International Journal of Obesity (2012) 36, 195–200
& 2012 Macmillan Publishers Limited All rights reserved 0307-0565/12
www.nature.com/ijo
ORIGINAL ARTICLE
Novel genes and cellular pathways related to infection
with adenovirus-36 as an obesity agent in human
mesenchymal stem cells
H-N Na1,3, H Kim2,3 and J-H Nam1
1
Department of Biotechnology, The Catholic University of Korea, Bucheon, Korea and 2Graduate School of Biomedical
Science and Engineering/College of Medicine, Hanyang University, Seoul, Korea
Objective: The objective of this study was to investigate the molecular mechanisms underlying adenovirus-36 (Ad-36)-induced
obesity by the identification of novel genes and cellular pathways.
Design: Viral growth, intracellular lipid accumulation and gene expression profiles were determined in human mesenchymal
stem cells (hMSCs) infected with Ad-36 or Ad-2. A microarray assay and gene set enrichment analysis (GSEA) were performed to
assess alterations in global gene expression profiles.
Results: Ad-36, but not Ad-2, induced lipid accumulation and upregulated adipogenesis-related genes. There was no difference
in viral growth between Ad-36 infection and Ad-2 infection in hMSCs. GSEA revealed that Ad-36 infection was more frequently
associated with activation of novel pathways, including the PPAR-gamma signaling pathway, and inflammation compared with
Ad-2 infection, raising the possibility that these pathways may be key regulators of Ad-36-induced adipogenesis.
Conclusion: This study may help foster a better understanding of the roles of several cellular factors in Ad-36-induced obesity.
International Journal of Obesity (2012) 36, 195–200; doi:10.1038/ijo.2011.89; published online 3 May 2011
Keywords: Ad-36; human mesenchymal stem cell; PPAR-gamma signaling; inflammation; gene set enrichment analysis
Introduction
Obesity is one of the top health problems in the world. The
causes of obesity include heredity, food intake, exercise,
environment and psychology. Recently, it was revealed
that obesity is associated with several pathogens, such as
adenovirus-36 (Ad-36), Ad-37, SMAM-1, scrapie and Chlamydia
pneumonia.1,2 Most importantly, the relationship between
Ad-36 and human obesity, as well as obesity in mice, chickens,
rhesus monkeys, marmosets and rats, is well known.3,4
Moreover, a study performed on American adult human
samples, using serology to determine previous exposure to
Ad-36, showed that 30% of the obese individuals were
antibody-positive compared with 11% of the non-obese
subjects.5 A pediatric study in Korea revealed that a larger
number of obese subjects tested positive for the Ad-36
Correspondence: Professor J-H Nam, Department of Biotechnology, The
Catholic University of Korea, 43-1 Yeokgok-dong, Wonmi-gu, Bucheon,
420-743, Korea.
E-mail: [email protected]
3
These authors contributed equally to this work.
Received 13 January 2011; revised 10 March 2011; accepted 13 March 2011;
published online 3 May 2011
antibody than non-obese subjects (28.57 vs 13.56%,
respectively; P ¼ 0.0174).6,7 In addition, other studies using
the Italian and the US children and adolescents demonstrated
the association between the Ad-36 antibody and obesity.8,9
Currently, little is known about the cellular and molecular
mechanisms underlying Ad-36-induced obesity. Recently,
Ad-36 E4 orf-1 was identified as a novel inducer of the rodent
and human adipocyte differentiation process.10 In addition,
it increases glucose uptake via the upregulation of the
glucose transporter 4 in human skeletal muscle cells and
human adipose tissue-derived stem cells.11,12 However, it
remains unclear which cellular and viral factors are involved
in Ad-36-induced obesity or adipogenesis. The lack of proper
model systems to analyze the signal pathways and genetic
profiles involved in Ad-36-induced obesity may be one of the
reasons for this uncertainty.
Human preadipocytes derived from adipose tissue are
widely used to study human adipogenesis.12 However, these
cells are already committed to the adipogenic lineage.
Moreover, human preadipocytes have limited proliferative
ability, an unforeseeable variability (based on different
donors and anatomical sites) and confined availability.12
Human bone marrow mesenchymal stem cells (hMSCs) are
multipotent stem cells that can be induced to differentiate
Molecular mechanism of Ad-36-induced obesity
H-N Na et al
196
into adipocytes, osteoblasts, chondrocytes and myoblasts
when cultured under defined in vitro conditions.13,14
Previous reports showed the specific gene profiles during
the process of differentiating hMSCs to adipocytes using
adipogenic induction medium.15
In this study, we found that hMSCs differentiated to
adipocytes when cells were infected with Ad-36 without the
use of adipogenic induction medium. We also identified
entire gene expression profiles using microarray analysis. To
explore novel cellular pathways that are activated by Ad-36,
we performed gene set enrichment analysis (GSEA). It
showed that infection with Ad-36 activated pathways of
PPAR-gamma signaling and inflammation in hMSCs, providing novel insights into Ad-36-induced obesity. Overall, our
results suggest that hMSCs represent an optimal surrogate
module to study Ad-36-induced adipogenesis. Moreover,
several of the cellular gene sets that were upregulated in
Ad-36-infected hMSCs may provide clues regarding the
cellular mechanisms involved in Ad-36-induced obesity.
Materials and methods
Human bone marrow mesenchymal stem cells
Human bone marrow mesenchymal stem cells were acquired
from the Ewha Womans University Medical School (Dr Jo
Inho, Department of Molecular Medicine) and cultured in
Dulbecco’s Modified Eagle’s Medium (Hyclone, Rockford, IL,
USA) containing 10% fetal bovine serum (Hyclone) and 1%
antibiotics (Hyclone) at 37 1C and 5% CO2. Cells from
passages 2 to 4 were used for experiments.
Viruses and infection
Ad-36 and Ad-2 were obtained from the American Type
Culture Collection (Manassas, VA, USA; Cat no. VR-913 and
VR-846), plaque purified and propagated in A549 cells
(a human lung cancer cell line), as described previously.5
Viral titers were determined using a plaque assay.16 Samples
for microarray assay were prepared as follows: B80%
confluent hMSCs were starved overnight and infected with
a multiplicity of infection of 4 of Ad-36 and Ad-2 in serumfree Dulbecco’s Modified Eagle’s Medium for 1.5 h. hMSCs
were switched to completed Dulbecco’s Modified Eagle’s
Medium (10% fetal bovine serum and 1% antibiotics). At 1
and 2 days later, hMSCs were washed twice with phosphatebuffered saline, lifted using cell scrapers, collected by
centrifugation (12 000 r.p.m., 15 min, 4 1C) and subjected to
RNA isolation as described below.
Neutral lipid staining
hMSCs (80% confluent) cultured in six-well plates (Corning,
New York, NY, USA) were infected with multiplicity of
infection 4 of Ad-36 or Ad-2 and fixed 2 days later. The mock
group was treated with phosphate-buffered saline. The cells
International Journal of Obesity
were rinsed twice with phosphate-buffered saline and fixed
with 4% paraformaldehyde for 10 min. The fixed cells were
subjected to BODIPY using a modification of a protocol
reported previously,17 with BODIPY 493/503 (Invitrogen,
Carlsbad, CA, USA; dilution, 1:1000). For Oil Red O staining,
we discarded media and added 2 ml of 10% formalin,
followed by 30 min incubation at room temperature. The
Oil Red O stock solution was prepared by dissolving 300 mg
of Oil Red O powder (Sigma, Ronkonkoma, NY, USA) in
100 ml of 99% isopropanol. The fixed cells grown in six-well
plates were treated with 60% isopropanol (2 ml per well) for
5 min. The isopropanol was removed, 2 ml of the Oil Red O
working solution was added to each well for 2 h, and cells
were incubated at room temperature and then washed with
tap water.
RNA isolation and cDNA synthesis
All procedures were performed according to those of
previous reports.11 RNA was extracted from cells using the
TRIzol reagent (Invitrogen). Purified RNAs were stored at
80 1C until use. For cDNA synthesis, 5 ml RNA, 1 ml oligo-dT,
2 ml DTT, 4 ml distilled water and 8 ml of a Master Mix for
the Reverse Transcription step (RT&GO, MP Biomedicals,
Solon, OH, USA) were mixed and RT-PCR was performed
(Thermocycler; Biometra, Goettingen, Germany).
Microarray assay
Human Expression BeadChips were designed using Illumina
BeadArray technology (Illumina, San Diego, CA, USA). A
mock chip (non-infected hMSCs) was selected as a standard.
Gene expression profiles from test samples were compared
with the standard. For analysis, we compared the Ad-36 and
mock gene sets at 1 and 2 days after infection. We also
carried out experiments for Ad-2 using the same conditions
and identified changes in gene expression.
Gene set enrichment analysis
To explore pathways that were regulated differentially by
Ad-36 and Ad-2 in hMSCs, we performed GSEA using the
microarray data, as described previously18 using a GSEA
program (http://www.broad.mit.edu/gsea/msigdb/index.jsp).
We used C2-curated gene sets (http://www.broadinstitute.
org/gsea/msigdb/collections.jsp#C2, version 2.5) with a size
of 15–1000 genes. Po0.05 was considered significant.
Results
Absence of difference in viral growth between Ad-36- and
Ad-2-infected hMSCs
First, we evaluated viral replication in hMSCs, as we had to
demonstrate that all phenotypic differences observed
between Ad-36- and Ad-2-infected cells stemmed from
intrinsic characteristics of the viruses and not from efficiency
Molecular mechanism of Ad-36-induced obesity
H-N Na et al
197
Figure 1 Similar viral expansion rates of Ad-36 and Ad-2 in hMSCs.
Supernatants and hMSCs were collected at 6, 12, 24 and 48 h after infection
with multiplicity of infection of 4 of viruses to determine the extracellular and
intracellular virus titers, respectively. The hMSCs collected were washed with
phosphate-buffered saline and subjected to three cycles of freeze–thaw to
release intracellular viruses. The titer of extracellular and intracellular viruses
was quantified using a plaque assay.
of viral replication or viral infection. There were no
differences between the extracellular and intracellular
growth of the Ad-36 and Ad-2 viruses in hMSCs (Figure 1),
suggesting that any differential phenotypes observed in cells
was not caused by differences in viral growth between Ad-36
and Ad-2.
Ad-36, but not Ad-2, induced adipogenic differentiation
of hMSCs
Cell morphology and lipid accumulation were assessed at 6,
24 and 48 h after infection of hMSCs with multiplicity of
infection of 4 of Ad-36 and Ad-2. Only Ad-36-infected, but
not Ad-2- or mock-infected, hMSCs underwent morphological changes into more rounded shapes. In addition, there
was an increase in the number of cells containing lipid
droplets, exclusively in Ad-36-infected hMSCs, which was
confirmed by BODIPY and Oil Red O staining (Figure 2a).
Next, we quantified adipogenic differentiation by counting
BODIPY-positive cells. The number of BODIPY-positive cells
was two-, three- and fourfold higher at 6, 24 and 48 h,
respectively, in hMSCs infected with Ad-36 compared with
the mock (Po0.012, Po0.001 and Po0.0003, respectively),
whereas there was no difference in the number of BODIPYpositive cells between the mock and Ad-2-infected cells
(Figure 2b). These data suggest that Ad-36, but not Ad-2,
induces the adipogenic differentiation of hMSCs.
Ad-36 infection upregulated adipogenesis-related genes
in hMSCs
Next, we performed a microarray assay to determine the
effect of Ad-36 and Ad-2 infection on gene expression in
hMSCs, especially regarding genes involved in adipogenesis.
No global difference was observed in terms of the total gene
Figure 2 Ad-36, but not Ad-2, induced the adipogenic differentiation
of hMSCs. (a) Infection with Ad-36 (48 h), but not Ad-2, led to the
morphological change of spindle-shaped hMSCs to a round shape; moreover,
intracellular lipid droplets were observed. Intracellular lipid accumulation was
confirmed by BODIPY and Oil Red O staining. Scale bar, 50 mm. (b) To
quantify adipogenic differentiation, cells were stained with BODIPY, and
BODIPY-positive cells were counted by a blinded investigator (n ¼ 3; *Po0.05,
**Po0.01 and ***Po0.001).
expression profiles between Ad-36- and Ad-2-infected hMSCs
(Supplementary Figure 1). However, the profiles of obesityrelated genes were more up- and downregulated after
infection with Ad-36 than with Ad-2; 296 genes related to
obesity showed changes in expression during adipogenesis
(Supplementary Figure 2). The microarray data revealed that
several obesity-related genes were upregulated during Ad-36
infection compared with Ad-2 infection (Table 1). The
upregulation of some genes after Ad-36 infection was
confirmed using semi-quantitative real-time RT-PCR (data
not shown). Interestingly, the comparison of the microarray
International Journal of Obesity
Molecular mechanism of Ad-36-induced obesity
H-N Na et al
198
Table 1 Microarray data showing the obesity-related genes that were
upregulated after Ad-36 infection
Gene
Uncoupling protein 1 (UCP1)
Fatty acid binding protein 4 (FABP4)
Sorbin and SH3 domain containing 1 (SORBS1)
Suppressor of cytokine signaling 4 (SOCS4)
Peroxisome proliferator-activated receptor gamma
coactivator 1-alpha (PPARGC1A)
CCAAT/enhancer binding protein alpha (C/EBPa)
General regulatory factor 2 (GRF2)
Ad-36/Ad-2 ratio
1D
2D
11.65
1.49
6.22
7.20
0.91
116.88
20.63
19.58
11.29
8.33
72.42
2.83
0.58
1.62
and inflammation pathways (Po0.01) compared with Ad-2
(Figure 4), which raises the possibility that Ad-36 activates
these two pathways selectively compared with Ad-2. These
two pathways may be key regulators of Ad-36-induced
adipogenesis in hMSCs. Subsequently, we performed a
leading-edge subset analysis to identify the core genes
involved in the PPAR-gamma signaling pathway and in the
inflammation pathway. Activation of PPAR-gamma pathways
was associated with selective upregulation of eight genes,
including uncoupling protein 1 (UCP-1), and the activation of
inflammation pathways was linked with the upregulation of
three genes, including interleukin-11 (Figure 4).
Abbreviation: Ad, adenovirus.
Discussion
Figure 3 Comparison of gene profiles between induction medium and
Ad-36-induced adipogenesis in hMSCs. The gene profiles observed after
treatment with induction medium were obtained from a previous report.20
Induction medium and Ad-36 infection led to a twofold induction compared
with mock (non-infected hMSCs). Induction medium data were collected at 1,
3, 5, 7, 9 and 14 days after treatment, and Ad-36 data were obtained at 1 and
2 days after infection. LPL; lipoprotein lipase; ATF3, activating transcription
factor 3; FABP4, fatty acid-binding protein 4; C/EBPa, CCAAT/enhancer
binding protein a; C/EBPb, CCAAT/enhancer binding protein b; CXCL12,
chemokine (C-X-C motif) ligand 12; TIMP3, TIMP metallopeptidase
inhibitor 3; ITGA3, integrin alpha 3.
data obtained in this study with gene profiles reported
previously, which were determined after inducing mediatreated hMSCs to adipocytes,15 revealed that 35 genes were
similarly upregulated in Ad-36 infection and in induced
adipogenesis; these included LPL, ATF3, FABP4, C/EBPa and
C/EBPg (Figure 3). This indicated that the 35 upregulated
genes (listed in Supplementary Table 1) may be essential for
the differentiation of hMSCs to adipocytes.
GSEA identified novel pathways that were regulated selectively
by Ad-36 compared with Ad-2
We explored novel pathways that were regulated selectively
by Ad-36 compared with Ad-2 using GSEA as previously
described.18 Interestingly, GSEA revealed that Ad-36 significantly increased PPAR-gamma signaling pathways (Po0.05)
International Journal of Obesity
Many previous studies showed that Ad-36 infection increases
body weight and visceral, epididymal, inguinal and retroperitoneal fats, as well as the number of fat cells in rats,19
chickens,4 and rhesus and marmoset monkeys.20 According
to the results of an in vitro study, Ad-36 E4 orf-1 is one of the
viral factors that are involved in Ad-36-induced adipogenesis.10 However, the exact cellular and molecular mechanisms involved in this phenomenon have not been identified.
Here, we found that hMSCs had the ability to differentiate
into adipocytes after Ad-36 infection (Figure 2a). Despite the
absence of differences in viral growth between cells infected
with Ad-36 or Ad-2 (Figure 1), only Ad-36-infected hMSCs
differentiated into adipocytes. Given that undifferentiated
hMSCs can be induced to differentiate into adipocytes,
hMSCs may represent a good model for the study of Ad-36induced adipogenesis.
Ad-36 infection upregulated several obesity-related genes
(Table 1). Among them, UCP-1 is a member of the
mitochondrial anion-carrier protein family and functions
to dissipate undue energy.21 Therefore, it is possible that
Ad-36 may regulate the energy balance via UCP-1 to energy
expenditure for obesity. In addition, it is well known that
C/EBPa and PPARg have a decisive role in the differentiation
of hMSCs to adipocytes.22 Interestingly, Ad-36 infection also
upregulated C/EBPa and PPARg in hMSCs (Table 1, Supplementary Figure 2), suggesting that upregulation of these
two genes contributes to Ad-36-induced adipogenesis.21
Moreover, FABP4, which encodes a lipid-transport protein
expressed in adipocytes,22 SORBS1, which encodes an
insulin-signaling molecule,23 and ADIPOQ, which encodes
the insulin-sensitizing hormone adiponectin, were upregulated in Ad-36-infected hMSCs (Table 1, Figure 4b). Previous
reports showed that SORBS1 affects insulin sensitivity and
glucose/lipid metabolism.24,25 In addition, adiponectin
induces the PPAR-gamma pathway and increases hepatic
insulin sensitivity, which translates into systemic improvements.26–28 Therefore, these genes may have a function in
the adipogenesis, energy expenditure, lipid transport and
insulin sensitivity involved in Ad-36-induced obesity, thus
Molecular mechanism of Ad-36-induced obesity
H-N Na et al
199
Figure 4 Gene set enrichment analysis (GSEA) identified novel pathways that were activated by Ad-36 infection compared with Ad-2 infection. GSEA was
performed using the microarray data to compare gene expression profiles between Ad-36 infection and Ad-2 infection. (a) GSEA showed that the PPAR-gamma
signaling pathway and the inflammation pathway were upregulated in Ad-36 infection compared with Ad-2 infection. (b) Leading-edge analysis revealed the
upregulation of eight and three genes that are important for the selective activation of the PPAR-gamma signaling and inflammation pathways, respectively.
representing good clues regarding the mechanisms that
underlie this phenomenon. However, further studies are
necessary to identify the function of the genes that were
regulated in Ad-36-induced obesity.
In this study, we used GSEA to explore novel pathways that
were regulated differentially after infection of Ad-36 compared with Ad-2. As many biological processes, such as
inflammation and adipogenesis, occur as a network of
changes in the expression levels of many genes, the analysis
of a few genes may not be sufficient to predict biological
phenomena. The strategy described herein for the analysis of
Ad-36-induced obesity aimed to identify individual genes
that showed differences of expression between Ad-36 and
Ad-2 infection. However, it is difficult to detect biological
processes, which are distributed across entire networks of
genes, and subtle changes at the level of individual genes.
GSEA, a tool used to analyze entire genetic networks,
revealed the upregulation of the PPAR-gamma signaling
pathway and that of the inflammation pathway (Figure 4).
It was expected to identify changed signals for the PPARgamma and inflammation pathways after Ad-36 infection,
as previous reports showed that these two pathways are
involved in obesity.29,30 The identification of the involvement of the inflammation pathway in Ad-36-induced
obesity was interesting, given that inflammation in adipocytes is important for the maintenance of obesity states.30
Moreover, many viruses lead to chronic inflammation states
in infected hosts.25–27 Interestingly, it has been reported that
Ad-36 can infect human adipocytes.12 Therefore, it is
possible that Ad-36 induces inflammation, which in turn
leads to obesity. In addition, we generated data that shows
that Ad-36 infection increased some inflammatory factors in
adipocytes (Na and Nam, unpublished data). Therefore, we
can expect that the inflammation mechanisms involved in
Ad-36 infection may provide new insights into Ad-36induced obesity. Finally, GSEA seems very useful for the
prediction of the cellular mechanisms involved in Ad-36induced obesity and other diseases.
International Journal of Obesity
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200
Taken together, our results showed that Ad-36 induced the
differentiation of hMSCs into an adipocyte lineage via the
activation of specific genes and novel cellular pathways
(PPAR-gamma and inflammation). Our study provides novel
insights into Ad-36-induced obesity and suggests that hMSCs
are a useful tool for the study of Ad-36-induced adipogenesis.
12
13
14
Conflict of interest
15
The authors declare no conflict of interest.
16
Acknowledgements
This study was supported by a grant from the GRRC of the
Catholic University of Korea, the Biogreen21 Program
(PJ007186) of Rural Development of Administration, MKE
and KOTEF through the Human Resource Training Project
for Strategic Technology, and Medical Research Center
(2010-0029474) programs funded by the Ministry of Science
and Technology, Republic of Korea. H-N Na was supported
by Hi Seoul Science (Humanities) Fellowship funded by the
Seoul Scholarship Foundation.
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Supplementary Information accompanies the paper on International Journal of Obesity website (http://www.nature.com/ijo)
International Journal of Obesity