Towards a definite mouse model of NAFLD
Authors: Rui E. Castro, Anna Mae Diehl
Journal: Journal of Hepatology (2018)

A simple diet- and chemical-induced murine NASH model with rapid progression of steatohepatitis, fibrosis and liver cancerJournal of HepatologyVol. 69Issue 2PreviewNon-alcoholic fatty liver disease (NAFLD) is a rising cause of chronic liver disease worldwide. Although the majority of patients with NAFLD have only steatosis without progression, a sizeable fraction develop non-alcoholic steatohepatitis (NASH), which can lead to cirrhosis, hepatocellular carcinoma (HCC), and increased liver-related mortality.1 The prevalence of NAFLD in the US population is estimated at ∼24% (or ∼65 million) and up to a third of these individuals have NASH.2,3 The prevalence of NAFLD is steadily increasing in parallel with the rising prevalence of obesity. Full-Text PDF The worldwide prevalence of non-alcoholic fatty liver disease (NAFLD) is estimated to be 24% and continues to increase significantly because of the rise in obesity and other metabolic disorders.[1]Younossi Z. Anstee Q.M. Marietti M. Hardy T. Henry L. Eslam M. et al.Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.Nat Rev Gastroenterol Hepatol. 2018; 15: 11-20Crossref PubMed Scopus (2436) Google Scholar Further, the number of patients quickly progressing to more severe stages of the disease is also increasing; in the US alone, non-alcoholic steatohepatitis (NASH) is already considered the second most common indication for liver transplantation, with prevalent NASH cases forecasted to increase to 27 million by 2030.[2]Estes C. Razavi H. Loomba R. Younossi Z. Sanyal A.J. Modeling the epidemic of nonalcoholic fatty liver disease demonstrates an exponential increase in burden of disease.Hepatology. 2018; 67: 123-133Crossref PubMed Scopus (984) Google Scholar This is paralleled by rises in advanced liver disease, cirrhosis and hepatocellular carcinoma (HCC). Altogether, it is not surprising that the burden of NASH on patient reported outcomes and the economy are also increasing.[1]Younossi Z. Anstee Q.M. Marietti M. Hardy T. Henry L. Eslam M. et al.Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.Nat Rev Gastroenterol Hepatol. 2018; 15: 11-20Crossref PubMed Scopus (2436) Google Scholar Because there is currently no approved pharmacotherapy for NASH, pre-clinical and clinical studies need to transform and adapt to respond to this huge, unmet need. Unfortunately, this has proven to be a difficult task because of the asymptomatic nature of NASH, its heterogeneous etiology and pathogenesis, the need for sequential biopsies and lack of a validated surrogate endpoint during clinical trials, among other factors.[3]Filozof C. Chow S.C. Dimick-Santos L. Chen Y.F. Williams R.N. Goldstein B.J. et al.Clinical endpoints and adaptive clinical trials in precirrhotic nonalcoholic steatohepatitis: Facilitating development approaches for an emerging epidemic.Hepatol Commun. 2017; 1: 577-585Crossref PubMed Scopus (29) Google Scholar In pre-clinical studies, most NAFLD researchers use mouse models to generate and test hypotheses on disease molecular triggers and drivers of progression towards NASH, cirrhosis and HCC; as well as to identify and exploit molecular targets or test putative pharmacological agents. Knowledge gained from these studies can then be used to fuel clinical trials in developing novel diagnostic, prognostic, preventative, and therapeutic approaches. Although many different dietary and genetic animal models of NAFLD have been described,4Santhekadur P.K. Kumar D.P. Sanyal A.J. Preclinical models of non-alcoholic fatty liver disease.J Hepatol. 2018; 68: 230-237Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 5Oseini A.M. Cole B.K. Issa D. Feaver R.E. Sanyal A.J. Translating scientific discovery: the need for preclinical models of nonalcoholic steatohepatitis.Hepatol Int. 2018; 12: 6-16Crossref PubMed Scopus (19) Google Scholar, 6Ibrahim S.H. Hirsova P. Malhi H. Gores G.J. Animal models of nonalcoholic steatohepatitis: eat, delete, and inflame.Dig Dis Sci. 2016; 61: 1325-1336Crossref PubMed Scopus (143) Google Scholar, 7Michelotti G.A. Machado M.V. Diehl A.M. NAFLD, NASH and liver cancer.Nat Rev Gastroenterol Hepatol. 2013; 10: 656-665Crossref PubMed Scopus (696) Google Scholar most researchers agree that a single model, recapitulating all features of human disease, is still lacking. In this issue of Journal of Hepatology, Tsuchida et al. attempt to fill this gap; their study sought to establish a mouse NASH model with rapid progression of extensive fibrosis and HCC by merging features of currently available, complementary models, namely a western diet (WD) combined with carbon tetrachloride (CCl4) injections. The WD contained 21.1% fat, 41% sucrose and 1.25% cholesterol, and animals had their drinking water supplemented with 23.1 g/L d-fructose and 18.9 g/L d-glucose. CCl4 was injected once a week intraperitoneally at 0.32 μg/g of body weight, for 12 and 24 weeks. WD feeding models incorporating fructose likely rank among those of highest relevance to human NAFLD; the EASL–EASD–EASO Clinical Practice Guidelines for the management of NAFLD highlight that a high-calorie diet, including excess saturated fats and refined carbohydrates, together with sugar-sweetened beverages, with a high fructose concentration, predispose individuals to disease development.[8]European Association for the Study of the Liver (EASL)European Association for the Study of Diabetes (EASD)European Association for the Study of Obesity (EASO)EASL-EASD-EASO clinical practice guidelines for the management of non-alcoholic fatty liver disease.J Hepatol. 2016; 64: 1388-1402Abstract Full Text Full Text PDF PubMed Scopus (2513) Google Scholar Further, high fructose consumption increases the risk of NASH and advanced fibrosis.[9]Jensen T. Abdelmalek M.F. Sullivan S. Nadeau K.J. Green M. Roncal C. et al.Fructose and sugar: A major mediator of non-alcoholic fatty liver disease.J Hepatol. 2018; 68: 1063-1075Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar At the same time, NAFLD is characterized by excessive hepatic fat accumulation in association with insulin resistance (IR). In fact, the metabolic complications of over-nutrition during NASH typically revolve around IR, which is further believed to fuel obesity-related carcinogenesis.[7]Michelotti G.A. Machado M.V. Diehl A.M. NAFLD, NASH and liver cancer.Nat Rev Gastroenterol Hepatol. 2013; 10: 656-665Crossref PubMed Scopus (696) Google Scholar In this regard, although Tsuchida and co-workers show that WD-fed animals are insulin resistant, WD/CCl4 animals display a more moderate phenotype, which the authors claim is likely caused by blunted weight gain. And while transcriptomic data also supported engagement of IR pathways in this group, an insulin and glucose infusion clamp test on fasting animals should help to provide more insight about this issue in the future. CCl4-induced liver damage is a well-established general model for liver fibrosis but does not otherwise resemble human NAFLD. Kubota et al. were among the first to show that a combined model of high-fat diet (HFD) feeding and CCl4 administration induced hepatic steatosis, inflammatory cell accumulation, hepatocellular ballooning, fibrosis, and increased aminotransferase levels.[10]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: 422-430Crossref PubMed Scopus (47) Google Scholar The current study of WD/CCl4 mice reported by Tsuchida recapitulates those findings and shows that after 24 weeks of combined WD/CCl4 treatment, some mice have developed F4 fibrosis (i.e., cirrhosis). Interestingly, both Tsuchida et al. and Kubota et al. noted that mice subjected to these fatty diet + CCl4 protocols developed progressive NASH-like histology despite the fact that adding CCl4 treatment to HFD/WD feeding attenuated typical HFD/WD-induced increases in body weight, cholesterol and insulin/glucose levels. Indeed, some mitigation of HFD-induced obesity and metabolic syndrome features has also been reported to occur with other protocols that induce progressive NASH in mice, based on studies of mice that develop fibrosing NASH when fed an HFD that is deficient in choline and restricted in methionine (CDAA-HFD diet model).11Matsumoto M. Hada N. Sakamaki Y. Uno A. Shiga T. Tanaka C. et al.An improved mouse model that rapidly develops fibrosis in non-alcoholic steatohepatitis.Int J Exp Pathol. 2013; 94: 93-103Crossref PubMed Scopus (242) Google Scholar, 12Ikawa-Yoshida A. Matsuo S. Kato A. Ohmori Y. Higashida A. Kaneko E. et al.Hepatocellular carcinoma in a mouse model fed a choline-deficient, L-amino acid-defined, high-fat diet.Int J Exp Pathol. 2017; 98: 221-233Crossref PubMed Scopus (42) Google Scholar Importantly however, in all the aforementioned NASH models, mice do not lose sufficient weight to fall below the weight of chow-fed controls or become hypoglycemic, as occurs when mice are fed strict methionine/choline-deficient diets for 6–8 weeks to rapidly induce pathology that resembles fibrosing NASH in humans. Further, all of the more metabolically-temperate models that gradually induce fibrosing NASH in mice activate hepatic stellate cells, evoke a ductular reaction, and eventually promote adenomatous hyperplasia and HCC development. Tsuchida et al. demonstrated HCC in 30% of WD/CCl4 mice at 24 weeks, a time point when all WD/CCl4 mice had at least F3 fibrosis. It will be interesting to elucidate whether WD/CCl4 mice continue to accumulate liver damage after 24 weeks of feeding, or after transitioning back to a normal diet. Whether or not liver damage persists (or progresses) after withdrawal of WD/CLL4 is particularly pertinent for researchers who plan to screen potential NASH therapies using this model because it might afford an opportunity to avoid confounding influences of CCl4 on drug-metabolizing enzymes. Interestingly, 100% of mice fed CDAA-HFD for 36 weeks were reported to have NASH, advanced fibrosis, and hepatic adenomas without overt HCCs and despite switching back to a standard diet at 37 weeks, many of these mice exhibited fibrosis progression, and ∼67% had demonstrable HCCs at 48 weeks.11Matsumoto M. Hada N. Sakamaki Y. Uno A. Shiga T. Tanaka C. et al.An improved mouse model that rapidly develops fibrosis in non-alcoholic steatohepatitis.Int J Exp Pathol. 2013; 94: 93-103Crossref PubMed Scopus (242) Google Scholar, 12Ikawa-Yoshida A. Matsuo S. Kato A. Ohmori Y. Higashida A. Kaneko E. et al.Hepatocellular carcinoma in a mouse model fed a choline-deficient, L-amino acid-defined, high-fat diet.Int J Exp Pathol. 2017; 98: 221-233Crossref PubMed Scopus (42) Google Scholar Hence, some NASH protocols seem to evoke pathology that persists/progresses after NASH-inducing insults are withdrawn and thus, may model the subgroup of patients with NAFLD and inherently progressive liver damage. Last but not least, Tsuchida and colleagues performed global transcriptome profiling of the mouse liver and HCC tumor tissues by RNA-Seq and compared dysregulated molecular pathways from KEGG and HALLMARK gene sets with those of two human NASH cohorts and 16 previously published diet, chemical, and/or genetic NASH mouse models. Results showed that the dysregulated molecular pathways in WD/CCl4 mice at 12 weeks were similar to those of early/mild human NASH. Finally, tumors from WD/CCl4 mice at 24 weeks were similar to human HCC molecular subclasses, highlighting the model’s parallelism with human disease progression. The STAM™ mouse model was another NASH-HCC model included in this transcriptomic comparison. STAM mice are treated with drug that kills pancreatic beta cells shortly after birth and sequentially develop steatohepatitis, fibrosis and HCC in a short period of time when fed HFD later in life (after 8, 16 and 20 weeks of HFD, respectively). These mice have a reduced mass of pancreatic beta cells (similar to type 1 diabetics) and do not become obese.[13]Fujii M. Shibazaki Y. Wakamatsu K. Honda Y. Kawauchi Y. Suzuki K. et al.A murine model for non-alcoholic steatohepatitis showing evidence of association between diabetes and hepatocellular carcinoma.Med Mol Morphol. 2013; 46: 141-152Crossref PubMed Scopus (216) Google Scholar Other models of NAFLD-HCC have been described, like the American lifestyle-induced obesity syndrome model which reproduces IR and other features of human NASH, but consistent tumor development is apparent only after 12 months of feeding,[14]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: 1550-1561Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar similar to mice fed a high-fat, high-cholesterol, high-sucrose diet (HF-HC-HSD).[15]Ganz M. Bukong T.N. Csak T. Saha B. Park J.K. Ambade A. et al.Progression of non-alcoholic steatosis to steatohepatitis and fibrosis parallels cumulative accumulation of danger signals that promote inflammation and liver tumors in a high fat-cholesterol-sugar diet model in mice.J Transl Med. 2015; 13: 193Crossref PubMed Scopus (81) Google Scholar Charlton et al. had proposed a modification of the HF-HC-HSD diet (dubbed the high fat, fructose and cholesterol [FFC] diet). This diet also recapitulates features of the metabolic syndrome (e.g., obesity, IR) and efficiently causes hepatic steatosis; steatohepatitis and fibrosis develop, but only after 24 weeks and with no evidence of pre-neoplastic nodules.[16]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-G834Crossref PubMed Scopus (275) Google Scholar It was later shown that the FFC diet also induces muscle IR[17]Rodrigues P.M. Afonso M.B. Simao A.L. Carvalho C.C. Trindade A. Duarte A. et al.MiR-21 ablation and obeticholic acid ameliorate nonalcoholic steatohepatitis in mice.Cell Death Dis. 2017; 8e2748Crossref PubMed Scopus (65) Google Scholar and adipose tissue inflammation,[18]Idrissova L. Malhi H. Werneburg N.W. LeBrasseur N.K. Bronk S.F. Fingas C. et al.TRAIL receptor deletion in mice suppresses the inflammation of nutrient excess.J Hepatol. 2015; 62: 1156-1163Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar a key feature of human NASH. It will be interesting to evaluate whether the WD + CCl4 diet also induces features of the metabolic syndrome in the muscle and, particularly, in the adipose tissue. More recently, Asgharpour et al. described a new model of NAFLD – the Diet-Induced Animal Model Of NAFLD (DIAMOND) – based on an isogenic strain of C57Bl/J and S129/V mice fed an HFD with ad lib glucose-fructose in drinking water.[19]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: 579-588Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar Mice sequentially developed steatosis and IR (8–16 weeks), steatohepatitis with hepatocellular ballooning (24 weeks), and progressive fibrosis, resulting in bridging fibrosis by 36 weeks and nodule formation by 52 weeks, with a transcriptomic profile similar to NASH patients. However, the duration of feeding needed to achieve advanced NASH is significantly longer in the DIAMOND model than in the WD/CCl4 model. Further, the breeding of knock-out or transgenic mice into the mixed background of the DIAMOND mice may be challenging. Curiously, and despite the different concentration of cholesterol in the diets (1.25% in the WD vs. 0.1% in the DIAMOND diet), both the WD/CCl4 and DIAMOND models display significant disturbances in pathways related to cholesterol homeostasis, which are not observed in human NASH, an issue that deserves further exploration. In sum, currently available animal models of NASH, particularly those recapitulating NASH progression towards HCC, have inherent specific advantages and disadvantages. The continuous improvement of these models will eventually enable a complete understanding of human NASH-HCC pathogenesis, while allowing for the development of much needed therapeutic options. In this issue of J Hepatology, Tsuchida et al. propose a novel mouse NASH model that exhibits rapid progression to advanced fibrosis, cirrhosis, and HCC, while mimicking histological and transcriptomic features of human NASH. As such, the new WD/CCl4 model meets most of the required criteria to be considered an ideal animal model for human NASH.[5]Oseini A.M. Cole B.K. Issa D. Feaver R.E. Sanyal A.J. Translating scientific discovery: the need for preclinical models of nonalcoholic steatohepatitis.Hepatol Int. 2018; 12: 6-16Crossref PubMed Scopus (19) Google Scholar However, achieving these positive attributes exacted a price, i.e., chronically exposing mice to CCl4, a potent hepatotoxin that has no known role in human NASH pathogenesis and which induces drug-metabolizing enzymes. The latter response might confound efforts to use the model for drug discovery. Further, chronic CCl4 treatment seems to somewhat mitigate key metabolic parameters that are implicated in the pathogenesis of human NASH (e.g., susceptibility to diet-induced obesity, IR and glucose tolerance). These caveats reinforce the concept that for the time being, NASH investigators must have a very clear understanding of what pathology they wish to study, and choose the animal model that best suits their research goals. Dr Diehl reports research grants from Immuron, Metabolomics, Cellgene and Prometheus. She also reports funding for clinical trials from Immuron, Gilead, Conatus, Galmed, NGM Pharmaceuticals, Bristol Myer Squibb, Madrigal, Galectin, Exalenze, Shire, Intercept and Genfit. She received consulting fees (all less than $10,000 each) from Pfizer, Novartis, Lumena, Cellgene, and Boerhinger-Ingleheim and an honoraria for a sponsored lecture from Allergan. She is funded by the NIH via the NIDDK-supported NASH CRN UO1 grant and is developing a patent application related to a potential caspase target for NASH. Dr. Castro reports grants from Gilead Sciences, grants from Intercept, outside the submitted work. Please refer to the accompanying ICMJE disclosure forms for further details. Download .pdf (.1 MB) Help with pdf files Supplementary data