of labelled substrates, especially in clinical studies. An alternative isotope is 14C, which has an extremely low natural abundance (ca. one in a trillion). In combination with analysis by extremely sensitive accelerator mass spectrometry (AMS), this enables the detection of very small amounts of 14C-labeled product/biomarker at very low (microtrace) amounts of labelled substrate administration.

  De Novo Lipogenesis (DNL)
  HDL funcitionality / Reverse Cholesterol Transport
  Muscle Protein Synthesis & Breakdown (combined 14C and D2O analysis)
  Glucose Metabolism (DNL, conversion to fluctose)

  Technical info:
●   Flux Analysis using 14C and AMS

Liver-on-a-Chip・ Cell model for liver fibrosis


We have developed a liver-on-a-chip model that exhibits MASH / liver fibrosis. By adding oleate and palmitate and later fructose, steatosis and steatohepatitis are induced. Adding also LPS leads to the induction of fibrosis. This 3D liver spheroid model uses human hepatocytes, stellate cells and Kupffer cells. The model is sensitive to reference compounds such as pioglitazone and fenofibrate.

・A diet-induced disease-mimicking 3D in vitro model, closely resembling the pathophysiology of liver steatosis and early fibrosis was developed;
・Liver spheroids composed of primary hepatocytes, stellate and Kupffer cells exhibit expected cell function for at least 21 days;
・The steatosis and steatohepatitis induced by fatty acids and fructose can be modulated by model drugs;

●   Development of a disease-mimicking model for NASH and fibrosis in a triple cell-type, spheroid-based liver-on-chip platform with microfluidics.(Poster)

Biomarkers for MASLD・MASH

Fibrosis is the main determinant for mortality and is therefore an important clinical readout. Detection of fibrosis via a liver biopsy is still the golden standard but this is an invasive procedure with risk for complications, sampling and reading errors. Fibrosis is a dynamic process and currently no validated diagnostic/prognostic methods exist, especially biomarkers which can detect the early onset and progression of fibrosis. We aim to identify a mechanism-based biomarker panel which is prognostic for MASLD-related fibrosis and enables early identification of people at risk with active fibrogenesis. By integration of mechanistic mouse studies and human clinical samples we identified a set of circulating proteins related to the active fibrogenesis process.

Hepatic molecular signature in a translational NASH model as an early screening tool for novel NASH therapeutics.(Poster)

(New) Early prediction of progressive fibrogenesis in NAFLD-NASH patients using blood-based biomarkers.(Poster)

Optimized assays in Plasma, Serum, Breast milk, Fecal water, Saliva, etc validated in healthy subject or in patient groups.