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Citation: Kulkarni, A.; Ibrahim, S.;
Haider, I.; Basha, A.; Montgomery, E.;
Ermis, E.; Mirmira, R.G.; Anderson,
R.M. A Novel 2-Hit Zebrafish Model
to Study Early Pathogenesis of
Non-Alcoholic Fatty Liver Disease.
Biomedicines 2022,10, 479. https://
doi.org/10.3390/biomedicines
10020479
Academic Editors: François
R. Jornayvaz, Karim Gariani and
Albrecht Piiper
Received: 23 January 2022
Accepted: 15 February 2022
Published: 17 February 2022
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biomedicines
Article
A Novel 2-Hit Zebrafish Model to Study Early Pathogenesis of
Non-Alcoholic Fatty Liver Disease
Abhishek Kulkarni 1, † , Sara Ibrahim 2, †, Isra Haider 2, Amina Basha 1, Emma Montgomery 1, Ebru Ermis 1,
Raghavendra G. Mirmira 1, * and Ryan M. Anderson 1,*
1Department of Medicine, The University of Chicago, Chicago, IL 60637, USA;
abhikulkarni@medicine.bsd.uchicago.edu (A.K.); amina.basha@uchospitals.edu (A.B.);
ejmontgomery@uchicago.edu (E.M.); eermis@uchicago.edu (E.E.)
2The Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine,
Indianapolis, IN 46202, USA; saibrahi@iupui.edu (S.I.); inhaider@iu.edu (I.H.)
*Correspondence: mirmira@uchicago.edu (R.G.M.); ryananderson@uchicago.edu (R.M.A.)
† These authors contributed equally to this work.
Abstract:
Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in
adults. NAFLD progresses from benign liver fat accumulation to liver inflammation and cirrhosis, and
ultimately leads to liver failure. Although several rodent models have been established for studying
NAFLD, they have limitations that include cost, speed of disease development, key dissimilarities,
and poor amenability to pharmacological screens. Here, we present a novel 2-hit zebrafish model to
replicate aspects of NAFLD pathogenesis. We fed zebrafish larvae a high-fat diet (HFD) to drive liver
fat accumulation (first hit). Next, we exacerbated liver-specific inflammation using a transgenic line
(fabp10-CETI-PIC3) that induces the expression of proinflammatory cytokines following induction
with doxycycline (second hit). These hits promoted fat accumulation and liver inflammation, as
demonstrated by the high expression of inflammatory cytokines, macrophage infiltration, stress
induction, and hepatic lipid droplet accumulation. Furthermore, zebrafish in this paradigm showed
deranged glucose metabolism. To validate a small-molecule screening approach, we treated HFD-
fed fish with pioglitazone, a drug shown to be beneficial for NAFLD in humans, and measured a
sharp reduction in liver lipid accumulation. These results demonstrate new utility for zebrafish in
modeling early NAFLD pathogenesis and demonstrate their feasibility for
in vivo
screening of new
pharmacological interventions.
Keywords: NAFLD; inflammation; zebrafish; obesity; PIC3; macrophages; models; screening;
pioglitazone
1. Introduction
In the United States, the number of NAFLD cases is projected to expand to 100.9 million
in 2030, and the global prevalence is estimated at around 25% [
1
]. NAFLD comprises a broad
spectrum of liver damage, which can range from macrovesicular steatosis to steatohepatitis
(NASH), fibrosis and liver injury to cirrhosis, and hepatocellular carcinoma (HCC) [
2
].
Compared to the incidence of HCC in other liver diseases, a larger percentage of HCCs
that arise in NASH occur before patients are cirrhotic, leading to larger and less treatable
tumors [
3
]. Although there has been a growing interest in learning the pathogenesis of
NAFLD and identifying key steps in development that could help stop the progression of
the disease, no cure and few therapies have been developed.
There is a multiple-hit theory for the pathogenesis of NAFLD [
4
,
5
]. An initial “hit”
comprises an insulin-resistant state that results from a predisposition to inflammation in
the adipose tissue in the setting of a hypercaloric diet rich in fats and carbohydrates. The
development of insulin resistance drives an increase in circulating free fatty acids, which
may be sequestered in the liver via lipogenesis. This lipid accumulation, along with the
Biomedicines 2022,10, 479. https://doi.org/10.3390/biomedicines10020479 https://www.mdpi.com/journal/biomedicines
Biomedicines 2022,10, 479 2 of 14
presence of chronic inflammation, comprises a second “hit” in the pathogenesis of NAFLD.
Recent studies in mice have revealed that hepatocyte inflammation may be an important
link between the initial metabolic stress and subsequent hepatocyte death and stimulation
of fibrosis in NASH [
6
,
7
]. Hepatic fibrosis is caused by the activation of hepatic stellate
cells and myofibroblasts that leads to the subsequent deposition of a fibrous extracellular
matrix [
8
]. Finally, through the expression of proinflammatory cytokines, such as IL-1
β
and
IL-18, apoptosis is promoted.
To unravel the mechanisms of NAFLD pathogenesis, the majority of animal studies
have utilized rodent models, such as mice and rats [
9
]. While these models have indeed
provided a means to a deeper understanding of the roles of diet and overnutrition in the
pathogenesis of NAFLD, they come with many limitations. These include cost, time and
scale limitations, difficulty of
in vivo
pathological studies, and impracticality of
in vivo
small molecule screening. Recent studies have shown that lipid metabolism in zebrafish is
similar to that in humans [
10
]. The benefits of using zebrafish for studying NAFLD include
low cost, fast maturation, ease of genetic modification, and feasibility of experimental
manipulations with respect to treatment paradigms [11].
Zebrafish serve as a physiologically relevant model system to study NAFLD owing
to the similarity of the zebrafish hepato-pancreatic-biliary anatomy to humans [
12
]. Fur-
thermore, at early developmental stages, the yolk strongly expresses apolipoproteins to
metabolize the yolk [
13
]. Similar to mammals, microsomal triglycerides transfer protein
play a critical role in lipid transport in zebrafish [
14
]. The zebrafish also express cholesteryl
ester transfer protein (CETP) and are susceptible to atherosclerosis, making them attrac-
tive models for studying this disorder [
15
]. Apart from these physiological similarities,
zebrafish also possess orthologues of critical lipid metabolic genes including microsomal
TG transfer protein (mttp), fatty acid transport protein (slc27a), and acyl-CoA synthetase
(acsl) gene families, as well as the LDL receptor (ldlr). In addition, the expression pattern of
these genes appear to be comparable to humans [16].
In addition, the transparency and whole-body real-time monitoring make the larval
zebrafish a promising model to study NAFLD [
17
]. Although there have been a few studies
that have tried to utilize zebrafish for studying NAFLD pathogenesis using treatment
paradigms similar to rodent models [
11
,
18
], there are no zebrafish models that demonstrate
how inflammation plays a role in the pathogenesis of this disease. Recently, we established
a model for tissue-specific, titratable cytokine induction in zebrafish [
19
], which may serve
as a powerful animal model for studying diseases with a maladaptive inflammatory com-
ponent. The Cre-Enabled Tetracycline Inducible transgenic system (CETI-PIC3) provides
for liver-restricted overexpression of the cytokines IL1
β
, TNF
α
, and IFN
γ
, when crossed to
the liver-specific fabp10:Cre zebrafish line. This system is novel and expedient because it
models the inflammatory component on NAFLD and studies the downstream effects of
cytokine induction of NAFLD.
2. Materials and Methods
2.1. Animal Experiments
Zebrafish (Danio rerio) were spawned and raised at 28.5
◦
C under standard laboratory
conditions approved by the University of Chicago and the Indiana University Institu-
tional Animal Care and Use Committees. The following established transgenic zebrafish
lines were utilized: Tg(mpeg:GFP)
gl22
,Tg(fabp10:dsred,ela3l:EGFP)
gz12
(referred to as 2CLIP
throughout for 2-Color Liver and Pancreas), and Tg(CETI-PIC3)
iu15
. A novel Tg(fabp10:cre)
line was generated using transgene constructs described previously [
20
]. Tg(fabp10:cre) and
Tg(CETI-PIC3)
iu15
fish were used together to generate “Li-PIC3” zebrafish for liver-specific
induction of zebrafish orthologues of the cytokines IL1
β
, TNF
α
, and IFN
γ
.Tg(mpeg:GFP)
fish were utilized for macrophage visualization and Tg(fabp10:dsRed) fish were used for
liver visualization. All embryos were collected at spawning and incubated at 28.5
◦
C
in egg water-filled (0.1% instant ocean salt, 0.0075% calcium sulfate) petri dishes. After
Biomedicines 2022,10, 479 3 of 14
gastrulation stages, 0.003% 1-Phenyl-2-thiourea (PTU; Acros #207250250, New Jersey, USA)
supplementation in egg water was used to prevent pigmentation in all embryos and larvae.
2.2. Inflammatory Liver Model
Tg(CETI-PIC3)
iu15
fish were crossed with Tg(fabp10:cre) fish to generate CETI-PIC3/fabp10:cre
embryos that overexpress cytokines IL-1
β
, TNF
α
, and IFN
γ
following treatment with doxy-
cycline. Ten CETI-PIC3/fabp10:cre embryos and ten Tg(fabp10:cre) control larvae at 7 days
post fertilization (dpf) were incubated in 3 mL of 2.5
µ
g/mL doxycycline in egg water for
24 h. For time-point studies, 7 dpf larvae were treated for 0 h, 3 h, 6 h, 12 h, or 24 h. Larvae
were fixed at 8 dpf with 3% formaldehyde in PEM buffer (0.21M PIPES, 1 mM MgSO
4
,
2 mM EGTA, and pH 7) at 4 ◦C overnight.
2.3. High-fat Diet Feeding and Lipid Staining
A 5% chicken egg solution in egg water (High-fat diet, HFD) was prepared by mixing
1 mL chicken egg in 19 mL egg water. BODIPY analog (Invitrogen # D3922, Carlsbad,
CA, USA) was added to 10 mL of the 5% chicken egg solution and vortexed for 30 s for
a final concentration of 6.4
µ
M of BODIPY-FL. Twenty 5 dpf larvae were incubated in
4 mL of the chicken egg solution for 2 h in the dark at 28.5
◦
C and washed in egg water
following feeding. Feeding was repeated at 7 dpf, and larvae were fixed at 8 dpf with 3%
formaldehyde in a PEM buffer at 4 ◦C overnight.
2.4. Pioglitazone Treatment
Pioglitazone (PIO; Thermo Fisher Scientific # 41-245-0, Waltham, MA, USA) was added
to egg water at 5 or 10
µ
M along with the HFD. After 2 h of HFD treatment, the larvae were
washed with egg water and incubated again with the same concentration of PIO until the
following HFD treatment, and then to the end of the experiment. PIO-treated embryos and
controls were stained with BODIPY dissolved at 40
µ
M in 0.1% DMSO for 1 h, then washed
extensively for 1 h and fixed in 3% formaldehyde in buffered saline at 4
◦
C overnight.
Larvae were micro-dissected to remove skin and any residual yolk, then mounted in
Vectashield (Vector Labs, Burlingame, CA, USA) and imaged with confocal microscopy.
One microliter optical sections were collected and analyzed by NIH FIJI software using
thresholding, contiguous area measurement, and analyze particles functions. The index for
comparing fatty liver conditions was calculated by dividing the number of lipid droplets by
the pixel area marked by fabp10:dsRed (2CLIP) fluorescence in each sample, and is displayed
in arbitrary units.
2.5. Free Glucose Assay
Glucose colorimetric assays were performed to measure free glucose levels in larvae.
Glucose colorimetric assays (BioVision #K686, Milpitas, CA, USA) were performed by ho-
mogenizing 20 larvae within 500
µ
L of assay buffer and then following the manufacturer’s
protocol. Results were measured using a SpectraMax M5 multiwell plate reader (Molecular
Devices, Sunnyvale, CA, USA).
2.6. Combination Inflammatory Liver and High-Fat Diet Feeding Model
Li-PIC3 larvae were fed HFD at 5 dpf and 7 dpf as described above. Following HFD
feeding at 7 dpf, larvae were incubated in 3 mL of 2.5
µ
g/mL Doxycycline in egg water for
24 h. Larvae were then fixed with 3% formaldehyde in a buffered saline at 4
◦
C overnight.
2.7. Detection of Reactive Oxidative Species
To detect Reactive Oxidative Species (ROS), zebrafish were incubated in 10
µ
M CellRox
Deep Red (Invitrogen #C10422, Carlsbad, CA, USA) in egg water in the dark at 28.5
◦
C for
1 h. Larvae were washed with PBS and fixed immediately after with 3% formaldehyde in
buffered saline at 4 ◦C overnight, as described previously [21].
Biomedicines 2022,10, 479 4 of 14
2.8. Immunofluorescence Staining
Antibody staining was performed as described [
22
]. Briefly, whole mount zebrafish
samples were stained with primary antibodies: mouse anti-TNF
α
(1:50; Abcam #52B83,
Cambridge, UK), guinea pig anti-insulin (1:200, Invitrogen #180067, Carlsbad, CA, USA),
rabbit anti-cleaved caspase 3 (1:100; Cell Signaling Technologies #9661S, Danvers, MA,
USA), mouse anti-Prox1 (1:50, Developmental Studies Hybridoma Bank, AB_2619013, Iowa
City, IA, USA), and chicken anti-GFP (1:200, Aves labs #GFP-1010, Davis, CA, USA). Primary
antibodies were detected with complementary Alexa-conjugated secondary antibodies
(1:500, Jackson ImmunoResearch, West Grove, PA, USA). DNA was stained with TO-PRO3
(1:500, Thermo Fisher #T3605, Waltham, MA, USA) or DAPI (1:400, Thermo Fisher #62248,
Waltham, MA, USA). After staining, larvae were mounted on slides in VECTASHIELD
(Vector Labs H-1000). Confocal imaging was performed with a Zeiss LSM700 or Nikon A1
microscope. Images of livers were analyzed using NIH Fiji software. Images following
immunofluorescence staining were used to verify the presence of TNF
α
and H2BGFP in
livers and to count infiltrating macrophages.
2.9. Quantitative Real-Time PCR
RNA isolation and reverse transcription were performed using miRNeasy and miScript
II RT kits according to the manufacturer’s protocol (Qiagen, Hilden, Germany). Quantita-
tive real-time PCR (qPCR) was performed using the miScript SYBR Green PCR Kit (Qiagen,
Hilden, Germany) and a Mastercycler realplex instrument (Eppendorf, Hauppage, NY,
USA). Using the comparative Ct method as described [
23
], mRNA levels of IL-1
β
, TNF
α
,
and IFNγwere determined relative to mRNA levels of β-actin.
2.10. Statistical Analysis
Statistical analyses were performed using GraphPad Prism Version 9.3 (GraphPad Soft-
ware, La Jolla, CA, USA). Student’s t-tests were used for comparison between experimental
and control groups. One-way ANOVA with Tukey’s post-test for multiple comparisons
was used when comparing more than two groups. A p-value of
≤
0.05 was considered
significant (indicated with *) for all analyses. Lesser p-values of
≤
0.01,
≤
0.001, and
≤
0.0001
were indicated with **, ***, and ****, respectively.
3. Results
3.1. Models Exhibit Cytokine Expression and Lipid Accumulation in the Hepatocytes
First, to produce a state of overnutrition in our model, we used a high-fat feeding
paradigm (depicted in Figure 1A) wherein zebrafish larvae were immersed in a 5% ho-
mogenized chicken egg solution for 2 h, at both 4 days post-fertilization (dpf) and 6 dpf.
This high-fat diet (HFD) treatment of larvae resulted in fat accumulation in the liver, as
revealed by BODIPY labeling of lipid droplets in hepatocytes (Figure 1B,C). To determine
if an inflammatory state was promoted, we measured the expression of the zebrafish or-
thologues of the pro-inflammatory cytokines IFN
γ
(ifng), TNF
α
(tnfa), and IL-1
β
(il1b)
using quantitative PCR (qPCR) of whole embryo cDNA preparations and found a mild but
statistically significant increase in tnfa only (Figure 1D). These results indicate that dietary
lipids are being consumed by the larvae, that these are processed and trafficked to the
hepatocytes, and this may induce a mildly inflamed state.
Biomedicines 2022,10, 479 5 of 14
Biomedicines 2022, 10, 479 5 of 14
Figure 1. High fat diet treatment. (A) The schematic for high-fat diet feedings of the zebrafish larvae.
(B,C) BODIPY labeling of unfed (B) and 5% High-fat Diet (HFD) fed larvae (C). There is minimal
accumulation of lipid droplets in the liver of unfed larvae (outlined by dashed white line), while
HFD treatment resulted in significant accumulation of lipid droplets in hepatocytes. (D) The larval
mRNA level of tnfa only is significantly increased in response to HFD. Expression analyses were
performed on whole larvae. Statistical analyses by students t-test, n = 4–5 clutches with at least 15
embryos per clutch. ns—not significant,* p < 0.05.
Next, we sought to elicit a hyper-inflammatory response in the liver by directly in-
ducing the misexpression of IFNγ, TNFα, and IL1β using the Cre-Enabled Tetracycline
Inducible transgenic zebrafish line (CETI-PIC3) that we previously developed [19]. For
this approach, CETI-PIC3 transgenic zebrafish were bred with fabp10:cre transgenic ani-
mals to generate fabp10:Cre
+
; CETI-PIC3
+
(henceforth Li-PIC3), which express cre only in
hepatocytes [24] (Figure 2A). Thus, in these doubly transgenic animals, the hepatocyte-
restricted transcription of cytokines and a translationally-linked fluorescent H2B-GFP
marker located downstream from the cytokines, is enabled after recombination. Treat-
ment with 2.5 µg/mL doxycycline from 6 dpf to 7 dpf (schematized in Figure 2B) induced
the expression of the three cytokines and the H2B-GFP marker in the liver (Figure 2C–E).
Hepatocyte expression of H2B-GFP and TNFα was confirmed via immunofluorescent
staining (Figure 2C,D). Furthermore, expression of the cytokines and H2B-GFP was ex-
amined by qPCR in whole embryo cDNA preparations, and each showed significantly
elevated expression relative to fabp10:cre controls.
Figure 1.
High fat diet treatment. (
A
) The schematic for high-fat diet feedings of the zebrafish larvae.
(
B,C
) BODIPY labeling of unfed (
B
) and 5% High-fat Diet (HFD) fed larvae (
C
). There is minimal
accumulation of lipid droplets in the liver of unfed larvae (outlined by dashed white line), while HFD
treatment resulted in significant accumulation of lipid droplets in hepatocytes. (
D
) The larval mRNA
level of tnfa only is significantly increased in response to HFD. Expression analyses were performed
on whole larvae. Statistical analyses by students t-test, n = 4–5 clutches with at least 15 embryos per
clutch. ns—not significant,* p< 0.05.
Next, we sought to elicit a hyper-inflammatory response in the liver by directly in-
ducing the misexpression of IFN
γ
, TNF
α
, and IL1
β
using the Cre-Enabled Tetracycline
Inducible transgenic zebrafish line (CETI-PIC3) that we previously developed [
19
]. For this
approach, CETI-PIC3 transgenic zebrafish were bred with fabp10:cre transgenic animals to
generate fabp10:Cre
+
; CETI-PIC3
+
(henceforth Li-PIC3), which express cre only in hepato-
cytes [
24
] (Figure 2A). Thus, in these doubly transgenic animals, the hepatocyte-restricted
transcription of cytokines and a translationally-linked fluorescent H2B-GFP marker located
downstream from the cytokines, is enabled after recombination. Treatment with 2.5
µ
g/mL
doxycycline from 6 dpf to 7 dpf (schematized in Figure 2B) induced the expression of the
three cytokines and the H2B-GFP marker in the liver (Figure 2C–E). Hepatocyte expression
of H2B-GFP and TNF
α
was confirmed via immunofluorescent staining (Figure 2C,D).
Furthermore, expression of the cytokines and H2B-GFP was examined by qPCR in whole
embryo cDNA preparations, and each showed significantly elevated expression relative to
fabp10:cre controls.
Lastly, we combined these Li-PIC3 and HFD-feeding approaches to generate a multiple-
hit model of NAFLD (Li-PIC3+HFD, schematized in Figure 2F). Larvae were immersed in
5% chicken egg solution for 2 h at 4 and 6 dpf, then treated with 2.5
µ
g/mL doxycycline
from 6–7 dpf. Again, we used qPCR to measure transcriptional changes in cytokine
expression and found that the levels of ifng, tnfa, and il1b mRNA were all significantly
increased as compared to Tg(fabp10:cre) control larvae that were not fed HFD (Figure 2G).
Biomedicines 2022,10, 479 6 of 14
Biomedicines 2022, 10, 479 6 of 14
Figure 2. Hepatic induction of proinflammatory cytokines. (A) Diagram of the CETI-PIC3 transgene
that is enabled in a hepatocyte-specific manner by fabp10:cre. (B) Experimental scheme for induction
of the PIC3 cytokines at 6 dpf. (C–D’’’) Immunostaining of 7 dpf Tg(fabp10:cre) control larvae (C)
and Li-PIC3 larvae (D) for GFP (green, C,D), TNFα (magenta, C’,D’), DNA (blue, C’’,D’’), and the
merged images (C’’’,D’’’). The PIC3 cassette was strongly induced by doxycycline, as evidenced by
both GFP and TNFα labeling in the liver (outlined by dashed white line). (E) The levels of ifng, tnfa,
il1b, and H2B-GFP are all increased after doxycycline induction in the Li-PIC3 embryos as compared
to Tg(fabp10:cre) control embryos. (F) Diagram for combined HFD followed by dox induction in zebrafish
larvae. (G) The levels of ifng, tnfa, and il1b are all increased in Li-PIC3/HFD larvae as compared to
Tg(fabp10:cre) control embryos. Expression analyses were performed on whole larvae. Statistical analyses
by students t-test, n = 4–5 clutches with at least 15 embryos per clutch. ** p < 0.01, *** p < 0.001, **** p <
0.0001.
Lastly, we combined these Li-PIC3 and HFD-feeding approaches to generate a mul-
tiple-hit model of NAFLD (Li-PIC3+HFD, schematized in Figure 2F). Larvae were im-
mersed in 5% chicken egg solution for 2 h at 4 and 6 dpf, then treated with 2.5 µg/mL doxycy-
Figure 2.
Hepatic induction of proinflammatory cytokines. (
A
) Diagram of the CETI-PIC3 transgene
that is enabled in a hepatocyte-specific manner by fabp10:cre. (
B
) Experimental scheme for induction
of the PIC3 cytokines at 6 dpf. (
C
–
D”’
) Immunostaining of 7 dpf Tg(fabp10:cre) control larvae (
C
)
and Li-PIC3 larvae (
D
) for GFP (green, C,D), TNF
α
(magenta,
C’
,
D’
), DNA (blue,
C”
,
D”
), and the
merged images (
C”’
,
D”’
). The PIC3 cassette was strongly induced by doxycycline, as evidenced by
both GFP and TNF
α
labeling in the liver (outlined by dashed white line). (
E
) The levels of ifng,tnfa,
il1b, and H2B-GFP are all increased after doxycycline induction in the Li-PIC3 embryos as compared
to Tg(fabp10:cre) control embryos. (
F
) Diagram for combined HFD followed by dox induction in
zebrafish larvae. (
G
) The levels of ifng,tnfa, and il1b are all increased in Li-PIC3/HFD larvae as
compared to Tg(fabp10:cre) control embryos. Expression analyses were performed on whole larvae.
Statistical analyses by students t-test, n = 4–5 clutches with at least 15 embryos per clutch. ** p< 0.01,
*** p< 0.001, **** p< 0.0001.
Biomedicines 2022,10, 479 7 of 14
3.2. NAFLD Induction Promotes Infiltration of Macrophages
NAFLD progression is characterized by the infiltration of macrophages [
25
]. To study
the phenotype of macrophage infiltration in our NAFLD model, we bred the Tg(mpeg:GFP)
allele into our Li-PIC3 transgenic fish to mark macrophages with green fluorescence.
Whereas macrophages were rarely observed within the liver boundaries at 7 dpf under
control conditions (Figure 3A), they were often observed in the livers of HFD, Li-PIC3, and
Li-PIC3+HFD larvae (Figure 3B–D). When infiltrating macrophages were quantified, we
calculated a statistically significant elevation in hepatic macrophage infiltration for HFD
and Li-PIC3+HFD larvae relative to controls (Figure 3E).
Biomedicines 2022, 10, 479 7 of 14
cline from 6–7 dpf. Again, we used qPCR to measure transcriptional changes in cytokine ex-
pression and found that the levels of ifng, tnfa, and il1b mRNA were all significantly increased
as compared to Tg(fabp10:cre) control larvae that were not fed HFD (Figure 2G).
3.2. NAFLD Induction Promotes Infiltration of Macrophages
NAFLD progression is characterized by the infiltration of macrophages [25]. To study
the phenotype of macrophage infiltration in our NAFLD model, we bred the
Tg(mpeg:GFP) allele into our Li-PIC3 transgenic fish to mark macrophages with green flu-
orescence. Whereas macrophages were rarely observed within the liver boundaries at 7
dpf under control conditions (Figure 3A), they were often observed in the livers of HFD,
Li-PIC3, and Li-PIC3+HFD larvae (Figure 3B–D). When infiltrating macrophages were
quantified, we calculated a statistically significant elevation in hepatic macrophage infil-
tration for HFD and Li-PIC3+HFD larvae relative to controls (Figure 3E).
Figure 3.
Macrophage infiltration is increased in HFD, Li-PIC3, and Li-PIC3+HFD
larvae. (A–D) 7 dpf
larvae labeled with GFP immunostaining to reveal macrophages and either a 2CLIP transgene
(
A,B
) or immunolabeled with Prox1 antibody (
C,D
) to reveal hepatocytes. Infiltrating macrophages
are indicated by white arrows. (
E
) Quantification of macrophages within the liver boundary reveals
a statistically significant increase with HFD and Li-PIC3+HFD treatments. Statistical analysis by
one-way ANOVA, ns-non significant, ** p< 0.01, *** p< 0.001.
Biomedicines 2022,10, 479 8 of 14
3.3. NAFLD Induction Leads to ROS Accumulation and Liver Damage
To assess reactive oxygen species (ROS)-mediated stress in the liver, which is a common
feature of NAFLD, cohorts of HFD-fed, Li-PIC3, and Li-PIC3+HFD larvae, and controls
were each stained with CellRox Deep Red, an indicator of ROS (Figure 4A–D). When
the intensity of CellRox staining was quantified, we detected no increase with either of
the single treatments, HFD or Li-PIC3 (Figure 4E). However, we detected nearly a 2-fold
increase in ROS under the combined Li-PIC3+HFD experimental conditions. Next, in
humans with liver damage, gamma-glutamyl transferase (GGT) and lactate dehydrogenase
(LDH) proteins are released into circulation [
26
,
27
], and their levels may be used as a
diagnostic benchmark for determining the extent of the injury. Accordingly, we reasoned
that ggt and ldh1a mRNA expression levels would be correspondingly elevated in the
hepatocytes of damaged livers, mirroring their corresponding elevated serum protein
levels; this would permit measurement of ggt and ldh1a in zebrafish larvae as a surrogate
approach to quantifying the extent of hepatocyte injury where it is not possible to assay
serum. Thus, we measured the mRNA expression levels of ldh1a and ggt using qPCR.
(Figure 4F,G). Whereas larvae subjected to each of the single experimental stresses showed
no significant evidence of liver injury, those in the combined Li-PIC3+HFD condition
showed striking elevations of ggt (>50-fold) and ldh1a (>230-fold).
Biomedicines 2022, 10, 479 8 of 14
Figure 3. Macrophage infiltration is increased in HFD, Li-PIC3, and Li-PIC3+HFD larvae. (A–D) 7
dpf larvae labeled with GFP immunostaining to reveal macrophages and either a 2CLIP transgene
(A,B) or immunolabeled with Prox1 antibody (C,D) to reveal hepatocytes. Infiltrating macrophages
are indicated by white arrows. (E) Quantification of macrophages within the liver boundary reveals
a statistically significant increase with HFD and Li-PIC3+HFD treatments. Statistical analysis by
one-way ANOVA, ns-non significant, ** p < 0.01, *** p < 0.001.
3.3. NAFLD Induction Leads to ROS Accumulation and Liver Damage
To assess reactive oxygen species (ROS)-mediated stress in the liver, which is a com-
mon feature of NAFLD, cohorts of HFD-fed, Li-PIC3, and Li-PIC3+HFD larvae, and con-
trols were each stained with CellRox Deep Red, an indicator of ROS (Figure 4A–D). When
the intensity of CellRox staining was quantified, we detected no increase with either of
the single treatments, HFD or Li-PIC3 (Figure 4E). However, we detected nearly a 2-fold
increase in ROS under the combined Li-PIC3+HFD experimental conditions. Next, in hu-
mans with liver damage, gamma-glutamyl transferase (GGT) and lactate dehydrogenase
(LDH) proteins are released into circulation [26,27], and their levels may be used as a di-
agnostic benchmark for determining the extent of the injury. Accordingly, we reasoned
that ggt and ldh1a mRNA expression levels would be correspondingly elevated in the
hepatocytes of damaged livers, mirroring their corresponding elevated serum protein lev-
els; this would permit measurement of ggt and ldh1a in zebrafish larvae as a surrogate
approach to quantifying the extent of hepatocyte injury where it is not possible to assay
serum. Thus, we measured the mRNA expression levels of ldh1a and ggt using qPCR. (Fig-
ure 4F,G). Whereas larvae subjected to each of the single experimental stresses showed no
significant evidence of liver injury, those in the combined Li-PIC3+HFD condition showed
striking elevations of ggt (>50-fold) and ldh1a (>230-fold).
Figure 4. ROS mediated stress is induced in livers of zebrafish fed a high-fat diet and liver-specific
cytokine induced embryos. (A–D) Cell Rox Deep Red staining used to visualize ROS-mediated
stress in the livers of control, HFD-fed, Li-PIC3, and Li-PIC+HFD larvae. (E) Quantification of Cell
Rox intensity demonstrated an increase in only Li-PIC3+HFD embryos as compared to Tg(fabp10:cre)
controls, while the comparison to controls trended towards significance (p = 0.776). (F) Quantifica-
tion of ldh1a mRNA levels by qPCR (G) Quantification of ggt mRNA levels by qPCR. Both ldh1a
and ggt are elevated in the combined Li-PIC3+HFD condition. Statistical analysis was by one-way
ANOVA, n = 4–5 per condition; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
3.4. Association of NAFLD with Insulin Resistance
In humans, NAFLD is strongly associated with insulin resistance, obesity, and type
2 diabetes [28]. We hypothesize that treatment with HFD, Li-PIC3, and their combination
Figure 4.
ROS mediated stress is induced in livers of zebrafish fed a high-fat diet and liver-specific
cytokine induced embryos. (
A
–
D
) Cell Rox Deep Red staining used to visualize ROS-mediated stress
in the livers of control, HFD-fed, Li-PIC3, and Li-PIC+HFD larvae. (
E
) Quantification of Cell Rox
intensity demonstrated an increase in only Li-PIC3+HFD embryos as compared to Tg(fabp10:cre)
controls, while the comparison to controls trended towards significance (p= 0.776). (
F
) Quantification
of ldh1a mRNA levels by qPCR (
G
) Quantification of ggt mRNA levels by qPCR. Both ldh1a and ggt
are elevated in the combined Li-PIC3+HFD condition. Statistical analysis was by one-way ANOVA,
n= 4–5 per condition; * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001.
3.4. Association of NAFLD with Insulin Resistance
In humans, NAFLD is strongly associated with insulin resistance, obesity, and type 2
diabetes [
28
]. We hypothesize that treatment with HFD, Li-PIC3, and their combination is
associated with increasing levels of pancreatic
β
cell dysfunction/insufficiency as reflected
by increasing systemic glucose levels. To measure glucose levels in zebrafish larvae, cohorts
of like-treated larvae were pooled and homogenized, and free glucose levels were measured
via colorimetric assay as previously described [
22
]. We found that glucose was elevated
relative to controls under all conditions tested (Figure 5A), though the highest levels were
measured in larvae subjected to the Li-PIC3+HFD combined treatment. To determine
if this relative deficit in
β
cell function was related to a change in the absolute quantity
of
β
cells, we counted
β
cells using confocal microscopy. No differences were observed
Biomedicines 2022,10, 479 9 of 14
among any of the treatments (Figure 5B). Next, we measured the expression of mRNA
to determine if insulin levels produced by the
β
cells were responsible for the change in
glucose levels. We found small but significant variation in insulin expression, with the
lowest levels in the single Li-PIC3 larvae (Figure 5C). Lastly, we measured the expression of
phosphoenolpyruvate carboxykinase (pck1) in zebrafish as a measure of insulin signaling;
pck1 is expressed in the liver, where it catalyzes the first committed step in gluconeogenesis,
and its expression is suppressed by insulin. We found that pck1 levels remained unchanged
with the single perturbations of HFD and Li-PIC3 but were elevated nearly 10-fold with
the combination Li-PIC3+HFD treatment (Figure 5D).
Biomedicines 2022, 10, 479 9 of 14
is associated with increasing levels of pancreatic β cell dysfunction/insufficiency as re-
flected by increasing systemic glucose levels. To measure glucose levels in zebrafish lar-
vae, cohorts of like-treated larvae were pooled and homogenized, and free glucose levels
were measured via colorimetric assay as previously described [22]. We found that glucose
was elevated relative to controls under all conditions tested (Figure 5A), though the high-
est levels were measured in larvae subjected to the Li-PIC3+HFD combined treatment. To
determine if this relative deficit in β cell function was related to a change in the absolute
quantity of β cells, we counted β cells using confocal microscopy. No differences were
observed among any of the treatments (Figure 5B). Next, we measured the expression of
mRNA to determine if insulin levels produced by the β cells were responsible for the
change in glucose levels. We found small but significant variation in insulin expression,
with the lowest levels in the single Li-PIC3 larvae (Figure 5C). Lastly, we measured the
expression of phosphoenolpyruvate carboxykinase (pck1) in zebrafish as a measure of in-
sulin signaling; pck1 is expressed in the liver, where it catalyzes the first committed step
in gluconeogenesis, and its expression is suppressed by insulin. We found that pck1 levels
remained unchanged with the single perturbations of HFD and Li-PIC3 but were elevated
nearly 10-fold with the combination Li-PIC3+HFD treatment (Figure 5D).
Figure 5. Hyperglycemia is induced without loss of β cells in HFD, Li-PIC3, and Li-PIC3+HFD lar-
vae. (A) Blood glucose levels for Tg(fabp10:cre), HFD, Li-PIC3, and Li-PIC3+HFD larvae. (B) β cell
count in Tg(fabp10:cre), HFD, Li-PIC3, and Li-PIC3+HFD larvae. (C) Transcript levels for insulin
Tg(fabp10:cre), HFD, Li-PIC3, and Li-PIC3+HFD larvae. (D) Levels of pck1, a marker of gluconeogen-
esis, in Tg(fabp10:cre), HFD, Li-PIC3, and Li-PIC3+HFD larvae. Statistical analysis was by one-way
ANOVA, n = 3–9; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
3.5. Pioglitazone Treatment Abates Steatosis
The thiazolidinedione PPARγ agonist Pioglitazone (PIO) stimulates hepatic fatty
acid oxidation and improves insulin sensitivity and reduces steatosis in NAFLD patients
[29,30]. Thus, to test the utility of our zebrafish model in screening for pharmacological
interventions, we treated HFD-fed zebrafish larvae in combination with PIO between 4
and 7 dpf (Figure 6A). BODIPY-labeled neutral lipid droplets were quantified per pixel
area of liver tissue in confocal optical sections using FIJI software. As expected, HFD treat-
ment significantly increased fat deposits in the liver (511.0 ± 315.2 in control unfed larvae
versus 1174 ± 230.7 in HFD-fed, p = 0.03) (Figure 6B,C,E). While a lower dose of 1µM PIO
showed no significant effect on lipid accumulation versus HFD alone (1104 ± 430.7 p =
Figure 5.
Hyperglycemia is induced without loss of
β
cells in HFD, Li-PIC3, and Li-PIC3+HFD
larvae. (
A
) Blood glucose levels for Tg(fabp10:cre), HFD, Li-PIC3, and Li-PIC3+HFD larvae. (
B
)
β
cell count in Tg(fabp10:cre), HFD, Li-PIC3, and Li-PIC3+HFD larvae. (
C
) Transcript levels for insulin
Tg(fabp10:cre), HFD, Li-PIC3, and Li-PIC3+HFD larvae. (
D
) Levels of pck1, a marker of gluconeogene-
sis, in Tg(fabp10:cre), HFD, Li-PIC3, and Li-PIC3+HFD larvae. Statistical analysis was by one-way
ANOVA, n = 3–9; * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001.
3.5. Pioglitazone Treatment Abates Steatosis
The thiazolidinedione PPAR
γ
agonist Pioglitazone (PIO) stimulates hepatic fatty acid
oxidation and improves insulin sensitivity and reduces steatosis in NAFLD patients
[29,30]
.
Thus, to test the utility of our zebrafish model in screening for pharmacological inter-
ventions, we treated HFD-fed zebrafish larvae in combination with PIO between 4 and
7 dpf (Figure 6A). BODIPY-labeled neutral lipid droplets were quantified per pixel area of
liver tissue in confocal optical sections using FIJI software. As expected, HFD treatment
significantly increased fat deposits in the liver (511.0
±
315.2 in control unfed larvae versus
1174
±
230.7 in HFD-fed, p= 0.03) (Figure 6B,C,E). While a lower dose of 1
µ
M PIO showed
no significant effect on lipid accumulation versus HFD alone (1104
±
430.7 p= 0.982),
treatment with 5
µ
M PIO significantly reduced the quantity of lipid droplets (275.5
±
215.5,
p= 0.0022), returning them to control levels (Figure 6D,E).
Biomedicines 2022,10, 479 10 of 14
Biomedicines 2022, 10, 479 10 of 14
0.982), treatment with 5 µM PIO significantly reduced the quantity of lipid droplets (275.5
± 215.5, p = 0.0022), returning them to control levels (Figure 6 D,E).
Figure 6. Pioglitazone treatment reduces lipid droplet accumulation in the liver. (A) Experimental
scheme of PIO treatment. Larvae were immersed in PIO+ 0.1% DMSO for 1 h prior to the first HFD
treatment and remained in PIO throughout. (B–D) Representative optical slices of BODIPY-stained
2CLIP zebrafish liver depicting (B) untreated controls, (C) HFD-fed larvae, and (D) HFD-fed + PIO
hi (5 µM) treated larvae; hepatocytes are labeled in red via expression of the transgene fabp10:dsRed
and examples of green-labeled lipid droplets are indicated by arrows. (E) Quantification of lipid
droplets in hepatocytes area as executed with FIJI software. Statistical analysis was by one-way
ANOVA; * p < 0.05, ** p < 0.01. Scale bar = 50 µm.
4. Discussion
Lipid accumulation and inflammation in the liver both play a critical role in the path-
ogenesis of NAFLD [31]. Most in vivo studies of NAFLD pathogenesis using animal mod-
els have been performed in rodent models and have been largely dependent on diet and
obesity [11]. The methionine and choline-deficient (MCD) diet is high in sucrose and fat
but lacks methionine and choline, which are essential for hepatic β-oxidation; rats and
mice fed this diet develop features of NAFLD, including steatosis and fibrosis [32]. How-
ever, one major disadvantage to the MCD model is that insulin, leptin, and glucose levels
lead to greater insulin sensitivity, the opposite of that seen in NAFLD patients [33]. An-
other study demonstrated that mice fed a high-fat diet to the point of obesity developed
insulin resistance and hepatic damage. However, these mice did not have as severe liver
damage as those in the MCD model [34]. Thus, although many models can lead to lipid
accumulation in the liver, not all replicate aspects of the pathology of human NAFLD.
In this study, we utilized the strengths of the zebrafish model to address these limitations.
Due to substantial genetic, anatomical, and physiological similarities between the hepato-pan-
creatic systems of humans and zebrafish, these aquatic organisms are quite suitable for stud-
ying a metabolic disorder such as NAFLD. Previous studies by others have used zebrafish
embryos, larvae, and adults to model NAFLD through diets with high cholesterol, high fruc-
tose, or by simply excessively feeding for 10 days. Interestingly, the development of steatosis
has been found to be most severe in the excessive feeding models [11]. Other zebrafish studies
have shown that feeding zebrafish larvae a high-fat diet and a high-fat plus high-cholesterol
diet induces simple steatosis, with the high-fat plus high-cholesterol diet causing more severe
steatosis [18]. However, these models do not completely mimic human NAFLD pathogenesis
in which hyper-inflammation plays a critical role as well [35].
In our study, we subjected zebrafish larvae to a “two-hit” paradigm, wherein they were
first fed a lipid-rich diet to excess, followed by induction of cytokines using our Liver-CETI-
PIC3 system. We studied macrophage infiltration using the Tg(mpeg:GFP) line that we have
well-characterized for studying migration and infiltration of these immune cells in other in-
flammation models [36,37]. Curiously, while the animals subjected to the HFD alone exhibited
a clear inflammatory response, those only mis-expressing cytokines hepatically via Li-PIC3
Figure 6.
Pioglitazone treatment reduces lipid droplet accumulation in the liver. (
A
) Experimental
scheme of PIO treatment. Larvae were immersed in PIO+ 0.1% DMSO for 1 h prior to the first HFD
treatment and remained in PIO throughout. (
B
–
D
) Representative optical slices of BODIPY-stained
2CLIP zebrafish liver depicting (
B
) untreated controls, (
C
) HFD-fed larvae, and (
D
) HFD-fed + PIO hi
(5
µ
M) treated larvae; hepatocytes are labeled in red via expression of the transgene fabp10:dsRed and
examples of green-labeled lipid droplets are indicated by arrows. (
E
) Quantification of lipid droplets
in hepatocytes area as executed with FIJI software. Statistical analysis was by one-way ANOVA;
*p< 0.05, ** p< 0.01. Scale bar = 50 µm.
4. Discussion
Lipid accumulation and inflammation in the liver both play a critical role in the
pathogenesis of NAFLD [
31
]. Most
in vivo
studies of NAFLD pathogenesis using animal
models have been performed in rodent models and have been largely dependent on diet
and obesity [
11
]. The methionine and choline-deficient (MCD) diet is high in sucrose and
fat but lacks methionine and choline, which are essential for hepatic
β
-oxidation; rats
and mice fed this diet develop features of NAFLD, including steatosis and fibrosis [
32
].
However, one major disadvantage to the MCD model is that insulin, leptin, and glucose
levels lead to greater insulin sensitivity, the opposite of that seen in NAFLD patients [
33
].
Another study demonstrated that mice fed a high-fat diet to the point of obesity developed
insulin resistance and hepatic damage. However, these mice did not have as severe liver
damage as those in the MCD model [
34
]. Thus, although many models can lead to lipid
accumulation in the liver, not all replicate aspects of the pathology of human NAFLD.
In this study, we utilized the strengths of the zebrafish model to address these lim-
itations. Due to substantial genetic, anatomical, and physiological similarities between
the hepato-pancreatic systems of humans and zebrafish, these aquatic organisms are quite
suitable for studying a metabolic disorder such as NAFLD. Previous studies by others
have used zebrafish embryos, larvae, and adults to model NAFLD through diets with
high cholesterol, high fructose, or by simply excessively feeding for 10 days. Interestingly,
the development of steatosis has been found to be most severe in the excessive feeding
models [
11
]. Other zebrafish studies have shown that feeding zebrafish larvae a high-fat
diet and a high-fat plus high-cholesterol diet induces simple steatosis, with the high-fat
plus high-cholesterol diet causing more severe steatosis [
18
]. However, these models do
not completely mimic human NAFLD pathogenesis in which hyper-inflammation plays a
critical role as well [35].
In our study, we subjected zebrafish larvae to a “two-hit” paradigm, wherein they
were first fed a lipid-rich diet to excess, followed by induction of cytokines using our
Liver-CETI-PIC3 system. We studied macrophage infiltration using the Tg(mpeg:GFP) line
that we have well-characterized for studying migration and infiltration of these immune
cells in other inflammation models [
36
,
37
]. Curiously, while the animals subjected to the
HFD alone exhibited a clear inflammatory response, those only mis-expressing cytokines
hepatically via Li-PIC3 did not. Importantly, the addition of Li-PIC3 cytokine expression to
the HFD treatment led to an enhanced character of the inflammation in the liver, modeling
Biomedicines 2022,10, 479 11 of 14
a more severe state of steatohepatitis. For instance, increased ROS, decreased activity of
detoxifying enzymes, and an increase in net oxidative stress are strongly associated with
NAFLD (reviewed in [
38
,
39
]). The combination of HFD with Li-PIC3 resulted in increased
ROS in the liver as compared to HFD alone, while other conditions were indistinguishable
from one another. Additionally, expression of ldh1a and ggt, whose expression has been
shown to be elevated in some liver diseases [
40
,
41
] and used here as surrogate measures of
liver cell dysfunction, were unchanged from controls with either HFD or Li-PIC3 treatments
alone. However, expression of these markers was dramatically upregulated (200- and
50-fold for ldh1a and ggt, respectively) in the Li-PIC3+HFD animals. Lastly, we observed
elevated systemic free glucose levels in larvae under all experimental conditions, and whose
levels were highest in Li-PIC3+HFD larvae despite normal numbers of pancreatic beta cells
and normal insulin expression. This, together with the sharply increased expression of
phosphoenolpyruvate carboxykinase (pck1) only under Li-PIC3+HFD conditions, suggests
a deregulation of glucose metabolism mirroring aspects of human metabolic syndrome. As
insulin potently suppresses the transcription of pck1 within minutes, and as pck1 is a key
rate-limiting enzyme for gluconeogenesis, this finding is consistent with the interpretation
that Li-PIC3+HFD larvae are highly insulin resistant. Taken together, our data suggest
that excess dietary lipids are trafficked to the liver and elicit a generally benign immune
infiltration of macrophages. Upon hepatic expression of proinflammatory cytokines by
Li-PIC3, a local milieu is generated, which drives oxidative stress and triggers insulin
resistance and likely other cellular dysfunctions. It will be important in future studies to
characterize the infiltrating macrophages and to define their activation states, i.e., are they
classically activated, and how are the macrophages of any state of activation integral to the
observed NAFLD phenotypes in this model.
As presented in this study, our model system is unique and powerful for studying
NAFLD in that it combines fat accumulation with targeted inflammation in the liver. In the
future, our model can be modified to examine different degrees of steatosis. Furthermore,
because the CETI-PIC3 system is titratable, different levels of severity of NAFLD such as
Non-Alcoholic Steatohepatitis can be modeled through increased and or chronic cytokine
induction. In addition, we fed zebrafish larvae a high-fat diet consisting of 5% chicken
egg in PTU water, but alternative diets may be fed to zebrafish to model NAFLD-like
outcomes. These might include combination high-fat and high-cholesterol diets, or high-
fat/low-protein diets. Protein deficiency is associated with NAFLD and NASH because
proteins are important for hepatocyte regeneration and the prevention of fat deposits in
the liver [
42
]. Finally, in this study, while a HFD is used as the first “hit” and cytokine-
mediated inflammation the second parallel “hit” for pathogenesis of NAFLD, a model in
which cytokines are first induced and a HFD is fed afterward, can also be studied. It is
important to note that NAFLD pathogenesis is a complex metabolic disorder that involves
other factors including hormonal regulation [
43
], gut microbiota dysbiosis, and bile acid
abnormalities [
44
] that are not easily recapitulated at the larval stages of zebrafish. For that,
supplementary studies in more complex physiological conditions including adult staged
zebrafish or mammalian models are necessary.
Lastly, we have also demonstrated that zebrafish can be utilized as an efficient and
facile platform for investigating pharmacological agents that may be translatable to treat
NAFLD patients. Here, we used pioglitazone as a proof of this concept, as it has been
shown to improve NAFLD measures in human studies [
45
,
46
]. Pioglitazone binds to the
PPAR
γ
, a member of the nuclear receptor superfamily. PPAR
γ
is known to play a role in
glucose regulation and lipid metabolism [
47
]. Pioglitazone is metabolized by hydroxylation
and oxidation in the liver to form different metabolites [
48
]. Further studies could reveal
whether NAFLD outcomes might be modulated by titrating the dosages of pioglitazone.
Biomedicines 2022,10, 479 12 of 14
5. Conclusions
In conclusion, we have demonstrated that our zebrafish model can be utilized for
studying the early pathogenesis of NAFLD, especially for testing the 2-hit theory of NAFLD
pathogenesis. Our studies show the efficiency and feasibility of zebrafish as a platform
in vivo
for unbiased testing of novel small molecules that could impact the pathogenesis
of NAFLD.
Author Contributions:
A.K., S.I., R.G.M. and R.M.A., conceived the study; A.K., S.I., A.B., I.H., E.M.,
E.E. and R.M.A., provided methodology and performed experiments; A.K., S.I. and R.M.A., analyzed
the data; A.K., S.I., R.G.M. and R.M.A., wrote the original draft. All authors reviewed and edited the
manuscript. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was supported by the NIH, grants R01 DK060581 and R01 DK105588 (both
to R.G.M.) and F30 DK122681 (to S.I.). This work utilized core facility resources provided by NIH
grants P30 DK020595 (to the University of Chicago) and P30 DK097512 (to Indiana University).
Institutional Review Board Statement:
Experiments involving zebrafish were performed under a
protocol (#72636) approved on 24 April 2020 by the Institutional Animal Care and Use Committee
of the University of Chicago and a protocol (#11242) approved on 8 March 2017 by the Institutional
Animal Care and Use Committee of Indiana University.
Informed Consent Statement: Not applicable.
Data Availability Statement:
All of the primary data in this manuscript are available on Mendeley
Data (Mirmira, Raghavendra (2022), “A Novel 2-Hit Zebrafish Model to Study Early Pathogenesis of
Non-Alcoholic Fatty Liver Disease”, Mendeley Data, V1, doi:10.17632/tw458tcx88.1).
Conflicts of Interest: The authors declare no conflict of interest.
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