A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer - PubMed (original) (raw)

doi: 10.1016/j.jhep.2016.05.005. Epub 2016 May 31.

Sophie C Cazanave 2, Tommy Pacana 1, Mulugeta Seneshaw 1, Robert Vincent 1, Bubu A Banini 1, Divya Prasanna Kumar 1, Kalyani Daita 1, Hae-Ki Min 1, Faridoddin Mirshahi 1, Pierre Bedossa 3, Xiaochen Sun 4, Yujin Hoshida 4, Srinivas V Koduru 5, Daniel Contaifer Jr 6, Urszula Osinska Warncke 7, Dayanjan S Wijesinghe 7, Arun J Sanyal 8

Affiliations

A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer

Amon Asgharpour et al. J Hepatol. 2016 Sep.

Abstract

Background & aims: The lack of a preclinical model of progressive non-alcoholic steatohepatitis (NASH) that recapitulates human disease is a barrier to therapeutic development.

Methods: A stable isogenic cross between C57BL/6J (B6) and 129S1/SvImJ (S129) mice were fed a high fat diet with ad libitum consumption of glucose and fructose in physiologically relevant concentrations and compared to mice fed a chow diet and also to both parent strains.

Results: Following initiation of the obesogenic diet, B6/129 mice developed obesity, insulin resistance, hypertriglyceridemia and increased LDL-cholesterol. They sequentially also developed steatosis (4-8weeks), steatohepatitis (16-24weeks), progressive fibrosis (16weeks onwards) and spontaneous hepatocellular cancer (HCC). There was a strong concordance between the pattern of pathway activation at a transcriptomic level between humans and mice with similar histological phenotypes (FDR 0.02 for early and 0.08 for late time points). Lipogenic, inflammatory and apoptotic signaling pathways activated in human NASH were also activated in these mice. The HCC gene signature resembled the S1 and S2 human subclasses of HCC (FDR 0.01 for both). Only the B6/129 mouse but not the parent strains recapitulated all of these aspects of human NAFLD.

Conclusions: We here describe a diet-induced animal model of non-alcoholic fatty liver disease (DIAMOND) that recapitulates the key physiological, metabolic, histologic, transcriptomic and cell-signaling changes seen in humans with progressive NASH.

Lay summary: We have developed a diet-induced mouse model of non-alcoholic steatohepatitis (NASH) and hepatic cancers in a cross between two mouse strains (129S1/SvImJ and C57Bl/6J). This model mimics all the physiological, metabolic, histological, transcriptomic gene signature and clinical endpoints of human NASH and can facilitate preclinical development of therapeutic targets for NASH.

Keywords: Drug therapy; Fibrosis; Hepatocellular carcinoma; Hepatocyte ballooning; Steatosis.

Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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Conflict of interest statement

Dr. Sanyal reports grants from NIH, during the conduct of the study; In addition, Dr. Sanyal has a patent VCU pending and Virginia Commonwealth University and Dr. Sanyal has established a biotechnology company (SanyalBio) which may test compounds being evaluated for NASH in the future. All the other authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Figures

Fig. 1

Fig. 1. DIAMOND mice develop obesity, liver injury, dyslipidemia and insulin resistance

B6/129 mice were fed a chow diet (CD NW) or high fructose/glucose, high fat Western Diet (WD SW) for up to 52 weeks. (A) Body weight change over time, (B) Liver weight, (C) serum ALT and AST levels, (D) serum cholesterol, LDL-c and triglycerides levels, (E) Insulin tolerance test (ITT) and (F) glucose tolerance test (GTT). Data are expressed as the mean ± SEM for 6–10 mice per group;*p <0.05 and **p <0.001, WD SW compared to CD NW.

Fig. 2

Fig. 2. DIAMOND mice sequentially develop a fatty liver, steatohepatitis, advanced fibrosis and liver tumors

(A) Gross liver from B6/129 mice fed a chow diet (CD NW) or high fructose/glucose, high fat Western Diet (WD SW) for 8 (a, b), 16–24 (c, d) and 52 weeks (e, f). In mice fed a high fat Western Diet for 52 weeks, multiple foci of tumors were observed at the time of necropsy (h–j) as compared to CD NW mice (g); areas of hemorrhage were seen in larger tumors (i). (B) Microscopic views of livers from CD NW or WD SW mice at 8 (a, d, g, j), 16–24 (b, e, h, k) or 52 weeks (c, f, i, l) of diet. Representative liver sections stained with hematoxylin-eosin (H&E) (a–f) or Picrosirius Red (g–l) are shown. Original magnification, ×20. (C) Histology score for steatosis, hepatocyte ballooning, lobular inflammation, NAFLD Activity Score and fibrosis were quantified. Data are expressed as the mean ± SEM for 6–10 mice per group; *p <0.05. (D) Representative images of hematoxylin-eosin (H&E) (a, c–e) and CK-18 (b) staining of liver tissue from mice fed a WD SW for 52 weeks depicting the individual component of steatohepatitis (as indicated by large arrow): (a) hepatocyte ballooning, (c) Mallory-Denk bodies (MDB), (d) lobular inflammation and (e) apoptotic bodies. (b) A marked CK-18 staining is present in the cytoplasm of normal hepatocytes (as indicated by small arrow), whereas in ballooned hepatocyte (as indicated by large arrow), a reduction (almost a complete loss) in CK-18 staining in their cytoplasm is noted. Lipid vacuoles are indicated by a star. Original magnification, ×40.

Fig. 3

Fig. 3. Main histological features of DIAMOND mice are comparable to human NASH

Representative images of liver histology from DIAMOND mice (a,c,e,g,i,k) or human NASH (b, d, f, h, j, l) depicting steatosis (a, b; H&E; original magnification, ×5), Hepatocyte ballooning (c, d; H&E; original magnification, ×40), Mallory-Denk bodies (as indicated by large arrow) (e, f; H&E; original magnification, ×40), Stage 1 fibrosis (g, h; Sirius Red; original magnification, ×20), Stage 2 fibrosis (i, j; Sirius Red; original magnification, ×20), Stage 3 fibrosis (k, l; Sirius Red; original magnification, ×10). CV, central vein; PT, portal tract.

Fig. 4

Fig. 4. Activation of signaling pathways relevant to human NASH in the liver of DIAMOND mice

(A) Whole cell lysates were prepared from liver tissue from B6/129 mice fed a chow diet (CD NW) or high fructose/glucose, high fat Western Diet (WD SW) for 8 or 52 weeks. Immunoblot analysis were performed for FAS-N, acetyl-CoA carboxylase (ACC), phosphorylated and total JNK (p-JNK and t-JNK), phosphorylated and total p42/p44 (p-p42/p44 and t-p42/p44), PUMA, BIM, PARP displaying cleaved PARP product p89 and caspase-3 (C3) displaying cleaved caspase-3 product p18. The cleaved form of C3 was only visualized after long exposure times. β-actin was used as a control for protein loading. Bands were cut and combined from the same radiograph. (B–F) Transcriptome analysis was performed on liver tissues from CD NW or WD SW mice after 8 weeks of diet (n = 5 per group). The data are presented as: (B) volcano plot; (C) Heat map demonstrating deregulated genes. Red and blue colors indicate high and low gene expression, respectively; (D) Gene ontology (GO) processes; (E) Gene set Enrichment Analysis (GSEA); and (F) Process Networks analysis. The top rank ordered processes, maps and networks are based on statistical significance.

Fig. 5

Fig. 5. Hepatic gene expression dataset in DIAMOND mice at 52 weeks concords with a human liver cirrhosis and NASH-associated gene signature

Transcriptome analysis was performed on liver tissues from B6/129 mice fed a chow diet (CD NW) or a high fructose/glucose, high fat Western diet (WD SW) mice for 52 weeks (A–F) and 8 weeks (E) (n = 5 per group). (A) Gene ontology (GO) processes; (B) Gene set Enrichment Analysis (GSEA); (C) Process Networks analysis. The top rank ordered processes, maps and networks are based on statistical significance; (D) Heat map demonstrating deregulated genes. Red and blue colors indicate high and low gene expression, respectively. (E) Similarity between global liver transcriptome of mice fed a WD SW for 8 or 52 weeks and global liver transcriptome in liver biopsy tissues from 18 human NASH patients and 41 normal/healthy obese individuals using subclass mapping algorithm (see Supplementary materials and methods). Numbers on heat map indicate FDR values for the transcriptome similarity. (F) Concordance by GSEA between a 186-gene signature prognostic (73 poor prognosis-correlated and 113 good prognosis-correlated genes) in human liver cirrhosis and HCC from mixed etiologies and the pattern of gene expression in DIAMOND mice at 52 weeks NES, normalized enrichment score; FDR, false discovery rate.

Fig. 6

Fig. 6. Tumors gene signature in DIAMOND mice at 52 weeks

(A) Microscopic views of adenomas (a, b, c) and hepatocarcinomas (HCC) tumors (d, e, f) from B6/129 mice fed a high fructose/glucose, high fat Western diet (WD SW) for 52 weeks. (a) adenoma, (hematoxylin-eosin (H&E), original magnification, ×2.5); (b) cords of hepatocytes with mild atypia and trabecular organization (H&E, original magnification, ×20); (c) unpaired artery between hepatocytes with mild anisocaryosis (H&E, original magnification, ×40); (d) a basophilic well-demarcated tumor with a satellite nodule and with steatosis in the background liver (H&E, original magnification, ×2); (e) interface between malignant tumor (top half) and non-tumoral liver. The lobules of tumoral cells show marked anisocaryosis, eosinophilic cytoplasm, irregular basophilic nuclei and loss of sinusoidal architecture (H&E, original magnification, ×40); (f) satellite nodules made of clusters of tumoral cells (white arrow) and dysplastic foci with multinucleated irregular hepatocytes (black arrow) (H&E, original magnification, ×20). (B) Concordance by gene set enrichment analysis (GSEA) between the gene signatures of human HCC subclasses S1, S2, and S3 and WD SW-induced HCCs in mice at 52 weeks. NES, normalized enrichment score; FDR, false discovery rate. (C–G) Transcriptome analysis was performed on liver adjacent to tumors (WD SW) or tumor tissue (HCC) from WD SW mice at 52 weeks (n = 5). (C) volcano plot; (D) Heat map demonstrating deregulated genes. Red and blue colors indicate high and low gene expression, respectively; (E) Gene ontology (GO) processes; (F) Gene set Enrichment Analysis (GSEA); and (G) Process Networks analysis. The top rank ordered processes, maps and networks are based on statistical significance.

Fig. 7

Fig. 7. Only the B6/129 background mice, but not the parent strains C57BL/6J (B6) or 129 S1/SvImJ (S129) fed a high fructose/glucose, high fat Western diet (WD SW) for 16–22 weeks depicts the full phenotypic, biological and histological parameters associated with human NAFLD

C57BL/6J (B6), 129 S1/SvImJ (S129) or B6/129 mice were fed for 16–22 weeks either a chow diet (CD NW) or a high fructose/sucrose, high fat Western diet (WD SW). (A) Gross liver pictures of WD SW-fed mice for 16–22 weeks, (B) insulin tolerance test (ITT), (C) representative liver sections stained with hematoxylin-eosin (H&E) (a, c, e) or Picrosirius Red (b, d, f) are shown (original magnification, ×10), (D) Histology score for steatosis, hepatocyte ballooning, lobular inflammation, NAFLD activity score, fibrosis and steatosis-activity-fibrosis score. Data represent mean ±SEM for 4–6 mice per CD NW group and 7–13 mice per WD SW group; ***p<0.001, **p <0.01, *p<0.05 WD SW B6/129 vs. WD SW B6 or WD SW S129. (E) Liver n3- and n6-polyunsaturated fatty acids (PUFAs) were measured from B6/129 mice fed a chow diet (CD NW) or high fructose/glucose, high fat Western Diet (WD SW) for 8,16–24 and 52 weeks as described in the Supplementary materials and methods section. Data represent mean ± SEM for 4 mice per group; *p <0.05 WD SW vs. CD NW.

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