A preclinical model of severe NASH-like liver injury by chronic administration of a high-fat and high-sucrose diet in mice

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease, affecting 32% of adults globally. If left untreated, NAFLD may progress to more advanced forms of the disease, including non-alcoholic steatohepatitis (NASH), liver cirrhosis, and fibrosis. Early NAFLD detection is critical to prevent disease progression. Using an obesogenic high-fat and high-sucrose (HF/HS) diet, we characterized the progression of NAFLD in male and female Collaborative Cross CC042 mice at 20-, 40-, and 60-week intervals of chronic HF/HS diet feeding. The incidence and severity of liver steatosis, inflammation, and fibrosis increased in both sexes over time, with male mice progressing to a NASH-like disease state faster than female mice, as indicated by earlier and more pronounced changes in liver steatosis. Histopathological indication of macrovesicular steatosis and gene expression changes of key lipid metabolism genes were found to be elevated in both sexes after 20 weeks of HF/HS diet. Measurement of circulating markers of inflammation (CXCL10 and TNF-α), histopathological analysis of immune cell infiltrates, and gene expression changes in inflammation-related genes indicated significant liver inflammation after 40 and 60 weeks of HF/HS diet exposure in both sexes. Liver fibrosis, as assessed by Picosirius red and Masson’s trichrome staining and changes in expression of key fibrosis related genes indicated significant changes after 40 and 60 weeks of HF/HS diet exposure. In conclusion, we present a preclinical animal model of dietary NAFLD progression, which recapitulates human pathophysiological and pathomorphological changes, that could be used to better understand the progression of NAFLD and support development of new therapeutics.

Introduction

The prevalence of nonalcoholic fatty liver disease (NAFLD) is continuing to increase worldwide^{1, 2} and parallels the growing epidemic of obesity³. A recently conducted meta-analysis study of NAFLD prevalence shows that one-third of the world’s population has NAFLD, and its rate has increased by 13%, rising from 25% to 38% over the past three decades². This signals an urgent need for a better understanding of the underlying mechanisms associated with NAFLD development and progression, which is critical for effective disease prevention and treatment.

The pathogenesis of NAFLD is a complex heterogenous process of dysregulated metabolic, cellular, and molecular events⁴. Although the hepatic pathomorphological features of different NAFLD forms, such as simple hepatic steatosis or nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), NASH-fibrosis, and NASH-cirrhosis, are well established and characterized^{5, 6}, the molecular alterations driving disease progression are less understood. Investigating these NAFLD-related molecular alterations in humans only after the occurrence of clinical liver-related changes provides a snapshot of disease-specific stage alterations rather than their dynamics. This approach identifies general specific abnormalities at each NAFLD stage but does not uncover the role of these abnormalities in the disease pathogenesis. An incomplete understanding of the mechanisms driving the progression of NAFLD from early, generally benign disease stages, toward advanced life-threatening NAFLD phenotypes, hampers the accurate detection/staging of the disease, as well as slows the development of the mechanism based NAFLD pharmacological treatments and preventive approaches. In this respect, relevant animal models exhibiting similar pathohistological and pathophysiological characteristics of human NAFLD^{7, 8} have been shown to be useful preclinical tools to investigate the pathogenesis of NAFLD and overcome the limitations of human NAFLD studies.

In a previous study⁹, we demonstrated that maintaining 25 male and 24 female strains of Collaborative Cross (CC) mice on an obesogenic high-fat and high-sucrose (HF/HS) diet for 12 weeks resulted in the development of NAFL in all animals; however, the severity was strain- and sex-dependent, with male CC042/GeniUnc (CC042) mice exhibiting the most pronounced histopathological effects marked by significant increases in liver and body weight, excessive liver lipid accumulation, and differential gene expression of key lipid regulating genes⁹. Based on these findings, the goal of the present study was to investigate molecular mechanisms linked to the progression of diet-induced NAFLD in male and female CC042 mice. To achieve this goal, we fed male and female CC042 mice a HF/HS diet and evaluated the serial physiological, histological, and molecular effects in the liver and serum after 20, 40, and 60 weeks of feeding.

Materials and Methods

Animals, diets, and experimental design

Male and female CC042 mice were obtained from the University of North Carolina Chapel Hill (Chapel Hill, NC, USA) and acclimated for one week upon arrival at the National Center for Toxicological Research (NCTR). The mice were housed in sterilized cages in a temperature-controlled room (24°C) with a 12-h light/dark cycle and ad libitum access to water and NIH-41 irradiated pelleted diet. At 8 weeks of age, mice were allocated randomly into either an experimental or control group (n = 18 per diet group per sex). Mice in the experimental group were maintained on a HF/HS diet (Teklad Custom Diet - TD.88137, Envigo, Indianapolis, IN, USA) for up to 60 weeks. This diet contained 42% kcal from fat (21.2% by weight sourced from milkfat high in palmitic acid including 0.2% by weight of total cholesterol), 42.7% kcal carbohydrates (sourced from 34% sucrose by weight), and 15.2% kcal protein (17.3% by weight). Mice in the control group received an ingredient-matched low-fat and reduced sucrose control diet (Teklad Custom Diet – TD.08485, Envigo) containing 13% kcal from fat and 12% kcal from sucrose. The body weights of the mice were recorded weekly. Mice were euthanized with isoflurane in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines 20, 40, and 60 weeks after diet initiation. The livers were excised, and a slice of the median lobe was fixed in neutral buffered formalin for 48 h for histopathological examination. The remaining liver was snap-frozen immediately in liquid nitrogen and stored at −80°C for subsequent analyses. All experimental procedures were reviewed and approved by the NCTR’s IACUC and conducted in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Blood biochemistry

Blood samples were collected in serum separator tubes by cardiac puncture at necropsy between 8:00 and 11:00 am. The blood was allowed to clot at room temperature and serum was obtained by centrifugation at 1000 x g for 10 min. Clinical chemistry analyses were conducted on the Randox Daytona+ (Randox Laboratories-US, Kearneysville, WV, USA). Randox reagents were used for glucose, alanine transaminase (ALT), aspartate transaminase (AST), cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). Insulin testing was performed using mouse-specific ELISA methods (ALPCO, Salem, NH, USA). The plates were read on an Epoch Universal Microplate Reader (Bio-Tek, Winooski, VT, USA). A standard curve was run with each batch of samples and results were calculated by the reader’s software.

Quantitation of serum proinflammatory cytokines

CXCL10 and TNF-α concentrations were quantified in 25 μL of undiluted mouse serum using Luminex ProcartaPlex multiplex immunoassay custom mix (PPX-36-MX323Z9: Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s protocol. The analyte data were acquired on a Luminex Intelliflex (Thermo Fisher Scientific), and concentrations were quantified using the Thermo Fisher Scientific ProcartaPlex analysis application.

Liver tissue processing and histological analysis

The section of the median lobe of the liver that had been fixed in 10% neutral-buffered formalin for 48 – 96 h was trimmed, processed, and embedded in infiltrating media Surgipath Formula R^® (Leica Biosystems, Richmond, IL, USA), sectioned at approximately 5 microns, mounted on a glass slide, and stained with hematoxylin and eosin. The hematoxylin and eosin-stained sections were evaluated for steatosis, inflammation, fibrosis, and glycogen-type hepatocellular cytoplasmic vacuolation. In addition, the hematoxylin and eosin-unstained liver sections were examined for lipid droplets using an osmium tetroxide staining method; images were obtained with an Aperio Scanscope System (Leica Biosystems, Vista, CA, USA). The Positive Pixel Count Algorithm (Positive Pixel Count Algorithm User’s Guide, Leica Biosystems) was followed to evaluate the osmium tetroxide-stained area. Settings of the algorithm were adjusted to recognize osmium tetroxide-stained lipid droplets as strong positive (dark brown) and negative (dark blue) pixels. The surrounding area was recognized as weak positive (orange) and positive (light brown) pixels. The osmium tetroxide-stained area (%) was calculated using the following formula:

$$\text{Osmium-Stained}\text{Area}(\%)=\frac{(\text{Strong}\text{positive}+\text{negative}\text{pixels})\times 100}{\text{Total}\text{number}\text{of}\text{pixels}}$$

where the total number of pixels was the sum of the strong positive, positive, weak positive, and negative pixels. The osmium-staining scores were checked against the pathologist’s evaluation, and the two were well-correlated.

Hepatic lipid fatty acid composition

The analysis of esterified lipid fatty acid composition in the livers of control mice and mice fed the HF/HS diet was conducted by ultra-high performance liquid chromatography/electrospray tandem mass spectrometry as described previously¹¹.

Quantitative reverse transcription polymerase chain reaction

Total RNA was extracted from liver tissue samples using miRNeasy Mini kits (Qiagen, Valencia, CA, USA). Total RNA (2 μg) was reverse transcribed using random primers and High-Capacity cDNA Reverse Transcription kits (Life Technologies, Grand Island, NY, USA). Gene expression was determined by quantitative reverse transcription polymerase chain reaction PCR (qRT-PCR) using the TaqMan gene expression assays that are listed in Supplementary Table 1. The glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene was used as an endogenous control. The relative amount of each mRNA transcript was determined with the 2^−ΔΔCt method¹².

Statistical analysis

The effect of a HF/HS diet on body weight, as a function of time, was assessed by a two-way repeated-measures mixed-effects model. When there was a significant diet effect, pairwise comparisons were conducted by a Holm-Sidak multiple comparisons test. Percentage changes in body and the liver weights, clinical chemistry data, and gene expression data were assessed by a two-way analysis of variance, using diet and time points as fixed factors. When necessary, the data were natural log transformed before the analysis to maintain an equal variance and/or normal data distribution. Histopathological incidence data were assessed by a one-tailed Fisher’s Exact test; histopathological severity data were assessed by a Mann-Whitney Rank Sum test. P-Values < 0.05 were considered significant.

Results

Chronic feeding a HF/HS diet results in obesity and metabolic dysfunction in male and female CC042 mice

Feeding CC042 mice the HF/HS diet significantly increased body weight in both male and female CC042 mice. In male mice fed a HF/HS diet, there was a significant increase in body weight gain beginning from week 5 to week 40 as compared to male mice fed the control diet. In HF/HS diet-fed and control male mice, body weight increases plateaued after 30–40 weeks, and there was no significant increase in body weight or percent weight gain found at 60 weeks in male mice. In contrast, the body weight in female mice steadily increased at all time points throughout the 60 weeks on the diet (Figures 1a and 1b). The body weight gain in female mice at 20, 40, and 60 weeks was 83%, 69%, and 104%, respectively, which was markedly greater than the body weight gain in male mice of 41%, 45%, and 17% at the same time points, (Figures 1c and 1d). Liver weight also increased at 20, 40, and 60 weeks in both sexes of mice (Figures 1e and 1f); however, when adjusted for changes in body weight, the increase in liver weight was only significant for male mice after feeding the HF/HS diet for 40 and 60 weeks.

Figure 1. Body and liver weight in CC042 mice fed a chronic HF/HS diet for 20, 40, or 60 weeks.

Average body weight of CC042 male (a) mice and female (b) mice fed a control diet or HF/HS diet for up to 60 weeks. Error bars denote standard error of the mean. * Denotes significant difference from control diet-fed mice determined by Holm-Sidak multiple comparisons test p<0.05. Average percent weight gain in CC042 male (c) and female (d) mice fed a control diet or HF/HS diet for 20, 40, and 60 weeks. Percent weight gain was calculated by subtracting body weight of individual animal at the beginning of the experiment (0 weeks) from the body weight of that animal at the specified time point. Average liver weight in CC042 male (e) and female (f) mice fed a control or HF/HS diet for 20, 40, or 60 weeks. Average ratio of body weight/liver weight in CC042 male (g) and female (h) mice fed a control of HF/HS diet for 20, 40, or 60 weeks. N=6/sex/diet/time point. Error bars denote standard deviation. * Denotes significant difference (p < 0.05) from control diet-fed mice at the same time point.

The increased body weight in HF/HS diet-fed male and female mice was accompanied by significant metabolic alterations indicative of liver injury, as evidenced by the elevated activity of ALT (Figure 2a), and AST (Figure 2b), which tended to increase with time. The levels of triglycerides (Figure 2c), glucose (Figure 2e), and insulin (Figure 2f) were not affected appreciably. In contrast, the levels of cholesterol (Figure 2d) and HDL-C (Figure 2g) in HF/HS diet-fed male and female mice were increased at 20, 40, and 60 weeks (only in female mice), whereas the levels of LDL-C (Figure 2h) were elevated at 40 and 60 weeks in both sexes of mice. Markers of inflammation were also measured in serum of mice fed the HF/HS or control diet. Feeding the HF/HS diet resulted in increased total circulating levels of CXCL10 in male mice at 20 and 40 weeks and in female mice at 40 and 60 weeks (Figure 2i). TNF-α levels were significantly increased in male mice only after 40 weeks of HF/HS diet (Figure 2j).

Figure 2. Progression of serum biochemical indicators of a NASH-like phenotype in CC042 mice after feeding a HF/HS diet.

Averages for the serum alanine transaminase (ALT) (a), aspartate transaminase (AST) (b), triglycerides (c), cholesterol (d), glucose (e), insulin (f), high-density lipoprotein (HDL-C) (g), low-density lipoprotein (LDL-C) (h), and inflammatory cytokines CXCL10 (i) and TNFα (j) in male and female mice fed control diet or HF/HS diet for 20, 40, or 60 weeks. N=6/sex/diet/time point. Error bars denote standard deviation. * Denotes significant difference (p < 0.05) from control diet-fed mice at the same time point.

Progressive NASH-like histopathological alterations in the livers of mice fed a HF/HS diet

Feeding male and female CC042 mice the HF/HS diet for up to 60 weeks resulted in the development of a NAFLD-like state that steadily progressed to a severe NASH-like disease, an advanced and life-threatening form of NAFLD, as evidenced by the presence of classic histopathological features of human NASH including steatosis, inflammation, and liver fibrosis (Table 1). Hematoxylin and eosin- and osmium tetroxide-stained liver sections (Table 1, Figure 3, and Supplementary Figure 1) revealed that the earliest and most prominent histological finding in male and female HF/HS diet-fed mice was macrovesicular steatosis, defined as large, single, or multiple intracytoplasmic clear vacuoles in enlarged cells with displaced nucleus (Figure 3, arrows). The incidence of macrovesicular steatosis at 20 weeks was similar in the livers of male and female mice; however, its severity in the livers of male mice at 20 weeks was more pronounced. Interestingly, the severity of macrovesicular steatosis at 40 weeks and, especially at 60 weeks, in the livers of male and female HF/HS diet-fed mice was similar. In control male and female mice, the incidence of macrovesicular steatosis increased with time, reaching its very high incidence at 60 weeks of the experiment (Table 1, Figure 3, and Supplementary Figure 1), but with a lower severity. Lobular and portal inflammation, another landmark feature of NASH, was evident in the livers of both male and female mice fed the HF/HS diet (Figure 3, arrowheads). The incidence of lobular inflammation was high at all time points, whereas portal inflammation increased with time on the HF/HS diet (Table 1). Another important histopathological finding was the development of hepatic fibrosis in the livers of both sexes of HF/HS diet-fed mice, with the incidence and severity increasing with time (Table 1, Supplementary Figure 2, arrows).

Table 1.

Summary of the type and extent of hepatic lesions in CC042 mice fed the control and high fat and high sucrose (HF/HS) diet for 20, 40 and 60 weeks^{^}.

Time on diet	20 weeks		40 weeks		60 weeks
Diet	Control	HF/HS diet	Control	HF/HS diet	Control	HF/HS diet
Males
Steatosis
Macrovesicular	0/6 (0.0)	5/6* (2.5*)	2/6 (0.0)	6/6 (3.0*)	5/5 (1.0)	6/6 (2.5)
Microvesicular	1/6 (0.0)	0/6 (0.0)	0/6 (0.0)	6/6* (1.0*)	1/5 (0.0)	2/6 (0.0)
Inflammation
Lobular	0/6 (0.0)	5/6 (1.0*)	2/6 (0.0)	6/6* (3.0*)	4/5 (1.0)	6/6 (2.0)
Portal	0/6 (0.0)	0/6 (0.0)	1/6 (0.0)	6/6* (1.5*)	1/5 (0.0)	4/6 (1.0)
Fibrosis	0/6 (0.0)	2/6 (0.0)	0/6 (0.0)	6/6* (2.0*)	1/5 (0.0)	6/6* (2.5*)
Vacuolation	6/6 (1.5)	5/6 (3.0)	4/6 (1.0)	6/6 (3.0*)	5/5 (4.0)	6/6 (4.0)
Females
Steatosis
Macrovesicular	0/6 (0.0)	5/6* (1.0*)	1/6 (0.0)	6/6* (2.0*)	4/5 (2.0)	6/6 (2.5)
Microvesicular	0/6 (0.0)	0/6 (0.0)	0/6 (0.0)	6/6* (1.0*)	0/5 (0.0)	0/6 (0.0)
Inflammation
Lobular	0/6 (0.0)	5/6 (1.0*)	3/6 (0.5)	6/6 (2.0*)	3/5 (1.0)	6/6 (1.0)
Portal	0/6 (0.0)	1/6 (0.0)	0/6 (0.0)	6/6* (1.0*)	1/5 (0.0)	6/6* (1.0*)
Fibrosis	0/6 (0.0)	3/6 (0.5)	0/6 (0.0)	5/6* (1.5*)	0/5 (0.0)	6/6* (3.0*)
Vacuolation	6/6 (3.0)	6/6 (4.0*)	6/6 (4.0)	6/6 (4.0)	5/5 (3.0)	6/6 (4.0)

The data are presented as the incidence and median severity.

^*p < 0.017

^{^}- Histological scoring of the severity of histological lesions was performed on a scale of “0–4”, where “0” – no significant findings; “1” – minimal alterations; “2” – mild alterations; “3” – moderate alterations, and “4” -severe alterations as described previously (10).

Figure 3. Progression of NASH-like histopathological alterations in the livers of CC042 mice fed a HF/HS diet.

Representative images of hematoxylin and eosin staining in liver sections in CC042 male (a), and female (b) mice fed a control diet or HF/HS diet for 20, 40, or 60 weeks. Hepatic macrovesicular steatosis (large, clear-staining vacuoles) indicated by arrows and inflammation indicated by arrowheads.

The hepatic profile of esterified fatty acid composition in the livers of mice fed a HF/HS diet

Feeding male and female CC042 mice a HF/HS diet for up to 60 weeks resulted in distinct alterations in the hepatic esterified acid lipid composition as compared to the control diet-fed mice (Figure 4). Principal component analysis (PCA) demonstrated clear difference between male and female mice fed the HF/HS diet and the control diet. PCA also showed clustering of male and female mice fed the HF/HS diet for 20, 40, and 60 weeks (Figures 4a and 4b).

Figure 4. Hepatic profile of esterified fatty acid composition in the livers of CC042 mice fed a HF/HS diet.

Principal component analysis illustrating the differences in the composition of esterified fatty acids in the liver of CC042 male (a) and female (b) mice fed a HF/HS diet or a control diet for 20, 40, or 60 weeks. Percent composition of esterified fatty acids in the liver of CC042 male (c) and female (d) mice fed a HF/HS diet or a control diet. Quantified fatty acids include oleic/vaccenic acid (C18:1), palmitic acid (C16:0), linoleic acid (C18:2), palmitoleic/7-hexadecenoic acid (C16:1), stearic acid (C18:0), ω-3/ω-6-arachidonic acid (C20:4), docosahexaenoic acid (C22:6), and other fatty acids (myristic acid (C14:0), heptadecanoic acid (C17:0), α/γ-linolenic acid (C18:3), 11-eicosenoic acid (C20:1), eicosatrienoic acid (C20:3), eicosapentanoic acid (C20:5), and docosapentaenoic acid (C22:5)). The ratio of esterified polyunsaturated fatty acids (PUFA) to monounsaturated fatty acids (MUFA) in CC042 male (e) and female (f) mice fed a control diet or HF/HS diet for 20, 40, or 60 weeks. Error bars denote standard deviation. * Denotes significant difference (p < 0.05) from control diet-fed mice at the same time point.

Analysis of fatty acid composition demonstrated that the majority of esterified fatty acids in control diet-fed mice were composed of monounsaturated oleic/vaccenic acid (C18:1), ranging from 31% at 20 weeks to 48% at 60 weeks in male mice, and from 40% at 20 weeks to 47% at 60 weeks in female mice; saturated palmitic acid (C16:0), ranging from 20% to 24% in male mice, and from 22% to 23% in female mice; and polyunsaturated linoleic acid (C18:2), ranging from 8% to 14% in male mice, and from 7% to 9% in female mice (Figures 4c and 4d).

Feeding mice the HF/HS diet-induced changes in the hepatic esterified fatty acid composition characterized by an increased percentage of oleic/vaccenic acid (C18:1) in the livers of male mice at 20 and 40 weeks and female mice at 20, 40, and 60 weeks, and decreased percentage of linoleic acid (C18:2) at 20, 40, and 60 weeks in the livers of both sexes of mice (Figures 4c and 4d). Saturated fatty acids palmitic (C16:0) and stearic acid (C18:0) levels were unchanged after HF/HS diet in male and female mice (Supplementary Figure 3a), whereas the levels of monounsaturated fatty acids (MUFAs; oleic/vaccenic acid (C18:1) and palmitoleic/7 hexadecenoic acid (C16:1)) increased after HF/HS diet at 20 and 40 weeks in male mice, and 20, 40, and 60 weeks in female mice (Supplementary Figure 3b). Polyunsaturated fatty acids (PUFAs; ω-3/ω-6-arachidonic (C20–4) and docosahexaenoic acid (C22:6)) decreased after 20 and 40 weeks of HF/HS diet in male mice, and decreased after 20, 40, and 60 weeks of HF/HS diet in female mice (Supplementary Figure 3c). These changes resulted in a reduction of the cumulative PUFA/MUFA molecule ratios in the livers of HF/HS diet-fed male mice at 20 and 40 weeks in male and female mice as compared to mice fed the control diet (Figures 4e and 4f).

Development of NAFLD-like liver injury in the livers of mice fed a HF/HS diet is associated with dynamic transcriptomic alterations

The expression of genes involved in lipid metabolism, inflammation, and fibrogenesis was investigated to determine the underlying molecular mechanisms associated with the development of severe NASH-like liver injury in mice fed the HF/HS diet. In male mice, there was increased expression of lipid metabolism genes Cd36 and Pparg after 20 and 40 weeks, Acaa1b after 40 weeks, Fabp2 and Gdf15 after 20 weeks on the HF/HS diet. In female mice, increased expression of lipid metabolism genes Cd36 and Pparg was observed after 20, 40, and 60 weeks and Acaa1b, and Fabp2 after 20 and 40 weeks on the HF/HS diet. Interestingly, no significant changes to lipid metabolism-related gene expression were observed in male mice after 60 weeks of diet exposure (Figure 5a).

Figure 5. Expression of genes involved in lipid metabolism, inflammation, and fibrogenesis in the livers of CC042 mice fed a HF/HS diet.

Analysis of genes involved in (a) hepatic lipid metabolism pathways, (b) NASH-related inflammation, and (c) fibrosis in CC042 male and female mice fed a control diet or HF/HS diet for 20, 40, or 60 weeks. Gene expression was measured by qRT-PCR and the relative amount of each mRNA transcript was determined using the 2^−ΔΔCt method. N=6/sex/diet/time point. Error bars denote standard deviation. * Denotes significant difference (p < 0.05) from control diet-fed mice at the same time point.

The development of NASH-like liver injury in male and female CC042 mice maintained on the HF/HS diet for up to 60 weeks was accompanied by changes to proinflammatory cytokines (Figure 5b). The expression of the Cd44 gene and the proinflammatory CXC motif chemokine ligand 2 (Cxcl2) gene was found to be increased in males after 20, 40, and 60 weeks of HF/HS diet exposure. Cxcl10 and tumor necrosis factor (Tnf-ɑ) were found to be increased in male after 20 and 40 weeks of HF/HS diet. In female mice, there were no changes in Cd44 expression, but there were increases in Cxcl10, Cxcl2, and Tnf-ɑ expression after 20, 40, and 60 weeks of exposure. The expression of intercellular adhesion molecule-1 (Icam-1) was increased after 40 and 60 weeks of HF/HS diet in male mice; however, the expression was decreased in female mice after 20 and 60 weeks.

The expression of the fibrosis-related collagen (Col) 1a1 and 1a2 genes was increased in both sexes at all time points (Figure 5c). Col6a3 expression was increased at 20, 40, and 60 weeks in male mice and at 40 and 60 weeks in female mice. The level of keratin (Krt) 8/18 was elevated at 20 and 40 weeks on a HF/HS diet in both sexes, whereas feeding a HF/HS diet did not affect the expression the α-smooth muscle actin (Acta) gene.

Discussion

NAFLD comprises several pathological states ranging from simple hepatic steatosis or NAFL to NASH, NASH-fibrosis, and NASH-cirrhosis. NAFL is a benign state; however, some NAFL patients may gradually progress to develop NASH, NASH-fibrosis, and NASH-cirrhosis. Currently there are no approved drugs for the treatment of NASH. Considering the growing burden of NASH, more recently being termed as metabolic dysfunction-associated steatotic liver disease (MASLD), the challenge of identifying molecular drivers to inform therapies to slow, halt, or reverse this debilitating and prevalent disease condition is a critical unmet medical need of great importance. To address this unmet need, the U.S. FDA has emphasized and encouraged the use of relevant models of NASH to screen and identify potential investigational drugs¹³ for the treatment of non-cirrhotic NASH with liver fibrosis to improve the fibrotic condition or prevent progression to cirrhosis, and/or resolve steatohepatitis. In this regard, the development of additional relevant preclinical models for NASH is crucial and was the goal of this study.

The present study demonstrates that chronically feeding male and female CC042 mice an obesogenic HF/HS diet for 60 weeks induced the development of NASH-like liver injury in both male and female mice. This was evidenced by increased body and liver weights, elevated serum ALT, AST, cholesterol, HDL-C, and LDL-C, and histological findings, detected by hematoxylin and eosin, osmium tetroxide, Masson’s trichrome, and Picrosirius red staining. Feeding a HF/HS diet to CC042 mice induced NAFL, evidenced by steady intracellular lipid accumulation in the livers, which gradually progressed to NASH-like fibrosis in both sexes of CC042 mice. The incidence of steatosis was the same in male and female HF/HS diet-fed mice; however, there was sex-specific difference in the severity of steatosis between male and female mice at 20 weeks with the greater severity in male mice. This difference in steatosis severity diminished with time (at 40 and 60 weeks) on a HF/HS diet and, more importantly, histopathological measures of the severity of NASH-like fibrosis did not differ between male and female mice at 40 and 60 weeks. In general, it is believed that women have a lower risk of NAFLD than men². However, a recent systematic review and meta-analysis of patient studies clearly established that once NAFLD develops, women have a greater risk for the development of fibrosis¹⁴. Our findings of a markedly lower severity of hepatic steatosis in female mice at 20 weeks compared with their male counterparts with similar or greater severity of fibrosis by 60 weeks are consistent with these human studies.

It is also widely regarded that intracellular hepatic lipid accumulation and altered lipid composition is the initiating step in the pathogenesis of NAFLD¹⁵. In the present study, we demonstrated significant sex-independent alterations in the esterified fatty acid composition in the livers of both male and female mice fed a HF/HS diet at all time points. Specifically, we observed elevations of oleic/vaccenic acid (C18:1) and total MUFA and a reduction of linoleic acid (C18:2) and total PUFA resulting in substantial reduction of PUFA/MUFA ratios as the duration of feeding the HF/HS diet increased. In our previous study⁹, we reported an inverse correlation between the total PUFA/MUFA ratio and the severity of hepatic steatosis across a spectrum of genetically diverse CC mouse strains. In that study, the CC042 strain demonstrated most pronounced signs of NASH-like fibrosis in both male and female mice fed a HF/HS diet for 20 weeks. This strain also demonstrated recognizable NASH-like related alterations in the esterified fatty acid composition profiles and markedly reduced PUFA/MUFA ratios. A similar observation of MUFA and PUFA alterations has been reported during the progression of NAFL to NASH in humans^{16, 17}.

Another important finding in this study is a correspondence between the steps in the histopathologic progression of the NASH-like phenotypes, and the changes in expression of genes that are known to be associated with different stages of the liver disease. This is evidenced by consistent changes between expression of NAFL-related genes, e.g., Cd36 and Pparg, inflammation-related genes, e.g., Cxcl2, Cxcl10, Cd44, and Tnf-ɑ, fibrosis-related genes, Col1a1, Col1a2, and Col6a3, and histomorphological characteristics of NAFLD progression. Furthermore, we report evidence of associations between the gene expression changes and the clinical chemistry data. For example, serum levels of CXCL10, a proinflammatory cytokine encoded by the Cxcl10 gene (one of the upregulated genes in the livers of HF/HS diet-fed male and female mice), were substantially higher in HF/HS diet-fed mice than in mice on the control diet. Previously, Zhang et al.¹⁸ reported a key role of CXCL10 in the pathogenesis of NASH. More importantly, the level of circulating CXCL10 was also increased in NASH patients, suggesting its potential role as a non-invasive predictor of NASH progression.

While we report several similarities of this model to human NASH-like disease, the shortcomings in the preclinical models of NASH are well established and our study is not without limitations. On the one hand, the experimental animal models that mimic NASH in humans have proven to be useful tools to investigate disease pathogenesis, characterize molecular mechanisms, and identify potential druggable targets. On the other hand, obesity and other metabolic dysfunction-associated steatotic changes are very challenging to reproduce in mice or these effects are only transient. Preclinical models of NASH include genetic modifications (e.g., apolipoprotein E knock-out and knock-in and major urinary protein urokinase plasminogen activator models), dietary factors (e.g., various methionine- and choline-deficient, and/or obesogenic-diet induced models), and combined diet- and chemical-induced models (e.g., Stelic animal mouse model, diet-CCl₄, diet-DEN)^{19, 20}. Still, none of these experimental models of NASH has been shown to recapitulate the full spectrum of complex pathological, biochemical, and molecular phenotypes associated with NASH in humans^{21 22}.

A recent comprehensive comparative study aligned a large number of preclinical models with human clinical outcomes and assessed inter-species similarities in MASLD. The authors identified induction of obesity and dyslipidemia, severity of liver damage, histological evidence of steatosis, lobular inflammation and/or ballooning, as well as hallmark gene expression as key features that make an animal model more similar to human NASH. This study concluded that obesogenic (“Western”) diet-induced models most closely resemble the natural progression and various features of NASH development in humans²³. In this regard, our study that used a HF/HS dietary model of NASH in a sensitive genetic background without specific mutation/polymorphisms may represent an additional option among other preclinical models. Previous studies^{24, 25} have shown that feeding an obesogenic HF/HS diet to male isogenic mice obtained by crossing C57BL/6J and 129S1/SvImJ mice²⁴ or male C57BL/6NJ mice²⁵ recapitulates many pathophysiological and pathomorphological milestones of NASH that have been observed in humans. The present study using male and female CC042 mice showed hepatic NASH-specific pathological changes, including steatosis, inflammation, and fibrosis, in both sexes – all histopathological features that are relevant to human NASH. Still, it is important to acknowledge that a number of observations in our study are not concordant with known key features of human NASH. These include differences in histopathology – hepatocyte ballooning and bridging fibrosis were not detected in our model, and steatotic changes – we found increased HDL levels while this marker is more frequently decreased in patients with NAFLD. Additionally, while we observed elevation in serum ALT, AST, cholesterol, and LDL-C levels, no major changes in the levels of glucose, triglycerides, and insulin, which are considered as clinical hallmarks of human NASH, were detected. Also, additional comprehensive studies of transcriptional changes in the HF/HS dies in CC042 strain are needed to enable a more comprehensive comparison to other models, similar to the analyses detailed by Vacca et al²³.

In conclusion, our study provides a comprehensive dataset of time-specific changes in a chronic preclinical model of NASH-like liver injury. We show that in CC042 male and female mice fed HF/HS diet for up to 60 weeks, develop progressive severe liver fibrosis in both sexes. While this model does not reflect the full spectrum of pathophysiological and pathomorphological changes associated with NASH development in humans, it could be used as a preclinical model to investigate potential candidate drugs for the treatment of non-cirrhotic NASH with liver fibrosis, a current major public health issue²⁶.

Supplementary Material

Supplementary Figure 3

Supplementary Figure 3. Hepatic profile of saturated, monounsaturated, and polyunsaturated esterified fatty acid composition in the livers of CC042 mice fed a HF/HS diet. Average percent composition of saturated esterified fatty acids (SFA) (a), monounsaturated esterified fatty acids (MUFA) (b), and polyunsaturated esterified fatty acids (PUFA) (c) in the liver of CC042 male and female mice fed a HF/HS diet or a control diet for 20, 40, or 60 weeks. N=6/sex/diet/time point. Error bars denote standard deviation. * Denotes significant difference (p < 0.05) from control diet-fed at the same time point.

Supplementary Table 1

Supplementary Table 1: Primer list for qRT-PCR

Supplementary Figure 1

Supplementary Figure 1. Histopathological finding of lipid accumulation in the livers of CC042 mice fed a HF/HS diet. Representative images from osmium staining in live sections from control mice or mice fed a HF/HS for 20, 40, or 60 weeks. (a) male and (b) female mice. Globular black staining (arrows) represents intracytoplasmic hepatocellular lipid. (c) Quantitation of average percent osmium staining. N=6/sex/diet/time point. Error bars denote standard deviation. * Denotes significant difference (p < 0.05) from control diet-fed mice at the same time point.

Supplementary Figure 2

Supplementary Figure 2. Histopathological finding of fibrosis in the livers of CC042 mice fed a HF/HS diet. Representative images of Masson’s Trichrome (blue stain; arrows) staining in liver sections in CC042 male (a) and female (b) mice fed a control diet or HF/HS diet for 20, 40, or 60 weeks.

Highlights.

• Chronically feeding mice a high-fat and high-sucrose diet induced changes consistent with clinical NAFLD

• The model exhibits histological indication of steatosis, inflammation, and fibrosis in both sexes

• Gene expression changes support NASH-like histopathological progression

• This preclinical NASH model could be used for future drug therapy studies