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MOUSE MODELS OF NONALCOHOLIC STEATOHEPATITIS: A SYSTEMATIC CRITIQUE OF RECENT LITERATUREFahrettin Haczeyni 1, Matthew M. Yeh 2, George N. Ioannou 3, Isabelle A. Leclercq 4, Robert Goldin 5, Yock Young Dan 6, Jun Yu 7, Narcissus C. Teoh 1, Geoffrey C. Farrell 11 Liver Research Group, Australian National University Medical School at the Canberra Hospital, Canberra, ACT, Australia; 2 Department of Pathology, University of Washington, Seattle, WA, USA; 3 Division of Gastroenterology, Department of Medicine, VA Puget Sound Healthcare System and University of Washington, Seattle, WA, USA; 4 Institute of Experimental and Clinical Research, Université catholique de Louvain (UCL), Brussels, Belgium; 5 Department of Cellular Pathology, Imperial College London, London, UK; 6 Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 7 Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong SAR, ChinaRunning title: Mouse Models in Preclinical NASH ResearchCorrespondence:Professor Geoffrey C Farrell, MD, FRACPAustralian National University Medical SchoolThe Canberra Hospital, Level 5, Building 10Yamba Drive, Garran 2605, ACT, AustraliaTel: +61 (0) 2 6244 2595 Fax: +61 (0) 2 6244 3235E-mail: geoff.farrell@anu.edu.auKeywords: Non-alcoholic fatty liver disease; liver pathology; inflammation; fibrosis; overnutrition; insulin resistance; animal models.Word count: Abstract 281, full text 5908.Financial support: Australian National Health and Medical Research Council (NHMRC) project grants: 1044288 and 102818.Disclosure: The authors declare no conflict of interest regarding this manuscript.Contributors: FH assisted with study design, performed the literature search and data analyses, and wrote the manuscript. MMY, GNI, IAL, RG, YYD, JY, and NCT contributed intellectual input and reviewed and edited the manuscript. GCF conceptualized and directed the study, obtained research grant support and wrote and edited the manuscript.Abbreviations: ALT, alanine aminotransferase; CCl4, carbon tetrachloride; CDAA, choline-deficient amino acid-defined diet; DIO, diet-induced obesity; foz/foz, Alms1 mutant Fat Aussie mouse; HFD, high fat diet; LDL, low-density lipoprotein; MCD, methionine and choline-deficient; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD activity score; NASH, nonalcoholic steatohepatitis; PTEN, phosphatase and tensin homolog; SO, steatosis only; STZ, streptozotocin.ABSTRACTNonalcoholic steatohepatitis (NASH) is a pathological form of nonalcoholic fatty liver disease (NAFLD), strongly associated with overnutrition, insulin resistance, and predisposition to type 2 diabetes. It is now timely to critique the translational significance of currently-used animal models of NASH. We surveyed journals during the last three years for papers that studied NASH pathogenesis using mouse models. Among 146 articles, 34 (23%) studies (32% of genetic, 20% dietary) used models in which overnutrition was reported, and 36 (25%) studies (26% of genetic, 24% dietary) demonstrated insulin resistance, with or without glucose intolerance. Half the articles contained no information on whether the models exhibited overnutrition, insulin sensitivity or metabolic complications. 75 papers (52%) reported >2-fold increase of serum/plasma alanine aminotransferase (ALT) compared to controls; ALT levels were near normal or not reported in the remainder. Liver pathology was assessed by a pathologist with an interest in liver pathology in 53% of articles published in gastroenterology/hepatology journals, versus 43-44% in metabolism, pharmacology or other journals. Conclusions: While there appears to be a trend to use models that are potentially relevant to the pathogenesis of human NASH, major journals currently publish articles using data on mouse models in which overnutrition and insulin resistance do not occur, without ALT increase or appropriate analysis of NASH pathology, claiming the results are relevant to NASH. Conducting and publishing experimental studies using animal models that are unlikely to have translational relevance for human health is both a scientific and bioethical concern. We recommend that investigators, reviewers and journal editors carefully consider the validity of NASH models in current use, and that moves are made to reach a consensus on what the minimal criteria of such models should be.INTRODUCTIONNonalcoholic fatty liver disease (NAFLD) affects one-third of the world’s population ADDIN EN.CITE <EndNote><Cite><Author>Younossi</Author><Year>2016</Year><RecNum>53</RecNum><DisplayText>(1, 2)</DisplayText><record><rec-number>53</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507260138">53</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Younossi, Zobair M</author><author>Blissett, Deirdre</author><author>Blissett, Robert</author><author>Henry, Linda</author><author>Stepanova, Maria</author><author>Younossi, Youssef</author><author>Racila, Andrei</author><author>Hunt, Sharon</author><author>Beckerman, Rachel</author></authors></contributors><titles><title>The economic and clinical burden of nonalcoholic fatty liver disease in the United States and Europe</title><secondary-title>Hepatology</secondary-title></titles><periodical><full-title>Hepatology</full-title></periodical><pages>1577-1586</pages><volume>64</volume><number>5</number><dates><year>2016</year></dates><isbn>1527-3350</isbn><urls></urls></record></Cite><Cite><Author>Farrell</Author><Year>2013</Year><RecNum>54</RecNum><record><rec-number>54</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507260169">54</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Farrell, Geoffrey C</author><author>Wong, Vincent Wai-Sun</author><author>Chitturi, Shiv</author></authors></contributors><titles><title>NAFLD in Asia—as common and important as in the West</title><secondary-title>Nature Reviews Gastroenterology and Hepatology</secondary-title></titles><periodical><full-title>Nature Reviews Gastroenterology and Hepatology</full-title></periodical><pages>307-318</pages><volume>10</volume><number>5</number><dates><year>2013</year></dates><isbn>1759-5045</isbn><urls></urls></record></Cite></EndNote>(1, 2). It results from oversupply of energy in relation to bodily needs (hereafter referred to as overnutrition), with or without obesity, especially in those with a genetic predisposition to or established type 2 diabetes ADDIN EN.CITE <EndNote><Cite><Author>Loomba</Author><Year>2012</Year><RecNum>50</RecNum><DisplayText>(3)</DisplayText><record><rec-number>50</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507259224">50</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Loomba, Rohit</author><author>Abraham, Maria</author><author>Unalp, Aynur</author><author>Wilson, Laura</author><author>Lavine, Joel</author><author>Doo, Ed</author><author>Bass, Nathan M</author></authors></contributors><titles><title>Association between diabetes, family history of diabetes, and risk of nonalcoholic steatohepatitis and fibrosis</title><secondary-title>Hepatology</secondary-title></titles><periodical><full-title>Hepatology</full-title></periodical><pages>943-951</pages><volume>56</volume><number>3</number><dates><year>2012</year></dates><isbn>1527-3350</isbn><urls></urls></record></Cite></EndNote>(3). The development of NAFLD is associated with visceral obesity, insulin resistance, hyperglycemia, atherogenic dyslipidemia, hypoadiponectemia, and arterial hypertension ADDIN EN.CITE <EndNote><Cite><Author>Marchesini</Author><Year>2007</Year><RecNum>51</RecNum><DisplayText>(4, 5)</DisplayText><record><rec-number>51</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507259772">51</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Marchesini, Giulio</author><author>Marzocchi, Rebecca</author></authors></contributors><titles><title>Metabolic syndrome and NASH</title><secondary-title>Clinics in liver disease</secondary-title></titles><periodical><full-title>Clinics in liver disease</full-title></periodical><pages>105-117</pages><volume>11</volume><number>1</number><dates><year>2007</year></dates><isbn>1089-3261</isbn><urls></urls></record></Cite><Cite><Author>Yki-J?rvinen</Author><Year>2014</Year><RecNum>52</RecNum><record><rec-number>52</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507259805">52</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Yki-J?rvinen, Hannele</author></authors></contributors><titles><title>Non-alcoholic fatty liver disease as a cause and a consequence of metabolic syndrome</title><secondary-title>The Lancet Diabetes & Endocrinology</secondary-title></titles><periodical><full-title>The Lancet Diabetes & Endocrinology</full-title></periodical><pages>901-910</pages><volume>2</volume><number>11</number><dates><year>2014</year></dates><isbn>2213-8587</isbn><urls></urls></record></Cite></EndNote>(4, 5). For this reason, NAFLD is often described as the “hepatic manifestation” of the metabolic syndrome, although from a pathogenic standpoint, the development of fatty liver may contribute to the development of type 2 diabetes and atherogenic dyslipidemia.NAFLD encompasses a spectrum of pathological severity ranging from steatosis only (SO) through nonalcoholic steatohepatitis (NASH) with or without fibrosis, to cirrhosis and hepatocellular carcinoma (HCC) ADDIN EN.CITE <EndNote><Cite><Author>Rinella</Author><Year>2015</Year><RecNum>44</RecNum><DisplayText>(6)</DisplayText><record><rec-number>44</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507248254">44</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Rinella, Mary E</author></authors></contributors><titles><title>Nonalcoholic fatty liver disease: a systematic review</title><secondary-title>Jama</secondary-title></titles><periodical><full-title>Jama</full-title></periodical><pages>2263-2273</pages><volume>313</volume><number>22</number><dates><year>2015</year></dates><isbn>0098-7484</isbn><urls></urls></record></Cite></EndNote>(6). NASH is the pathological form of NAFLD in which steatosis is complicated by lobular mixed inflammation (pattern seen in adults, whereas portal inflammation is typically seen in children), hepatocellular injury and cell death, often with inflammation-driven fibrosis ADDIN EN.CITE <EndNote><Cite><Author>Brunt</Author><Year>2011</Year><RecNum>62</RecNum><DisplayText>(7, 8)</DisplayText><record><rec-number>62</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507263831">62</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Brunt, Elizabeth M</author><author>Kleiner, David E</author><author>Wilson, Laura A</author><author>Belt, Patricia</author><author>Neuschwander‐Tetri, Brent A</author></authors></contributors><titles><title>Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings</title><secondary-title>Hepatology</secondary-title></titles><periodical><full-title>Hepatology</full-title></periodical><pages>810-820</pages><volume>53</volume><number>3</number><dates><year>2011</year></dates><isbn>1527-3350</isbn><urls></urls></record></Cite><Cite><Author>Takahashi</Author><Year>2014</Year><RecNum>63</RecNum><record><rec-number>63</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507264139">63</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Takahashi, Yoshihisa</author><author>Fukusato, Toshio</author></authors></contributors><titles><title>Histopathology of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis</title><secondary-title>World journal of gastroenterology: WJG</secondary-title></titles><periodical><full-title>World journal of gastroenterology: WJG</full-title></periodical><pages>15539</pages><volume>20</volume><number>42</number><dates><year>2014</year></dates><urls></urls></record></Cite></EndNote>(7, 8). The processes additional to steatosis, such as substantial inflammation and hepatocyte injury and cell death, are likely pathological responses to “liver lipotoxicity”, triggered by hepatic accumulation of toxic lipids and lipid-metabolites in addition to triglycerides only PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5NYWNoYWRvPC9BdXRob3I+PFllYXI+MjAxNjwvWWVhcj48
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ADDIN EN.CITE.DATA (9-12), whereas SO is attributable to triglyceride accumulation.Until the last decade, NASH pathogenesis was conceptualized by a “two-hit” hypothesis ADDIN EN.CITE <EndNote><Cite><Author>Day</Author><Year>1998</Year><RecNum>42</RecNum><DisplayText>(13)</DisplayText><record><rec-number>42</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507247589">42</key></foreign-keys><ref-type name="Generic">13</ref-type><contributors><authors><author>Day, Christopher P</author><author>James, Oliver FW</author></authors></contributors><titles><title>Steatohepatitis: a tale of two “hits”?</title></titles><dates><year>1998</year></dates><publisher>Elsevier</publisher><isbn>0016-5085</isbn><urls></urls></record></Cite></EndNote>(13). Accordingly, the first hit would reflect excessive hepatic lipid partitioning, whereas the second hit induces a pro-injurious and pro-inflammatory response in the liver as the result of unrelated processes, such as oxidative stress and cytokines driven, for example, by gut microbiota ADDIN EN.CITE <EndNote><Cite><Author>Sanyal</Author><Year>2001</Year><RecNum>43</RecNum><DisplayText>(14)</DisplayText><record><rec-number>43</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507247721">43</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Sanyal, Arun J</author><author>Campbell–Sargent, Carol</author><author>Mirshahi, Faridoddin</author><author>Rizzo, William B</author><author>Contos, Melissa J</author><author>Sterling, Richard K</author><author>Luketic, Velimir A</author><author>Shiffman, Mitchell L</author><author>Clore, John N</author></authors></contributors><titles><title>Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities</title><secondary-title>Gastroenterology</secondary-title></titles><periodical><full-title>Gastroenterology</full-title></periodical><pages>1183-1192</pages><volume>120</volume><number>5</number><dates><year>2001</year></dates><isbn>0016-5085</isbn><urls></urls></record></Cite></EndNote>(14). Today, researchers in the field consider the two-hit hypothesis insufficient to describe the multistep pathogenesis of NASH. The newer concept by which metabolic factors lead to NASH is supported by the stronger association of NASH than SO with prediabetes and metabolic syndrome. By this concept, insulin resistance and resultant hyperinsulinemia could drive the hepatic accumulation of lipotoxic lipids, such as free cholesterol, saturated free fatty acids, diacyglycerol, and lysophosphatidylcholine PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5UaWxnPC9BdXRob3I+PFllYXI+MjAxMDwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA (15-19). These more reactive lipid molecules can injure hepatocytes, with activation of innate immunity to initiate an inflammatory response.For two decades, investigators have addressed questions about NASH pathogenesis using animal models, and increasingly these are being used to test putative therapeutic molecules PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5IYWN6ZXluaTwvQXV0aG9yPjxZZWFyPjIwMTc8L1llYXI+
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ADDIN EN.CITE.DATA (20, 21). Rodent (especially mouse) models have been most used; the excellent research on larger animals, such as miniature pigs ADDIN EN.CITE <EndNote><Cite><Author>Lee</Author><Year>2009</Year><RecNum>97</RecNum><DisplayText>(22)</DisplayText><record><rec-number>97</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507950988">97</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Lee, Lydia</author><author>Alloosh, Mouhamad</author><author>Saxena, Romil</author><author>Van Alstine, William</author><author>Watkins, Bruce A</author><author>Klaunig, James E</author><author>Sturek, Michael</author><author>Chalasani, Naga</author></authors></contributors><titles><title>Nutritional model of steatohepatitis and metabolic syndrome in the Ossabaw miniature swine</title><secondary-title>Hepatology</secondary-title></titles><periodical><full-title>Hepatology</full-title></periodical><pages>56-67</pages><volume>50</volume><number>1</number><dates><year>2009</year></dates><isbn>1527-3350</isbn><urls></urls></record></Cite></EndNote>(22) and opossums ADDIN EN.CITE <EndNote><Cite><Author>Chan</Author><Year>2012</Year><RecNum>100</RecNum><DisplayText>(23)</DisplayText><record><rec-number>100</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1508927681">100</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Chan, Jeannie</author><author>Sharkey, Francis E</author><author>Kushwaha, Rampratap S</author><author>VandeBerg, Jane F</author><author>VandeBerg, John L</author></authors></contributors><titles><title>Steatohepatitis in laboratory opossums exhibiting a high lipemic response to dietary cholesterol and fat</title><secondary-title>American Journal of Physiology-Gastrointestinal and Liver Physiology</secondary-title></titles><periodical><full-title>American Journal of Physiology-Gastrointestinal and Liver Physiology</full-title></periodical><pages>G12-G19</pages><volume>303</volume><number>1</number><dates><year>2012</year></dates><isbn>0193-1857</isbn><urls></urls></record></Cite></EndNote>(23), will not be considered further here. Earlier research used animal models showing SO because the focus was on interventions that could convert SO to steatohepatitis. In rodents, production of SO occurs with a high carbohydrate or a high fat diet (HFD), as well as with several genetic eating defects, such as ob/ob or db/db mice or fa/fa rats ADDIN EN.CITE <EndNote><Cite><Author>Reid</Author><Year>2015</Year><RecNum>35</RecNum><DisplayText>(24)</DisplayText><record><rec-number>35</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507246807">35</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Reid, DT</author><author>Eksteen, B</author></authors></contributors><titles><title>Murine models provide insight to the development of non-alcoholic fatty liver disease</title><secondary-title>Nutrition research reviews</secondary-title></titles><periodical><full-title>Nutrition research reviews</full-title></periodical><pages>133-142</pages><volume>28</volume><number>2</number><dates><year>2015</year></dates><isbn>0954-4224</isbn><urls></urls></record></Cite></EndNote>(24). In 1996 to 2000, work from Storr Liver Unit in Sydney, Australia focussed on a rodent model of steatohepatitis (not caused by alcohol) ADDIN EN.CITE <EndNote><Cite><Author>Weltman</Author><Year>1996</Year><RecNum>101</RecNum><DisplayText>(25)</DisplayText><record><rec-number>101</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1508927965">101</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Weltman, MARTIN D</author><author>Farrell, GEOFFREY C</author><author>Liddle, CHRISTOPHER</author></authors></contributors><titles><title>Increased hepatocyte CYP2E1 expression in a rat nutritional model of hepatic steatosis with inflammation</title><secondary-title>Gastroenterology</secondary-title></titles><periodical><full-title>Gastroenterology</full-title></periodical><pages>1645-1653</pages><volume>111</volume><number>6</number><dates><year>1996</year></dates><isbn>0016-5085</isbn><urls></urls></record></Cite></EndNote>(25), and characterized the methionine and choline deficient (MCD) dietary model.In 2008, Larter and Yeh reviewed the metabolic features and histopathology that animal models should exhibit to reflect the human context and liver pathology of NASH ADDIN EN.CITE <EndNote><Cite><Author>Larter</Author><Year>2008</Year><RecNum>71</RecNum><DisplayText>(26)</DisplayText><record><rec-number>71</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507348155">71</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Larter, Claire Z</author><author>Yeh, Matthew M</author></authors></contributors><titles><title>Animal models of NASH: getting both pathology and metabolic context right</title><secondary-title>Journal of gastroenterology and hepatology</secondary-title></titles><periodical><full-title>Journal of Gastroenterology and Hepatology</full-title></periodical><pages>1635-1648</pages><volume>23</volume><number>11</number><dates><year>2008</year></dates><isbn>1440-1746</isbn><urls></urls></record></Cite></EndNote>(26). 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ADDIN EN.CITE.DATA (24, 27-33); however, while multiple etiological factors can cause steatohepatitis pathology, the relevance of some of these models to NASH as a clinical entity is questionable (Table 1), and some fail to demonstrate the defining liver pathology.The recent determination of biomedical journal editors to publish results of animal model research only when studies conform to agreed international standards (ARRIVE) is an important advance ADDIN EN.CITE <EndNote><Cite><Author>Omary</Author><Year>2016</Year><RecNum>98</RecNum><DisplayText>(34)</DisplayText><record><rec-number>98</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1508107225">98</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Omary, M Bishr</author><author>Cohen, David E</author><author>El‐Omar, Emad M</author><author>Jalan, Rajiv</author><author>Low, Malcolm J</author><author>Nathanson, Michael H</author><author>Peek, Richard M</author><author>Turner, Jerrold R</author></authors></contributors><titles><title>Not all mice are the same: standardization of animal research data presentation</title><secondary-title>Hepatology</secondary-title></titles><periodical><full-title>Hepatology</full-title></periodical><pages>1752-1754</pages><volume>63</volume><number>6</number><dates><year>2016</year></dates><isbn>1527-3350</isbn><urls></urls></record></Cite></EndNote>(34). ARRIVE recommends reporting details of animals used, such as strain/genetic fidelity, use of littermates, specifics of diet/nutrients, and other elements of humane and healthy animal husbandry. Beyond this, the basic precepts for experimental use of animals in research demand attention be given to their minimalization and effective use: replacement (by other technologies), reduction (of mouse numbers), and refinement (precision of use for stated purpose), are together summarized as “the three Rs”. Anecdotally, we frequently raise concerns about model relevance when acting as journal reviewers. It is therefore timely this issue is raised and discussed in an informed manner in the field of hepatology. To initiate this discussion, we reviewed all studies that employed mouse models claimed to represent NASH over the last 3 years. We sought data about how often these models met what we regard as key preconditions, such as overnutrition, insulin resistance and glucose intolerance, liver injury, and a specialist pathologist’s assessment of liver pathology.METHODSSearch StrategyWe performed a systematic literature search of all studies that report on NASH mouse models, using the criteria shown in Fig. 1. Electronic databases (MEDLINE, SCOPUS) were interrogated for original research articles published in English. Articles were selected if they contained the keywords “steatohepatitis, NASH, NAFLD, mouse, mice, model” in the title, abstract, and/or keywords. The operators “AND” and “OR” were used in the search algorithm to ensure that article abstracts included at least 3 of these keywords.Study Selection and Eligibility CriteriaFrom 01 January 2015 to 01 September 2017, 270 articles were found. Secondary criteria were then applied (Fig. 1), to include only journals with impact factor ≥3, and to exclude studies uniquely reporting lipodystrophy-associated disorders or HCC. After eliminating articles that did not meet the secondary criteria, 146 (unique) full text articles were downloaded. Two authors (FH, GCF) confirmed that each study provided sufficient information to be included in the detailed analysis (Supporting Table 1). To establish whether the number of research articles in this field is changing, we applied the same primary search criteria to articles published in 2012-2014, but without further detailed analysis.Outcome MeasuresJournalsJournals were grouped as “gastroenterology/hepatology” (e.g. Hepatology, Journal of Hepatology, Gastroenterology), “metabolism” (e.g. Metabolism, The Journal of Lipid Research, Obesity), “pharmacology” (e.g. Journal of Pharmacology and Experimental Therapeutics, Acta Pharmacologica Sinica), and “other” (e.g. The Journal of Clinical Investigation, Clinical Science, Oncotarget).Classification of Model TypeIn this review, models with a genetic manipulation were called “genetic models” regardless of the diet(s) employed, whereas models that have no genetic defect or manipulation were considered as “dietary models”.Genetic mouse models of NASH included defects of a) appetite regulation, and b) metabolic regulation. In the first group, mice carry a defect in hypothalamic regulation of appetite that results in increased food intake and early-onset obesity akin to monozygotic childhood obesity in humans. Examples include melanocortin 4 receptor knockout (Mc4r-/-) ADDIN EN.CITE <EndNote><Cite><Author>Itoh</Author><Year>2011</Year><RecNum>79</RecNum><DisplayText>(35)</DisplayText><record><rec-number>79</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507421572">79</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Itoh, Michiko</author><author>Suganami, Takayoshi</author><author>Nakagawa, Nobutaka</author><author>Tanaka, Miyako</author><author>Yamamoto, Yukio</author><author>Kamei, Yasutomi</author><author>Terai, Shuji</author><author>Sakaida, Isao</author><author>Ogawa, Yoshihiro</author></authors></contributors><titles><title>Melanocortin 4 Receptor–Deficient Mice as a Novel Mouse Model of Nonalcoholic Steatohepatitis</title><secondary-title>The American journal of pathology</secondary-title></titles><periodical><full-title>The American journal of pathology</full-title></periodical><pages>2454-2463</pages><volume>179</volume><number>5</number><dates><year>2011</year></dates><isbn>0002-9440</isbn><urls></urls></record></Cite></EndNote>(35) and Alms1-mutant (foz/foz) PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5BcnNvdjwvQXV0aG9yPjxZZWFyPjIwMDY8L1llYXI+PFJl
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ADDIN EN.CITE.DATA (36-38) mice. In such models, hyperinsulinemia and diabetes occur due to metabolic dysregulation, and not direct effect of genetic manipulation. With a balanced rodent (chow) diet, animals develop SO, whereas feeding atherogenic diet causes NASH. With metabolic defects, leptin-deficient ob/ob (Lepob) and leptin receptor-deficient db/db (Leprdb) mice have impaired leptin responses and develop early-onset obesity, hyperglycemia, hyperinsulinemia, and NAFLD, especially when fed high calorie diets ADDIN EN.CITE <EndNote><Cite><Author>Trak‐Smayra</Author><Year>2011</Year><RecNum>86</RecNum><DisplayText>(39)</DisplayText><record><rec-number>86</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507425415">86</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Trak‐Smayra, Viviane</author><author>Paradis, Valérie</author><author>Massart, Julie</author><author>Nasser, Selim</author><author>Jebara, Victor</author><author>Fromenty, Bernard</author></authors></contributors><titles><title>Pathology of the liver in obese and diabetic ob/ob and db/db mice fed a standard or high‐calorie diet</title><secondary-title>International journal of experimental pathology</secondary-title></titles><periodical><full-title>International journal of experimental pathology</full-title></periodical><pages>413-421</pages><volume>92</volume><number>6</number><dates><year>2011</year></dates><isbn>1365-2613</isbn><urls></urls></record></Cite></EndNote>(39), although hepatic injury and inflammatory infiltration remain mild. Other genetic NASH models include phosphatase and tensin homolog (PTEN)-deficient and low-density lipoprotein receptor knockout (Ldlr-/-) mice. In these models, metabolic regulation differs from that of human NASH, but development of steatohepatitis could provide insights into inflammatory recruitment and liver outcomes.There are several diet-induced mouse models of steatohepatitis. Diet-induced obesity (DIO) (obesogenic) models are based on provision of excess calorie intake without measures of correspondingly increased physical activity. Because studies of hepatic lipid fractions in human NASH show excessive accumulation of free cholesterol PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5WYW4gUm9veWVuPC9BdXRob3I+PFllYXI+MjAxMTwvWWVh
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ADDIN EN.CITE.DATA (40-42), researchers have also used diets high in both fat and cholesterol, variously referred to as atherogenic, western, or fast food diet. Nutrient-deficient models are based on the absence of choline and methionine, which causes dysregulation of β-oxidation and lipoprotein production to increase hepatic lipid partitioning ADDIN EN.CITE <EndNote><Cite><Author>Leclercq</Author><Year>2000</Year><RecNum>76</RecNum><DisplayText>(43, 44)</DisplayText><record><rec-number>76</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507363154">76</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Leclercq, Isabelle A</author><author>Farrell, Geoffrey C</author><author>Field, Jaqueline</author><author>Bell, David R</author><author>Gonzalez, Frank J</author><author>Robertson, Graham R</author></authors></contributors><titles><title>CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis</title><secondary-title>Journal of Clinical Investigation</secondary-title></titles><periodical><full-title>Journal of Clinical Investigation</full-title></periodical><pages>1067</pages><volume>105</volume><number>8</number><dates><year>2000</year></dates><urls></urls></record></Cite><Cite><Author>Zeisel</Author><Year>2009</Year><RecNum>75</RecNum><record><rec-number>75</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507362941">75</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Zeisel, Steven H</author><author>Da Costa, Kerry‐Ann</author></authors></contributors><titles><title>Choline: an essential nutrient for public health</title><secondary-title>Nutrition reviews</secondary-title></titles><periodical><full-title>Nutrition reviews</full-title></periodical><pages>615-623</pages><volume>67</volume><number>11</number><dates><year>2009</year></dates><isbn>1753-4887</isbn><urls></urls></record></Cite></EndNote>(43, 44); these diets may include high sucrose and/or fat content to mitigate reductions in body weight. Although we do not favour use of drug or toxin toxicity to develop steatohepatitis, hepatotoxic models have been included in the dietary group since most are supported by high calorie diets. In the STAM mouse model, streptozotocin (STZ) deletes β cells to impair insulin production, and STZ-injected HFD-fed mice develop liver damage and fibrosis. However, STZ also has direct effects on hepatocytes ADDIN EN.CITE <EndNote><Cite><Author>Bolzán</Author><Year>2002</Year><RecNum>78</RecNum><DisplayText>(45)</DisplayText><record><rec-number>78</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507365261">78</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Bolzán, Alejandro D</author><author>Bianchi, Martha S</author></authors></contributors><titles><title>Genotoxicity of streptozotocin</title><secondary-title>Mutation Research/Reviews in Mutation Research</secondary-title></titles><periodical><full-title>Mutation Research/Reviews in Mutation Research</full-title></periodical><pages>121-134</pages><volume>512</volume><number>2</number><dates><year>2002</year></dates><isbn>1383-5742</isbn><urls></urls></record></Cite></EndNote>(45). Carbon tetrachloride (CCl4) has also been used to generate fatty liver with cirrhosis in mice; some authors have described the pathology as NASH.Metabolic FactorsMetabolic factors recognized to be “established associations to NASH development” by the three largest regional liver societies are listed in Table 2 PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5Xb25nPC9BdXRob3I+PFllYXI+MjAxNzwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA (46-48). We focused on overnutrition, insulin resistance and impaired glucose metabolism in this review.Liver PathologyBecause NASH (vs. SO) is associated with liver injury and frequent increases in serum alanine aminotransferase (ALT), we surveyed serum/plasma levels of ALT, noted as increased >2-fold, increased <2-fold, data not presented or not mentioned, or uninterpretable data (when there was no appropriate control for comparison). The distinction of NASH from SO is clinically important because NASH (with hepatocyte injury, i.e. ballooning) is more often associated with fibrosis than SO. While expert pathologists have developed different histological scoring systems, the NAFLD activity score (NAS) developed by Kleiner, Brunt and colleagues of the Pathology Subcommittee of the NIH NASH Clinical Research Network has been most used in clinical trials ADDIN EN.CITE <EndNote><Cite><Author>Kleiner</Author><Year>2005</Year><RecNum>69</RecNum><DisplayText>(49)</DisplayText><record><rec-number>69</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507337756">69</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Kleiner, David E</author><author>Brunt, Elizabeth M</author><author>Van Natta, Mark</author><author>Behling, Cynthia</author><author>Contos, Melissa J</author><author>Cummings, Oscar W</author><author>Ferrell, Linda D</author><author>Liu, Yao‐Chang</author><author>Torbenson, Michael S</author><author>Unalp‐Arida, Aynur</author></authors></contributors><titles><title>Design and validation of a histological scoring system for nonalcoholic fatty liver disease</title><secondary-title>Hepatology</secondary-title></titles><periodical><full-title>Hepatology</full-title></periodical><pages>1313-1321</pages><volume>41</volume><number>6</number><dates><year>2005</year></dates><isbn>1527-3350</isbn><urls></urls></record></Cite></EndNote>(49). NAS was not devised for semi-quantification of NAFLD characteristics in mouse models, but has the advantage of considering all three key criteria required for definition of NASH: steatosis, hepatocyte injury (as ballooning), and lobular inflammation (Table 2). Fibrosis can be scored separately by the system devised by Elizabeth Brunt, as modified by the above committee ADDIN EN.CITE <EndNote><Cite><Author>Brunt</Author><Year>2011</Year><RecNum>62</RecNum><DisplayText>(7)</DisplayText><record><rec-number>62</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507263831">62</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Brunt, Elizabeth M</author><author>Kleiner, David E</author><author>Wilson, Laura A</author><author>Belt, Patricia</author><author>Neuschwander‐Tetri, Brent A</author></authors></contributors><titles><title>Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings</title><secondary-title>Hepatology</secondary-title></titles><periodical><full-title>Hepatology</full-title></periodical><pages>810-820</pages><volume>53</volume><number>3</number><dates><year>2011</year></dates><isbn>1527-3350</isbn><urls></urls></record></Cite></EndNote>(7). Finally, Brunt and Kleiner have emphasized the difference between the NAS ADDIN EN.CITE <EndNote><Cite><Author>Kleiner</Author><Year>2005</Year><RecNum>69</RecNum><DisplayText>(7, 49)</DisplayText><record><rec-number>69</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507337756">69</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Kleiner, David E</author><author>Brunt, Elizabeth M</author><author>Van Natta, Mark</author><author>Behling, Cynthia</author><author>Contos, Melissa J</author><author>Cummings, Oscar W</author><author>Ferrell, Linda D</author><author>Liu, Yao‐Chang</author><author>Torbenson, Michael S</author><author>Unalp‐Arida, Aynur</author></authors></contributors><titles><title>Design and validation of a histological scoring system for nonalcoholic fatty liver disease</title><secondary-title>Hepatology</secondary-title></titles><periodical><full-title>Hepatology</full-title></periodical><pages>1313-1321</pages><volume>41</volume><number>6</number><dates><year>2005</year></dates><isbn>1527-3350</isbn><urls></urls></record></Cite><Cite><Author>Brunt</Author><Year>2011</Year><RecNum>62</RecNum><record><rec-number>62</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507263831">62</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Brunt, Elizabeth M</author><author>Kleiner, David E</author><author>Wilson, Laura A</author><author>Belt, Patricia</author><author>Neuschwander‐Tetri, Brent A</author></authors></contributors><titles><title>Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings</title><secondary-title>Hepatology</secondary-title></titles><periodical><full-title>Hepatology</full-title></periodical><pages>810-820</pages><volume>53</volume><number>3</number><dates><year>2011</year></dates><isbn>1527-3350</isbn><urls></urls></record></Cite></EndNote>(7, 49), in which values ≥5 (of max 8 points) are usually associated with NASH, versus the diagnosis of NASH that can only be made after global assessment of the liver pathology by an experienced liver pathologist. We scrutinized all reports for methods of assessing liver pathology, whether NAS (or a similar scoring system) was used, and whether a pathologist was involved in global assessment of liver pathology for possible NASH.RESULTSPublication CharacteristicsDetails of the 146 studies reviewed are in Supporting Table 1. 53 (36%) were published in gastroenterology/hepatology journals, 30 (21%) in metabolism, 9 (6%) pharmacology, and 54 (37%) other journal categories. By experimental design, 108 studies (74%) reported data on dietary models, while 38 studies (26%) incorporated a genetic model. Use of genetic models was highest among studies published in gastroenterology/hepatology journals (genetic 34% vs. 66% dietary) compared to 30% in metabolism, 20% other, and none among articles published in pharmacology journals. A brief survey of earlier years (using only the primary descriptors) indicated an apparent recent increase in the number of articles employing NASH mouse models (Supp Fig. 1).Among 146 articles, only 34 (23%) studies used models in which overnutrition was reported (Fig. 2), and 36 (25%) studies demonstrated insulin resistance, with or without glucose intolerance (Fig. 2). The largest proportion of articles contained no information on whether the models exhibited overnutrition, insulin sensitivity or metabolic complications that are associated with human NASH. 75 papers (52%) reported >2-fold increase of serum/plasma alanine aminotransferase (ALT) levels compared to controls (Fig. 2).Characteristics of Genetic ModelsThe most commonly used was ob/ob mice (11 articles, 29% of genetic model articles) followed by db/db (4 articles, 11%) and foz/foz (6 articles, 16%). Others included PTEN-KO and Ldlr-/- mice. Among genetic model studies, 12 (32%) reported body weight >40g, 8 (21%) exhibited body weight <40g, and nearly half (47%) failed to report body weight data (Fig. 3). Some studies did mention weight gain after the start of the diet, but failed to mention final body weight. Insulin resistance alone or with glucose intolerance and diabetes was reported in 10 (26%) genetic model studies (Fig. 3). Again, 11 (29%) showed no evidence of IR/diabetes and nearly half (45%) did not provide results about insulin sensitivity in the model used. Nineteen studies (50%) reported elevated serum/plasma ALT levels >2-times higher than no NASH controls; in 6 studies (16%), ALT was not increased or the increase failed to exceed 2-fold compared to controls. There was no information about ALT in 10 studies (26%), and no relevant control (e.g. HFD-fed vs. chow-fed) in 3 (8%) studies (Fig. 3).Characteristics of Dietary ModelsA wide range of different dietary regimes was used. The most common was the MCD diet (42 articles, 39%), choline-deficient amino acid-defined diet (CDAA) (8 articles, 7%), high fat only diet (18 articles, 17%), and high fat with cholesterol diet (16 articles, 15%). Several other studies (n=22) incorporated a HFD with cholesterol and/or high carbohydrate, variously referring to the diet as “Western”, “fast food”, or “atherogenic”. The cholesterol content range from 0.02% to 2% by weight, or remains undefined.Only 22 (20%) of dietary studies reported body weight >40g, and in the majority of dietary models (39%), mice did not gain excessive body weight (Fig. 4). Further, 44 studies (41%) failed to report final body weight after the dietary regime. Only 26 studies (24%) showed evidence of insulin resistance and/or diabetes in the model used, 40 studies (37%) found no such evidence, and 42 studies (39%) contained no relevant information (Fig. 4). The number of studies employing a dietary model in which blood ALT levels increased >2-fold control values was 56 (53%), whereas 9 studies (8%) showed normal or very mild ALT increase. Thirty-four studies (32%) contained no information about blood ALT, and 7 (7%) reported ALT without reference to a control group, which we deemed uninterpretable data (Fig. 4).Analyses of Liver HistopathologyWe analyzed the 146 articles as to whether they contained a pathologist’s assessment of NAFLD pathology. Among studies published in gastroenterology/hepatology journals, 53% provided a pathologist’s analysis of NASH. This proportion was less (43-44%) in metabolism, pharmacology, and other journals. In terms of model type, studies with a diet-induced NASH models were more likely to contain an appropriate analysis of liver pathology than those employing a genetic model (Fig. 5). An additional finding was that some authors had “invented” their own version of NAS, mis-naming it as the “NASH activity score”, or in some instances, eliminating the hepatocellular injury component conferred by scoring hepatocyte ballooning.DISCUSSIONThe need for a detailed understanding of NASH pathogenesis so as to design effective pharmacological intervention is among the highest priorities in contemporary hepatology. The nexus between overnutrition, usually that causing overweight or obesity, and NASH is supported by a wealth of clinical studies and underscored mechanistically by the complete reversal of NASH pathology that occurs with 10% weight loss ADDIN EN.CITE <EndNote><Cite><Author>Vilar-Gomez</Author><Year>2015</Year><RecNum>27</RecNum><DisplayText>(50, 51)</DisplayText><record><rec-number>27</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507245421">27</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Vilar-Gomez, Eduardo</author><author>Martinez-Perez, Yadina</author><author>Calzadilla-Bertot, Luis</author><author>Torres-Gonzalez, Ana</author><author>Gra-Oramas, Bienvenido</author><author>Gonzalez-Fabian, Licet</author><author>Friedman, Scott L</author><author>Diago, Moises</author><author>Romero-Gomez, Manuel</author></authors></contributors><titles><title>Weight loss through lifestyle modification significantly reduces features of nonalcoholic steatohepatitis</title><secondary-title>Gastroenterology</secondary-title></titles><periodical><full-title>Gastroenterology</full-title></periodical><pages>367-378. e5</pages><volume>149</volume><number>2</number><dates><year>2015</year></dates><isbn>0016-5085</isbn><urls></urls></record></Cite><Cite><Author>Klebanoff</Author><Year>2017</Year><RecNum>107</RecNum><record><rec-number>107</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1510809404">107</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Klebanoff, Matthew J</author><author>Corey, Kathleen E</author><author>Chhatwal, Jagpreet</author><author>Kaplan, Lee M</author><author>Chung, Raymond T</author><author>Hur, Chin</author></authors></contributors><titles><title>Bariatric surgery for nonalcoholic steatohepatitis: A clinical and cost‐effectiveness analysis</title><secondary-title>Hepatology</secondary-title></titles><periodical><full-title>Hepatology</full-title></periodical><pages>1156-1164</pages><volume>65</volume><number>4</number><dates><year>2017</year></dates><isbn>1527-3350</isbn><urls></urls></record></Cite></EndNote>(50, 51). For this reason, the 2017 Asia-Pacific Guidelines on NAFLD, broadly in accord with EASL and AASLD Guidelines, define NAFLD and its complication of NASH as a disorder that can reasonably be attributed to overnutrition and its complications, such as weight gain, central obesity, development of insulin resistance with glucose intolerance and metabolic syndrome ADDIN EN.CITE <EndNote><Cite><Author>Wong</Author><Year>2017</Year><RecNum>13</RecNum><DisplayText>(46)</DisplayText><record><rec-number>13</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507243663">13</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Wong, Vincent Wai‐Sun</author><author>Chan, Wah‐Kheong</author><author>Chitturi, Shiv</author><author>Chawla, Yogesh</author><author>Dan, Yock Young</author><author>Duseja, Ajay</author><author>Fan, Jiangao</author><author>Goh, Khean‐Lee</author><author>Hamaguchi, Masahide</author><author>Hashimoto, Etsuko</author></authors></contributors><titles><title>The Asia‐Pacific Working Party on Nonalcoholic Fatty Liver Disease Guidelines 2017 Part 1: Definition, risk factors and assessment</title><secondary-title>Journal of Gastroenterology and Hepatology</secondary-title></titles><periodical><full-title>Journal of Gastroenterology and Hepatology</full-title></periodical><dates><year>2017</year></dates><isbn>1440-1746</isbn><urls></urls></record></Cite></EndNote>(46). Lipotoxicity is now a favoured concept for NASH pathogenesis. However, there is an outstanding need to elucidate details about what appears to be a network of mal-adaptive responses in the liver and whole body that lead to the liver pathology of NASH. Since it is difficult to study dynamic tissue changes in the human liver, investigators have developed and used animal models, especially mouse models for this purpose.The number of articles reporting studies into the pathogenesis or treatment of NASH using mouse models has increased from ~40/year in 2012 to currently over 100/year (projected from studies to September 2017). Twenty years ago the central tenant about NASH pathogenesis was that obesity and metabolic factors (e.g. diabetes) are simply the setting for NASH – a separate pro-injurious and pro-inflammatory “hit” was required to transform steatosis to steatohepatitis. The more integrated idea of lipotoxicity resulting from “unhealthy obesity” (overnutrition complicated by insulin resistance) requires different animal models for study, ones that reflect a direct metabolic causation of NASH. Our anecdotal experience as reviewers of articles submitted to biomedical journals suggested that investigators and journals have not yet established standards of what should be the minimal requirements for a relevant animal model of NASH, such as those we offer in Table 3. As articulated by Larter and Yeh in 2008 ADDIN EN.CITE <EndNote><Cite><Author>Larter</Author><Year>2008</Year><RecNum>71</RecNum><DisplayText>(26)</DisplayText><record><rec-number>71</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507348155">71</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Larter, Claire Z</author><author>Yeh, Matthew M</author></authors></contributors><titles><title>Animal models of NASH: getting both pathology and metabolic context right</title><secondary-title>Journal of gastroenterology and hepatology</secondary-title></titles><periodical><full-title>Journal of Gastroenterology and Hepatology</full-title></periodical><pages>1635-1648</pages><volume>23</volume><number>11</number><dates><year>2008</year></dates><isbn>1440-1746</isbn><urls></urls></record></Cite></EndNote>(26), we consider that models that reflect both the metabolic context and appropriate liver pathology of human NASH would seem most relevant. To establish a decade later how widespread is the use of such models, we conducted a systematic review of articles about the pathogenesis or drug treatment of NASH, noting whether the animals used became over-nourished (body weight ≥40 g), developed insulin resistance and/or glucose intolerance/diabetes, and recording how liver pathology was assessed.While we are unable to make a value judgement about the quality of articles or their likely contribution to knowledge, we were surprised that less than one quarter of them reported on animal body weight attaining a value that reflects mouse obesity; about the same proportion reported body weights in the normal range, and fully one half failed to report any data on body weight. Some mouse model studies mentioned weight gain after commencement of dietary regimens but failed to mention the final body weight. There is current debate about the proportion of patients with NASH who are not obese, up to 25% in some Asian series, but most of these cases have exhibited weight gain, they often show central adiposity, commonly give a family history of type 2 diabetes, and, if tested, they virtually always have insulin resistance ADDIN EN.CITE <EndNote><Cite><Author>Liu</Author><Year>2012</Year><RecNum>104</RecNum><DisplayText>(52)</DisplayText><record><rec-number>104</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1510031955">104</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Liu, Chun‐Jen</author></authors></contributors><titles><title>Prevalence and risk factors for non‐alcoholic fatty liver disease in Asian people who are not obese</title><secondary-title>Journal of gastroenterology and hepatology</secondary-title></titles><periodical><full-title>Journal of Gastroenterology and Hepatology</full-title></periodical><pages>1555-1560</pages><volume>27</volume><number>10</number><dates><year>2012</year></dates><isbn>1440-1746</isbn><urls></urls></record></Cite></EndNote>(52). We therefore propose that use of models where at least half of mice are not overweight, up to 75% if the absence of data in 50% of the papers is because there was no weight gain, is a substantive difficulty. The metabolic complications of over-nutrition pertinent to NASH pathogenesis centre around insulin resistance. This may be evident with fasting hyperinsulinemia and calculated (by also considering fasting blood glucose) using the homeostatic model assessment score (HOMA-IR), or by a physiological challenge such as an intraperitoneal glucose tolerance test which is simpler to perform on mice than the technically more demanding “gold standard” of an insulin and glucose infusion clamp test. One quarter (10, 26%) of studies employing genetic models (usually with a dietary regimen) demonstrated insulin resistance alone or with glucose intolerance and diabetes, 11 (29%) found no insulin resistance, and 18 (45%) provided no relevant information. Rather similarly, 26 (24%) of studies in purely dietary models reported insulin resistance and/or diabetes, 39 (36%) found no evidence of insulin resistance and 42 (39%) failed to provide the relevant information. As for over-nutrition, if somewhere between 26% and 74% (worst case scenario if failure to provide pertinent information always reflected insulin resistance was not present) of articles are about fatty liver without insulin resistance, the literature on NASH pathogenesis using mouse models could be seriously flawed as at least 98% of humans with NASH have insulin resistance ADDIN EN.CITE <EndNote><Cite><Author>Chitturi</Author><Year>2002</Year><RecNum>46</RecNum><DisplayText>(53)</DisplayText><record><rec-number>46</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507258286">46</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Chitturi, Shivakumar</author><author>Abeygunasekera, Shehan</author><author>Farrell, Geoffrey C</author><author>Holmes‐Walker, Jane</author><author>Hui, Jason M</author><author>Fung, Caroline</author><author>Karim, Rooshdiya</author><author>Lin, Rita</author><author>Samarasinghe, Dev</author><author>Liddle, Christopher</author></authors></contributors><titles><title>NASH and insulin resistance: insulin hypersecretion and specific association with the insulin resistance syndrome</title><secondary-title>Hepatology</secondary-title></titles><periodical><full-title>Hepatology</full-title></periodical><pages>373-379</pages><volume>35</volume><number>2</number><dates><year>2002</year></dates><isbn>1527-3350</isbn><urls></urls></record></Cite></EndNote>(53).We respect that not all authors may concur with the proposal that overnutrition with insulin resistance is central to NASH pathogenesis. However, less circumspection should surround the requirement for liver injury and a specific pattern of liver pathology to distinguish between NASH and SO. Despite this, we found only 50% of studies using genetic models documented blood ALT increase ≥2-fold versus control values, 53% using a dietary model, while up to 16% of genetic model and 8% of dietary model research reported normal ALT values, and 13-25% failed to mention any ALT (or AST) data. In addition, 7% of studies failed to use an appropriate control (e.g. wildtype mice in genetic studies, chow-fed mice in dietary studies). If recommendations for the interpretation of liver pathology required for the diagnosis of human NASH are considered, attempts to document the phenotype and severity of fatty liver pathology in mouse model research were also deficient, particularly in non-specialist gastroenterology/hepatology journals. Accordingly, among all 146 articles surveyed, 53% of those published in gastroenterology/hepatology journals used an experienced liver pathologist to interpret liver pathology, versus 43-44% in other journals.An insurmountable problem with use of animal models for NASH research is that no model can overcome the species differences between humans and mice, and operation of genetic polymorphisms is known to play a major role in pathogenesis of the human condition. Emerging data from hepatic transcriptome analysis indicate that some models exhibit changes in gene expression that are more similar than other models to those found in human NASH livers. On the other hand, the cellular morphology of liver injury, such as the pattern of hepatocyte ballooning and Mallory-Denk bodies, seems to differ, at least subtly, between humans and mice, as well as between mouse models ADDIN EN.CITE <EndNote><Cite><Author>Ibrahim</Author><Year>2016</Year><RecNum>38</RecNum><DisplayText>(30)</DisplayText><record><rec-number>38</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507246879">38</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Ibrahim, Samar H</author><author>Hirsova, Petra</author><author>Malhi, Harmeet</author><author>Gores, Gregory J</author></authors></contributors><titles><title>Animal models of nonalcoholic steatohepatitis: eat, delete, and inflame</title><secondary-title>Digestive diseases and sciences</secondary-title></titles><periodical><full-title>Digestive diseases and sciences</full-title></periodical><pages>1325-1336</pages><volume>61</volume><number>5</number><dates><year>2016</year></dates><isbn>0163-2116</isbn><urls></urls></record></Cite></EndNote>(30). Until agreement is reached on which measures of hepatocyte injury best reflect lipotoxic (or NASH-related) hepatocyte injury in mice, it would be inappropriate to rule out the potential value of existing and new models, particularly if they possess other attributes of special pathogenic interest – such as genetic polymorphisms identical to those found in human NASH. Differing requirements for macronutrients are also important differences between humans and rodents. A relevant example in NASH pathogenesis studies is dietary cholesterol; some dietary studies have used 1 or 2% cholesterol (wt/wt), which as Ginsberg noted in 2004 represents the human equivalent of eating more than 100 Big Macs a day ADDIN EN.CITE <EndNote><Cite><Author>Ginsberg</Author><Year>2006</Year><RecNum>108</RecNum><DisplayText>(54)</DisplayText><record><rec-number>108</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1510809493">108</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Ginsberg, Henry N</author></authors></contributors><titles><title>Is the slippery slope from steatosis to steatohepatitis paved with triglyceride or cholesterol?</title><secondary-title>Cell metabolism</secondary-title></titles><periodical><full-title>Cell metabolism</full-title></periodical><pages>179-181</pages><volume>4</volume><number>3</number><dates><year>2006</year></dates><isbn>1550-4131</isbn><urls></urls></record></Cite></EndNote>(54)! On the other hand, 0.2% cholesterol in diet is ideally high for a mouse, and transcriptome analyses of a mouse strain (B6/129) fed 0.1% dietary cholesterol showed good analogy with human NASH ADDIN EN.CITE <EndNote><Cite><Author>Asgharpour</Author><Year>2016</Year><RecNum>110</RecNum><DisplayText>(55)</DisplayText><record><rec-number>110</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1511234907">110</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Asgharpour, Amon</author><author>Cazanave, Sophie C</author><author>Pacana, Tommy</author><author>Seneshaw, Mulugeta</author><author>Vincent, Robert</author><author>Banini, Bubu A</author><author>Kumar, Divya Prasanna</author><author>Daita, Kalyani</author><author>Min, Hae-Ki</author><author>Mirshahi, Faridoddin</author></authors></contributors><titles><title>A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer</title><secondary-title>Journal of hepatology</secondary-title></titles><periodical><full-title>Journal of Hepatology</full-title></periodical><pages>579-588</pages><volume>65</volume><number>3</number><dates><year>2016</year></dates><isbn>0168-8278</isbn><urls></urls></record></Cite></EndNote>(55).Given the differing views of authors in this field and the dearth of consensus about what might or might not be appropriate animal models to study NASH pathogenesis or test putative therapeutic molecules, it is premature to call for a moratorium on specific mouse models provided their use otherwise complies with international and local institutional standards for ethical experimentation. That stated, we are concerned with the number of articles that still use the MCD model, which we introduced to this field in 1996 ADDIN EN.CITE <EndNote><Cite><Author>Weltman</Author><Year>1996</Year><RecNum>101</RecNum><DisplayText>(25)</DisplayText><record><rec-number>101</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1508927965">101</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Weltman, MARTIN D</author><author>Farrell, GEOFFREY C</author><author>Liddle, CHRISTOPHER</author></authors></contributors><titles><title>Increased hepatocyte CYP2E1 expression in a rat nutritional model of hepatic steatosis with inflammation</title><secondary-title>Gastroenterology</secondary-title></titles><periodical><full-title>Gastroenterology</full-title></periodical><pages>1645-1653</pages><volume>111</volume><number>6</number><dates><year>1996</year></dates><isbn>0016-5085</isbn><urls></urls></record></Cite></EndNote>(25), and refined for mice in 2000 ADDIN EN.CITE <EndNote><Cite><Author>Leclercq</Author><Year>2000</Year><RecNum>76</RecNum><DisplayText>(43)</DisplayText><record><rec-number>76</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507363154">76</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Leclercq, Isabelle A</author><author>Farrell, Geoffrey C</author><author>Field, Jaqueline</author><author>Bell, David R</author><author>Gonzalez, Frank J</author><author>Robertson, Graham R</author></authors></contributors><titles><title>CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis</title><secondary-title>Journal of Clinical Investigation</secondary-title></titles><periodical><full-title>Journal of Clinical Investigation</full-title></periodical><pages>1067</pages><volume>105</volume><number>8</number><dates><year>2000</year></dates><urls></urls></record></Cite></EndNote>(43). Between 2015 and 2017, the MCD model was used by authors of 42 articles, 39% of all dietary models. When it was stated, the time of dietary exposure varied from 2 to 20 weeks, most typically 4 weeks, whereas we have noted it takes ~3 weeks for steatohepatitis pathology to evolve, and at least 5 weeks for histologically apparent fibrosis ADDIN EN.CITE <EndNote><Cite><Author>Leclercq</Author><Year>2002</Year><RecNum>84</RecNum><DisplayText>(56)</DisplayText><record><rec-number>84</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507425215">84</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Leclercq, Isabelle A</author><author>Farrell, Geoffrey C</author><author>Schriemer, Rixt</author><author>Robertson, Graham R</author></authors></contributors><titles><title>Leptin is essential for the hepatic fibrogenic response to chronic liver injury</title><secondary-title>Journal of hepatology</secondary-title></titles><periodical><full-title>Journal of Hepatology</full-title></periodical><pages>206-213</pages><volume>37</volume><number>2</number><dates><year>2002</year></dates><isbn>0168-8278</isbn><urls></urls></record></Cite></EndNote>(56). Authors rarely if ever stated the reason for such short duration of their studies, but we note that after 3-4 weeks MCD-fed mice lose ~20% of their starting body weight, and by 8 weeks have generally lost 40% of body weight (the smallest mouse we noted in the reviewed studies weighed 12 g). Whether Institutional Review Boards are not permitting animals to lose this weight, as might be expected, it is problematic when authors fail to indicate such a key factor of experimental design in their article. Further, mice fed MCD diet are unusually insulin sensitive rather than the contrary, and the claim (made initially by us) that the development of steatohepatitis pathology makes the model of interest to NASH may not be strictly correct if livers do not show ballooning ADDIN EN.CITE <EndNote><Cite><Author>Ibrahim</Author><Year>2016</Year><RecNum>38</RecNum><DisplayText>(30)</DisplayText><record><rec-number>38</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1507246879">38</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Ibrahim, Samar H</author><author>Hirsova, Petra</author><author>Malhi, Harmeet</author><author>Gores, Gregory J</author></authors></contributors><titles><title>Animal models of nonalcoholic steatohepatitis: eat, delete, and inflame</title><secondary-title>Digestive diseases and sciences</secondary-title></titles><periodical><full-title>Digestive diseases and sciences</full-title></periodical><pages>1325-1336</pages><volume>61</volume><number>5</number><dates><year>2016</year></dates><isbn>0163-2116</isbn><urls></urls></record></Cite></EndNote>(30). The CDAA model avoids extreme weight loss in a choline-deficient model that is less restricted in essential amino acids, while liver pathology progresses over a predictable and reasonable time frame through phases of steatosis only to steatohepatitis with fibrosis. However, authors have yet to report on physiological studies of insulin sensitivity, and rarely provide metabolic indices such as fasting serum insulin, blood glucose and serum lipids.Other dietary models can produce over-nutrition, insulin resistance and NASH pathology as documented by NAS ≥ 5 and/or experienced liver pathologist assessment. Those that contain both high fat and high cholesterol (for a rodent) seem most likely to provide all these dimensions, although many such diets use in-house dietary formulation without documenting the source and nutrient content of ingredients (e.g. lard, milk fat). Diets better described as high fat or high carbohydrate (alone) rarely produce liver injury with ALT elevation and convincing liver pathology showing steatohepatitis. Finally, we encountered articles that used a toxic regimen, usually repeated CCl4 administration, to produce fatty liver pathology that some authors claimed represented NASH. While forms of drug-induced liver injury (DILI) such as that due to tamoxifen, amiodarone and nifedipine can cause steatohepatitis pathology, this is better regarded as a different condition (drug-induced steatohepatitis), not NASH ADDIN EN.CITE <EndNote><Cite><Author>Farrell</Author><Year>2002</Year><RecNum>111</RecNum><DisplayText>(57)</DisplayText><record><rec-number>111</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1511235189">111</key></foreign-keys><ref-type name="Conference Proceedings">10</ref-type><contributors><authors><author>Farrell, Geoffrey C</author></authors></contributors><titles><title>Drugs and steatohepatitis</title><secondary-title>Seminars in liver disease</secondary-title></titles><pages>185-194</pages><volume>22</volume><number>02</number><dates><year>2002</year></dates><publisher>Copyright? 2002 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel.:+ 1 (212) 584-4662</publisher><isbn>0272-8087</isbn><urls></urls></record></Cite></EndNote>(57).CONCLUSIONSThis systematic review found that a large range of mouse models is currently being used to study NASH pathogenesis and treatment. Some are purely dietary, others genetic or genetic manipulations with or without exposure to one or more diets. We are not convinced that all such models provide data of sound validity for a better understanding of NASH and its treatment, and there is need for international consensus in this area. It is first important that journals insist authors provide the basic data set required to consider the nature of such a model. A minimum data set is summarized in Table 3. Ideally we wonder whether all models should show weight gain (or indicate reasons why this is not relevant to the study objective), liver injury (serum enzymes and/or ballooning and/or measures of cell death) as well as substantial liver inflammation. We acknowledge that there may be subtle differences, such as the morphology and frequency of ballooning in even what seem otherwise excellent models, but we recommend that a pathologist with experience in human fatty liver disease be integrally involved with at least the initial description of a new animal model of NASH. In addition, an informed use of NAS would allow pathology between articles to be compared, and arbitrary modification (such as omitting ballooning scores) should be discouraged. Observed from an ethical viewpoint, many details in animal experimentation (as in “ARRIVE”) are now regarded as an essential prerequisite for publication of an article in most major biomedical journals, including HEPATOLOGY ADDIN EN.CITE <EndNote><Cite><Author>Omary</Author><Year>2016</Year><RecNum>98</RecNum><DisplayText>(34)</DisplayText><record><rec-number>98</rec-number><foreign-keys><key app="EN" db-id="99t55dzebrrpfpew50gprxabe0zed9t0z9d2" timestamp="1508107225">98</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Omary, M Bishr</author><author>Cohen, David E</author><author>El‐Omar, Emad M</author><author>Jalan, Rajiv</author><author>Low, Malcolm J</author><author>Nathanson, Michael H</author><author>Peek, Richard M</author><author>Turner, Jerrold R</author></authors></contributors><titles><title>Not all mice are the same: standardization of animal research data presentation</title><secondary-title>Hepatology</secondary-title></titles><periodical><full-title>Hepatology</full-title></periodical><pages>1752-1754</pages><volume>63</volume><number>6</number><dates><year>2016</year></dates><isbn>1527-3350</isbn><urls></urls></record></Cite></EndNote>(34). In our view, such thinking should extend to whether it is logistically or ethically justified to publish articles in which the authors purport to be studying NASH when there is insufficient evidence provided to support such a claim. Our purpose in pointing out this concern is to improve experimental planning so that that quality of biomedical research into NASH pathogenesis and treatment is improved. Only in this way can it ultimately bear fruit in terms of the much needed breakthrough in drug treatment.REFERENCES ADDIN EN.REFLIST 1.Younossi ZM, Blissett D, Blissett R, Henry L, Stepanova M, Younossi Y, et al. The economic and clinical burden of nonalcoholic fatty liver disease in the United States and Europe. Hepatology. 2016;64(5):1577-86.2.Farrell GC, Wong VW-S, Chitturi S. NAFLD in Asia—as common and important as in the West. Nature Reviews Gastroenterology and Hepatology. 2013;10(5):307-18.3.Loomba R, Abraham M, Unalp A, Wilson L, Lavine J, Doo E, et al. 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The American journal of pathology. 2011;179(5):2454-63.36.Arsov T, Silva DG, O’bryan MK, Sainsbury A, Lee NJ, Kennedy C, et al. Fat aussie—a new Alstrom syndrome mouse showing a critical role for ALMS1 in obesity, diabetes, and spermatogenesis. Molecular endocrinology. 2006;20(7):1610-22.37.Haczeyni F, Barn V, Mridha AR, Yeh MM, Estevez E, Febbraio MA, et al. Exercise improves adipose function and inflammation and ameliorates fatty liver disease in obese diabetic mice. Obesity. 2015;23(9):1845-55.38.Haczeyni F, Poekes L, Wang H, Mridha AR, Barn V, Geoffrey Haigh W, et al. Obeticholic acid improves adipose morphometry and inflammation and reduces steatosis in dietary but not metabolic obesity in mice. Obesity. 2017;25(1):155-65.39.Trak‐Smayra V, Paradis V, Massart J, Nasser S, Jebara V, Fromenty B. Pathology of the liver in obese and diabetic ob/ob and db/db mice fed a standard or high‐calorie diet. 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Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41(6):1313-21.50.Vilar-Gomez E, Martinez-Perez Y, Calzadilla-Bertot L, Torres-Gonzalez A, Gra-Oramas B, Gonzalez-Fabian L, et al. Weight loss through lifestyle modification significantly reduces features of nonalcoholic steatohepatitis. Gastroenterology. 2015;149(2):367-78. e5.51.Klebanoff MJ, Corey KE, Chhatwal J, Kaplan LM, Chung RT, Hur C. Bariatric surgery for nonalcoholic steatohepatitis: A clinical and cost‐effectiveness analysis. Hepatology. 2017;65(4):1156-64.52.Liu CJ. Prevalence and risk factors for non‐alcoholic fatty liver disease in Asian people who are not obese. Journal of gastroenterology and hepatology. 2012;27(10):1555-60.53.Chitturi S, Abeygunasekera S, Farrell GC, Holmes‐Walker J, Hui JM, Fung C, et al. NASH and insulin resistance: insulin hypersecretion and specific association with the insulin resistance syndrome. Hepatology. 2002;35(2):373-9.54.Ginsberg HN. Is the slippery slope from steatosis to steatohepatitis paved with triglyceride or cholesterol? Cell metabolism. 2006;4(3):179-81.55.Asgharpour A, Cazanave SC, Pacana T, Seneshaw M, Vincent R, Banini BA, et al. A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer. Journal of hepatology. 2016;65(3):579-88.56.Leclercq IA, Farrell GC, Schriemer R, Robertson GR. Leptin is essential for the hepatic fibrogenic response to chronic liver injury. Journal of hepatology. 2002;37(2):206-13.57.Farrell GC, editor Drugs and steatohepatitis. Seminars in liver disease; 2002: Copyright? 2002 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel.:+ 1 (212) 584-4662.FIGURE LEGENDSFigure 1. Search processes for inclusion of relevant studiesFigure 2. Reported body weight, insulin resistance and diabetes, and blood alanine transaminase (ALT) results of mouse models of NASH from all reviewed (146) articles.Figure 3. Reported body weight, insulin resistance and diabetes, and blood alanine transaminase (ALT) results in genetic models of NASH.Figure 4. Reported body weight, insulin resistance and diabetes, and blood alanine transaminase (ALT) results in dietary models of NASH.Figure 5. Liver histology analysis by a pathologist per experimental NASH model. ................
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