Editorial

DOI: 10.1111/j.1478-3231.2011.02463.x

Animal models in nonalcoholic steatohepatitis research: utility and clinicaltranslation

Nonalcoholic fatty liver disease (NAFLD) represents aspectrum of metabolic syndrome-associated liver diseaseprogressing from simple steatosis, through nonalcoholicsteatohepatitis (NASH) and fibrosis to cirrhosis andhepatocellular carcinoma (1). Increasingly sedentary life-styles and changing dietary patterns have allowedNAFLD/NASH to become the most common cause ofliver dysfunction in many countries worldwide. Despitethis, our understanding of disease pathogenesis remainsincomplete and as yet specific therapies to delay diseaseprogression or treat fibrosis are limited.

Experience has shown that murine biological processesare similar to those in man and that rodent models areboth genetically tractable and generally reliable surro-gates for human biology. Although some research may beundertaken at the cellular level in vitro, in order to dissectmultisystem pathophysiological processes such as thoseassociated with NAFLD/NASH, it is necessary to studyeffects at the whole-organism level. Established inbredstrains permit both genetic background (which maycontain susceptibility alleles) and environmental factorsto be controlled and therefore facilitate study of complexdisease traits such as NAFLD/NASH. The contribution ofmurine models to this field already extends from basicresearch through to preclinical testing of novel therapeu-tics. Indeed, the ‘Two-Hit’ hypothesis was first proposedby Day and James in an editorial that accompanied apaper by Berson and colleagues describing a link betweendrug-induced oxidative stress and steatohepatitis in rats(2, 3). More recently, hepatocyte fatty acid (FFA) fluxrather than histologically evident steatosis was shown tobe the driver of progression to steatohepatitis in a mousemodel of fibrosing NASH (4). In this important studysiRNA techniques were used to block diacylglycerolacyltransferase 2 expression showing that, while trigly-ceride accumulation was reduced, hepatocellular oxida-tive stress, inflammation and necrotic/apoptotic celldeath were increased (4). It is through these and otherstudies using rodent models that our understanding ofdisease pathogenesis has developed. Specifically, insulinresistance promotes greater hepatic FFA flux which leadsto direct hepatocyte lipotoxicity, hepatocellular oxidativestress secondary to free radical production during b- ando-FFA oxidation, endotoxin/TLR4-induced inflamma-tion, cytokine release and endoplasmic reticulum stress(5). Consequent cellular damage triggers a mixture ofimmune-mediated hepatocellular injury and both necro-

tic and apoptotic cell death pathways which culminatesin tissue fibrosis. This greater mechanistic understandingis now informing strategies for therapeutic interventionwhich can be tested preclinically in these same rodentmodels and may one day be applied in the clinic.

Research models of NAFLD may be divided into twobroad categories, those caused by either spontaneous orinduced genetic mutation and those with an acquiredNAFLD phenotype (1). The latter group may be pro-duced by either dietary or pharmacological manipula-tion. Despite their proven utility, an apparent‘phenotype-gap’ exists between some of the currentlyavailable models and the condition encountered at thebedside (1, 6). While the literature contains numerousdifferent rodent models that exhibit histological evidenceof hepatic steatosis and mild–moderate steatohepatitis,the features of more advanced steatohepatitis (includingballooning hepatocyte degeneration and an inflamma-tory infiltrate) combined with insulin resistance, are lessfrequently demonstrated (1). Depending on the back-ground strain studied, a chronic (45 cal%) high-fat dietor sustained high fructose consumption provide accep-table models of the metabolic syndrome with histologicalfeatures of a mild-moderate steatohepatitis with which tostudy effects of dietary/environmental manipulation, aswell as hepatocellular lipid handling, fatty acid metabo-lism and associated cellular stresses (7, 8). However, thelack of progression to fibrosing steatohepatitis has lim-ited such models’ utility for therapeutics discovery inprogressive disease. Attempts at using higher dietary fatlevels (9) and/or hyperalimentation by gastrostomy feed-ing (10) have produced some increase in steatohepatitisseverity but have not been widely adopted due totechnical difficulties and the modest increase in diseaseseverity generated. A more severe fibrosing steatohepati-tis may be induced by feeding a diet deficient inmethionine and choline (MCD). The metabolic conse-quences of MCD feeding are complex (11, 12) and animportant caveat remains: steatohepatitis is inducedthrough a nonphysiological process. Indeed, MCD diet-ary manipulation alone does not produce insulin resis-tance (12), but instead causes weight loss which can limitthe extent to which disease may be allowed to progress.

Several groups have attempted to bridge the pheno-type gap by combining genetic and acquired (dietary)manipulation (or multiple separate dietary/pharmacolo-gical challenges) to produce a more severe, accelerated or

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fibrosing steatohepatitis. These include Abcb11 mutantmice fed MCD (13), apolipoprotein E mutant mice fed ahigh-fat diet (14) and PPARa null mice fed MCD (15).Currently, one of the most promising and more widelyadopted models available for studying more advanceddisease at the steatohepatitis–fibrosis interface is gener-ated by feeding genetically hyperphagic, obese/diabeticdb/db mice an MCD diet (4, 16, 17). The combinedgenetic and dietary manipulation provides a prosteatotic,metabolic syndrome background with histological fea-tures of severe fibrosing steatohepatitis evidenced at thetranscriptome level by a 10-fold increase in hepaticprocollagen type 1 expression in MCD fed db/db micewhile their nonsteatotic db/m littermates have only afour-fold rise after MCD feeding (17).

The publication of another rodent model of NAFLD/NASH prompts us to consider what are the specificcharacteristics required in a good fatty liver diseasemodel (Table 1)? It is a given that a model must be easyto use, offer a genetically stable background on which tostudy disease modifiers and produce a reliable andpredictable disease progression within a relatively shorttime frame so that studies deliver results in a timelyfashion and do not consume excessive resources. Therecognition that insulin resistance and FFA flux are at theheart of the disease process would suggest that a back-ground insulin resistant, obese phenotype is required;however, what remains unresolved is whether a modelmust over time recapitulate every aspect of the clinicaldisease spectrum or whether it must simply be sufficientto the research question and stage of disease studied.What is certain is that the more faithful a preclinical

model is to the clinical condition, the more likely thatwhat we learn from it will be clinically relevant.

In the current edition of the journal, Ogasawara et al.(18) describe a new ‘combined model’ generated byexploiting two acquired challenges in the search for abetter NASH model. Rather than relying on geneticmutations such as leptin deficiency (ob/ob mice) orleptin receptor deletion (db/db mice and fa/fa rats) toinduce a hyperphagic state, the authors have used theability of gold-thioglucose (GTG) to induce hyperphagia,obesity and NASH through selective ablation of a ‘satietycentre’ within the venteromedial hypothalamic nucleusof C57BL/6 mice (19). This technique obviates concernsregarding absence of leptin signalling and the consequentdownstream changes in innate and acquired immuneresponse and fibrogenesis that may be levelled at leptin-deficient models such as the ob/ob mouse (1). Here, thecentral effects of GTG, first reported in Nature over 50years ago (20), have been combined with a 12-weekperiod of extreme high-fat diet (82 cal% fat) consump-tion to exacerbate the obese, metabolic syndrome phe-notype and produce greater oxidative stress and a moresevere histological steatohepatitis with mild zone 3 andpericellular hepatic fibrosis. Although the authors do notshow progression to advanced fibrosis or cirrhosis, thismodel could facilitate the study of the transition fromsimple steatosis to steatohepatitis and the initiation offibrogenesis while remaining faithful to many of thefundamental aetiological processes which underpinNAFLD/NASH and the metabolic syndrome. Howwidely this model will be adopted will be determined inpart by how consistently reproducible the model is foundto be. However, a model exhibiting a predictable, obesity-related, mild-moderate fibrosing steatohepatitis pro-duced through voluntary hyperalimentation could serveas a useful platform for basic science and therapeuticinvestigation across an important spectrum of clinicaldisease.

The evolution of rodent NASH models from those,such as the Ob mouse, that were limited to a single stepin disease pathogenesis, to models such as the Db/MCDmodel (17) and the one discussed here (18), that blur theonce ridged boundaries between steatosis, steatohepatitisand fibrosis are a welcome development in the transla-tional investigation of NASH.

Quentin M. AnsteeInstitute of Cellular Medicine, Newcastle University,

Newcastle-upon-Tyne, UK

Acknowledgements

Q. M. A. is the recipient of a Clinical Senior LectureshipAward from the Higher Education Funding Council forEngland (HEFCE). Q. M. A. is also the recipient of anMRC Programme Grant to identify and study novelmurine models of liver disease.

Table 1. Desirable features in a nonalcoholic steatohepatitis model

Phenotype Metabolic syndrome (obesity, insulin resistance,dyslipidaemia)Biochemical and histological features of steatohepatitis,including a necroinflammatory infiltrate and hepatocyteballooning degenerationEvidence of histological progression with stellate cellactivation and hepatic fibrosisUpregulation of mitochondrial and extramitochondrialFFA oxidation pathwaysIncreased tissue oxidative stress and lipid peroxidationAppropriate transcriptional changes consistent withinflammation, necrotic/apoptotic activity andfibrogenesis

Practicalissues

Readily available background strain with well-characterised genetic and metabolic featuresNAFLD/NASH easy to induce without need for specialistsurgical techniques or equipmentPredictable rate of disease progression over timeMinimal stress to animalDuration of disease progression to fibrosis within lessthan 3–4 months so that studies are not excessivelyresource intensive

NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis.

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Anstee Editorial

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Editorial Anstee