How reproducible are rat steatosis models using high-fat diets?
Authors: Hendrik A. Marsman, Rana L. Al-Saady, Michal Heger, Thomas M. van Gulik
Journal: Journal of Hepatology (2009)

Non-alcoholic fatty liver disease (NAFLD) is a disease on the increase in the Western world with vast clinical implications. Patients with NAFLD are susceptible to develop NASH and are at risk of complications when undergoing a liver resection, particularly in living donor liver transplantation procedures [[1]Vetelainen R. van Vliet A. Gouma D.J. van Gulik T.M. Steatosis as a risk factor in liver surgery.Ann Surg. 2007; 245: 20-30Crossref PubMed Scopus (218) Google Scholar]. In this setting moderate and severe steatosis are considered exclusion criteria. Consequently, clinically representative animal models yielding an adequate steatosis degree are imperative to conduct mechanistic and interventional investigations. Presently, most models are based on genetic or dietary adaptations. With respect to the former, ob/ob mice lacking the leptin gene and Zucker fa/fa rats lacking the leptin receptor exhibit hyperphagia resulting in obesity, insulin resistance, and steatosis. However, the drawback of these models includes the dissimilar etiology of steatosis induction vs. its clinical counterpart (i.e., genes vs. diet). Moreover, experimental studies show that liver regeneration in these models appears to be impaired not due to steatosis per se, but due to the leptin deficiency. A clinically representative model for experimental steatosis would thus be of great value. In 2004 Lieber et al. [[2]Lieber C.S. Leo M.A. Mak K.M. Xu Y. Cao Q. Ren C. et al.Model of nonalcoholic steatohepatitis.Am J Clin Nutr. 2004; 79: 502-509Crossref PubMed Scopus (361) Google Scholar] introduced a model using a liquid high-fat diet for the induction of NASH in Sprague–Dawley rats, referred to as the Lieber–Decarli (LDC) diet. Within 3 weeks the animals purportedly developed panlobular steatosis with inflammatory foci, in association with insulin resistance and increased hepatic TNF-α levels and lipid peroxidation products, i.e., clinical features of NASH. In a recent study published in this Journal, Lieber et al. [[3]Lieber C.S. Decarli L.M. Leo M.A. Mak K.M. Ponomarenko A. Ren C. et al.Beneficial effects versus toxicity of medium-chain triacylglycerols in rats with NASH.J Hepatol. 2008; 48: 318-326Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar] used medium chain triglycerides to prevent LDC diet-induced NASH. In this letter we would like to address several irreproducibility issues regarding the LDC diet and its solid high-fat equivalent. We have attempted to set up the LDC model in Wistar rats following the authors' exact methodology [[2]Lieber C.S. Leo M.A. Mak K.M. Xu Y. Cao Q. Ren C. et al.Model of nonalcoholic steatohepatitis.Am J Clin Nutr. 2004; 79: 502-509Crossref PubMed Scopus (361) Google Scholar]. However, after a 3-week LDC diet none of the livers (n = 5) showed any histological evidence of steatosis (Fig. 1A). Our findings were recently corroborated by Akin et al. [[4]Akin H. Deniz M. Tahan V. Can G. Kedrah A E. Celikel C. et al.High-fat liquid "Lieber–DeCarli" diet for an animal model of nonalcoholic steatohepatitis: does it really work?.Hepatol Internat. 2007; 23;1: 449-450Crossref Google Scholar] in Sprague–Dawley rats, where only 20% developed 1–2% microvesicular steatosis after a 3-week LDC diet (Fig. 1B). In both studies, animals had gained significantly less weight (98 and 76 g, respectively) after 3 weeks than the animals in the original study (173 g). Alternatively, we attempted to induce steatosis with a solid high-fat diet in accordance with Svegliati-Baroni et al. [[5]Svegliati-Baroni G. Candelaresi C. Saccomanno S. Ferretti G. Bachetti T. Marzioni M. et al.A model of insulin resistance and nonalcoholic steatohepatitis in rats: role of peroxisome proliferator-activated receptor-alpha and n-3 polyunsaturated fatty acid treatment on liver injury.Am J Pathol. 2006; 169: 846-860Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar]. A 3-, 5-, and 7-week diet resulted in a mean ± SD histological steatosis degree of 10 ± 5%, 5 ± 3%, and 10 ± 9%, respectively, in Wistar rats (n = 5/group) and 14 ± 11%, 2 ± 1%, and 0%, respectively, in Sprague–Dawley rats (n = 5/group). Next, the reproducibility of the LDC diet was evaluated against other studies referring to the LDC diet. Using the ISI Web of Science database (accessed 01/15/2008), 53 articles citing the original work were retrieved: 38 experimental studies, 9 reviews, 5 clinical studies, and 1 editorial paper. Of the 38 experimental studies, 6 articles (Fig. 1C) reported the use of the LDC diet, of which 2 actually followed its original experimental 3-week diet protocol; both by Lieber et al. [3Lieber C.S. Decarli L.M. Leo M.A. Mak K.M. Ponomarenko A. Ren C. et al.Beneficial effects versus toxicity of medium-chain triacylglycerols in rats with NASH.J Hepatol. 2008; 48: 318-326Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 6Lieber C.S. Leo M.A. Mak K.M. Xu Y. Cao Q. Ren C. et al.Acarbose attenuates experimental non-alcoholic steatohepatitis.Biochem Biophys Res Commun. 2004; 315: 699-703Crossref PubMed Scopus (42) Google Scholar]. In another study by Lieber et al. [[7]Lieber C.S. Leo M.A. Cao Q. Mak K.M. Ren C. Ponomarenko A. et al.The combination of S-adenosylmethionine and dilinoleoylphosphatidylcholine attenuates non-alcoholic steatohepatitis produced in rats by a high-fat diet.Nutr Res. 2007; 27: 565-573Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar] the diet was employed during 3 and 6 weeks. Interestingly, histological steatosis degrees were not reported in any of the studies by Lieber et al. Yalniz et al. [[8]Yalniz M. Bahcecioglu I.H. Kuzu N. Celebi S. Ataseven H. Ustundag B. et al.Amelioration of steatohepatitis with pentoxifylline in a novel nonalcoholic steatohepatitis model induced by high-fat diet.Dig Dis Sci. 2007; 52: 2380-2386Crossref PubMed Scopus (31) Google Scholar] used a solid high-fat diet with the same fat composition for 4 weeks, resulting in mild steatosis, whereas Wang et al. [[9]Wang Y. Ausman L.M. Russell R.M. Greenberg A.S. Wang X.D. Increased apoptosis in high-fat diet-induced nonalcoholic steatohepatitis in rats is associated with c-Jun NH2-terminal kinase activation and elevated proapoptotic Bax.J Nutr. 2008; 138: 1866-1871Crossref PubMed Scopus (74) Google Scholar] employed the LDC diet for 6 weeks and observed mild steatosis. Thus, albeit frequently cited, the original LDC protocol for NASH induction is in fact sparsely employed. Instead, the LDC diet is either completely modified to achieve a greater degree of steatosis or applied for longer time periods to induce mild steatosis. These findings make the utility of liquid and solid high-fat diets for NASH-related studies highly questionable. Furthermore, interventional studies [[3]Lieber C.S. Decarli L.M. Leo M.A. Mak K.M. Ponomarenko A. Ren C. et al.Beneficial effects versus toxicity of medium-chain triacylglycerols in rats with NASH.J Hepatol. 2008; 48: 318-326Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar] in this model are to be interpreted with due caution. It is well known that mild steatosis (0–33%), as was observed in the studies listed in Fig. 1C, have no major clinical implications in contrast to moderate (33–66%) or severe (>66%) steatosis. Consequently, it is paramount to use experimental models that produce high degrees of histological steatosis in order to adequately test therapeutic and nutritional regimes or surgical interventions. Unfortunately, no reports exist achieving clinically relevant high degrees of steatosis with liquid or solid high-fat diets. In the final analysis, a reproducible and effective means is needed to induce clinically relevant NASH. A suitable method comprises the methionine–choline-deficient (MCD) diet. This diet metabolically deprives the liver from crucial components required for the synthesis of very low density lipoprotein, which is responsible for triglyceride export from the liver [[1]Vetelainen R. van Vliet A. Gouma D.J. van Gulik T.M. Steatosis as a risk factor in liver surgery.Ann Surg. 2007; 245: 20-30Crossref PubMed Scopus (218) Google Scholar]. In contrast to the LDC diet, the MCD diet is highly efficient, resulting in mild steatosis after 1–2 weeks (Fig. 1D), moderate steatosis after 3 weeks, and severe steatosis after 5 weeks (Fig. 1E). This model is also associated with inflammatory foci as well as increased TNF-α and lipid peroxidation levels. Most importantly, this model has been used by many different research groups, all reporting the same degrees of histological steatosis, and has therefore proven to be highly reproducible. In conclusion, the difficulties with reproducing high-fat steatosis models is underexposed in literature and should be taken into account when performing experimental NAFLD studies.