Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Jun 8, 2016; 8(16): 673-684
Published online Jun 8, 2016. doi: 10.4254/wjh.v8.i16.673
Obese diet-induced mouse models of nonalcoholic steatohepatitis-tracking disease by liver biopsy
Maria Nicoline Baandrup Kristiansen, Sanne Skovgård Veidal, Kristoffer Tobias Gustav Rigbolt, Kirstine Sloth Tølbøl, Jonathan David Roth, Jacob Jelsing, Niels Vrang, Michael Feigh
Maria Nicoline Baandrup Kristiansen, Sanne Skovgård Veidal, Kristoffer Tobias Gustav Rigbolt, Kirstine Sloth Tølbøl, Jacob Jelsing, Niels Vrang, Michael Feigh, Gubra Aps, 2970 Hørsholm, Denmark
Jonathan David Roth, Intercept Pharmaceuticals, Inc., San Diego, CA 9212, United States
Author contributions: Kristiansen MNB, Veidal SS, Rigbolt KTG, Tølbøl KS and Feigh M performed the experiments and analyzed the data; Rigbolt KTG performed the molecular investigations; Kristiansen MNB and Veidal SS performed the histological analysis; Veidal SS, Rigbolt KTG, Roth JD, Jelsing J, Vrang N and Feigh M designed and coordinated the research; Kristiansen MNB, Veidal SS, Rigbolt KTG, Tølbøl KS, Roth JD, Jelsing J, Vrang N and Feigh M wrote the paper.
Institutional review board statement: This study includes no data or material from patients. We confirm that all of the required permissions for this study were obtained from our local authorities as mentioned in the Institutional animal care and use committee statement.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Danish Committee for animal research and covered by a personal license for Jacob Jelsing (2013-15-2934-00784). All of the institutional and national guidelines for the care and use of laboratory animals were followed.
Conflict-of-interest statement: There are no patents, products in development or marked products to declare.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: Michael Feigh, PhD, Gubra Aps, Hørsholm Kongevej 11B, 2970 Hørsholm, Denmark.
Telephone: +45-31522651
Received: February 11, 2016
Peer-review started: February 12, 2016
First decision: March 9, 2016
Revised: April 1, 2016
Accepted: April 20, 2016
Article in press: April 22, 2016
Published online: June 8, 2016

AIM: To characterize development of diet-induced nonalcoholic steatohepatitis (NASH) by performing liver biopsy in wild-type and genetically obese mice.

METHODS: Male wild-type C57BL/6J (C57) mice (DIO-NASH) and male Lepob/Lepob (ob/ob) mice (ob/ob-NASH) were maintained on a diet high in trans-fat (40%), fructose (22%) and cholesterol (2%) for 26 and 12 wk, respectively. A normal chow diet served as control in C57 mice (lean chow) and ob/ob mice (ob/ob chow). After the diet-induction period, mice were liver biopsied and a blinded histological assessment of steatosis and fibrosis was conducted. Mice were then stratified into groups counterbalanced for steatosis score and fibrosis stage and continued on diet and to receive daily PO dosing of vehicle for 8 wk. Global gene expression in liver tissue was assessed by RNA sequencing and bioinformatics. Metabolic parameters, plasma liver enzymes and lipids (total cholesterol, triglycerides) as well as hepatic lipids and collagen content were measured by biochemical analysis. Non-alcoholic fatty liver disease activity score (NAS) (steatosis/inflammation/ballooning degeneration) and fibrosis were scored. Steatosis and fibrosis were also quantified using percent fractional area.

RESULTS: Diet-induction for 26 and 12 wk in DIO-NASH and ob/ob-NASH mice, respectively, elicited progressive metabolic perturbations characterized by increased adiposity, total cholesterol and elevated plasma liver enzymes. The diet also induced clear histological features of NASH including hepatosteatosis and fibrosis. Overall, the metabolic NASH phenotype was more pronounced in ob/ob-NASH vs DIO-NASH mice. During the eight week repeated vehicle dosing period, the metabolic phenotype was sustained in DIO-NASH and ob/ob-NASH mice in conjunction with hepatomegaly and increased hepatic lipids and collagen accumulation. Histopathological scoring demonstrated significantly increased NAS of DIO-NASH mice (0 vs 4.7 ± 0.4, P < 0.001 compared to lean chow) and ob/ob-NASH mice (2.4 ± 0.3 vs 6.3 ± 0.2, P < 0.001 compared to ob/ob chow), respectively. Furthermore, fibrosis stage was significantly elevated for DIO-NASH mice (0 vs 1.2 ± 0.2, P < 0.05 compared to lean chow) and ob/ob NASH (0.1 ± 0.1 vs 3.0 ± 0.2, P < 0.001 compared to ob/ob chow). Notably, fibrosis stage was significantly (P < 0.001) increased in ob/ob-NASH mice, when compared to DIO-NASH mice.

CONCLUSION: These data introduce the obese diet-induced DIO-NASH and ob/ob-NASH mouse models with biopsy-confirmed individual disease staging as a preclinical platform for evaluation of novel NASH therapeutics.

Keywords: Nonalcoholic steatohepatitis, Liver biopsy, Diet-induced obesity, Nonalcoholic fatty liver disease, Fibrosis

Core tip: We characterize the development and progression of diet-induced nonalcoholic steatohepatitis (NASH) in a wild-type and a genetically obese mouse model. We confirm that a diet high in trans-fat, fructose and cholesterol, develops key histological hallmarks of NASH (steatosis, inflammation, ballooning degeneration) in conjunction with fibrosis. Concomitantly, marked alterations in NASH associated gene expression pathways can be evaluated by RNAseq analysis. In addition, we describe that performing a baseline liver biopsy enables individual disease staging for subsequent stratified randomization of animals into study groups. Finally, we show these models′ utility for a chronic repeated dosing study to evaluate pharmacological intervention.