A male mouse model for metabolic dysfunction-associated steatotic liver disease and hepatocellular carcinoma

Abstract

The lack of an appropriate preclinical model of metabolic dysfunction-associated steatotic liver disease (MASLD) that recapitulates the whole disease spectrum impedes exploration of disease pathophysiology and the development of effective treatment strategies. Here, we develop a mouse model (Streptozotocin with high-fat diet, STZ + HFD) that gradually develops fatty liver, metabolic dysfunction-associated steatohepatitis (MASH), hepatic fibrosis, and hepatocellular carcinoma (HCC) in the context of metabolic dysfunction. The hepatic transcriptomic features of STZ + HFD mice closely reflect those of patients with obesity accompanying type 2 diabetes mellitus, MASH, and MASLD-related HCC. Dietary changes and tirzepatide administration alleviate MASH, hepatic fibrosis, and hepatic tumorigenesis in STZ + HFD mice. In conclusion, a murine model recapitulating the main histopathologic, transcriptomic, and metabolic alterations observed in MASLD patients is successfully established.

Fig. 1. HFD feeding after STZ administration induces the whole spectrum of MASLD.

a–n Seven-week-old B6J mice were treated with STZ and fed SCD (STZ + SCD) or HFD (STZ + HFD) for 6-60 weeks. a Representative liver gross image of STZ-treated mice. Scale bar, 1 cm. b Liver weight; Before STZ treatment, n = 10; STZ + SCD, n = 5, 6, 6, 5, 10, and 6, from 14 to 68-week-old, respectively; STZ + HFD, n = 7, 9, 9, 6, 9, and 5, from 14 to 68-week-old, respectively. c Hepatic triglyceride levels; Before STZ treatment, n = 4; STZ + SCD, n = 4, 6, 6, 5, 5, and 4, from 14 to 68-week-old, respectively; STZ + HFD, n = 6, 5, 4, 5, 6, and 4, from 14 to 68-week-old, respectively. Representative liver histology evaluated via (d) H&E staining and (e) Masson’s trichrome staining for hepatic fibrosis in STZ-treated mice. Blue arrows indicate ballooning degeneration of hepatocytes. Red circles signify perisinusoidal fibrosis. The data are representative results of the biological replicates shown in f and g. Scale bar, 100 μm. f, g Nonalcoholic fatty liver disease score (NAS) and fibrosis stage graded using the NASH CRN scoring system in STZ-treated mice; Before STZ treatment, n = 10; STZ + SCD, n = 10, 6, 6, 5, 3, and 3, from 14 to 68-week-old, respectively; STZ + HFD, n = 7, 10, 10, 6, 9, and 6, from 14 to 68-week-old, respectively. Plasma (h) AST and (i) ALT levels; Before STZ treatment, n = 10; STZ + SCD, n = 10, 6, 6, 5, 5, and 6, from 14 to 68-week-old, respectively; STZ + HFD, n = 7, 10, 10, 6, 9, and 6, from 14 to 68-week-old, respectively. Scale bar, 100 μm. j–l Hepatic lipid droplets (red, k) and collagen fibrils (green, l) of STZ + HFD mice assessed via in vivo liver imaging; n = 5, 4, 5, 5, 5, 4, and 4, from 7 to 68-week-old, respectively. Hepatic tumor (m) incidence and (n) number in STZ + HFD mice; n = 10, 7, 10, 8, 10, 8, 6, 6, 9, and 6, from 7 to 68-week-old, respectively. o Comprehensive MASLD progression overview. Data are expressed as means ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001, compared with the age-matched group, Student’s t-test or Welch’s t-test (b, c, and f–i). ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.000, compared with the 7-week-old control group, two-sided Student’s t-test or Welch’s t-test (b, c, f–I, k, l, n). Source data are provided as a Source Data file.

Fig. 2. Molecular characteristics of MASH progression in STZ + HFD mice.

a–f Liver RNA-seq profiling of 7- to 56-week-old STZ + HFD mice. a Principal component analysis of RNA-seq data showing transcriptomic changes over time (red arrow). b K-means clustering of differentially expressed genes from 7- to 32-week-old STZ + HFD mice. Functional enrichment analyses using EnrichR with c KEGG and d GO biological process pathways for each gene cluster. Benjamini-Hochberg method and Fisher’s exact test were used. e Volcano plot comparing 32-week-old STZ + HFD mouse liver samples with 14 to 20-week-old mouse liver samples using DESeq2. Benjamini-Hochberg method and Fisher’s exact test were used. f Heatmap displaying significant molecular events during MASLD progression in STZ + HFD mice. g Submap clustering analyses with Fisher’s exact test comparing liver RNA-seq data from STZ + HFD mice and human MASLD patients at different NAS/fibrosis stages. Hierarchical clustering with a STRING-based protein interaction network of mutually dysregulated genes in human patients and STZ + HFD mouse liver samples during (h) early (human: NAS 5-6, fibrosis stage 1 versus healthy donors; mouse: 14 to 20-weeks-old STZ + HFD mice versus 7-weeks-old control mice) and (i) late (human: NAS 4-7, fibrosis stage 3-4 versus NAS 5-6, fibrosis stage 1-2; mouse: 32-weeks-old versus 14 to 20-weeks-old STZ + HFD mice) MASLD progression. j Submap clustering analyses with Fisher’s exact test comparing liver RNA-seq data from STZ + HFD mice and human MASH patients with or without coexisting type 2 diabetes.

Fig. 3. Histopathological and molecular characteristics of hepatic tumors in STZ + HFD mice.

a–n Hepatic tumors originating from STZ + HFD mice resembling human steatohepatitic hepatocellular carcinoma (HCC) (a–c, k–n), steatohepatitic hepatocellular adenoma (HCA) (g–j), or conventional HCC (d–f, o–r). Representative (a) gross and (b, c) microscopic H&E images of hepatic tumor resembling steatohepatitic HCC (Scale bar: a; 1 cm, b, c; 100 μm). The red circle indicates tumor. The data are representative of 6 independent biological replicates. Representative (d) gross and (e, f) microscopic images of hepatic tumor resembling conventional HCC (Scale bar: d; 1 cm, e, f; 100 μm). The red circle indicates tumor. The data are representative of 8 independent biological replicates. Immunohistochemical and histochemical staining of hepatic tumor resembling (g–j) steatohepatitic HCA, (k–n) steatohepatitic HCC, or (o–r) conventional HCC (g, k, o; Reticulin, h, l, p; Glutamine synthase (GS), i, m, q; Glypican-3 (GPC-3), j, n, r; Masson’s trichrome (MT), scale bar: 100 μm). The data are representative of (g–j) 11, (k–n) 6, or (o–r) 8 independent biological replicates. s–v Hepatic tumor and non-tumor liver tissues of 44 to 56-week-old STZ + HFD mice subjected to RNA-seq. s Principal component analyses of RNA-seq data, along with representative H&E images of different tumor groups (Scale bar:100 μm). t Heatmap displaying the top differentially enriched pathways in different tumor groups. u Submap clustering analyses with Fisher’s exact test comparing RNA-seq data of hepatic tumor and non-tumor liver tissues from STZ + HFD mice and human MASLD-related HCC patients (GSE164760). v Submap clustering analyses with Fisher’s exact test comparing RNA-seq data of hepatic tumor and non-tumor liver tissues from STZ + HFD mice and human HCC patients with viral or alcoholic hepatitis (GSE63898).

Fig. 4. Dietary changes ameliorate MASH, hepatic fibrosis, and hepatic tumorigenesis in STZ + HFD mice.

a–h Seven-week-old B6J mice were treated with STZ, fed HFD from 8 to 20 weeks, followed by HFD or SCD (HFD → SCD) until 26 weeks. a Schematic figure. b Liver weight; STZ + HFD, n = 8; HFD → SCD, n = 7. c Hepatic triglyceride levels; STZ + HFD, n = 7; HFD → SCD, n = 6. d Representative liver histology assessed via H&E staining (scale bar, 100 μm). e NAS grading using the NASH CRN scoring system; STZ + HFD, n = 8; HFD → SCD, n = 7. f Representative liver histology with Masson’s trichrome staining (scale bar, 100 μm). g Fibrosis stage grading with the NASH CRN scoring system; STZ + HFD, n = 8; HFD → SCD, n = 7. h Heatmap displaying the top differentially enriched pathways between two groups. i–s Seven-week-old B6J mice were treated with STZ and fed HFD from 8 to 33 weeks, followed by administration of HFD or SCD (HFD → SCD) until 51 weeks. i Schematic figure. j Liver weight; STZ + HFD, n = 8; HFD → SCD, n = 9. k Hepatic triglyceride levels; n = 8 per group. l Representative liver histology evaluation via H&E staining (Scale bar, 100 μm). m NAS grading based on the NASH CRN scoring system; STZ + HFD, n = 8; HFD → SCD, n = 9. n Representative liver histology via Masson’s trichrome staining (Scale bar, 100 μm). o Fibrosis stage grading via the NASH CRN scoring system; STZ + HFD, n = 8; HFD → SCD, n = 9. p Representative liver gross image (Scale bar, 1 cm). Red circles indicate hepatic tumors. Hepatic tumor (q) incidence and (r) number; STZ + HFD, n = 8; HFD → SCD, n = 9. s Heatmap displaying top differentially enriched pathways between two groups. Data are expressed as means ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001, compared with age-matched group, two-sided Student’s t-test or Welch’s t-test (b, c, e, g, j, k, m, o, r). Source data are provided as a Source Data file.

Fig. 5. Tirzepatide ameliorates MASH, hepatic fibrosis, and hepatic tumorigenesis in STZ + HFD mice.

a–l Seven-week-old B6J mice were treated with STZ, fed HFD from 8 weeks onwards, and administered vehicle or tirzepatide for 10–11 weeks from 21, 28, or 41 weeks of age. a Schematic illustration. b Liver weight; Vehicle, n = 6, 7, and 5, from 21–32 to 41–52 weeks, respectively; Tirzepatide, n = 6, 7, and 7, from 21–32 to 41–52 weeks, respectively. c Representative liver histology based on H&E staining (Scale bar, 100 μm). d NAFLD activity score grading using the NASH CRN scoring system; Vehicle, n = 6, 6, and 5, from 21–32 to 41–52 weeks, respectively; Tirzepatide, n = 6, 7, and 7, from 21–32 to 41–52 weeks, respectively. e Representative liver histology determined via Masson’s trichrome staining (Scale bar, 100 μm). f Fibrosis stage grading using the NASH CRN scoring system; Vehicle, n = 6, 6, and 5, from 21–32 to 41–52 weeks, respectively; Tirzepatide, n = 6, 7, and 7, from 21-32 to 41–52 weeks, respectively. g Representative liver gross images from STZ + HFD mice treated with vehicle or tirzepatide at 28–38 or 41–52 weeks of age (Scale bar, 1 cm). Hepatic tumor (h) incidence and (i) number of STZ + HFD mice treated with vehicle or tirzepatide at 28–38 or 41–52 weeks of age; Vehicle, n = 6 per group; Tirzepatide, n = 7 per group. j Principal component analyses of RNA-seq data. k Functional enrichment analyses using KEGG pathways for differentially expressed genes between age-matched groups subjected to tirzepatide treatment and l heatmap displaying the top enriched genes. Benjamini-Hochberg method and Fisher’s exact test were used. Data are expressed as means ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001, compared with age-matched group, two-sided Student’s t-test or Welch’s t-test (b, d, f, i). Source data are provided as a Source Data file.