Review Article

Commonly used animal models of non-alcoholic steatohepatitis Jian-Gao Fan and Liang Qiao Hong Kong, China

BACKGROUND: Animal models are an essential tool in non-alcoholic steatohepatitis (NASH) studies. Ideally, such models should reflect the etiology, disease progression, and the established pathology of human NASH. To date, no single animal model displays the range of histopathologic and pathophysiologic features associated with human NASH. The currently available models do not or only partially reflect the real picture of human NASH. In particular, insulin resistance and fibrosing steatohepatitis are rarely reproduced by the currently available models. Consequently, it is necessary to establish NASH models that can best mimic the real etiology, disease progression, and pathogenesis of human NASH. DATA SOURCES: We reviewed the major currently available animal models published in the literature (PubMed) and briefly commented on the pros and cons of these models. RESULT: Three major categories of animal models, genetic, dietary, and combination models, were reviewed and discussed. CONCLUSIONS: Animal models are not only useful in revealing the etiology of NASH, but also are important platforms for the assessment of therapeutic strategies. Currently available models do not reflect the full picture of NASH in patients. Better animal models are needed for a full understanding of human NASH and the development of efficient therapies for this condition. (Hepatobiliary Pancreat Dis Int 2009; 8: 233-240)

Author Affiliations: Department of Gastroenterology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China (Fan JG); and Division of Gastroenterology and Hepatology, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China (Qiao L) Corresponding  Author:  Liang Qiao, MD, PhD, Division of Gastroenterology and Hepatology, Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China (Tel: 852-2855 3830; Fax: 852-2872 5828; Email: [email protected]) Some contents of this manuscript have been published in Chinese at the Chinese Journal of Hepatology (CJH) 2008;16(11):806-808. Thus, there would be some similarities between this manuscript and the one published in the CJH. © 2009, Hepatobiliary Pancreat Dis Int. All rights reserved.

KEY WORDS: non-alcoholic fatty liver disease; non-alcoholic steatohepatitis; animal models

Introduction

N

on-alcoholic fatty liver disease (NAFLD) refers to a range of disorders associated with fatty liver, which occur in the absence of evident infection or significant consumption of alcohol.[1] Non-alcoholic steatohepatitis (NASH) is part of the spectrum of NAFLD,[2-6] characterized by steatosis, lobular inflammation and progressive pericellular fibrosis. NASH is now regarded as the most common cause of abnormal liver function tests worldwide. In the USA, the estimated prevalence of NAFLD is 20%-30% and that of NASH is 3.5%-5%,[7,  8] while in Asia, the prevalence of NAFLD is reported to be 12%-24%.[9] Owing to the increased trend of over-nutrition, decreased physical activity with disproportionate high-fat food intake, obesity, type 2 diabetes mellitus (T2DM), and the metabolic syndrome,[2, 4-6] the prevalence of NAFLD worldwide has witnessed a substantial increase over the past decades and is likely to further increase in the near future.[4, 10] Importantly, long-standing NASH may progress to hepatic fibrosis, cirrhosis, or even hepatocellular carcinoma (HCC).[11-15] Overall, the mechanisms responsible for the development and progression of human NASH are not well-defined, although the "two-hit" theory, oxidative stress, obesity, and insulin resistance (IR) are all widely studied.[3, 10, 16-22] In addition, very few effective therapeutic approaches for full-blown NAFLD/NASH are available at this stage.[9, 14, 22, 23] Understandably, development of efficient prevention and therapeutic options for NASH is hampered by a clear understanding of the etiology and mechanisms of this condition, which in turn is limited by the lack of a suitable study model.[3, 22, 23] Animal models of NASH can provide critical

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information leading to understanding its molecular mechanisms. Furthermore, animal models of NASH are an important platform for testing the therapeutic potency of certain agents against NASH.[24-29] In this article, we review the currently available animal models and briefly comment on their pros and cons.

Evaluation of currently available animal models of NASH In a simplistic way, NASH in animals can be reproduced by a wide variety of factors that lead to changes in hepatic fat disposition (more precisely, the imbalance between hepatic lipogenesis and uptake, and fatty acid oxidation or export). Major approaches to NASH induction can be classified as follows: (1) Genetic approach: animals that have naturally occurring or targeted over-expression or deletions of certain genes. Reported models include animals deficient in leptin and/or its receptors; animals with over-expression of Abcb11; and animals that lack peroxisome proliferatoractivated receptor-alpha (PPARα).[30-37] (2) Nutritional (dietary) approach: over-nutrition, overfeeding of unsaturated fat, and a methionine and choline deficient (MCD) diet.[32, 38-45] (3) A combination of genetic factors with others such as nutritional factors, oxidative stress, and drugs.[32, 33, 46-53]

Genetic models Acyl-coenzyme A oxidase null mice Acyl-coenzyme A oxidase (AOX) is the rate-limiting enzyme in peroxisomal fatty acid (FA) β-oxidation for the preferential metabolism of very long-chain FAs. AOX null (AOX-/-) mice have defective peroxisomal β-oxidation and exhibit steatohepatitis. Microvesicular fatty change in hepatocytes is evident at 7 days. At 2 months of age, livers show extensive steatosis and the presence in the periportal areas of clusters of hepatocytes with abundant granular eosinophilic cytoplasm rich in peroxisomes.[54] Increased PPARα, cytochrome P450 (Cyp) 4a10, and Cyp4a14 expression as well as raised H2O2 levels are observed by 4 to 5 months. By 6 to 7 months, however, there is a compensatory increase in FA oxidation and reversal of hepatic steatosis resulting from hepatocellular regeneration. The AOX-/- mice proceed to develop adenomas and carcinomas by 15 months of age.[3, 55] Methionine adenosyltransferase-1A null mice Methionine adenosyltransferase-1A (MAT1A) is a

liver-specific, rate-limiting enzyme for the metabolism of methionine that catalyzes the formation of S-adenosylmethionine, the principal biologic methyl donor. Mature liver expresses MAT1A. MAT1A null (MAT1A-/-) mice have reduced levels of antioxidants, such as glutathione and decreased expression of genes involved in lipid oxidation, such as Cyp4a10 and Cyp4a14. MAT1A-/- mice exhibit increased liver weights at 3 months of age. Spontaneous steatohepatitis and periportal inflammation occur after 8 months. Increased hepatic proliferation resulting in tumors is also observed after 8 months.[56]

Nuclear respiratory factor 1 null mice Nuclear respiratory factor 1 (NRF1) is a transcription factor believed to play a role in mediating the activation of genes responsive to oxidative stress through antioxidant response elements. Whole-animal NRF1 deletion is embryonically lethal. Liver-specific NRF1 knockout (NRF1-/-) mice exhibit steatosis, apoptosis, necrosis, inflammation, fibrosis and hepatic cancer. By 4 weeks of age, NRF1-/- mice develop steatohepatitis with raised levels of plasma triglycerides. Pericentral and pericellular fibrosis is observed by four months, and hepatic adenomas and carcinomas may develop by 10 months. Hepatocytes from NRF1-/- mice exhibit greater intracellular levels of reactive oxygen species and elevated amounts of lipid and DNA oxidation compared with wild-type control.[57] Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) null mice PTEN is a tumor suppressor gene encoding a lipid phosphatase whose major substrate is phosphatidylinositol-3, 4, 5-triphosphate (PIP3). PTEN is a negative regulator of several signaling pathways, such as phosphatidylinositol 3-kinase (PI3K) and serine-threonine protein kinase B (PKB, or Akt).[58] In almost half of the patients with liver cancer, expression of PTEN is reduced. Liver-specific PTEN knockout mice (PTEN flox/flox mice) develop extensive hepatomegaly and steatohepatitis with a histologic phenotype resembling human NASH.[59] Steatohepatitis is detectable at 10 weeks of age, and fibrosis at 35-40 weeks. Adenomas are present by 44 weeks, and by 74 to 78 weeks, all of the livers show adenomas, whereas 66% develop carcinomas. Hepatocytes lacking PTEN express adipocytespecific genes involved in lipogenesis and β-oxidation, such as AOX, connective tissue growth factor (CTGF), apolipoprotein B, steroyl Co-A desaturase,

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adiponectin, suppressors of cytokine (SOCS)-3, and secreted phosphoprotein 1.

signaling

Ob/ob mice Ob/ob mice carry a spontaneous mutation in the leptin gene (leptin deficient). The mice are hyperphagic, inactive, extremely obese and severely diabetic, with marked hyper-insulinemia and hyperglycemia. Ob/ob mice develop NASH spontaneously,[60] but unlike human NAFLD, ob/ob mice do not spontaneously progress from steatosis to steatohepatitis. Ob/ob mice require other stimuli such as an MCD diet or a high fat diet to trigger progression to steatohepatitis.[49] Db/db mice Db/db mice have a natural mutation in the leptin receptor (Ob-Rb) gene. The mice are obese and IR, and are able to develop macrovesicular hepatic steatosis. These mice readily develop symptoms of NASH upon induction with a second hit, such as feeding with an MCD diet.[52] Common features of the genetic models of NASH The genetic factors present in mice can cause disturbances in fat metabolism and oxidative stress. The mice possess a predisposition to spontaneously develop hepatic adenoma and HCC, and this is especially true with PTEN-/-, AOX-/-, MAT1A-/-, and NRF1-/- mice. These models do not generally have a distinctive phase of steatosis before the development of steatohepatitis. These mice may be more suitable for studying the progression of NASH to HCC. Although models developed in these mice display the pathogenic features of human NAFLD, such as obesity, steatosis, and IR, they do not develop a fibrosing steatohepatitis. The use of these mice in NASH studies is limited by lack of widespread availability and high costs for longterm maintainance.

Dietary models of NASH Methionine- and choline-deficient model This is the classical dietary model for NASH. The MCD diet contains high sucrose and fat (40% sucrose and 10% fat) but lacks two components, methionine and choline, which are essential factors for hepatic β-oxidation and very low-density lipoprotein (VLDL) production.[61] Mice fed a MCD diet may develop hepatic inflammation as early as 3 days after feeding. Severe pericentral steatosis may develop by 1 to

2 weeks, and necroinflammation may occur after 2 weeks, followed by progressive pericellular and pericentral fibrosis. Markedly enhanced oxidative stress can be observed from 3 weeks after intake of the MCD diet. The development and severity of MCD-induced NASH in rodents may depend on the gender, strain, and species used, reflecting the large inter-individual phenotypic variation seen in NASH patients. For example, MCD diet-induced steatohepatitis evolves more slowly in Sprague-Dawley rats than in other rodents. The reported mechanisms of the induction of NASH by a MCD diet include the following: (1) disruption of phosphatidylcholine synthesis caused by methionine and choline deficiency; (2) increased potential for oxidative stress; (3) increased potential for fibrogenesis; (4) increased production of proinflammatory and pro-fibrosis molecules such as interleukin 6, transforming growth factor-beta, and tumor necrosis factor alpha (TNF-α); and (5) activation of NF-κB as an important link between oxidative stress, chronic inflammation and hepatic fibrogenesis.[62] The MCD NASH model is one of the bestestablished models to study the evolution of inflammation, oxidant stress, and fibrotic changes associated with NASH. Compared with other nutritional models, the MCD model causes greater inflammation, oxidant stress, mitochondrial damage, and apoptosis in conjunction with fibrogenesis.[3] This model is easily established and is readily available to all research laboratories worldwide. However, the MCD NASH model is associated with several disadvantages. Rats and mice fed a MCD diet lose significant amounts of muscle and fat weight (up to 40% loss in 10 weeks). Unlike human NASH, animals on a MCD diet do not develop peripheral IR but instead have lower fasting plasma glucose and seem to be insulin-sensitive after 4 weeks of treatment.[45] However, mice fed a MCD diet for 4 weeks exhibit hepatic IR despite whole-body insulin sensitivity. These changes do not reflect the pathophysiology of NASH in humans.

Other nutritional (dietary) models of NASH Apart from the MCD diet, diets containing high fat have been widely used to induce steatosis and NASH in animals. High fat diet (HFD)-induced NASH is analogous to human NAFLD in terms of oxidant stress and fibrosis. For example, rats fed with a HFD (71% fat, 11% carbohydrates, and 18%

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proteins) show histopathological changes typical of human NASH, including IR and significantly raised plasma insulin.[63] Intragastric overfeeding of mice with a HFD (up to 85% in excess of standard intake for 9 weeks) replicates the histopathologic and pathogenic and features of NASH.[40] The HFD appears to induce NASH reliably in rats. Intragastric feeding of a high-fat emulsion to SpragueDawley rats induces changes closely resembling human NASH.[42] Following 6 weeks of feeding, rats develop obesity, and exhibit abnormal ALT, hepatic steatosis, inflammation, hyperlipoidemia, hyperinsulinemia, hyperglycemia and IR. However, other studies have reported that HFD models fail to develop histologic evidence of steatohepatitis.[64, 65] In addition, animals do not show an increase in body weight or plasma ALT levels after 3 weeks on the diet, and features that are not consistent with the changes seen in human NASH. Overall, the HFD-induced NASH model produces variable results with respect to the levels of steatosis, inflammation, and fibrosis. The rodent species, fat content in the diet, duration of treatment, and the composition of dietary fat may all play a role in NASH induction.

Combination models: animal models induced by the combination of genetic modification and nutritonal/dietary challenges As neither genetic nor nutritional models fully reflect the real picture of human NAFLD/NASH, certain mouse models make use of naturally occurring genetic mutations or targeted gene modifications plus a dietary or chemical challenge to mimic more closely the pathogenesis of human NAFLD/NASH. The following are reported examples of the combinations: (1) db/db mice+MCD;[35] (2) ob/ob mice+MCD;[49] (3) ob/ob mice+MCD+HFD;[46] (4) fa/fa (defective long-form leptin receptor) rats+HFD (60% lard);[43] (5) foz/foz mice+HFD;[48] (6) low-density lipoprotein receptor-deficient (ldlr-/-) and apolipoprotein E2 knock-in (APOE2ki) mice+HFD containing high cholesterol (HFC diet).[53] Generally, these models may induce various degrees of fibrosis, IR, obesity, steatosis, and

steatohepatitis. Inflammation may vary between models. For example, db/db+MCD induces a NASH model with much higher serum ALT levels and greater inflammation and fibrosis than the ob/ob+MCD. These models are also associated with various drawbacks. For example, foz/foz mice do not exhibit increased hepatic lipoperoxides but may have significantly reduced serum adiponectin levels. In addition, a long period of induction is needed for foz/foz mice to develop steatohepatitis and fibrosis (-300 days). The ldlr-/- and APOE2ki+HFC diet mouse model was recently reported,[53] in which ldlr-/and APOE2ki mice are fed a HFC diet (21% milk butter, 0.2% cholesterol) for up to 21 days. These mice develop steatosis with severe inflammation after 7 days. Interestingly, the degree of hepatic inflammation is linked to increased plasma levels of VLDL-cholesterol. Omitting cholesterol from the HFC diet markedly reduces the plasma VLDL-cholesterol and prevents the development of hepatic steatosis and inflammation. This study indicates that, unlike what had been held previously, dietary cholesterol is an important risk factor for the progression to hepatic inflammation in diet-induced NASH. The role of cholesterol in the induction of NASH has been supported by a dietary model established by the author's group,[66, 67] in which a HFD containing 2% (w/w) cholesterol is used to induce NASH in male Sprague-Dawley rats. Following 48 weeks of exposure, all experimental rats develop hepatic changes typical of human NASH. Grade Ⅰ hepatic steatosis (i.e., steatosis of less than 33% of the hepatic parenchyma) occurs at week 4, and fatty liver (steatosis of more than 33% of the hepatic parenchyma) develops by week 8. By weeks 12 through 48, steatohepatitis develops and abnormal liver function occurs. Masson staining reveals that a small percentage of rats develop mild perisinusoidal fibrosis by week 16. At week 24, all the rats develop perisinusoidal fibrosis. At weeks 36 and 48, the hepatic fibrosis is more severe. Bridging fibrosis is found in some rats. The sequence of events (simple fatty liver, steatohepatitis, and steatohepatitis with progressive fibrosis) in our experimental rats closely mimics that of the human spectrum of NASH. Further, these pathological features are predominant

Week 4 Week 8 Week 12 Weeks 16-24 Moderate to severe Greater degree of hepatic steatosis Diffusely mixed Hepatic steatosis and steatosis, marked hepatic and lobular inflammation, hepatic steatosis mild hepatic lobular inflammation some necrotic foci inflammation Fig. The sequence of events.

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in zone 3, and the lobular inflammation is more severe than portal inflammation. Another pathological feature reproduced by this model is the presence of mitochondrial lesions such as swelling, rounding up, and loss of cristae.[26-29, 66, 67] The sequence of events is illustrated in Fig.

Discussion Animal models are essential tools for studies of disease pathogenesis. Ideally, animal models should not only reproduce the established pathology of the human disease, but also reenact the context within which a disease develops and progresses. To date, there is no single animal model that displays the range of histopathologic and pathophysiologic features associated with NASH.[3, 22, 63, 68] The currently available animal models are associated with various drawbacks in that they do not or only partially reflect the real picture of human NASH in terms of etiology, pathogenesis, and disease mechanisms.[3, 68, 69] These models either only mimic the histopathologic features or the pathogenic correlates of human NASH. Peripheral IR and fibrosing steatohepatitis, the major pathophysiologic features associated with human NASH, are not faithfully reflected by any single animal model so far.[3] Those models that are claimed to mimic all of the major characteristics of human NASH (obesity, IR, fibrosis, steatosis, and steatohepatitis) are genetically modified animal models fed either HFD or MCD diets.[3] In addition, the majority of animal models with IR are defective in leptin or adiponectin signaling and do not develop steatohepatitis spontaneously with the significant levels of fibrosis seen in the clinical setting. Animal models of fibrosing steatohepatitis are usually under high levels of oxidant stress as a result of increased hepatic fat load in the setting of reduced levels of antioxidants, resulting in lipotoxicity. These models usually do not exhibit peripheral IR and have other limitations, such as excessive weight loss, which is not usually seen in the human disorder.[3, 24, 63, 68] Lack of disease progression or etiological factors that are usually seen in human NASH is also a drawback of some animal models. Spontaneous transition to significant steatohepatitis does not occur in nutritional and metabolic models of steatosis.[69] Although the MCD diet induces changes typical of NASH in rodents, and a liquid HFD (LHFD) also induces pathology typical of NASH, including steatosis, hepatic inflammation, and fibrosis in Sprague-Dawley rats.[63] These nutritional models do

not reflect the diet features seen in NASH patients, as MCD is not commonly seen in human beings, and the MCD model can cause severe weight loss and cachexia which are not characteristics of NASH seen in patients.[24,  38,  44] Besides, the FA composition of the LHFD model is vastly different from that of the diet of NASH patients in whom a high level of saturated FAs and a low level of polyunsaturated FAs are typical.[38, 44] In our high fat and high cholesterol diet model, the experimental rats develop NAFLD, visceral obesity and hyperlipidemia after 24 weeks of exposure.[26-29,  66,  67] In addition, these rats also exhibit increased serum levels of free FAs and TNF-α by week 24. These changes are mechanistically linked to the development of metabolic syndrome and IR. Conventional concepts hold that visceral obesity and IR, mostly mediated by adipokines such as TNF-α, result in increased free FA release from visceral adipose tissue with the consequent enhancement of lipid delivery to the liver. Furthermore, free FAs stimulate TNF-α expression in hepatocytes and in adipocytes and thus are closely implicated in the etiology of IR. From weeks 36 to 48, HFD-fed rats develop hyperglycemia and an increased IR index. These phenomena occur later than that of NAFLD and visceral obesity. We think our HFD model mimics the real process of human metabolic syndrome.[26-29, 66, 67] According to the well-known "single gateway hypothesis", hepatic steatosis and hepatic IR are early events that usually precede pancreatic insulin secretion decompensation and hyperglycemia. The connection between NASH and hyperglycemia is indicated by the epidemiological finding of others that NAFLD significantly increases the risk of type 2 diabetes mellitus and cardiovascular complications, thus making NAFLD a pro-diabetic/pro-atherogenic state.[1-3] The genetic rodent models of NASH are also limited as discussed above and pointed out by others.[36, 37] The availability of these genetic models is limited, and the cost of animal upkeep is high. More importantly, NASH models induced in these animals do not reflect the natural etiological background of NAFLD/NASH in patients, and these animals are generally resistant to the development of steatohepatitis and fibrosis. They are prone to develop liver cancer, and thus may be good models to investigate how NASH evolves into HCC. Pharmacological models of NASH are also frequently used but they have a major limitation of not reflecting the obesity and IR which are critically important in the development of human NASH. Clearly, all these models do not reflect the multi-

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factorial features of NASH observed in patients and thus are "non-physiological". Consequently, it is necessary to establish NASH models that can best mimic the actual etiology, disease progression, and pathogenesis of human NASH.[3, 68] Nevertheless, currently available animal models provide valuable tools to test various mechanistic hypotheses and to conduct interventional studies. One important factor that has to be considered in establishing animal models of NASH is that there are inter-strain differences in the susceptibility to develop NASH. For example, in MCD-induced NASH in rodents, the severity and progression of NASH depend on the gender, strain, and species used. MCD diet-induced steatohepatitis evolves more slowly in Sprague-Dawley rats than in other rodents. C57BL/6N mice are more susceptible than C3H/HeN mice to the development of NASH following a MCD diet.[70] In summary, animal models of NASH are particularly useful in understanding the relationship between lipid metabolism, host defenses, environmental triggers, genetic variability, inflammatory recruitment, and fibrogenesis. Such models are also important platforms for the assessment of therapeutic strategies. However, currently available models do not reflect the full picture of the disease in patients. They provide, however, valuable tools to test mechanistic hypotheses and to conduct interventional studies. With continuing improvement of the current models and the advent of novel models, scientists will be able to accumulate sufficient knowledge of disease pathogenesis which will eventually lead to a full understanding of human NASH and development of efficient therapy for this condition. Funding:  None. Ethical approval: Not needed. Contributors: FJG proposed the study. QL wrote the first draft. All authors contributed to the further drafts. QL is the guarantor. Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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