A simple diet- and chemical-induced murine NASH model with rapid progression of steatohepatitis, fibrosis and liver cancer

Published:March 20, 2018DOI:


      • A mouse model developed that recapitulates the progressive stages of human fatty liver disease.
      • The functional pathways of gene expression and immune abnormalities in this model closely resemble human disease.
      • The ease and reproducibility of this model makes it ideal to study disease pathogenesis and test new treatments.

      Background and Aims

      Although the majority of patients with non-alcoholic fatty liver disease (NAFLD) have only steatosis without progression, a sizeable fraction develop non-alcoholic steatohepatitis (NASH), which can lead to cirrhosis and hepatocellular carcinoma (HCC). Many established diet-induced mouse models for NASH require 24–52 weeks, which makes testing for drug response costly and time consuming.


      We have sought to establish a murine NASH model with rapid progression of extensive fibrosis and HCC by using a western diet (WD), which is high-fat, high-fructose and high-cholesterol, combined with low weekly dose of intraperitoneal carbon tetrachloride (CCl4), which serves as an accelerator.


      C57BL/6J mice were fed a normal chow diet ± CCl4 or WD ± CCl4 for 12 and 24 weeks. Addition of CCl4 exacerbated histological features of NASH, fibrosis, and tumor development induced by WD, which resulted in stage 3 fibrosis at 12 weeks and HCC development at 24 weeks. Furthermore, whole liver transcriptomic analysis indicated that dysregulated molecular pathways in WD/CCl4 mice and immunologic features were similar to those of human NASH.


      Our mouse NASH model exhibits rapid progression of advanced fibrosis and HCC, and mimics histological, immunological and transcriptomic features of human NASH, suggesting that it will be a useful experimental tool for preclinical drug testing.

      Lay summary

      A carefully characterized model has been developed in mice that recapitulates the progressive stages of human fatty liver disease, from simple steatosis, to inflammation, fibrosis and cancer. The functional pathways of gene expression and immune abnormalities in this model closely resemble human disease. The ease and reproducibility of this model make it ideal to study disease pathogenesis and test new treatments.

      Graphical abstract


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        • Singh S.
        • Allen A.M.
        • Wang Z.
        • Prokop L.J.
        • Murad M.H.
        • Loomba R.
        Fibrosis progression in nonalcoholic fatty liver vs nonalcoholic steatohepatitis: a systematic review and meta-analysis of paired-biopsy studies.
        Clin Gastroenterol Hepatol. 2015; 13 (e649): 643-654
        • Sayiner M.
        • Koenig A.
        • Henry L.
        • Younossi Z.M.
        Epidemiology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in the United States and the rest of the world.
        Clin Liver Dis. 2016; 20: 205-214
        • Younossi Z.M.
        • Koenig A.
        • Abdelatif D.
        • Fazel Y.
        • Henry L.
        • Wymer M.
        Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes.
        Hepatology. 2016; 64: 73-84
        • Wong R.J.
        • Cheung R.
        • Ahmed A.
        Nonalcoholic steatohepatitis is the most rapidly growing indication for liver transplantation in patients with hepatocellular carcinoma in the U.S..
        Hepatology. 2014; 59: 2188-2195
        • Ibrahim S.H.
        • Hirsova P.
        • Malhi H.
        • Gores G.J.
        Animal models of nonalcoholic steatohepatitis: eat, delete, and inflame.
        Dig Dis Sci. 2016; 61: 1325-1336
        • Machado M.V.
        • Michelotti G.A.
        • Xie G.
        • Almeida Pereira T.
        • Boursier J.
        • Bohnic B.
        • et al.
        Mouse models of diet-induced nonalcoholic steatohepatitis reproduce the heterogeneity of the human disease.
        PLoS One. 2015; 10e0127991
        • Itagaki H.
        • Shimizu K.
        • Morikawa S.
        • Ogawa K.
        • Ezaki T.
        Morphological and functional characterization of non-alcoholic fatty liver disease induced by a methionine-choline-deficient diet in C57BL/6 mice.
        Int J Clin Exp Pathol. 2013; 6: 2683-2696
        • Charlton M.
        • Krishnan A.
        • Viker K.
        • Sanderson S.
        • Cazanave S.
        • McConico A.
        • et al.
        Fast food diet mouse: novel small animal model of NASH with ballooning, progressive fibrosis, and high physiological fidelity to the human condition.
        Am J Physiol Gastrointest Liver Physiol. 2011; 301: G825-G834
        • Asgharpour A.
        • Cazanave S.C.
        • Pacana T.
        • Seneshaw M.
        • Vincent R.
        • Banini B.A.
        • et al.
        A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer.
        J Hepatol. 2016; 65: 579-588
        • Scholten D.
        • Trebicka J.
        • Liedtke C.
        • Weiskirchen R.
        The carbon tetrachloride model in mice.
        Lab Anim. 2015; 49: 4-11
        • Kubota N.
        • Kado S.
        • Kano M.
        • Masuoka N.
        • Nagata Y.
        • Kobayashi T.
        • et al.
        A high-fat diet and multiple administration of carbon tetrachloride induces liver injury and pathological features associated with non-alcoholic steatohepatitis in mice.
        Clin Exp Pharmacol Physiol. 2013; 40: 422-430
        • De Minicis S.
        • Agostinelli L.
        • Rychlicki C.
        • Sorice G.P.
        • Saccomanno S.
        • Candelaresi C.
        • et al.
        HCC development is associated to peripheral insulin resistance in a mouse model of NASH.
        PLoS One. 2014; 9e97136
        • Kleiner D.E.
        • Brunt E.M.
        • Van Natta M.
        • Behling C.
        • Contos M.J.
        • Cummings O.W.
        • et al.
        Design and validation of a histological scoring system for nonalcoholic fatty liver disease.
        Hepatology. 2005; 41: 1313-1321
        • Lee S.
        • Muniyappa R.
        • Yan X.
        • Chen H.
        • Yue L.Q.
        • Hong E.G.
        • et al.
        Comparison between surrogate indexes of insulin sensitivity and resistance and hyperinsulinemic euglycemic clamp estimates in mice.
        Am J Physiol Endocrinol Metab. 2008; 294: E261-E270
        • Bowe J.E.
        • Franklin Z.J.
        • Hauge-Evans A.C.
        • King A.J.
        • Persaud S.J.
        • Jones P.M.
        • et al.
        Metabolic phenotyping guidelines: assessing glucose homeostasis in rodent models.
        J Endocrinol. 2014; 222: G13-G25
        • Engstrom P.G.
        • Steijger T.
        • Sipos B.
        • Grant G.R.
        • Kahles A.
        • Ratsch G.
        • et al.
        Systematic evaluation of spliced alignment programs for RNA-seq data.
        Nat Methods. 2013; 10: 1185-1191
        • Robinson M.D.
        • Oshlack A.
        A scaling normalization method for differential expression analysis of RNA-seq data.
        Genome Biol. 2010; 11: R25
        • Subramanian A.
        • Tamayo P.
        • Mootha V.K.
        • Mukherjee S.
        • Ebert B.L.
        • Gillette M.A.
        • et al.
        Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.
        Proc Natl Acad Sci U S A. 2005; 102: 15545-15550
        • Liberzon A.
        • Birger C.
        • Thorvaldsdottir H.
        • Ghandi M.
        • Mesirov J.P.
        • Tamayo P.
        • et al.
        The Molecular Signatures Database (MSigDB) hallmark gene set collection.
        Cell systems. 2015; 1: 417-425
        • Moylan C.A.
        • Pang H.
        • Dellinger A.
        • Suzuki A.
        • Garrett M.E.
        • Guy C.D.
        • et al.
        Hepatic gene expression profiles differentiate presymptomatic patients with mild versus severe nonalcoholic fatty liver disease.
        Hepatology. 2014; 59: 471-482
        • Ahrens M.
        • Ammerpohl O.
        • von Schonfels W.
        • Kolarova J.
        • Bens S.
        • Itzel T.
        • et al.
        DNA methylation analysis in nonalcoholic fatty liver disease suggests distinct disease-specific and remodeling signatures after bariatric surgery.
        Cell Metab. 2013; 18: 296-302
        • Nakagawa S.
        • Wei L.
        • Song W.M.
        • Higashi T.
        • Ghoshal S.
        • Kim R.S.
        • et al.
        Molecular liver cancer prevention in cirrhosis by organ transcriptome analysis and lysophosphatidic acid pathway inhibition.
        Cancer Cell. 2016; 30: 879-890
        • Hoshida Y.
        • Nijman S.M.
        • Kobayashi M.
        • Chan J.A.
        • Brunet J.P.
        • Chiang D.Y.
        • et al.
        Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma.
        Cancer Res. 2009; 69: 7385-7392
        • Mederacke I.
        • Hsu C.C.
        • Troeger J.S.
        • Huebener P.
        • Mu X.
        • Dapito D.H.
        • et al.
        Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology.
        Nat Commun. 2013; 4: 2823
        • Richardson M.M.
        • Jonsson J.R.
        • Powell E.E.
        • Brunt E.M.
        • Neuschwander-Tetri B.A.
        • Bhathal P.S.
        • et al.
        Progressive fibrosis in nonalcoholic steatohepatitis: association with altered regeneration and a ductular reaction.
        Gastroenterology. 2007; 133: 80-90
        • Gadd V.L.
        • Skoien R.
        • Powell E.E.
        • Fagan K.J.
        • Winterford C.
        • Horsfall L.
        • et al.
        The portal inflammatory infiltrate and ductular reaction in human nonalcoholic fatty liver disease.
        Hepatology. 2014; 59: 1393-1405
        • Maeda S.
        • Kamata H.
        • Luo J.L.
        • Leffert H.
        • Karin M.
        IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis.
        Cell. 2005; 121: 977-990
        • Salomao M.
        • Yu W.M.
        • Brown Jr., R.S.
        • Emond J.C.
        • Lefkowitch J.H.
        Steatohepatitic hepatocellular carcinoma (SH-HCC): a distinctive histological variant of HCC in hepatitis C virus-related cirrhosis with associated NAFLD/NASH.
        Am J Surg Pathol. 2010; 34: 1630-1636
        • Teufel A.
        • Itzel T.
        • Erhart W.
        • Brosch M.
        • Wang X.Y.
        • Kim Y.O.
        • et al.
        Comparison of gene expression patterns between mouse models of nonalcoholic fatty liver disease and liver tissues from patients.
        Gastroenterology. 2016; 151 (e510): 513-525
        • Kobori M.
        • Masumoto S.
        • Akimoto Y.
        • Oike H.
        Chronic dietary intake of quercetin alleviates hepatic fat accumulation associated with consumption of a Western-style diet in C57/BL6J mice.
        Mol Nutr Food Res. 2011; 55: 530-540
        • Dorn C.
        • Engelmann J.C.
        • Saugspier M.
        • Koch A.
        • Hartmann A.
        • Muller M.
        • et al.
        Increased expression of c-Jun in nonalcoholic fatty liver disease.
        Lab Invest. 2014; 94: 394-408
        • Kim S.C.
        • Kim C.K.
        • Axe D.
        • Cook A.
        • Lee M.
        • Li T.
        • et al.
        All-trans-retinoic acid ameliorates hepatic steatosis in mice by a novel transcriptional cascade.
        Hepatology. 2014; 59: 1750-1760
        • Kwon E.Y.
        • Shin S.K.
        • Cho Y.Y.
        • Jung U.J.
        • Kim E.
        • Park T.
        • et al.
        Time-course microarrays reveal early activation of the immune transcriptome and adipokine dysregulation leads to fibrosis in visceral adipose depots during diet-induced obesity.
        BMC Genomics. 2012; 13: 450
        • Hur W.
        • Lee J.H.
        • Kim S.W.
        • Kim J.H.
        • Bae S.H.
        • Kim M.
        • et al.
        Downregulation of microRNA-451 in non-alcoholic steatohepatitis inhibits fatty acid-induced proinflammatory cytokine production through the AMPK/AKT pathway.
        Int J Biochem Cell Biol. 2015; 64: 265-276
        • Kita Y.
        • Takamura T.
        • Misu H.
        • Ota T.
        • Kurita S.
        • Takeshita Y.
        • et al.
        Metformin prevents and reverses inflammation in a non-diabetic mouse model of nonalcoholic steatohepatitis.
        PLoS One. 2012; 7e43056
        • Tryndyak V.
        • de Conti A.
        • Kobets T.
        • Kutanzi K.
        • Koturbash I.
        • Han T.
        • et al.
        Interstrain differences in the severity of liver injury induced by a choline- and folate-deficient diet in mice are associated with dysregulation of genes involved in lipid metabolism.
        FASEB J. 2012; 26: 4592-4602
        • Frades I.
        • Andreasson E.
        • Mato J.M.
        • Alexandersson E.
        • Matthiesen R.
        • Martinez-Chantar M.L.
        • et al.
        Integrative genomic signatures of hepatocellular carcinoma derived from nonalcoholic Fatty liver disease.
        PLoS One. 2015; 10e0124544
        • Tsai W.C.
        • Hsu S.D.
        • Hsu C.S.
        • Lai T.C.
        • Chen S.J.
        • Shen R.
        • et al.
        MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis.
        J Clin Invest. 2012; 122: 2884-2897
        • Yang J.S.
        • Kim J.T.
        • Jeon J.
        • Park H.S.
        • Kang G.H.
        • Park K.S.
        • et al.
        Changes in hepatic gene expression upon oral administration of taurine-conjugated ursodeoxycholic acid in ob/ob mice.
        PLoS One. 2010; 5e13858
        • Ma C.
        • Kesarwala A.H.
        • Eggert T.
        • Medina-Echeverz J.
        • Kleiner D.E.
        • Jin P.
        • et al.
        NAFLD causes selective CD4(+) T lymphocyte loss and promotes hepatocarcinogenesis.
        Nature. 2016; 531: 253-257
        • Wallace M.C.
        • Friedman S.L.
        • Mann D.A.
        Emerging and disease-specific mechanisms of hepatic stellate cell activation.
        Semin Liver Dis. 2015; 35: 107-118
        • Lee Y.A.
        • Wallace M.C.
        • Friedman S.L.
        Pathobiology of liver fibrosis: a translational success story.
        Gut. 2015; 64: 830-841
        • Machado M.V.
        • Michelotti G.A.
        • Pereira T.A.
        • Xie G.
        • Premont R.
        • Cortez-Pinto H.
        • et al.
        Accumulation of duct cells with activated YAP parallels fibrosis progression in NonAlcoholic Fatty Liver Disease.
        J Hepatol. 2015; 63: 962-970
        • Clapper J.R.
        • Hendricks M.D.
        • Gu G.
        • Wittmer C.
        • Dolman C.S.
        • Herich J.
        • et al.
        Diet-induced mouse model of fatty liver disease and nonalcoholic steatohepatitis reflecting clinical disease progression and methods of assessment.
        Am J Physiol Gastrointest Liver Physiol. 2013; 305: G483-G495
        • Dowman J.K.
        • Hopkins L.J.
        • Reynolds G.M.
        • Nikolaou N.
        • Armstrong M.J.
        • Shaw J.C.
        • et al.
        Development of hepatocellular carcinoma in a murine model of nonalcoholic steatohepatitis induced by use of a high-fat/fructose diet and sedentary lifestyle.
        Am J Pathol. 2014; 184: 1550-1561
        • Kleiner D.E.
        • Makhlouf H.R.
        Histology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in adults and children.
        Clin Liver Dis. 2016; 20: 293-312
        • Urtasun R.
        • Lopategi A.
        • George J.
        • Leung T.M.
        • Lu Y.
        • Wang X.
        • et al.
        Osteopontin, an oxidant stress sensitive cytokine, up-regulates collagen-I via integrin alpha(V)beta(3) engagement and PI3K/pAkt/NFkappaB signaling.
        Hepatology. 2012; 55: 594-608
        • Savard C.
        • Tartaglione E.V.
        • Kuver R.
        • Haigh W.G.
        • Farrell G.C.
        • Subramanian S.
        • et al.
        Synergistic interaction of dietary cholesterol and dietary fat in inducing experimental steatohepatitis.
        Hepatology. 2013; 57: 81-92
        • Wang D.Q.
        • Lammert F.
        • Cohen D.E.
        • Paigen B.
        • Carey M.C.
        Cholic acid aids absorption, biliary secretion, and phase transitions of cholesterol in murine cholelithogenesis.
        Am J Physiol. 1999; 276: G751-G760
        • Puri P.
        • Baillie R.A.
        • Wiest M.M.
        • Mirshahi F.
        • Choudhury J.
        • Cheung O.
        • et al.
        A lipidomic analysis of nonalcoholic fatty liver disease.
        Hepatology. 2007; 46: 1081-1090
        • Caballero F.
        • Fernandez A.
        • De Lacy A.M.
        • Fernandez-Checa J.C.
        • Caballeria J.
        • Garcia-Ruiz C.
        • et al.
        Enhanced free cholesterol, SREBP-2 and StAR expression in human NASH.
        J Hepatol. 2009; 50: 789-796
        • Wang X.
        • Zheng Z.
        • Caviglia J.M.
        • Corey K.E.
        • Herfel T.M.
        • Cai B.
        • et al.
        Hepatocyte TAZ/WWTR1 promotes inflammation and fibrosis in nonalcoholic steatohepatitis.
        Cell Metab. 2016; 24: 848-862
        • Kristiansen M.N.
        • Veidal S.S.
        • Rigbolt K.T.
        • Tolbol K.S.
        • Roth J.D.
        • Jelsing J.
        • et al.
        Obese diet-induced mouse models of nonalcoholic steatohepatitis-tracking disease by liver biopsy.
        World J Hepatol. 2016; 8: 673-684