Received: 9 November 2022 | Accepted: 30 December 2022

DOI: 10.1002/ame2.12309

ORIGINAL ARTICLE

Establishment of a non-­alcoholic fatty liver disease model by

high fat diet in adult zebrafish

Xiang Li1,2 | Lei Zhou2 | Yuying Zheng2 | Taiping He1 | Honghui Guo1 |
Jiangbin Li3 | Jingjing Zhang2

1
Department of Nutrition, School of Public
Health, Guangdong Medical University, Abstract
Zhanjiang, China
Background: Non-­alcoholic fatty liver disease (NAFLD) has become the most common
2
Zhanjiang Key Laboratory of Zebrafish
Model for Development and Disease,
chronic liver disease in recent years, but the pathogenesis is not fully understood.
Affiliated Hospital of Guangdong Medical Therefore, it is important to establish an effective animal model for studying NAFLD.
University, Zhanjiang, China
3
Methods: Adult zebrafish were fed a normal diet or a high-­fat diet combined with
School of Medical Technology,
Guangdong Medical University, egg yolk powder for 30 days. Body mass index (BMI) was measured to determine
Dongguan, China overall obesity. Serum lipids were measured using triglyceride (TG) and total choles-
Correspondence terol (TC) kits. Liver lipid deposition was detected by Oil Red O staining. Liver injury
Jingjing Zhang, Zhanjiang Key Laboratory was assessed by measuring glutathione aminotransferase (AST) and glutamic acid
of Zebrafish Model for Development and
Disease, Affiliated Hospital of Guangdong aminotransferase (ALT) levels. Reactive oxygen species (ROS) and malondialdehyde
Medical University, Zhanjiang 524001, (MDA) were used to evaluate oxidative damage. The level of inflammation was as-
China.
Email: jingjing.zhang@live.com sessed by qRT-­PCR for pro-­inflammatory factors. H&E staining was used for patho-

Jiangbin Li, School of Medical Technology,

logical histology. Caspase-­3 immunofluorescence measured apoptosis. Physiological
Guangdong Medical University, disruption was assessed via RNA-­seq analysis of genes at the transcriptional level and
Dongguan, 523808, China.
Email: jiangbinli@gdmu.edu.cn
validated by qRT-­PCR.
Results: The high-­fat diet led to significant obesity in zebrafish, with elevated BMI, he-
Honghui Guo, Department of Nutrition,
School of Public Health, Guangdong patic TC, and TG. Severe lipid deposition in the liver was observed by ORO and H&E
Medical University, Zhanjiang 524023,
staining, accompanied by massive steatosis and ballooning. Serum AST and ALT levels
China.
Email: guohh1999@gdmu.edu.cn were elevated, and significant liver damage was observed. The antioxidant system in
the body was severely imbalanced. Hepatocytes showed massive apoptosis. RNA-­seq
Funding information
the Discipline Construction Project of results indicated that several physiological processes, including endoplasmic reticu-
Guangdong Medical University, Grant/
lum stress, and glucolipid metabolism, were disrupted.
Award Number: 4SG22258G; the Science
and Technology Planning Project of Conclusion: Additional feeding of egg yolk powder to adult zebrafish for 30 consecu-
Zhanjiang, China, Grant/Award Number:
tive days can mimic the pathology of human nonalcoholic fatty liver disease.
2022A01231

KEYWORDS
ER stress, high-­fat-­diet, metabolism, non-­alcoholic fatty liver disease, zebrafish

Xiang Li and Lei Zhou contributed equally to this work.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in
any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2023 The Authors. Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for
Laboratory Animal Sciences.

Anim Models Exp Med. 2023;00:1–10.  wileyonlinelibrary.com/journal/ame2 | 1

 |
2 LI et al.
1 | I NTRO D U C TI O N
Non-­alcoholic fatty liver disease (NAFLD) has become the most
common liver disease worldwide over the past few decades due to
1
the harmful effects of overweight and obesity on human health.
The progression of NAFLD includes a series of different degrees of
liver injuries, from nonalcoholic fatty liver (NAFL) to non-­alcoholic
steatohepatitis (NASH). 2 NAFL has a good prognosis, while NASH
may progress to advanced liver fibrosis, cirrhosis and even hepa-
tocellular carcinoma (HCC). 3 By 2018, the number of NAFLD pa-
tients in the world had reached 25% of the total population,4 and
the prevalence of NAFLD in China was even as high as 29.2%. 5 In
spite of this huge medical demand, there is still no specific drug
for the treatment of NAFLD/NASH in the clinic because of the
complex pathogenesis. 6 At present, the scientific community has
proposed more widely accepted pathogenesis mechanisms includ-
ing the two-­hit theory and the multiple hit theory, but the com-
plexity of NAFLD still cannot be fully summarized.7,8 Therefore,
the establishment of a fast and effective animal model of NAFLD
is of great significance for exploring the molecular mechanism of
NAFLD.
At present, the pathogenesis of NAFLD has been explored
mainly using rodent models in vivo and cell models in vitro.9 After
years of development, rodent models have yielded good results.
However, rodent models inevitably involve high feeding costs,
large individual differences, and long experimental times, and are
not suitable for large-­s ample screening.10 Most of the cell models
in vitro are single cell cultures, but the pathogenesis of NAFLD
is closely related to multiple tissues and organs, and because cell
models grow in artificial environment, their morphology or func-
tion shows different degrees of change compared with the in vivo
environment, meaning they cannot fully reflect real physiological
and pathological changes.
Zebrafish (Danio rerio) were first used in the laboratory
30 years ago. Due to its many advantages such as low mainte-
nance cost, rapid reproduction and development, and ease of in-
heritance control and gene editing, the zebrafish has attracted
more and more attention as an in vivo model in many fields such
as toxicology, developmental biology and drug screening.11 The
zebrafish has become a model animal for studying NAFLD in
recent years because of its metabolic consistency with humans
and the highly conserved molecular program of liver develop-
ment.12,13 At present, several models of nonalcoholic fatty liver
disease have been established in zebrafish, including genetic,
diet-­induced, and chemo-­induced models. 3,14,15,16The disad-
vantage of genetic models is that mutant genes rarely occur in
humans.17 In contrast, drug-­induced models show high animal
mortality. Diet-­induced models are widely used in the study of
molecular mechanisms because they are most similar to human
NAFLD pathogenesis. The MCD diet is a common model of NASH
and can rapidly cause steatosis, necrosis, inflammation, and fibro-
sis.18 However, its drawback is that as a nutrient-­d eficient diet,
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it does not induce the same pattern of NAFLD pathogenesis as
humans.19 High-­f at diet-­f ed mice are widely used since they pres-
ent similar histological features and metabolic properties to those
observed in humans. 20-­2 2 However, monitoring of rodents fed a
high-­f at diet involves high costs and time requirements, whereas
the use of zebrafish allows for the rapid construction of an eco-
nomical and effective model of NAFLD. An et al. induced lipid ac-
cumulation and oxidative stress by feeding a high-­c holesterol diet
to zebrafish larvae. 23 Sapp et al. showed that fructose induced
endoplasmic reticulum (ER) stress and oxidative stress in zebraf-
ish larvae and successfully established a model of nonalcoholic
fatty liver disease. 24 However, most zebrafish NAFLD models use
larvae. Although the age profile of NAFLD is getting younger, the
juvenile version of NAFLD simulated by zebrafish larvae cannot
fully reflect the pathogenesis in adults. An et al. established a
model of NAFLD using adult zebrafish fed a high cholesterol diet
for 8 weeks. 25 Although the model was successfully established,
it required a long experimental time.
In this study, we used adult male zebrafish as a vertebrate
model. We established NAFLD by additional feeding of egg yolk
powder, and evaluated the relationship between diet-­induced lipid
metabolism disorders and related pathways in the zebrafish. The
study aimed to more fully understand the effects of high-­f at diet
on basic physical characteristics, liver histology and lipid metab-
olism, thereby extending our understanding of the pathogenesis
of NAFLD.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Care and feeding of adult Zebrafish
Embryos of the wild-­t ype zebrafish AB strain were collected and
placed in embryonic water, and incubated at 28.5°C in a constant
temperature incubator until 5 days post fertilization (dpf). Paramecium
and prawns were fed in still water from 6 dpf, and cultured in the sys-
tem at 15 dpf until adulthood (3 months post fertilization, mpf). Adult
male zebrafish were randomly divided into a normal diet group (ND)
and a high-­fat diet group (HFD), with 15 fish in each group. The ND
group was fed with 30 mg of artemia per adult fish per day, while the
HFD group was fed with 30 mg of artemia (20% fat, 60% protein and
polyunsaturated fatty acids; Shangjia, Shandong, China) per adult fish
per day and 70 mg of egg yolk powder (containing 55.8% fat, 34.2%
protein, 17.2% saturated fatty acids; purchased from Yuanye Bio-­
Technology, Shanghai, China) per adult fish per day (egg yolk powder
thoroughly mixed with 2 L water until no precipitation was observed).
The feeding lasted for 30 days. After feeding, the animals were fasted
for a day before being killed. Based on standard laboratory guide-
lines, all zebrafish were fed in an environment at 28.5°C with a peri-
odic rhythm of 14 h of light and 10 h of darkness. The zebrafish were
treated according to the regulations of Guangdong Province on the
Administration of Experimental Animals (2010).

LI et al.
2.2 | Body mass index (BMI)
At the time of randomization (day 0), adult zebrafish were anesthe-
tized with embryonic water containing 0.02% Tricaine. After drying
the body surface water, the body weight (mg) was measured using
an analytical balance, and the length (cm) from the fish mouth to
the end of the tail fin was measured with a ruler. The BMI of adult
zebrafish was calculated at the beginning and end of the feeding ex-
periment using the formula: BMI = weight/length2.
2.3 | Liver to body ratio (LBR)
Adult zebrafish were anesthetized and the abdomen was opened
with scissors and forceps to remove the intact liver. Livers were
weighed using the analytical balance and the LBR was calculated
using the formula: LBR = liver weight/body weight *100%.
2.4 | Histology
The whole visceral mass of adult zebrafish sacrificed after anesthe-
sia was completely removed with scissors and forceps, embedded
in Tissue-­Tek O.C.T. compound (SAKURA, USA), and frozen on dry
ice. The samples were fixed on a Leica CM1860 UV cryostat for col-
lection of 8 μm thick serial sections on adhesive slides. After sam-
ple collection, the sections were fixed in frozen methanol for 5 min.
After rinsing off excess methanol and O.C.T., the sections were
stained with H&E staining kit (Solarbio, Beijing, China) according
to the manufacturer's instructions. Samples for ORO staining were
stained with 0.5% Oil Red O dye (Aladdin, Shanghai, China) solution
without methanol fixation. The staining results were analyzed under
a BX53 microscope (Olympus, Germany).
2.5 | Biochemical analysis
After adult fish were sacrificed, intact livers were removed with
scissors and forceps, and liver total cholesterol (TC), triglycerides
(TG), reactive oxygen species (ROS), and malondialdehyde (MDA)
were measured using relevant kits according to the manufacturer's
specifications. After centrifugation of blood taken from the tail of
the fish, serum alanine aminotransferase (ALT) and aspartate ami-
notransferase (AST) levels were measured using the appropriate kit
(Jiancheng, Nanjing, China). 26-­28
2.6 | Imaging
In order to obtain the liver changes in adult zebrafish, the abdomen
was exposed and imaged using a Leica M205 FA stereo microscope
(Leica, Germany).
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3
2.7 | Quantitative real-­time PCR
Fresh adult zebrafish liver tissue was used to extract total RNA using
RNAiso Plus (Takara Bio), and a total of 1000 ng of RNA was reverse
transcribed into cDNA using a reverse transcription kit (Yeasen
Biotech). The cDNA obtained was mixed with specific primer pairs,
the Hieff® qPCR SYBR Green Master Mix (Yeasen Biotech) with Low
Rox and then subjected to real-­time PCR using the ABI QuantStudio
6 Flex system (Applied Biosystems). The sequences of primers are
listed in Table S1.
2.8 | Immunofluorescence (IF)
Samples were sectioned to 8 μm thick using a freezing microtome,
fixed with acetone for 10 min on ice, and then washed with PBS.
The samples were incubated with 5% goat serum for 1 h at room
temperature, and incubated with primary antibody (1:150, ab13847,
Abcam, UK) diluted with 5% goat serum overnight at 4°C in a re-
frigerator. The next day, the residual primary antibody was washed
with PBS. The sections were incubated with diluted secondary an-
tibody (1:200, 111-­605-­0 03, Jackson, PA, USA) and DAPI (1:500,
Sigma, USA) for 2 h at room temperature, washed with PBS, sealed
with anti-­fluorescence quenched sealing (Beyotime Biotechnology,
Shanghai, China) solution, and fixed with nail polish. Images were
obtained with an Olympus FV3000 confocal microscope.
2.9 | RNA-­seq
The quality of RNA was examined using a Thermo Scientific
NanoPhotometer Spectrophotometer, Qubit2.0 Fluorameter and
an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara,
CA, USA). High quality RNA extracts from each tissue sample were
pooled together for cDNA synthesis and sequencing. Fastp was used
for quality control and the RNA-­seq reads were filtered to remove
low quality sequences. The processed reads were then mapped to a
reference zebrafish genome using HISAT2. The mRNAs and genes
with differential expression levels were screened out on the basis
of log2(fold change) >1 or < −1 and at p < 0.05 level. The subsequent
functional and signaling pathway analyses of the above-­screened
differential genes were conducted using Gene Ontology (GO; http://
curre​nt.geneo​ntolo​g y.org/) and Gene Set Enrichment Analysis
(GSEA; http://www.gsea-­msigdb.org/).
2.10 | Statistical analyses
All data were statistically analyzed using Prism software (version 8.0
GraphPad, San Diego, CA). The normality of data distribution was
analyzed using the Shapiro–­Wilk test. The unpaired Student's t test
was used to compare the differences between groups. Data were
 |
4 LI et al.
expressed in the form of mean ± standard error of mean. p < 0.05
was considered as statistically significant.
3 | R E S U LT S
3.1 | Establishment of NAFLD zebrafish model
In order to establish the zebrafish model of NAFLD, adult male
zebrafish were fed with artemia mixed with or without egg yolk
powder for 30 days (Figure 1A), and NAFLD was characterized by
observing the accumulation of lipids in the liver. 29 At the end of the
feeding assay, the obesity phenotype of zebrafish in HFD group
(Figure 1B, right) was investigated and was compared with that in
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ND group (Figure 1B, left). After exposing the zebrafish belly, the
livers of the HFD group (Figure 1C, right) presented a more obvious
yellow color than those of ND zebrafish (Figure 1C, left). The results
of H&E staining showed that the livers of zebrafish in the HFD group
had inflammatory lesions (black arrow: aggregation of large numbers
of nuclei), ballooning degeneration (with blank regions in cytoplasm,
or nucleus hanging in the center or squeezed to the side), and larger
areas of steatosis (p < 0.0001), indicating the progression of NAFLD
(Figure 1D). In the ND group fed with artemia at 50 mg/fish per day,
the steatosis area reached 30%–­45% of total liver area. In the HFD
group fed 50 mg artemia/fish and 150 mg/fish egg yolk powder per
day, the steatosis area reached 35%–­50% (Figure S1). Based on the
comparison of area of steatosis in the HFD group with that of the
ND group (Figure 1D), the following experiments were carried out.
F I G U R E 1 Feeding protocol, zebrafish
body size and liver phenotype. (A) Feeding
protocol. Adult zebrafish (3 mpf) were
randomly divided into two groups. The
normal diet (ND) group was fed 30 mg
artemia/fish/day, while the high-­fat diet
(HFD) group was fed 30 mg artemia/
fish/day and 70 mg egg yolk/fish/day
powder. feeding lasted for 30 days. (B)
After completion of feeding, the HFD
group (right) showed a significantly obese
body shape compared to the ND group
(left). (C) After exposing the abdomen, the
liver of zebrafish in the HFD group (right)
appears distinctly yellow, while the liver of
zebrafish in the ND group (left) is orange-­
red. Scale bar: 1 mm. (D) H&E staining
results demonstrating steatosis and
inflammatory cell infiltration. In the ND
group 10–­20% steatosis was observed,
while in the HFD group 20–­30% steatosis
with marked inflammatory cell infiltration
was observed. The black arrow indicates
the regions where large numbers of
nuclei are aggregated and the ballooning
degeneration is seen as blank regions in
the cytoplasm with the nucleus hanging
in the center or squeezed to the side. The
solid black box indicates the magnified
area, and the high magnification image is
shown below. Scale bars: yellow, 100 μm;
black, 20 μm. 5 fields of view per fish (ND:
n = 14; HFD: n = 11). The rate of steatosis
was counted by Image J. Bar graphs,
mean ± SEM. ****p < 0.0001.
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LI et al. 5

3.2 | HFD diet causes abnormal lipid accumulation 3.3 | HFD diet induced pathological changes in
in adult zebrafish adult zebrafish liver

At the beginning of the feeding experiment, there was no significant
difference in BMI between the two groups. At the end of the feed-
2
ing experiment, the BMI of the HFD group (25.60 ± 0.38 mg/cm )
was significantly higher than that of the ND group (23.12 ± 0.33 mg/
cm2, p < 0.001, Figure 2A), indicating that significant obesity oc-
curred in the fish of the HFD group. LBR was also significantly in-
creased in the HFD group (1.90 ± 0.08%) compared to the ND group
(1.56 ± 0.08%, p < 0.01, Figure 2B), suggesting lipid accumulation in
the liver. The results of Oil Red O staining showed that a large num-
ber of red lipid droplets were present in the hepatocytes of the HFD
group (13.12 ± 1.89%), while only a small number of lipid droplets
were present in the ND group (0.57 ± 0.12%, p < 0.0001, Figure 2C).
Hepatic TC and TG levels were significantly lower in the ND group
(0.06 ± 0.01 mmol/g protein and 0.18 ± 0.02 mmol/g protein, re-
spectively) compared with the HFD group (0.16 ± 0.01 mmol/g pro-
tein and 0.26 ± 0.02 mmol/g protein, respectively, p < 0.0001 and
p < 0.05, respectively; Figure 2D,E). These results indicate that the
HFD diet can lead to abnormal lipid accumulation in adult zebrafish.
The serum levels of AST (Figure 3A) and ALT (Figure 3B) in the
HFD group (300.90 ± 37.39% and 180.10 ± 11.05%, respectively)
were significantly higher than those in ND group (100.00 ± 5.00%
and 100.00 ± 13.11%, respectively, p < 0.01). In addition, we meas-
ured liver ROS levels, which were significantly higher in the HFD
group (6.52 ± 0.28) than in the control group (0.94 ± 0.18, p < 0.0001)
(Figure 3C). At the same time, we detected the level of MDA, the
damaging oxidative product of lipid metabolism, and also found
that the level of the ND group (1.91 ± 0.79 mmol/mg protein) was
significantly lower than that of the HFD group (6.91 ± 1.10 mmol/
mg protein, p < 0.01) (Figure 3D). qRT-­PCR results showed that the
expression levels of proinflammatory factors (tnf-­α, il-­1β, il-­6, il-­8) in
the HFD group were significantly higher than those in the ND group
(p < 0.01, p < 0.05 and p < 0.05, respectively) (Figure 3E). Caspase-­330
immunofluorescent staining results showed that severe apoptosis
occurred in the liver of the HFD group, while the level of apoptosis
was lower in the ND group (Figure 3F). These results suggest that
HFD diet can lead to pathological changes in adult zebrafish liver.

F I G U R E 2 High-­fat diet causes lipid

accumulation in zebrafish. (A) BMI at the
beginning and at the end of feeding. No
significant difference in BMI between
ND and HFD groups at the beginning of
feeding experiment was recorded (ND:
n = 14; HFD: n = 15), but at the end of
feeding, BMI was significantly higher in
the HFD group compared to the ND group
(ND: n = 14; HFD: n = 8). (B) Liver-­to-­
body ratio (LBR) was significantly higher
in the HFD group (ND: n = 13; HFD:
n = 8). (C) ORO staining showed large
lipid accumulation in the livers of the HFD
group. The solid black box indicates the
magnified area, and the high magnification
image is shown below. Scale bars: yellow
100 μm, black 20 μm. 5 fields of view per
fish (ND: n = 8; HFD: n = 6). The ratio of
red lipid droplets was counted by Image
J. (D) Zebrafish liver TC content (two
livers were combined into one sample,
ND: n = 4; HFD: n = 5). (E) Zebrafish liver
TG content (two livers pooled into one
sample, ND: n = 5; HFD: n = 5). Bar graphs
show mean ± SEM. n.s., not statistically
significant, *p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001.
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6 LI et al.

F I G U R E 3 High-­fat diet causes pathological changes in the liver of zebrafish. (A) Zebrafish serum AST levels (serum from 5 fish in one
sample, ND: n = 3; HFD: n = 4 repeats). (B) Zebrafish serum ALT levels (serum from 5 fish in one sample, ND: n = 3; HFD: n = 3 repeats).
(C) Zebrafish liver ROS levels (two livers pooled into one sample, ND: n = 4; HFD: n = 4 repeats). (D) Zebrafish liver MDA levels (two livers
pooled one sample, ND: n = 5; HFD: n = 5 repeats) (E) Liver RNA was extracted for qRT-­PCR assay to detect the expression levels of mRNAs
of inflammatory factors. (F) Hepatocyte apoptosis was observed by staining with Caspase-­3 protein. The expression level of Caspase-­3
was significantly increased in the HFD group compared with the ND group, and hepatocytes were extensively apoptotic. The solid white
box indicates the magnified area, and the high magnification image is shown below. Scale bars: white 100 μm, green 30 μm. 3 fields of view
per fish (ND: n = 4; HFD: n = 4 repeats). Mean grayscale values were counted by Image J. Bar graphs show mean ± SEM. n.s. means not
statistically significant, *p < 0.05, **p < 0.01, ****p < 0.0001.

3.4 | RNA-­seq analysis of changes in gene
expression levels during NAFLD progression
Following a high-­f at diet, a volcano map showed that the expres-
sion of 293 genes was significantly increased and 452 genes were
significantly decreased in the high-­f at group (Figure S2). The re-
sults of GSEA and GO analysis showed that the expression of
genes related to the response to endoplasmic reticulum stress
and genes related to glycolipid metabolism were upregulated in
the HFD group (Figure 4A,B). This is consistent with previously
published results. 20,31,32,33 The genes associated with NAFLD in
the GO analysis were networked and enriched with the STRING
database, and then the cytoHubba app in Cytoscape software
was used for module screening, and 20 core genes were ob-
tained in order of score (Figure 4C). The expression of ER stress
molecules (hspa5, dnajc3), fatty acid synthase (fasn) and insulin
substrate receptor 2 (irs2) were significantly elevated in the HFD
group as verified by qRT-­P CR (Figure 4D), demonstrating the oc-
currence of ER stress and disorders of glucolipid metabolism in
the model group.
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LI et al. 7

F I G U R E 4 RNA-­seq analysis of changes in gene transcript levels during NALFD progression. (A) GSEA analysis of differentially expressed
genes with significant upregulation of response pathways to endoplasmic reticulum stress. (B) Bubble plot of GO analysis restlt, lipid
synthesis, insulin response and endoplasmic reticulum stress pathways were significantly upregulated. (C) Screening results of cytoHubba
app module with the top 20 highest scoring genes. (D) qRT-­PCR validation of mRNA expression levels of endoplasmic reticulum stress
molecules, fatty acid synthase and insulin substrate receptor (more than three samples were tested for each gene). n.s., no statistical
significance, *p < 0.05, **p < 0.01, ***p < 0.001.

4 | DISCUSSION the water to reduce any differences in feeding. Second, to obtain

In this study, a model for studying NAFLD was constructed using
egg yolk powder fed to adult zebrafish; this model is more consist-
ent with human epidemiological characteristics than models using
juvenile fish. In addition, additional intake of high-­fat diet beyond
that required to meet basic physiological needs can better mimic
the pathogenesis and pathological characteristics of human NAFLD.
Importantly, the dose of egg yolk powder used in the study allowed
the model to be completed in a shorter period of time.
First, to ensure that each zebrafish consumed the same amount
of food, we selected zebrafish of similar body size as the experi-
mental subjects, and we dispersed the egg yolk powder evenly in
suitable feeding concentrations, we performed feeding experiments
using two concentrations and compared them by H&E staining. It
was found that the ND group fed 30 mg/fish/day had a lower area
of steatosis and did not produce inflammatory lesions, which better
mimicked conditions in the normal physiological state. Therefore,
30 mg artemia /fish per day was chosen as the basal feeding level in
subsequent experiments.
Under normal conditions, only a very small amount of TG is
stored in the lipid droplets of hepatocytes in the liver. However, ex-
cessive storage of TG leads to the development of lipotoxicity and
steatosis.34,35 TC in vivo includes cholesteryl esters and free choles-
terol (FC), and the accumulation of FC has been shown to correlate
 |
8 LI et al.
with inflammation and fibrosis, among other symptoms, in the pro-
36
gression of NASH. Elevated TG and TC were detected in our ex-
perimental results, and the results of Oil Red O staining of frozen
sections also showed the presence of a large number of lipid drop-
lets in the HFD group, confirming the excessive accumulation of TG.
In addition, the results of H&E staining also showed a large extent of
fatty degeneration and inflammatory lesions.
An imbalance between the bioavailability of ROS and the antiox-
idant system in the body can lead to the development of oxidative
stress. Low levels of ROS play physiological roles such as signaling in
vivo, but high levels of ROS can lead to DNA damage, which in turn
can cause alterations in the expression of certain genes, ultimately
leading to increased cell death. We detected a significant increase in
ROS levels in the HFD group, indicating that the antioxidant balance
was disrupted. In addition, high levels of ROS were able to induce
protein and lipid peroxidation, leading to functional alterations of
substances such as enzymes and cell membranes. We detected a
significant increase in MDA levels in the HFD group. Excessive accu-
mulation of ROS affects the mitochondrial depolarization potential
by inducing lipid peroxidation reactions to produce MDA. In the mi-
tochondrial depolarized state, the burden of mitochondrial autoph-
agy increases and the number of mitochondria decreases, promoting
further development of steatosis, inflammation and fibrosis.37-­39
Hepatocyte injury usually releases AST and ALT into the blood.
Therefore, biochemical assays of serum AST and ALT are widely
used as markers of liver injury.40 In zebrafish in the HFD group, sig-
nificantly elevated serum AST and ALT levels were detected, sug-
gesting that the high-­fat diet induced the process of liver injury.
In recent years, as the pathogenesis of NAFLD/NASH has been
increasingly studied, the role played by endoplasmic reticulum
(ER) stress has received wide attention and ER stress has been
shown to play a regulatory role in apoptosis of hepatocytes.41 In
the presence of ER stress, the unfolded protein response (UPR)
is initiated and undergoes repair. However, prolonged ER stress
leads to a shift from adaptive to terminal UPR, followed by in-
creased inflammation and activation of pro-­a poptotic pathways
in the liver, resulting in massive hepatocyte death.42,43 Using a
immunofluorescence assay, we detected a large amount of the
apoptotic marker Caspase-­3 in the HFD group, suggesting that
ER stress may have occurred in zebrafish in the HFD group. The
dynamic mechanisms regulating ER stress in NAFLD are not fully
understood, and although extensive studies have been conducted
and many relevant molecules have emerged as new therapeutic
targets, more in-­d epth experiments are needed to explore the
complete mechanisms.44
Analysis of the results of RNA-­seq and qRT-­P CR assays al-
lowed us to identify several biochemical processes that are most
altered in this model. First, fatty acid synthase (FASN) is a key
rate-­limiting enzyme for hepatic de novo lipogenesis (DNL),45,46
and we found that a high-­f at diet induced a rise in FASN expres-
sion, leading to increased lipid synthesis and further leading to
TG accumulation in the liver. Second, altered expression of insulin
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receptor substrate 2 (IRS2) regulates the AKT pathway and affects
NAFLD progression.47 Consistent with the results of Chi et al,48
we detected increased mRNA expression of irs2. Finally, the en-
doplasmic reticulum plays an important role in lipid biosynthesis
in vivo,49 and consistent with previous findings, 50,51 we detected
increased expression of the ER stress chaperone molecule heat
shock protein 5 (hspa5) and the DNAJ heat shock protein family
member C3 (danjc3) in the HFD group. In conclusion, a high-­f at diet
induces upregulation of the DNL pathway and increased lipogene-
sis, leading to excessive accumulation of lipid droplets in the liver.
Lipid accumulation produces lipotoxicity, leading to mitochondrial
dysfunction or endoplasmic reticulum stress, which, together with
oxidative stress, activation of pro-­inflammatory substances and
apoptotic pathways, leads to hepatocyte death and promotes the
development of NAFLD.
Currently, research on the pathogenesis of NAFLD is limited,
and no drugs have been approved for treatment.14 Previously pro-
posed theories such as the ‘double-­hit’ theory based on triglycer-
ide accumulation, inflammation, oxidative stress and autophagy,8
and the ‘three strike’ theory52 based on steatosis, lipotoxicity and
inflammation, have failed to summarize the complexity of human
NAFLD. In recent years, the ‘multiple-­hit’ theory, which focuses on
mitochondrial dysfunction, endoplasmic reticulum stress, inflamma-
tory response, lipotoxicity, genetic factors, nutritional excess and
intestinal flora disorders, has flourished.7 Here, we constructed a
short, convenient, and high-­volume experimental model of NAFLD
in adult zebrafish. 3mpf male zebrafish were additionally fed with
egg yolk powder for 30 days, and relevant indexes were tested to
verify the success of the model, which successfully mimicked the
pathological characteristics of NAFLD and will hopefully provide a
stable and reliable in vivo model for drug screening and the study of
NAFLD pathogenesis.
AU T H O R C O N T R I B U T I O N S
Xiang Li: carried out the animal experiment and drafted the manu-
script. Yuying Zheng: information collection and revised the manu-
script. LeiZhou: design methodology and revision of manuscript.
Honghui Guo, Jiangbin Li and Taiping He: supervision, extra sugges-
tions on this study. Jingjing Zhang: supervision, conceptualization,
writing -­review and editing.
AC K N OW L E D G M E N T S
We thank Yuezhuang Zheng, Qiumei Hong and Weinan Wu for ex-
pert technical assistance with the zebrafish facility. This work was
supported by thePublic ServicePlatform of South China Sea for the
R&D of Marine BiomedicineResource for support.
F U N D I N G I N FO R M AT I O N
This study was supported by the Science and Technology
Planning Project of Zhanjiang, China (2022A01231) and the
Discipline Construction Project of Guangdong Medical University
(4SG22258G).

LI et al.
C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare that they have no conflicts of interest.
Jingjing Zhang is an editorial board member of Animal Models and
Experimental Medicine and a coauthors of this article. To minimize
bias, he was excluded from all editorial decision making related to
the acceptance of this article for publication.
E T H I C S S TAT E M E N T
This study does not contain any studies with human subjects or
with rodent vertebrates. Handling of zebrafish was performed in ac-
cordance with Guangdong State Regulations on Laboratory Animal
Management (2010). The whole experimental procedures were ex-
amined by the Clinical Ethics Committee of the Affiliated Hospital of
Guangdong Medical University, China.
ORCID
Xiang Li https://orcid.org/0000-0002-8193-7342
Honghui Guo https://orcid.org/0000-0001-7837-0392
Jingjing Zhang https://orcid.org/0000-0002-8789-4638
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S U P P O R T I N G I N FO R M AT I O N
Additional supporting information can be found online in the
Supporting Information section at the end of this article.
How to cite this article: Li X, Zhou L, Zheng Y, et al.
Establishment of a non-­alcoholic fatty liver disease model by
high fat diet in adult zebrafish. Anim Models Exp Med.
2023;00:1-10. doi:10.1002/ame2.12309