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Background: Hepatocellular senescence may be a causal factor in the development and progression of non-alcoholic steatohepatitis (NASH). The most effective currently available treatment for NASH is lifestyle intervention, including dietary modification. This study aimed to evaluate the effects of dietary intervention on hallmarks of NASH and molecular signatures of hepatocellular senescence in the Gubra-Amylin NASH (GAN) diet-induced obese (DIO) and biopsy-confirmed mouse model of NASH., Methods: GAN DIO-NASH mice with liver biopsy-confirmed NASH and fibrosis received dietary intervention by switching to chow feeding (chow reversal) for 8, 16 or 24 weeks. Untreated GAN DIO-NASH mice and chow-fed C57BL/6J mice served as controls. Pre-to-post liver biopsy histology was performed for within-subject evaluation of NAFLD Activity Score and fibrosis stage. Terminal endpoints included blood/liver biochemistry, quantitative liver histology, mitochondrial respiration and RNA sequencing., Results: Chow-reversal promoted substantial benefits on metabolic outcomes and liver histology, as demonstrated by robust weight loss, complete resolution of hepatomegaly, hypercholesterolemia, elevated transaminase levels and hepatic steatosis in addition to attenuation of inflammatory markers. Notably, all DIO-NASH mice demonstrated ≥ 2 point significant improvement in NAFLD Activity Score following dietary intervention. While not improving fibrosis stage, chow-reversal reduced quantitative fibrosis markers (PSR, collagen 1a1, α-SMA), concurrent with improved liver mitochondrial respiration, complete reversal of p21 overexpression, lowered γ-H2AX levels and widespread suppression of gene expression markers of hepatocellular senescence., Conclusions: Dietary intervention (chow reversal) substantially improves metabolic, biochemical and histological hallmarks of NASH and fibrosis in GAN DIO-NASH mice. These benefits were reflected by progressive clearance of senescent hepatocellular cells, making the model suitable for profiling potential senotherapeutics in preclinical drug discovery for NASH., (© 2024. The Author(s).)

Non-alcoholic steatohepatitis (NASH) is emerging as a major cause of hepatocellular carcinoma (HCC), however, it is not resolved if compounds in late-stage clinical development for NASH may have additional therapeutic benefits in NASH-driven HCC (NASH-HCC). Here, we profiled monotherapy with semaglutide (glucagon-like-receptor-1 receptor agonist) and lanifibranor (pan-peroxisome proliferator-activated receptor agonist) in a diet-induced obese (DIO) mouse model of NASH-HCC. Disease progression was characterized in male C57BL/6 J mice fed the GAN (Gubra Amylin NASH) diet high in fat, fructose and cholesterol for 12-72 weeks (n = 15 per group). Other GAN DIO-NASH-HCC mice fed the GAN diet for 54 weeks and with biopsy-confirmed NASH (NAFLD Activity Score ≥ 5) and advanced fibrosis (stage F3) received vehicle (n = 16), semaglutide (30 nmol/kg, s.c., n = 15), or lanifibranor (30 mg/kg, p.o., n = 15) once daily for 14 weeks. GAN DIO-NASH-HCC mice demonstrated progressive NASH, fibrosis and HCC burden. Tumors presented with histological and molecular signatures of poor prognostic HCC. Consistent with clinical trial outcomes in NASH patients, both lanifibranor and semaglutide improved NASH while only lanifibranor reduced fibrosis in GAN DIO-NASH-HCC mice. Notably, only semaglutide reduced tumor burden in GAN DIO-NASH-HCC mice. In conclusion, the GAN DIO-NASH-HCC mouse is a clinical translational model of NASH-HCC. Semaglutide improves both NASH and tumor burden in GAN DIO-NASH-HCC mice, highlighting the suitability of this preclinical model for profiling novel drug therapies targeting NASH-HCC., (© 2023. The Author(s).)

Fibroblast growth factor 21 (FGF21) plays a key role in hepatic lipid metabolism and long-acting FGF21 analogs have emerged as promising drug candidates for the treatment of nonalcoholic steatohepatitis (NASH). It remains to characterize this drug class in translational animal models that recapitulate the etiology and hallmarks of human disease. To this end, we evaluated the long-acting FGF21 analog PF-05231023 in the GAN (Gubra Amylin NASH) diet-induced obese (DIO) and biopsy-confirmed mouse model of NASH. Male C57BL/6J mice were fed the GAN diet high in fat, fructose, and cholesterol for 34 wk before the start of the study. GAN DIO-NASH mice with biopsy-confirmed NAFLD Activity Score (NAS ≥5) and fibrosis (stage ≥F1) were biweekly administered with PF-05231023 (10 mg/kg sc) or vehicle (sc) for 12 wk. Vehicle-dosed chow-fed C57BL/6J mice served as healthy controls. Pre-to-post liver biopsy histopathological scoring was performed for within-subject evaluation of NAFLD Activity Score (NAS) and fibrosis stage. Terminal endpoints included quantitative liver histology and transcriptome signatures as well as blood and liver biochemistry. PF-05231023 significantly reduced body weight, hepatomegaly, plasma transaminases, and plasma/liver lipids in GAN DIO-NASH mice. Notably, PF-05231023 reduced both NAS (≥2-point improvement) and fibrosis stage (1-point improvement). Improvements in NASH and fibrosis severity were supported by reduced quantitative histological markers of steatosis, inflammation, and fibrogenesis as well as improvements in disease-associated liver transcriptome signatures. In conclusion, PF-05231023 reduces NASH and fibrosis severity in a translational biopsy-confirmed mouse model of NASH, supporting development of FGF21 analogs for the treatment of NASH. NEW & NOTEWORTHY It is unclear if long-acting FGF21 analogs have antifibrotic efficacy in NASH. We therefore profiled the clinically relevant FGF21 analog PF-05231023 in a translational diet-induced obese and biopsy-confirmed mouse model of NASH. We found PF-05231023 to exert hepatoprotective effects as indicated by notable improvements in plasma markers and histological hallmarks of NASH, including improved fibrosis stage. Collectively, the present study supports the continued exploration of long-acting FGF21 analogs for the treatment of NASH and other fibrotic diseases.

Non-invasive biomarkers of non-alcoholic fatty liver disease (NAFLD) supporting diagnosis and monitoring disease progression are urgently needed. The present study aimed to establish a bioinformatics pipeline capable of defining and validating NAFLD biomarker candidates based on paired hepatic global gene expression and plasma bioanalysis from individuals representing different stages of histologically confirmed NAFLD (no/mild, moderate, more advanced NAFLD). Liver secretome gene signatures were generated in a patient cohort of 26 severely obese individuals with the majority having no or mild fibrosis. To this end, global gene expression changes were compared between individuals with no/mild NAFLD and moderate/advanced NAFLD with subsequent filtering for candidate gene products with liver-selective expression and secretion. Four candidate genes, including LPA (lipoprotein A), IGFBP-1 (insulin-like growth factor-binding protein 1), SERPINF2 (serpin family F member 2) and MAT1A (methionine adenosyltransferase 1A), were differentially expressed in moderate/advanced NAFLD, which was confirmed in three independent RNA sequencing datasets from large, publicly available NAFLD studies. The corresponding gene products were quantified in plasma samples but could not discriminate among different grades of NAFLD based on NAFLD activity score. Conclusion: We demonstrate a novel approach based on the liver transcriptome allowing for identification of secreted hepatic gene products as potential circulating diagnostic biomarkers of NAFLD. Using this approach in larger NAFLD patient cohorts may yield potential circulating biomarkers for NAFLD severity., Competing Interests: U.L.K. has served on scientific advisory board panels, served as a consultant and/or been part of speaker’s bureau of AstraZeneca, Mundipharma, Boehringer Ingelheim, Novo Nordisk, Sanofi, Eli Lilly and Abbott. T.V. has served on scientific advisory panels, been part of speaker’s bureaus for, served as a consultant to and/or received research support from Amgen, AstraZeneca, Boehringer Ingelheim, Eli Lilly, Gilead, Mundipharma, MSD/Merck, Novo Nordisk, Sanofi and Sun Pharmaceuticals. F.K.K. has served on scientific advisory panels and/or been part of speaker’s bureaus for, served as a consultant to, and/or received research support from Amgen, AstraZeneca, Boehringer Ingelheim, Carmot Therapeutics, Eli Lilly, Gubra, MedImmune, MSD/Merck, Mundipharma, Norgine, Novo Nordisk, Sanofi, ShouTi, Zealand Pharma and Zucara. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Non-alcoholic steatohepatitis (NASH) has emerged as a major challenge for public health because of high global prevalence and lack of evidence-based therapies. Most animal models of NASH lack sufficient validation regarding disease progression and pharmacological treatment. The Gubra-Amylin NASH (GAN) diet-induced obese (DIO) mouse demonstrate clinical translatability with respect to disease etiology and hallmarks of NASH. This study aimed to evaluate disease progression and responsiveness to clinically effective interventions in GAN DIO-NASH mice. Disease phenotyping was performed in male C57BL/6J mice fed the GAN diet high in fat, fructose, and cholesterol for 28-88 weeks. GAN DIO-NASH mice with biopsy-confirmed NASH and fibrosis received low-caloric dietary intervention, semaglutide (30 nmol/kg/day, s.c.) or lanifibranor (30 mg/kg/day, p.o.) for 8 and 12 weeks, respectively. Within-subject change in nonalcoholic fatty liver disease (NAFLD) Activity Score (NAS) and fibrosis stage was evaluated using automated deep learning-based image analysis. GAN DIO-NASH mice showed clear and reproducible progression in NASH, fibrosis stage, and tumor burden with high incidence of hepatocellular carcinoma. Consistent with clinical trial outcomes, semaglutide and lanifibranor improved NAS, whereas only lanifibranor induced regression in the fibrosis stage. Dietary intervention also demonstrated substantial benefits on metabolic outcomes and liver histology. Differential therapeutic efficacy of semaglutide, lanifibranor, and dietary intervention was supported by quantitative histology, RNA sequencing, and blood/liver biochemistry. In conclusion, the GAN DIO-NASH mouse model recapitulates various histological stages of NASH and faithfully reproduces histological efficacy profiles of compounds in advanced clinical development for NASH. Collectively, these features highlight the utility of GAN DIO-NASH mice in preclinical drug development., (© 2022 Gubra. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Background: Animal models of non-alcoholic steatohepatitis (NASH) are important tools in preclinical research and drug discovery. Gubra-Amylin NASH (GAN) diet-induced obese (DIO) mice represent a model of fibrosing NASH. The present study directly assessed the clinical translatability of the model by head-to-head comparison of liver biopsy histological and transcriptome changes in GAN DIO-NASH mouse and human NASH patients., Methods: C57Bl/6 J mice were fed chow or the GAN diet rich in saturated fat (40%), fructose (22%) and cholesterol (2%) for ≥38 weeks. Metabolic parameters as well as plasma and liver biomarkers were assessed. Liver biopsy histology and transcriptome signatures were compared to samples from human lean individuals and patients diagnosed with NASH., Results: Liver lesions in GAN DIO-NASH mice showed similar morphological characteristics compared to the NASH patient validation set, including macrosteatosis, lobular inflammation, hepatocyte ballooning degeneration and periportal/perisinusoidal fibrosis. Histomorphometric analysis indicated comparable increases in markers of hepatic lipid accumulation, inflammation and collagen deposition in GAN DIO-NASH mice and NASH patient samples. Liver biopsies from GAN DIO-NASH mice and NASH patients showed comparable dynamics in several gene expression pathways involved in NASH pathogenesis. Consistent with the clinical features of NASH, GAN DIO-NASH mice demonstrated key components of the metabolic syndrome, including obesity and impaired glucose tolerance., Conclusions: The GAN DIO-NASH mouse model demonstrates good clinical translatability with respect to the histopathological, transcriptional and metabolic aspects of the human disease, highlighting the suitability of the GAN DIO-NASH mouse model for identifying therapeutic targets and characterizing novel drug therapies for NASH.

Background: The trans-fat containing AMLN (amylin liver non-alcoholic steatohepatitis, NASH) diet has been extensively validated in C57BL/6J mice with or without the Lep ob /Lep ob ( ob/ob ) mutation in the leptin gene for reliably inducing metabolic and liver histopathological changes recapitulating hallmarks of NASH. Due to a recent ban on trans-fats as food additive, there is a marked need for developing a new diet capable of promoting a compatible level of disease in ob/ob and C57BL/6J mice., Aim: To develop a biopsy-confirmed mouse model of NASH based on an obesogenic diet with trans-fat substituted by saturated fat., Methods: Male ob/ob mice were fed AMLN diet or a modified AMLN diet with trans-fat (Primex shortening) substituted by equivalent amounts of palm oil [Gubra amylin NASH, (GAN) diet] for 8, 12 and 16 wk. C57BL/6J mice were fed the same diets for 28 wk. AMLN and GAN diets had similar caloric content (40% fat kcal), fructose (22%) and cholesterol (2%) level., Results: The GAN diet was more obesogenic compared to the AMLN diet and impaired glucose tolerance. Biopsy-confirmed steatosis, lobular inflammation, hepatocyte ballooning, fibrotic liver lesions and hepatic transcriptome changes were similar in ob/ob mice fed the GAN or AMLN diet. C57BL/6J mice developed a mild to moderate fibrotic NASH phenotype when fed the same diets., Conclusion: Substitution of Primex with palm oil promotes a similar phenotype of biopsy-confirmed NASH in ob/ob and C57BL/6J mice, making GAN diet-induced obese mouse models suitable for characterizing novel NASH treatments., Competing Interests: Conflict-of-interest statement: Michelle L. Boland and James L. Trevaskis were previously employed by MedImmune, LLC. Taylor S. Cohen, David Tabor, Fiona Fernandes, Andrey Tovchigrechko, Paul Warrener, and Bret R. Sellman are employed by MedImmune LLC. All other authors have nothing to disclose.

Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of conditions ranging from simple steatosis (NAFL), over nonalcoholic steatohepatitis (NASH) with or without fibrosis, to cirrhosis with end-stage disease. The hepatic molecular events underlying the development of NAFLD and transition to NASH are poorly understood. The present study aimed to determine hepatic transcriptome dynamics in patients with NAFL or NASH compared with healthy normal-weight and obese individuals. RNA sequencing and quantitative histomorphometry of liver fat, inflammation and fibrosis were performed on liver biopsies obtained from healthy normal-weight ( n = 14) and obese ( n = 12) individuals, NAFL ( n = 15) and NASH ( n = 16) patients. Normal-weight and obese subjects showed normal liver histology and comparable gene expression profiles. Liver transcriptome signatures were largely overlapping in NAFL and NASH patients, however, clearly separated from healthy normal-weight and obese controls. Most marked pathway perturbations identified in both NAFL and NASH were associated with markers of lipid metabolism, immunomodulation, extracellular matrix remodeling, and cell cycle control. Interestingly, NASH patients with positive Sonic hedgehog hepatocyte staining showed distinct transcriptome and histomorphometric changes compared with NAFL. In conclusion, application of immunohistochemical markers of hepatocyte injury may serve as a more objective tool for distinguishing NASH from NAFL, facilitating improved resolution of hepatic molecular changes associated with progression of NAFLD. NEW & NOTEWORTHY Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in Western countries. NAFLD is associated with the metabolic syndrome and can progress to the more serious form, nonalcoholic steatohepatitis (NASH), and ultimately lead to irreversible liver damage. Using gold standard molecular and histological techniques, this study demonstrates that the currently used diagnostic tools are problematic for differentiating mild NAFLD from NASH and emphasizes the marked need for developing improved histological markers of NAFLD progression.

Aim: To evaluate the pharmacodynamics of compounds in clinical development for nonalcoholic steatohepatitis (NASH) in obese mouse models of biopsy-confirmed NASH., Methods: Male wild-type C57BL/6J mice (DIO-NASH) and Lep ob/ob ( ob/ob -NASH) mice were fed a diet high in trans-fat (40%), fructose (20%) and cholesterol (2%) for 30 and 21 wk, respectively. Prior to treatment, all mice underwent liver biopsy for confirmation and stratification of liver steatosis and fibrosis, using the nonalcoholic fatty liver disease activity score (NAS) and fibrosis staging system. The mice were kept on the diet and received vehicle, liraglutide (0.2 mg/kg, SC, BID), obeticholic acid (OCA, 30 mg/kg PO, QD), or elafibranor (30 mg/kg PO, QD) for eight weeks. Within-subject comparisons were performed on changes in steatosis, inflammation, ballooning degeneration, and fibrosis scores. In addition, compound effects were evaluated by quantitative liver histology, including percent fractional area of liver fat, galectin-3, and collagen 1a1., Results: Liraglutide and elafibranor, but not OCA, reduced body weight in both models. Liraglutide improved steatosis scores in DIO-NASH mice only. Elafibranor and OCA reduced histopathological scores of hepatic steatosis and inflammation in both models, but only elafibranor reduced fibrosis severity. Liraglutide and OCA reduced total liver fat, collagen 1a1, and galectin-3 content, driven by significant reductions in liver weight. The individual drug effects on NASH histological endpoints were supported by global gene expression (RNA sequencing) and liver lipid biochemistry., Conclusion: DIO-NASH and ob/ob -NASH mouse models show distinct treatment effects of liraglutide, OCA, and elafibranor, being in general agreement with corresponding findings in clinical trials for NASH. The present data therefore further supports the clinical translatability and utility of DIO-NASH and ob/ob -NASH mouse models of NASH for probing the therapeutic efficacy of compounds in preclinical drug development for NASH., Competing Interests: Conflict-of-interest statement: The authors declare no conflict of interest.

Aim: To characterize the efficacy of the dual FXR/TGR5 receptor agonist INT-767 upon histological endpoints in a rodent model of diet-induced and biopsy-confirmed non-alcoholic steatohepatitis (NASH)., Methods: The effects of INT-767 on histological features of NASH were assessed in two studies using Lep ob/ob ( ob/ob ) NASH mice fed the AMLN diet (high fat with trans-fat, cholesterol and fructose). In a proof-of-concept study, Lep ob/ob ( ob/ob ) NASH mice were first dosed with INT-767 (3 or 10 mg/kg for 8 wk). A second ob/ob NASH study compared INT-767 (3 and 10 mg/kg) to obeticholic acid (OCA) (10 or 30 mg/kg; 16 wk). Primary histological endpoints included qualitative and quantitative assessments of NASH. Other metabolic and plasma endpoints were also assessed. A comparative assessment of INT-767 and OCA effects on drug distribution and hepatic gene expression was performed in C57Bl/6 mice on standard chow. C57Bl/6 mice were orally dosed with INT-767 or OCA (1-30 mg/kg) for 2 wk, and expression levels of candidate genes were assessed by RNA sequencing and tissue drug levels were measured by liquid chromatography tandem-mass spectrometry., Results: INT-767 dose-dependently (3 and 10 mg/kg, PO, QD, 8 wk) improved qualitative morphometric scores on steatohepatitis severity, inflammatory infiltrates and fibrosis stage. Quantitative morphometric analyses revealed that INT-767 reduced parenchymal collagen area, collagen fiber density, inflammation (assessed by Galectin-3 immunohistochemistry) and hepatocyte lipid droplet area following INT-767 treatment. In a comparative study (16 wk), the FXR agonists OCA (10 and 30 mg/kg) and INT-767 (3 and 10 mg/kg) both improved NASH histopathology, with INT-767 exerting greater therapeutic potency and efficacy than OCA. Mechanistic studies suggest that both drugs accumulate similarly within the liver and ileum, however, the effects of INT-767 may be driven by enhanced hepatic, but not ileal, FXR function., Conclusion: These findings confirm the potential utility of FXR and dual FXR/TGR5 activation as disease intervention strategies in NASH., Competing Interests: Conflict-of-interest statement: Jonathan Roth and Mark Young are employed by and hold equity in Intercept Pharmaceuticals, Inc. All other authors have nothing to disclose.

Non‐alcoholic steatohepatitis (NASH) has emerged as a major challenge for public health because of high global prevalence and lack of evidence‐based therapies. Most animal models of NASH lack sufficient validation regarding disease progression and pharmacological treatment. The Gubra‐Amylin NASH (GAN) diet‐induced obese (DIO) mouse demonstrate clinical translatability with respect to disease etiology and hallmarks of NASH. This study aimed to evaluate disease progression and responsiveness to clinically effective interventions in GAN DIO‐NASH mice. Disease phenotyping was performed in male C57BL/6J mice fed the GAN diet high in fat, fructose, and cholesterol for 28–88 weeks. GAN DIO‐NASH mice with biopsy‐confirmed NASH and fibrosis received low‐caloric dietary intervention, semaglutide (30 nmol/kg/day, s.c.) or lanifibranor (30 mg/kg/day, p.o.) for 8 and 12 weeks, respectively. Within‐subject change in nonalcoholic fatty liver disease (NAFLD) Activity Score (NAS) and fibrosis stage was evaluated using automated deep learning‐based image analysis. GAN DIO‐NASH mice showed clear and reproducible progression in NASH, fibrosis stage, and tumor burden with high incidence of hepatocellular carcinoma. Consistent with clinical trial outcomes, semaglutide and lanifibranor improved NAS, whereas only lanifibranor induced regression in the fibrosis stage. Dietary intervention also demonstrated substantial benefits on metabolic outcomes and liver histology. Differential therapeutic efficacy of semaglutide, lanifibranor, and dietary intervention was supported by quantitative histology, RNA sequencing, and blood/liver biochemistry. In conclusion, the GAN DIO‐NASH mouse model recapitulates various histological stages of NASH and faithfully reproduces histological efficacy profiles of compounds in advanced clinical development for NASH. Collectively, these features highlight the utility of GAN DIO‐NASH mice in preclinical drug development. [ABSTRACT FROM AUTHOR]