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Alginate oligosaccharide (AOS) improves

immuno-metabolic systems by inhibiting
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Cite this: DOI: 10.1039/c9fo00982e

STOML2 overexpression in high-fat-diet-
induced obese zebrafish†
a,b c
Van Cuong Tran, Se-Young Cho, Joseph Kwon*c and Duwoon Kim *a,d

Received 7th May 2019,
Accepted 2nd July 2019
DOI: 10.1039/c9fo00982e
rsc.li/food-function
This study aimed to evaluate the potentially beneficial effects of alginate oligosaccharide (AOS) on the
modulation of immuno-metabolic pathways in high-fat-diet (HFD)-induced obese zebrafish and the
underlying mechanism. AOS showed a marked anti-obesity effect in that it reduced body weight, BMI,
and the blood glucose level. To understand the mechanisms of action of AOS, comparative proteomics
was performed through UPLC-HDMSE analysis between HFD vs. normal diet (NFD) and HFD + AOS vs.
HFD. Among 146 proteins differentially modulated by AOS in HFD-induced obesity zebrafish, STOML2
(Stomatin-like protein 2) was selected as a specific biomarker. AOS suppressed obesity and pathophysio-
logical disorders in HFD-fed zebrafish by modulating lipid metabolism, suppressing inflammation, down-
regulating apoptosis-related genes, and improving immune function by inhibiting STOML2. Our results
suggest that STOML2 can serve as a platform for further studies to discover novel treatments for meta-
bolic disorders. AOS might be useful as a dietary health supplement, especially for reducing obesity.

1. Introduction Zebrafish are a popular model for studying metabolic

Alginate oligosaccharide (AOS) is a depolymerization product
of sodium alginate that has attracted increasing attention
in a wide variety of industries, including the food,1,2
agricultural,3–5 and pharmaceutical industries.6 Sodium algi-
nate and its derivatives confer health benefits owing to their
diverse biological activities, such as antioxidant,7,8 antifungal,9
anti-apoptotic,10,11 anti-inflammatory,12 and anti-tumor
effects.13,14 AOS has shown anti-obesity effect in vitro and
in vivo.15 Nevertheless, there are no reports on the impact of
dietary AOS on the regulation of the immuno-metabolic path-
ways in zebrafish on a high-fat diet.
a
Department of Food Science and Technology, Chonnam National University,
Gwangju, 61186, Republic of Korea. E-mail: dwkim@jnu.ac.kr; Fax: +82 62 530 2149
; Tel: +82 62 530 2144
b
Department of Food Science and Post-harvest Technology, Tay Nguyen University,
Daklak, Vietnam
c
Biological Disaster Analysis Group, Korea Basic Science Institute, Daejeon, 34133,
Republic of Korea. E-mail: joseph@kbsi.re.kr; Fax: +82 42 865 3419;
Tel: +82 42 865 3446
d
Foodborne Virus Research Center, Chonnam National University, Gwangju, 61186,
Republic of Korea
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9fo00982e
disorders and infectious diseases, toxicological studies, and
drug screening.16–19 Zebrafish have been used as a model for
high-fat-diet-induced obesity,20–22 as lipid metabolism in
zebrafish is very similar to that in humans.20 The zebrafish
genome has been sequenced, and the zebrafish physiology is
very similar to that of mammals; therefore, in the present
study, we used zebrafish to investigate how dietary AOS regu-
lates immuno-metabolic pathways. We conducted a compara-
tive proteomics analysis of high-fat-diet (HFD)- and normal-
diet (NFD)-fed animals.
STOML2 (Stomatin-like protein 2) is a mitochondrial
protein that is considered a cancer-related gene.23,24 STOML2
is overexpressed in various cancers, including human
non-small cell lung cancer cells, laryngeal carcinoma,
breast cancer, and gastric cancer, cervical cancer.23,25–27
Mitochondrial dysfunction is known to be linked to obesity.28
STOML2 overexpression might contribute to metabolism and
inflammatory responses.29 Further, there is a strong corre-
lation between obesity and cancers.30 Therefore, STOML2 may
play a role in obesity development. However, its mechanism of
action is not well characterized, and effects of STOML2 on
metabolic syndromes, especially obesity, have not been docu-
mented. As STOML2 is predicted to be a critical regulatory
protein, we explored its involvement in various pathways
involved in HFD-induced obesity in zebrafish.

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2. Materials and methods
2.1. Preparation and characterization of alginate
oligosaccharide (AOS)
Sodium alginate (Wako, Osaka, Japan) was used to produce
depolymerized alginate (AOS) by using bacterial alginate lyase.
The sodium alginate in phosphate buffer ( pH 7.0) was mixed
with the lyase and incubated at 30 °C for 24 h. After centrifu-
gation at 12 000g for 15 min, AOS in the supernatant was con-
centrated by ultrafiltration through a polysulfone membrane
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(VivaFlow 50; Sartorius, AG, Germany) with a molecular weight
(MW) cut-off of 3000 Da, and lyophilized.
The MW of AOS was analyzed by gel-filtration chromato-
graphy. The samples were separated on Ultrahydrogel 120, 250,
500, and 1000 (1.8 × 300 nm) columns using a high-perform-
ance size-exclusion chromatography system (e2695 Separations
Module; Waters Corporation, Milford, MA, USA) with a built-in
refractive index detector, and 0.1 M sodium phosphate buffer
(pH 7.0) was used as an eluent. Calibration curves were obtained
from polyethylene glycol. The relative MW was calculated by
comparing the retention times of the standard and sample.
To determine the M/G ratio in AOS, circular dichroism
experiments were carried out with a J-1500 spectrometer
(JASCO, Tokyo, Japan). The absorbance of AOS dissolved in
phosphate-buffered solution (2.5 mg mL−1) was measured
from 190 to 260 nm at 25 °C using a 1 mm-path cell with the
following setup: bandwidth, 1 nm; time constant, 1 s; scan-
ning speed, 200 nm min−1. Three spectra corrected for the
background were averaged for the sample.
2.2. Zebrafish care and diets
Adult zebrafish (Danio rerio) with body weights of 200–300 mg
and approximately three months old were purchased from a
local aquarium shop in Gwangju, Korea and were kept under a
14 h light/10 h dark cycle (lights on: 9 am, lights off: 11 pm) at
28 ± 0.5 °C for a week. After acclimatization, 30 fish per group
were randomly assigned to three experimental groups: normal
diet (NFD), HFD, and HFD supplemented with AOS (HAOS).
Fish in each group were maintained in a 4 L aquarium tank.
During the 5 week experiment, fish in all groups were fed
twice daily (at 9:00 am and 5:00 pm; Fig. S2†). The NFD was a
commercial feed (Tetra Bits Complete), and for the HFD, 25%
of lard was added to the commercial diet. For HAOS, 2.5% AOS
was added to the HFD. All zebrafish experiments were con-
ducted according to the Zebrafish Book (visited at https://zfin.
org/zf_info/zfbook/zfbk.html) and in accordance with the insti-
tutional animal care guidelines and with supervision of the
relevant committees of Chonnam National University (Permit
number: CNU IACUC-YS20135).
2.3. Body weight and fasting blood glucose measurements
During the five-week feeding period, five zebrafish were ran-
domly collected from each group on day 1 and then weekly to
determine the body weight. At the same time, the body length
(mm) was recorded and the BMI (mg mm−2) was calculated as
described previously.20
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To determine blood glucose levels, three to four fish were
randomly collected from each group and fasted overnight
(∼12 h). Blood samples were collected from the individual zeb-
rafish at the dorsal artery and glucose was immediately
measured with a glucometer (CareSens-II Plus+; Informa Life
Sciences) twice for each zebrafish.
2.4. Protein isolation and relative quantification
After the five-week experiment, five fish from each group were
pooled, frozen in liquid nitrogen, and grounded to form a
powder. Proteins were extracted using Pierce RIPA buffer
(Thermo Fisher Scientific, Waltham, MA, USA) containing
1.0% Halt Protease Inhibitor Cocktail 100× (Thermo Fisher
Scientific, Waltham, MA, USA), 1.0% Phosphatase Inhibitor
Cocktail (Sigma-Aldrich, St Louis, MO, USA), and 1.0% EDTA
0.5 M (Thermo Fisher Scientific, Waltham, MA, USA). The pro-
teins were digested by tube-gel digestion,31 and subjected to
nano-ultra-performance liquid chromatography (UPLC) and
Synapt G2-Si High Definition Mass Spectrometry (HDMSE,
Waters Corp., USA), as previously described.32 Proteins were
identified and quantified using Progenesis QI for Proteomics
version 2.0 (Nonlinear Dynamics, Newcastle, UK).33 Protein
lists were first defined using the International Protein Index
database, and then re-identified using the UniProt database
for zebrafish (http://www.uniprot.org).
2.5. Bioinformatics and network analyses
After identification and relative quantification, only proteins
containing at least one unique peptide were considered valid
for identification. Identified proteins were hierarchically clus-
tered based on their expression (log base 2), and a heatmap of
significantly differentially expressed proteins was generated.
K-means clustering was performed based on Euclidean dis-
tance with average linkage, using Multi Experiment Viewer
version 4.9.0 (http://mev.tm4.org/).34 Ingenuity Pathway
Analysis (IPA version 9.0; Ingenuity Systems Inc., http://www.
ingenuity.com) was used to identify biological functions and
canonical pathways of related proteins derived from proteo-
mics data. Gene set enrichment analysis (GSEA) (http://software.
broadinstitute.org/gsea/index.jsp) was performed to identify func-
tional classes and biological pathways of target molecules.35
2.6. Inhibitor/activator treatments
To verify the roles of AOS and to clarify whether its effect on
immuno-metabolic pathways may be mediated by STOML2 in
zebrafish, we used various inhibitors or activators, including
AICAR, U0126, KNK437, and LPS (details are given in
Table S1†). Zebrafish were given NFD or HFD for four weeks
until obesity-related phenotypes were observed. Stock solu-
tions of the above compounds were prepared in DMSO (Sigma-
Aldrich, St Louis, MO, USA) at 1 mg mL−1 or 10 mg mL−1, per
the supplier’s recommendations. For administration to the
zebrafish, the stock solutions were diluted in phosphate-
buffered solution. AICAR and AOS were administered at 100
mg kg−1 day−1, and U0126, KNK437, and LPS at 10 mg kg−1 day−1
once daily in 10 μL doses using a micro-pipette. Zebrafish were
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Food & Function
treated for three days and then 3–4 zebrafish/treatment were
sampled for further analysis.
2.7. RNA isolation and cDNA transcription
Total RNA was extracted from the whole bodies of adult zebra-
fish using RNAiso Plus reagent (Takara Bio, Shiga, Japan)
according to the manufacturer’s instructions. cDNA was syn-
thesized from 10 μg of RNA in a reaction mixture containing
1 μL random primer, 2 μL dNTPs, 0.5 μL recombinant RNase
inhibitor (Takara Bio), 1 μL MMLV RT, 4 μL 5× MMLV RT
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buffer, and 2 μL 5× DTT (Beams Biotechnology, Seongnam,
Korea), on a Takara PCR Thermal Cycler Dice Real Time
System under the following conditions: 63 °C for 10 min,
37 °C for 60 min, and 95 °C for 5 min.
2.8. Quantitative real time (qRT-)PCR
Genes related to lipid metabolism, endoplasmic reticulum
(ER) stress, stress-induced apoptosis and inflammation, and
immune responses were targeted using gene-specific primers
(details in Table S2†). qRT-PCR was conducted in a Thermal
Cycler Dice Real Time System (Takara Bio) using 21 μL reaction
mixtures containing 10 μL SYBR Green Master Kit (Takara
Bio), 1 μL of each of forward and reverse primers, 2 μL cDNA,
and 7 μL RNase-free water. Thermal cycles were as follows: 30 s
hold at 95 °C, 45 cycles of 5 s at 95 °C, 10 s at 55 °C, and 20 s
at 72 °C, and a cycle of dissociation at 95 °C for 15 s, 60 °C for
30 s and 95 °C for 15 s. The β-actin gene was used for normali-
zation. Target mRNA levels were determined by the 2–ΔΔCt
method. All qRT-PCRs were run in triplicate.
2.9. Statistical analysis
Statistical analysis was carried out using SPSS 21.0. Data are
expressed as the mean ± standard deviation (SD) and were ana-
lyzed by one- or two-way ANOVA with post hoc analysis followed
by Tukey’s or Duncan’s tests. GraphPad Prism 5 (GraphPad, La
Jolla, CA, USA) was used to draw figures. Differences were con-
sidered significant at p < 0.05.
3. Results and discussion
3.1. Characterization of alginate oligosaccharide (AOS)
The chromatogram of sodium alginate showed two peaks,
corresponding with average MWs of 351 727 Da, and 749 Da,
while the depolymerized sodium alginate (AOS) prepared
using bacterial alginate lyase showed three major peaks,
corresponding with average MWs of 167 471 Da, 796 Da, and
213 Da (Fig. S1A†), indicating that the enzyme had degraded
the extended polymer structure of the alginate. The percentage
(%) of the area of each peak was 73.27%, 18.46%, and 8.27%,
respectively, and it was found that the total alginate oligosac-
charides usable were 26% in total fermented products (data
not shown). Circular dichroic spectra of alginate showed a
peak at 200.4 nm and a trough at 212.9 nm, while AOS showed
a peak at 199.8 nm and a trough at 213.4 nm, respectively
(Fig. S1B†). Similarly, a previous study reported on poly-man-
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nuronic acid obtained from sodium alginate reported peak at
200 nm and a trough at 215 nm.36 As shown in Fig. S1B,†
sodium alginate was composed of 58.02% mannuronate (M)
and 41.98% guluronate (G) as the M/G of 1.38 : 1.0, while the
M/G ratio of AOS was calculated to be 1.51 : 1.0 based on mdeg
values, and the depolymerized sodium alginate consisted of
60.1% mannuronate and 39.9% guluronate. Thus, the product
prepared from sodium alginate by bacterial alginate lyase was
a mannuronate-rich alginate, which we termed “AOS”. The
unsaturated AOS had a characteristic peak at 234 nm and
maximal ellipticity at 250 nm, which can be explained by the
properties of unsaturated alginate decomposed by alginate lyase.37
3.2. AOS suppresses HFD-induced obesity in zebrafish
Previous studies have reported that HFD/high cholesterol diet
or overfeeding induce obesity in adult zebrafish.20–22,38
Nakazono et al. reported that alginate oligomer exerted anti-
obesity effects in mice fed a HFD by lowering serum triglycer-
ide and pancreatic lipase and by inhibiting gastrointestinal
lipid absorption.15 To investigate the potentially beneficial
effects of AOS in the obese zebrafish model, adult zebrafish
were fed a NFD, HFD, or HAOS for five weeks (Fig. S2†), and
changes in body weight, BMI, and fasting blood glucose were
measured. As expected, after the five-week experiment, the fish
had significantly gained weight in all groups as compared to
their initial weights, indicating normal growth. The HFD
group showed a significantly higher increase (1.22-fold or
22.14%) in body weight than the NFD group (Fig. 1A–C). AOS
attenuated weight gain; in the HAOS-group, mean body weight
was only 1.10-fold (10.68%) higher than in the NFD group
after five weeks of feeding (Fig. 1C). Similarly, the BMI showed
a significant increase in all groups at the end of the experi-
ment, but again, the HFD group showed the highest BMI
among the three groups, whereas it did not significantly differ
between NFD and HAOS (Fig. 1D). HFD significantly elevated
the fasting blood glucose level (122.0 ± 15.1 mg dL−1 vs. 56.6 ±
13.8 mg dL−1 in NFD, p < 0.001), whereas AOS had a signifi-
cant suppressive effect (80.6 ± 17.8 mg dL−1, p < 0.05) (Fig. 1E).
Our data indicated that the zebrafish obesity model was suc-
cessfully established by five weeks of HFD. Our results clearly
indicated that dietary AOS has an anti-obesity effect as it atte-
nuated weight gain and BMI and decreased blood glucose
levels in HFD-fed zebrafish.
3.3. AOS suppresses lipogenesis and elevates energy
expenditure in obese zebrafish
To investigate the effects of HFD and the potentially regulatory
effect of AOS on lipid metabolism, we evaluated the expression
of genes encoding leptin (LEPTIN), fatty acid synthase (FAS),
and sterol regulatory element binding proteins (SREBP1),
which are lipid metabolism markers, by qRT-PCR. The results
are shown in Fig. 2A–C. As expected, HFD strongly induced the
expression of LEPTIN (>80-fold), FAS (>20-fold increased), and
SREBP1 (>2-fold), suggesting that HFD induced leptin pro-
duction and insulin resistance, leading to gain weight and
elevated blood glucose in zebrafish.39 LEPTIN was the most
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Fig. 1 Changes in body weight, BMI, and fasting blood glucose in zebrafish on the different diets. (A–C) Changes in body weight during the five-
week feeding period and representative images of zebrafish at the start and the end of the experiment. (D) BMI and (E) fasting blood glucose in adult
zebrafish after 5 weeks of NFD, HFD, and HAOS.
strongly upregulated gene in HFD-fed zebrafish. In agreement
with our results, Simonds et al. reported that leptin plays an
important role in the increase in blood pressure and body
weight associated with obesity in mice.40 In humans, leptin
mRNA concentrations in adipose tissue and serum positively
correlate with the amount of fat mass, and fasting or weight
loss lowers leptin levels.39 In line with our findings, overfeed-
ing strongly induced FAS and SREBP1 expression in adult zeb-
rafish.22 AOS significantly suppressed LEPTIN, FAS, and
SREBP1 mRNA expression induced by HFD, indicating that
AOS exerts anti-adipogenic activity.
Elevating energy expenditure is one of the best approaches
to fight obesity.39 We evaluated whether AOS enhances whole-
body energy expenditure in HFD, by measuring the expression
of energy metabolism-related genes encoding AMP-activated
protein kinase α (AMPKα), peroxisome proliferator-activated
receptor gamma coactivator 1 α (PGC-1α), sirtuin (SIRT1),
acetyl-CoA carboxylase A (ACCA), and uncoupling protein 1
(UCP1) by qRT-PCR. The results are shown in Fig. 2D–H. HFD
dramatically reduced the expression of all biomarkers tested.
Interestingly, AOS supplementation restored marker expression
to levels similar to those in the NFD group. Notably, AOS sup-
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plementation strongly enhanced UCP1 expression (by 18.4-fold
as compared to NFD, Fig. 2H). AMPK plays important roles in
energy homeostasis regulation and stimulates fatty acid
β-oxidation. Thus, activated AMPK is involved in the inhibition
of lipogenesis or elevation of lipolysis, inducing weight loss.41
PGC-1α, as a pivotal regulator of mitochondrial biogenesis and
oxidative metabolism, is another key regulator of cellular
energy metabolism.42 SIRT1 is considered a critical metabolic
sensor that directly regulates a number of targets involved in
lipid metabolism,43 whereas UCP1 is a predominant thermo-
genic marker in brown adipose tissue.44 AMPK can increase
PGC-1α and SIRT1 activity and UCP1 expression and activity.45
Hence, AMPKα, PGC-1α, SIRT1, and UCP1 form a metabolism-
energy sensing network that stimulates energy expenditure to
prevent metabolic dysfunction.45 AMPKα expression and
activity were measured by western blotting, which revealed
strong activation of AMPKα by dietary AOS (Fig. S3†), in line
with the qRT-PCR results. Our results suggest that dietary AOS
supplementation in zebrafish has a significant impact on
AMPKα, UCP1, SIRT1, and PGC-1α expression and AMPK
activity, which may explain its effect in preventing body fat
accumulation under HFD. Previous studies have also shown
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Fig. 2 Effect of HFD and AOS supplemented on the mRNA expression of target genes. AOS modulates mRNA expression of the lipid metabolism-
related genes FAS (A), LEPTIN (B), and SREBP1 (C), the energy metabolism-related genes AMPKα (D), PGC-1α (E), SIRT1 (F), ACCA (G), UCP1 (H), and
ATP2A1 (I). Results are means ± SD of 3–4 experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.

that HFD profoundly decreases AMPKα, PGC-1α, SIRT1, and
UCP1 expression levels in C57BL/6 mice.42,46 As AMPKα, SIRT1,
PGC-1α, and UCP1 are considered vital nutrients and energy
sensors, it is highly likely that AOS promotes energy expenditure
in HFD-fed zebrafish by elevating their activities. Thus, AOS is
promising for nutritional or pharmacological interventions in
the treatment of obesity and metabolic disorders.
3.4. AOS reverses HFD-induced inflammatory and apoptotic
gene expression
Given the impacts of inflammation on adipokine production,
we evaluated expression levels of inflammatory genes encoding
interleukins (IL-1β, IL-6), and tumor necrosis factor α (TNF-α)
in all treatment groups. The data are shown in Fig. 3A–C,
mRNA levels of all tested pro-inflammatory genes were
increased in the HFD group compared to the NFD group. AOS
significantly inhibited the expression of these pro-inflamma-
tory cytokines. These findings suggest that HFD induces
inflammation, which may further induce obesity in zebrafish.
Accordingly, previous in vivo studies have reported that the
inflammatory genes IL-1β, IL-6, MCP-1, and TNF-α are
increased in HFD-induced obesity.46,47 A study on zebrafish
reported that obesity was strongly associated with upregulation
of the inflammation-related genes TNF-α, IL-1β, MMP2, and
MMP9.48 The suppressive effect of AOS on inflammatory
cytokine production may be associated with decreases in

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Fig. 3 Effect of HFD and AOS supplemented on the mRNA expression of target genes. AOS modulates mRNA expression of the inflammatory cyto-
kines related genes IL-1β (A), IL-6 (B), and TNF-α (C), and the apoptosis-related genes BAX (D), BCL-2 (E), BAX/BCL-2 ratio (F), NR3C1 (G), TP53 (H),
and VDAC1 (I). Results are means ± SD of 3–4 experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.

energy metabolic and lipogenic gene expression, as mentioned
above.46
To evaluate the potentially inhibitory effect of AOS on HFD-
induced apoptosis in zebrafish, we measured the expression of
the apoptosis-related genes BAX and BCL-2 and genes encod-
ing the proapoptotic tumor protein p53 (TP53), voltage-depen-
dent anion-selective channel protein 1 (VDAC1), and nuclear
receptor subfamily 3, group C, member 1 (NR3C1) by
qRT-PCR. As shown in Fig. 3D–H, HFD highly induced
expression of BAX, TP53, and NR3C1 and downregulated BCL-2
transcript levels in zebrafish after the five-week feeding period,
indicating it induced apoptosis. AOS supplementation signifi-
cantly reversed HFD-induced apoptosis-related gene
expression. AOS significantly up- and downregulated BCL2 and
BAX expression, respectively, leading to a significant decrease
in the BAX/BCL2 ratio. BCL-2 is an important antiapoptotic
gene, whereas BAX is the most critical proapoptotic gene, and
the BAX/BCL-2 ratio strongly contributes to apoptosis.49 As
shown in Fig. 3G–I, AOS significantly attenuated the expression
of NR3C1, TP53, and VDAC1, to levels observed in the NFD

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group. BAX, TP53, and BCL-2 are apoptosis biomarkers, and
VDAC1, an outer mitochondrial membrane, is also a key player
in apoptosis and is involved in cytochrome c release and inter-
actions with antiapoptotic genes/proteins.50 NR3C1 is a corti-
sol receptor that is induced in the hypothalamus–pituitary–
adrenal axis by stress.51 Apoptosis induction by HFD may
occur via oxidative stress as reactive oxygen species (ROS) are
massively produced in obesity.28,50 Our findings suggest that
dietary AOS protects against HFD-induced apoptosis in zebra-
fish by modulating apoptosis-related genes. This is consistent
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and in vivo. Tusi et al. reported that AOS protected PC12 cells
from apoptosis by inducing BCL-2 expression and inhibiting
BAX expression and suppressing H2O2-induced caspase-3
activation.49
3.5. Comparative proteomics analysis of the effects of HFD
and AOS
To elucidate the mechanisms of dietary AOS in suppressing
obesity and modulating immuno-metabolic responses to HFD,
samples from the three treatment groups were subjected to

with previous findings that AOS decreased apoptosis in vitro proteomic analysis. In total, 519 proteins were identified in the

Fig. 4 Venn diagram and heatmaps of significantly differentially expressed proteins based on their relative expression ratios in zebrafish on HFD and
HAOS. (A) Common proteins (146) between HFD vs. NFD and HAOS vs. HFD. (B) Common proteins (14) between HFD vs. NFD (downregulated) and
HAOS vs. HFD (upregulated). (C) Common proteins (132) between HFD vs. NFD (upregulated) and HAOS vs. HFD (downregulated). Ratios are shown
as log2 fold change, with upregulated proteins having expression >0.0, red; downregulated proteins having expression <0.0, green.

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three groups (Table S3-1†). Of these, 308 and 177 proteins were
significantly ( p < 0.05) differently expressed in HFD vs. ND and
HAOS vs. HFD, respectively (Fig. 4A). Heatmaps based on the
expression of these proteins are shown in Fig. 4B and C.
Remarkably, 146 proteins were found in common between
HFD vs. NFD and HAOS vs. HFD (Fig. 4A and Table S3-2†); of
these, most (132 proteins) were upregulated by HFD and down-
regulated by AOS (Fig. 4C). Only 14 proteins were downregu-
lated in HFD vs. NFD, and AOS treatment promoted the upre-
gulation of these proteins (Fig. 4B). Remarkably, STOML2 was
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the most differentially regulated among the proteins identi-
fied; it was induced by >16.5-fold by HFD (Table S3-2†). AOS
significantly downregulated STOML2 by 0.52-fold when com-
pared to the level in the HFD group (Table S3-2†). IPA analysis
revealed that, among 146 common significantly differentially
expressed proteins, which were related to cancer progression,
tumorigenesis, cancer metastasis, inflammation, and apopto-
sis, as well as hepatic diseases and pathogenic diseases
(Table S4†). Network relationships and biological functions of
the proteins identified were analyzed by STRING analysis.
Among the 146 common proteins, 51 proteins strongly ( p <
0.05) interacted together and were differentially regulated
between HFD and AOS treatments and may thus be considered
as potential biomarkers of immune regulation and the anti-
obesity mechanism of AOS in zebrafish (Fig. S4A†). In
addition, according to IPA and KEGG pathway analysis
(Table S5 and Fig. S4B†), most of the identified proteins were
assigned to two major groups: metabolic pathways (glycolysis/
gluconeogenesis) and the immune system (unfolded protein
response, mitochondrial dysfunction, endoplasmic reticulum,
etc.). Remarkably, STOML2 seems to act as a central mediator
connecting metabolic pathways and the immune system
(Fig. S4A†). Glycolysis/gluconeogenesis is a fundamental and
highly conserved metabolic pathway in most organisms. Five
major glycolytic proteins (common proteins of glucose metab-
olism, gluconeogenesis, glycolysis, and glycogenolysis), includ-
ing aldolase b (ALDOB), aldolase c (ALDOCB), glyceraldehyde-
Table 1 Most enriched gene/gene sets determined by GSEA (http://software.broadinstitute.org) (with only FDR q-values < 0.05) in zebrafish on
HFD with AOS supplementation
FDR
Gene/gene set p-Value q-value
−12
GO small molecule 1.11 × 10 1.98 × 10
metabolic process [1767]
GO glycosyl compound 4.79 × 10−12 4.27 × 10−8
metabolic process [368]
GO purine containing compound 9.37 × 10−12 5.56 × 10−8
metabolic process [394]
GO organonitrogen compound 1.91 × 10−11 6.81 × 10−8
metabolic process [1796]
GO nucleoside triphosphate 1.03 × 10−10 2.3 × 10−7
metabolic process [228]
GO nucleoside monophosphate 1.5 × 10−10 2.45 × 10−7
metabolic process [239]
The p-value and FDR q-value were calculated using Fisher’s exact test by GSEA. FDR: false discovery rate. Only the most-significant terms are
shown.
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3-phosphate dehydrogenase 2 (GAPDHS), pyruvate kinase-
muscle, a (PKMA), and phosphoglycerate mutase 2 (PGAM2),
were upregulated in the HFD group, whereas enolase (ENO1b)
and L-lactate dehydrogenase B–B chain (LDHBB) were
decreased under HFD. AOS significantly reversed the
expression of these proteins (Fig. S4C†).
HFD strongly upregulated the expression of various ER-
related proteins, especially, HSPA8, HSP90b1, HSC70, and
HSPA5 by 4.4-, 3.3-, 1.9-, and 1.4-fold, respectively, when com-
pared to NFD (Fig. S4C†), whereas these proteins were downre-
gulated by AOS. HSP70 and HSP90 are molecular chaperones
whose expression is increased by various types of stress.49 We
confirmed the proteomics expression data for HSP70 and
HSPA8 by western blotting (Fig. S3†). The significant upregula-
tion of all HSPs upon HFD may indicate that zebrafish try to
adapt to the stress induced by HFD.52 GSEA showed that pro-
teins related to metabolic pathways were the most strongly
enriched among the proteins detected. Many of these proteins
are involved in both metabolic pathways and immune systems.
STOML2 has been shown to be involved in various metabolic
process (Table 1). Based on the proteomic data, STOML2 was
predicted to be a central regulator in HFD-induced obesity and
was selected for further investigation of its role in immuno-
metabolic systems.
3.6. STOML2 regulates crosstalk between immune systems
and metabolic pathways to control HFD-induced obesity
First, we confirmed the relative mRNA expression of STOML2
in zebrafish by qRT-PCR. HFD significantly increased STOML2
expression (8.9-fold) as compared to NFD. Meanwhile, AOS
strongly inhibited this increase (1.4-fold increase in HAOS vs.
NFD) (Fig. 5). This was in accordance with the results obtained
by proteomics analysis. Similarly, STOML2 reportedly was
upregulated in skeletal muscle biopsies from obese as com-
pared to non-obese subjects.53 While STOML2 expression is
induced under different conditions, the mechanism under-
lying inhibition/activation of STOML2 is still unknown.
Molecules
−8
HSPA8, AHCY, ATP5A1, GAPDHS, PGAM2, ALDOB, ATP5B, COX6C,
STOML2, BHMT, P4HB, ALDH4A1, APOA2, DDC, CS, GPD2, ME3
HSPA8, AHCY, ATP5A1, GAPDHS, PGAM2, ALDOB, ATP5B, COX6C,
STOML2, BHMT
HSPA8, AHCY, ATP5A1, GAPDHS, PGAM2, ALDOB, ATP5B, COX6C,
STOML2, BHMT
HSPA8, AHCY, ATP5A1, GAPDHS, PGAM2, ALDOB, ATP5B, COX6C,
STOML2, BHMT, P4HB, ALDH4A1, APOA2, DDC, RPL7, GSTP1
HSPA8, ATP5A1, GAPDHS, PGAM2, ALDOB, ATP5B, COX6C, STOML2
HSPA8, ATP5A1, GAPDHS, PGAM2, ALDOB, ATP5B, COX6C, STOML2
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Fig. 5 Effects of inhibitor/activator treatments on the expression of biomarkers in zebrafish. (A) Effect of AICAR treatment on mRNA expression of
AMPKα, PGC-1α, and STOML2 and FAS, LEPTIN, and IL-1β. (B) Effect of U0126 treatment on mRNA expression of AKT1, ERK1, and STOML2, and FAS,
LEPTIN, and AMPKα. (C) Effect of KNK437 treatment on the expression of HSPA5, HSP90b1, and STOML2, and FAS, LEPTIN, and AMPKα. (D) Effect of
LPS treatment on mRNA expression of IFN-γ, IL-6, and STOML2, and FAS, LEPTIN, and AMPKα. Results are means ± SD of 3–4 experiments. NS, not
significant *p < 0.05, **p < 0.01, and ***p < 0.001.

If STOML2 is a central factor that plays important roles in
preventing obesity by targeting multiple pathways in immuno-
metabolic interaction, STOML2 expression should be regulated
by these specific inhibitors or activators. To confirm whether a
possible crosstalk among those pathways modulating lipogen-
esis is linked to the regulation of STOML2, we used various
inhibitors/activators of these pathways (AMPKα/SIRT1/PGC1α,
energy expenditure signaling; HSP signaling; AKT/MAPK/ERK
signaling). Remarkably, U0126, a specific MEK/ERK inhibi-
tor,54 the most strongly reduced the HFD-induced STOML2
response (∼20-fold decrease as compared to HFD without
inhibitor treatment, Fig. 5C). Similarly, AICAR, an AMPK

This journal is © The Royal Society of Chemistry 2019 Food Funct.


Paper
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Fig. 6 Proposed effects of dietary AOS on high fat diet-induced obesity and pathophysiological disorders in zebrafish. AOS regulates immuno-
metabolic systems by regulating pro-inflammatory cytokines, inhibiting apoptosis, and modulating energy expenditure and lipid metabolism by regu-
lating STOML2 in high fat diet-induced obesity in zebrafish.
activator41 suppressed STOML2 expression by about 10-fold as
compared to HFD without AICAR treatment (Fig. 5B). KNK437,
a novel inhibitor of HSP,55 and LPS-induced inflammation12,56
in zebrafish had a positive effect on STOML2 expression. As
predicted, KNK437 treatment in the HFD group inhibited
STOML2 expression (∼5.5-fold decrease, Fig. 5C), whereas LPS
treatment for 3 days in the NFD group highly induced the
expression of inflammatory cytokines (IL-6 and IFN-γ) and
STOML2 (>3-fold vs. NFD, Fig. 5D). To evaluate whether
STOML2 may act as a regulator of lipogenesis and thermogen-
esis, we measured the expression AMPKα, PGC-1α, UCP1, ACCA,
FAS, and LEPTIN, biomarkers of metabolic abnormalities, after
the above treatments. The transcriptional levels of all these
markers were strongly correlated with STOML2 expression
(Fig. 5B–D and Fig. S5†). In line with decreased STOML2 tran-
scription, FAS and LEPTIN expression was reduced upon treat-
ment with AICAR, KNK437, and U0126 (Fig. 5A–C). In contrast,
LPS induced the upregulation of STOML2, as well as of FAS
and LEPTIN (Fig. 5D). The energy expenditure-related genes
AMPKα, PGC-1α, and UCP1 showed similar expression patterns
under all treatments in correlation to STOML2 expression
(Fig. 5A–D and Fig. S5†). Remarkably, AOS mimicked the
actions of several molecules, such as AICAR, KNK437, and
U0126, which strongly activated energy expenditure and inhib-
ited STOML2 expression in zebrafish, and this may represent a
novel mechanism of action for anti-obesity drugs (Fig. 6).
STOML2, a member of the human stomatin family, is a mem-
Food Funct.
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Food & Function
brane protein in human red blood cells that has been recently
revealed to be associated with a variety of diseases.57 Increased
STOML2 expression has been associated with increased risk
for asthma, suggesting that STOML2 may have a pro-inflamma-
tory effect in asthma.58 Song et al. have reported that downre-
gulation of STOML2 reduces NF-κB transactivity and inhibits
the NF-κB pathway by downregulating of BCL-xL, TNF-α, MYC,
CCND1, IL-6, and IL-8.57 Recently, Yang et al. found that
STOML2 directly affected annexin A2 and β-catenin
expression.27 Based on our findings in the current study, we
suggest that the changes in the immuno-metabolic system in
zebrafish in response to HFD-induced obesity are regulated by
STOML2 (Fig. 6). Recent evidence suggests mitochondria
provide a powerful strategy to modulate energy homeostasis as
well as metabolic health for the treatment of obesity and meta-
bolic disorders.28,59 Even though the exact function and
mechanism of STOML2 in metabolic syndromes remain
unclear, our study provided evidence that STOML2 might be a
promising novel target in the treatment of immuno-metabolic
disorders, as such obesity (Fig. 6).
4. Conclusions
This study is the first to report that AOS significantly prevents
HFD-induced immuno-metabolic dysfunction in zebrafish.
Dietary AOS modulated a range of zebrafish genes and proteins
This journal is © The Royal Society of Chemistry 2019

Food & Function
related to immuno-metabolic systems by inhibiting STOML2,
thus maintaining/improving health in zebrafish fed an HFD.
Our findings pave the way for future dietary interventions and
clinical recommendations. Especially, STOML2, identified in
this study as a key regulator, warrants further study to develop
a potential treatment for metabolic disorders.
Abbreviations
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AOS Alginate oligosaccharide
HFD High-fat-diet
NFD Normal diet
Author contributions
D. K. designed the study; T. V. C. performed most of the experi-
ments and statistical analysis; T. V. C., and D. K. wrote the
manuscript; S. Y. C., and J. K., conducted proteomic analysis;
T. V. C., and S. Y. C., contributed to data organization, or ana-
lysis; and J. K., and D. K. supervised the study. All authors con-
tributed to comments and revision on the manuscript and
approved the final manuscript.
Conflicts of interest
The authors declared no conflicts of interest.
Acknowledgements
This study was supported by a grant from the Korea Basic
Science Institute (No. C39712).
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