Road To Fitness, Uncovering Novel Biomarkers Controlling Obesity in Volunteer Groups Undergoing Exercise Regiments in Kuwait

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Road to Fitness, Uncovering Novel Biomarkers Controlling Obesity in Volunteer


Groups Undergoing Exercise Regiments in Kuwait

Conference Paper · June 2012

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Proteomics Analysis of Human Obesity Reveals the
Epigenetic Factor HDAC4 as a Potential Target for
Obesity
Mohamed Abu-Farha1, Ali Tiss1, Jehad Abubaker1, Abdelkrim Khadir1, Fahad Al-Ghimlas2, Irina Al-
Khairi1, Engin Baturcam1, Preethi Cherian1, Naser Elkum3, Maha Hammad1, Jeena John1, Sina
Kavalakatt1, Samia Warsame1, Kazem Behbehani1,2,3, Said Dermime4, Mohammed Dehbi1,5*
1 Department of Biomedical Research, Dasman Diabetes Institute, Kuwait, Kuwait, 2 Fitness and Rehabilitation Centre, Dasman Diabetes Institute, Kuwait,
Kuwait, 3 Department of Biostatistics & Epidemiology, Dasman Diabetes Institute, Kuwait, Kuwait, 4 Biomedical Research Facility, King Fahad Specialist
Hospital Dammam, Dammam, Kingdom of Saudi Arabia, 5 Genomic Medicine and Systems Biology Research Center, Qatar Biomedical Research Institute,
Education City, Doha, Qatar

Abstract

Sedentary lifestyle and excessive energy intake are prominent contributors to obesity; a major risk factors for the
development of insulin resistance, type 2 diabetes and cardiovascular diseases. Elucidating the molecular
mechanisms underlying these chronic conditions is of relevant importance as it might lead to the identification of
novel anti-obesity targets. The purpose of the current study is to investigate differentially expressed proteins between
lean and obese subjects through a shot-gun quantitative proteomics approach using peripheral blood mononuclear
cells (PBMCs) extracts as well as potential modulation of those proteins by physical exercise. Using this approach, a
total of 47 proteins showed at least 1.5 fold change between lean and obese subjects. In obese, the proteomic
profiling before and after 3 months of physical exercise showed differential expression of 38 proteins.
Thrombospondin 1 (TSP1) was among the proteins that were upregulated in obese subjects and then decreased by
physical exercise. Conversely, the histone deacetylase 4 (HDAC4) was downregulated in obese subjects and then
induced by physical exercise. The proteomic data was further validated by qRT-PCR, Western blot and
immunohistochemistry in both PBMCs and adipose tissue. We also showed that HDAC4 levels correlated positively
with maximum oxygen consumption (VO2 Max) but negatively with body mass index, percent body fat, and the
inflammatory chemokine RANTES. In functional assays, our data indicated that ectopic expression of HDAC4
significantly impaired TNF-α-dependent activation of NF-κB, establishing thus a link between HDAC4 and regulation
of the immune system. Together, the expression pattern of HDAC4 in obese subjects before and after physical
exercise, its correlation with various physical, clinical and metabolic parameters along with its inhibitory effect on NF-
κB are suggestive of a protective role of HDAC4 against obesity. HDAC4 could therefore represent a potential
therapeutic target for the control and management of obesity and presumably insulin resistance.

Citation: Abu-Farha M, Tiss A, Abubaker J, Khadir A, Al-Ghimlas F, et al. (2013) Proteomics Analysis of Human Obesity Reveals the Epigenetic Factor
HDAC4 as a Potential Target for Obesity. PLoS ONE 8(9): e75342. doi:10.1371/journal.pone.0075342
Editor: Stefan Bereswill, Charité-University Medicine Berlin, Germany
Received May 22, 2013; Accepted August 13, 2013; Published September 24, 2013
Copyright: © 2013 Abu-Farha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Kuwait Foundation for the Advancement of Sciences under project (RA-2010-003). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]

Introduction a key risk factor contributing to the overall burden of chronic


disease occurring over a wide geographic area and affecting
The increase in sedentary lifestyles and excessive food virtually all ages, genders and socioeconomic groups [2]. The
intake are considered as key contributing factors to a group of recent annual report from the International Association for the
obesity-associated disorders such as insulin resistance, Study of Obesity documented approximately 1.5 billion of
diabetes, dyslipidemia and cardiovascular complications that adults that are overweight, of whom around 525 million are
substantially contribute to a significant reduction of either life clinically obese (www.iaso.org). In Kuwait, the prevalence of
quality or expectancy, if not both [1]. Paradoxically coexisting in overweight and obesity is highly alarming among adult
developing countries with under-nutrition, obesity has become population with rates of 80.4% and 47.2%, respectively [3].

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Downregulation of HDAC4 by Obesity

Exercise is an important component of healthy lifestyle that has Using this approach, 47 proteins were found to be
long-time been prescribed as a part of the prophylactic differentially expressed between lean and obese subjects.
treatment and management of obesity, insulin resistance and Proteins that were upregulated in obese group included
their associated complications [4]. thrombospondin 1 (TSP1), whereas, the histone deacetylase 4
Obesity is characterized by a chronic, low-grade (HDAC4) protein levels were reduced in obese subjects. In
inflammatory response referred to as metaflammation and obese subjects, the proteomic profiling before and after 3
dysregulation of the stress response system in key metabolic months of physical exercise showed differential expression of
organs such as liver, muscle, adipose tissue and pancreas [5]. 38 proteins. Interestingly, the expression levels of both TSP1
The stress response; known also as metabolic stress, is highly and HDAC4 were corrected by physical exercise and became
complex and includes an impairment of the heat shock comparable to the levels initially observed in control group. In
response [6,7], excessive activation of the oxidative stress functional assays, our data indicated that overexpression of
versus a down-regulation of the cellular antioxidant defence HDAC4 significantly impaired TNF-α-dependent activation of
system [8,9], dysfunction of the mitochondria or defect in its NF-κB, establishing thus a link between HDAC4 and regulation
of the immune system. Together, our in vivo and in vitro data
biogenesis [10], hypoxia [11], and the emerging role of the
on HDAC4 suggest that it has a protective role against obesity
endoplasmic reticulum (ER)-mediated stress [12]. Recent
and could therefore represent a potential therapeutic target for
investigations indicated that the inflammatory and stress
management of obesity and presumably in insulin resistance
responses pathways are highly integrated and they work most-
and type 2 diabetes.
likely in vicious cycles, which largely explain the myriad of
disorders associated with obesity [13,14]. Metabolic stresses
are known to activate several stress kinases but the best
Materials and Methods
characterized are the c-Jun N-terminal kinase (JNK) and the
inhibitor of κB kinase-β (IKKβ), both of them can phosphorylate Study population and ethical statement
the insulin receptor substrate-1 (IRS-1) and rendering this The current investigation is part of a large cohort study
crucial intermediate a poor substrate for the activated insulin aimed at investigating the effect of physical exercise on the
receptor [15,16]. inflammatory, metabolic and stress responses in obese and
Several genomic and proteomic studies have been carried diabetic subjects. In the present study, we selected 48 adult
over the last decade on human and mouse adipose tissue and non-diabetic male participants consisting of 11 lean (20 ≤ BMI
the outcomes of such efforts were pivotal in gaining insight into < 25 kg/m2) and 37 obese (30 ≤ BMI < 40 kg/m2) to identify
differentially expressed proteins between the two groups.
the molecular mechanisms underlying obesity and its
Written informed consent was obtained from all subjects before
associated complications [17,18,19,20,21]. A recent proteomics
their participation in the study which was approved by the
study was carried out on two-major intra-abdominal human fat
Review Board of Dasman Diabetes Institute and carried out in
tissues, namely the subcutaneous and the omental fat to
line with the guideline ethical declaration of Helsinki. All
identify and validate obesity-related reference proteins for data
volunteers were subjected to a pre-screening assessment to
standardization [22]. Accordingly, only ENOA, PARK7 and β-
determine their eligibility to participate in the study. Glucose
actin proteins were reported as proper reference standards for
and lipid profiles were analyzed for all volunteers. Candidates
omental fat in obesity studies, while FAA was shown to be the that followed any physical exercise within the last 6 months
best control for both omental and subcutaneous adipose prior to this study, morbid obese (i.e. BMI >40 kg/m2) and those
tissues regardless of the obesity status [22]. The limited with prior major illness were excluded from the study. In
number of identified proteins was, in large part due to the addition, eligible candidates should not be taking any
inherent limitation of the technology (2D gel combined to Maldi- medication and/or supplement known to influence the body
Tof) and the technical challenges to extract soluble proteins composition or bone mass. The physical as well as the clinical
from fat adipose tissue in sufficient amounts. and biochemical parameters of the participating subjects are
In the present study, we applied a shot-gun proteomic shown in Table 1 and Table 2, respectively.
profiling approaches on PBMCs isolated from lean and obese
human male subjects to identify and quantify proteins Exercise Program
expressed in the two groups and established their possible All eligible subjects were enrolled to a supervised exercise
correlation with clinical outcomes. This study will provide a program at the Fitness and Rehabilitation Center (FRC) of
snapshot of the proteomic profile of lean and obese subject Dasman Diabetes Institute. Prior to exercise prescription, each
and the data generated can be used to formulate novel individual underwent a symptom-limited maximal incremental
hypotheses on the molecular mechanisms orchestrating cardiopulmonary exercise test “CPET” (COSMED Quark, Italy)
obesity and its management with physical exercise. PMBCs using an electromagnetically braked cycle ergometer. The
were chosen as surrogate tissue in this investigation based on CPET was primarily used to determine the maximum heart rate
their important inflammatory and stress response roles in a (max HR) as well as the response to aerobic exercise as
variety of diseases including obesity. Based on the beneficial measured by the maximum oxygen consumption (VO₂ Max) for
effect of physical exercise on improving the clinical outcomes each subject. Thereafter, a physical fitness assessment test
associated with obesity; we also investigated its impact on was performed to determine muscle strength and endurance
differentially expressed proteins in obese subjects. along with flexibility by performing grip strength

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Downregulation of HDAC4 by Obesity

at 65-80% of max HR. For the duration of the 3-months period,


Table 1. Physical characteristics of the study population at
participants exercised 3 to 5 times per week and they were
baseline.
instructed to reach and maintain the recommended heart rate
range. This was achieved by regular monitoring of the heart
Lean (n = 11) Obese (n = 37) P-value
rate during the aerobic training. Strength training was
Age (year) 42.73 ± 9.71 46.38 ± 12.50 0.37
performed 2 to 3 times a week according to the program plan.
BMI (kg/m2) 23.67 ± 1.47 34.13 ± 2.82 <0.0001
Exercise intensity, duration and blood pressures were recorded
PBF (%) 24.21 ± 2.27 34.65 ± 3.26 <0.0001
for each session. All trainings were supervised by qualified
Waist (cm) 82.35 ± 16.67 113.34 ± 8.53 <0.0001
fitness professionals and a consultant respirologist from FRC.
Hip (cm) 93.55 ± 6.07 113.92 ± 7.62 <0.0001
To assess the effectiveness of the exercise, the same physical
Data are presented as mean ± SD. Mann-Whitney t-test was used to compare
stress and fitness tests were performed for all subjects at the
differences between lean and obese subjects. BMI (body mass index), PBF
end of the exercise program.
(percent body fat).
doi: 10.1371/journal.pone.0075342.t001 Anthropometric and biochemical measurements
Anthropometric measurements were taken at the baseline
and after 3 months of exercise. Whole-body composition was
Table 2. Clinical and biochemical parameters of the study determined by dual-energy radiographic absorptiometry device
population at baseline. (Lunar DPX, Lunar radiation, Madison, WI). Glucose and lipid
profiles were measured on the Siemens Dimension RXL
chemistry analyzer (Diamond Diagnostics, Holliston, MA).
Lean (n=11) Obese (n=37) P-value HbA1C was determined using the VariantTM device (BioRad,
Resting HR (beat/min) 86.67 ± 19.54 73.78 ± 7.60 0.1 Hercules, CA). Plasma levels of inflammatory and metabolic
SBP (mmHg) 113.33 ± 12.11 131.67 ± 12.25 0.03 markers were measured using bead-based multiplexing
DBP (mmHg) 76.67 ± 5.16 86.00 ± 9.66 0.05 xMAP® technology (Luminex, Austin, TX) using the Bio-Plex
VO2 Max (ml/kg/min) 23.34 ± 3.21 18.8 ± 5.35 0.11 Pro™ human cytokine 27-plex assay and diabetes 10-plex
Cholesterol (mmol/l) 5.00 ± 0.59 5.12 ± 1.04 0. 76 assay kits (BioRad, Hercules, CA). Median fluorescence
HDL (mmol/l) 1.34 ± 0.78 1.04 ± 0.22 0.16 intensities were collected on a Bioplex-200 system using Bio-
LDL (mmol/l) 3.07 ± 1.00 3.38 ± 0.92 0.59 plex Manager software version 6 (BioRad, Hercules, CA). Lipid
TG (mmol/l) 1.14 ± 0.52 1.52 ± 80 0.13 peroxidation was assessed by measuring plasma levels of
Glucose (mmol/l) 5.20 ± 0.60 5.59 ± 0.95 0.29 malonaldehyde, using TBARs assay kit (Cayman Chemical
HbA1C (%) 5.56 ± 0.55 5.95 ± 0.64 0.07 Company, Ann Arbor, MI). Serum levels of ROS were
C-peptide (ng/ml) 2.79 ± 0.74 3.54 ± 1.35 0.18 determined using the OxiSelect™ ROS Assay Kit (Cell Biolabs
GLP-1 (ng/ml) 2.50 ± 0. 91 2.91 ± 1.60 0.96 Inc, San Diego, CA). All the above assays were carried out
Insulin (ng/ml) 2.55 ± 1.23 3.97 ± 2.21 0.08 according to the instructions of the manufacturers.
Leptin (ng/ml) 3.19 ± 1.37 6.60 ± 2.69 0.001
PAI-1 (ng/ml) 2.45 ± 0.72 3.98 ± 1.14 0.006
Blood and tissue sampling
TNF-α (pg/ml) 18.15 ± 1.86 31.91 ± 14.95 0.13
Venous peripheral blood and subcutaneous adipose tissue
IL-1β (pg/ml) 1.10 ± 0.46 1.28 ± 0.48 0.34
biopsies were obtained before starting the exercise (baseline)
IL-6 (pg/ml) 3.89 ± 1.20 5.41 ± 2.48 0.08
and after the 3 months of exercise period. Peripheral blood
IL-10 (pg/ml) 1.31 ± 0.99 2.43 ± 1.84 0.17
mononuclear cells (PBMCs) were prepared from 40 ml of blood
IP-10 (ng/ml) 0.39 ± 0.19 0.55 ± 0.22 0.07
using Ficoll-Hypaque density gradient centrifugation method
RANTES (ng/ml) 1.12 ± 0.42 1.61 ± 0.68 0.04
and then, resuspended in freezing media containing 10%
ROS (mM) 1.43 ± 0.35 1.44 ± 0.14 0.83
DMSO and stored in liquid nitrogen. Plasma and serum were
TBARS (μM) 1.01 ± 0.45 1.64 ± 0.54 0.006
prepared using vacutainer tubes and then aliquoted and stored
Data are presented as mean ± SD. Mann-Whitney t-test was used to compare
at -80°C until assayed. Subcutaneous superficial adipose
differences between lean and obese subjects. HR (heart rate), SBP (systolic blood
tissue biopsies (~0.5 g) were obtained by a surgeon from the
pressure), DBP (diastolic blood pressure), VO2 Max (maximum oxygen
periumbilical area by surgical biopsy after a local anesthesia.
consumption), HDL (high density lipoprotein), LDL (low density lipoprotein) and TG
Once removed, the biopsy was rinsed in cold PBS, divided into
(triglycerides).
4 pieces and stored appropriately until used for mRNA
doi: 10.1371/journal.pone.0075342.t002
extraction and immunohistochemical studies.

(dynamometer), push-ups (upper body strength), sit-ups and Preparation of protein extract for MS analysis
forward bending test (both upper and lower body flexibility). Frozen PBMCs collected from lean subjects (n=6), obese
The exercise training involves a combination of both moderate subjects before (n=6) and after exercise (n=6) were washed
intensity of aerobic exercise and resistance training using with ice-cold PBS and the total cell count and viability were
either treadmill or cycling. Each exercise session includes 10 determined by using Countess™ Automated Cell Counter as
minutes warming-up and cooling down steps at 50-60% of max instructed by the manufacturer (Invitrogen, Carlsbad, CA).
HR, along with 40 minutes of the prescribed exercise program Equal amount of cells (2.5x106) were pooled to make three

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Downregulation of HDAC4 by Obesity

pairs for each group and then, used to prepare whole cell analyzed using the Ingenuity pathways analysis (IPA) software
extracts. After centrifugation, the cell pellet was resuspended in (Ingenuity Systems; Qiagen, Inc., Valencia, CA).
200 µL of lysis buffer (6M urea, 4% CHAPS) supplemented
with a mini complete protease inhibitor cocktail (Roche Gene expression analysis
Diagnostics, Laval, Quebec) for 30 min at 4°C. Extracts were Total RNA extracted from PBMCs and adipose tissue using
centrifuged at 14,000 rpm for 30 minutes at 4°C. Protein AllPrep RNA/Protein and The RNeasy Lipid Tissue extraction
concentration was determined by Bradford method using γ- kits, respectively (Qiagen, Inc., Valencia, CA) was converted to
globulin as a standard and proteins were subjected to cDNA using High Capacity cDNA Reverse Transcription Kit
centrifugal proteomic reactor as described previously [23]. (Applied Biosystems, Foster City, CA) and analyzed by
Briefly, 20 µg of proteins were added to 10 µL of strong cationic quantitative real-time PCR using Rotor Gene Q-100 (Qiagen,
exchange (BcMag™ SCX Magnetic bead slurry (Bioclone, San Inc., Valencia, CA). Changes in gene expression of the
Diego, CA) in 1 ml of 5% formic acid and the mixture was selected genes between obese and controls subjects were
vigorously vortexed. After 2 min centrifugation at 10,000 g, the determined by the comparative ΔΔCT method [24] using
pellet was resuspended in 1 ml of 0.5% formic acid and then, GAPDH and β-actin as internal references. ΔΔCT = ΔCT
samples were reduced by adding 20 µl of reducing solution (obese group)-ΔCT (control group) for RNA samples.
(150 mM NH4HCO3, 20 mM DTT) for 15 min at 56°C with
constant rotation at 800 rpm and then alkylated by adding 20 µl Western blot analysis
of alkylating solution (150 mm NH4HCO3, 100 mm Western blots were carried out on whole PBMC extracts
iodoacetamide) for 15 min at room temperature. The alkylation prepared in RIPA buffer (50mM Tris-HCl pH 7.5, 150mM NaCl,
reaction was stopped by adding 1 ml of 0.5% formic acid. The 1% Triton x100, 1mM EDTA, 0.5% Sodium deoxycholate and
beads were centrifuged and proteins were digested with trypsin 0.1% SDS). Cytoplasmic and nuclear extracts were prepared
for overnight at 37°C with constant shaking and then, eluted from PBMCs using commercially available kit as recommended
with 1 ml of 5% formic acid at four different pH levels (2.5, 4, 8 by the manufacturer (BioRad, Hercules, CA). Protein
and 12). Fractions were lyophilized to complete dryness in a concentration was determined by Bradford method using
speed-vac and stored at -20°C until subjected to MS analysis. globulin as a standard. For Western blot, 20 µg of proteins
were resolved on 10% SDS-PAGE gels. Proteins were then
NanoLC-MS/MS Analysis transferred onto PVDF membranes, blocked with 5% non-fat
dried milk in Tris-buffered saline containing 0.05% Tween 20
The analysis of peptide digests was done by Liquid
(TBST) for 1 h at room temperature (RT) and then probed with
chromatography (Easy nanonLC; Proxeon Biosystems,
the primary antibody for overnight at 4°C. After washing, the
Denmark) coupled to tandem mass spectrometry (MS/MS;
membranes were incubated with horseradish peroxidase-
LTQ-Orbitrap Velos, Thermo Scientific, Germany). Briefly,
conjugated secondary antibody for 2 h at RT. Finally, protein
peptides samples were first dissolved in 5% formic acid and
bands were visualized by chemiluminescence and the images
then loaded on a C18-A1 easy column (Proxeon Biosystems,
were captured by using the Versadoc 5000 system (BioRad,
Denmark). Peptides were desalted with 5% acetonitrile/ 0.1%
Hercules, CA). Primary antibodies used in this study consisted
formic acid before their elution from the C18-A1 column. The of anti-HDAC4 (Catalog # ab1437, Abcam, Cambridge, CA),
peptides were then directed to a C18-A2 analytical easy anti-TSP1 (Catalog # ABIN749558, Antibodies online.com,
column (Proxeon Biosystems, Denmark). Peptides were finally Atlanta, GA) and anti-HSF1 (Catalog # 4356, Cell Signaling
eluted at a gradient of 5 to 35% acteonitrile with 0.1% formic Technology, Inc, Danvers, MA). Tubulin (Catalog # 05-661,
acid over 80 min and analyzed with MS. Millipore, Timecula, CA) and Lamin B (Catalog # 12586, Cell
The full MS spectra scan was performed at a resolution of Signaling Technology, Inc, Danvers, MA) were used as internal
60,000. MS/MS spectra were acquired in a data-dependant controls. For densitometric analysis, the intensity of the bands
acquisition mode that automatically selected and fragmented was determined using Quantity One Software (BioRad,
the fifteen most intense peaks from each MS spectrum Hercules, CA).
generated. Raw data files were analyzed using the Proteome
Discoverer 1.3 software (Thermo Scientific; Germany) using Immunohistochemistry
Sequest and Mascot search engine against the Homo sapiens Formalin fixed, paraffin embedded adipose tissue samples
International Protein Index (IPI) protein sequence database were prepared and used to make sections for
version 3.68 (European Bioinformatics Institute, United immunohistochemical studies as described previously [25].
Kingdom). Search parameters were set as follow: trypsin was Briefly, sections were deparaffinized and the antigens were
selected as digestive enzyme with 2 miss-cleavages allowed, retrieved at high-temperature using antigen unmasking solution
carbamidomethyl of cysteine as a fixed modification and (Dako, Denmark). The endogenous peroxidase was quenched
methionine oxidation as a variable modification. Peptide and using 3% H2O2 (Merck Schuchardt, Gemany) for 30 min at RT.
MS/MS mass tolerances were set at 7 ppm and 0.8 Da, Sections were blocked with 5% fat-free milk for 60 min at RT
respectively. Peptide confidence was set to high ensuring a 1% followed by 1% BSA for another 60 min and then, incubated at
false discovery rate (FDR). Sieve software version 1.3 (Thermo 4°C for overnight with the corresponding primary antibody.
Scientific, Germany) was used for quantification of proteins After washing, sections were stained with horseradish
identified. Molecular functions and protein networks were conjugated secondary antibody (Dako, Denmark) for 60

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Downregulation of HDAC4 by Obesity

minutes at RT. Colors were developed using DAB kit (Dako, Results
Denmark) and sections were counterstained with hematoxylin
(Sigma Aldrich, St. Louis, MO). All slides were scanned at 20x Baseline characteristics of study population
magnification and the quantification of the The physical characteristics of the study population baseline
immunohistochemical data was done using Aperio are displayed in Table 1. As the population was matched for
ImageScope software version 11.1 (Aperio, Vista, CA). The age, there was no significant age difference between the two
algorithm Positive Pixel Count v9 of this software provided the groups. As expected, body mass index (BMI), percent body fat
percentage of positive staining (number of stained pixels) as (PBF), waist and hip circumferences were all significantly
compared to the whole slide. Finally, each annotated picture higher in obese subjects than in the control (P < 0.0001). Table
was manually checked against the original slide picture to 2 shows that obese subjects had higher systolic and diastolic
ensure the correct matching between the positive staining and blood pressures (P = 0.03 and P = 0.05, respectively). No
the software annotation. significant difference between the two groups was observed in
the lipid profile as well as the levels of fasting glucose, HbA1C,
Plasmid constructs insulin and C-peptide. By contrast, the levels of leptin and
PAI-1 were significantly higher in obese subjects (P = 0.001
The expression vector encoding HDAC4 (pCMV-HDAC4)
and P = 0.006, respectively). Obese subjects had also higher
and the pCMV empty vector were purchased from OriGene
levels of the inflammatory chemokine RANTES (P = 0.04), but
(OriGene Technologies, Inc, Rockville, MD). Reporter plasmids
there was no significant difference with the remaining
carrying luciferase gene under the control of NF-κB were
inflammatory mediators including TNF-α and IL-6 (Table 2 and
described previously [26]. Briefly, plasmids 3xwt-κB-pGL3 and
data not shown). Both lean and obese subjects had
3xmut-κB-pGL3 were constructed by inserting three copies of
comparable levels of ROS, but TBARS levels were significantly
wild type (5’-AGTTGAGGGGACTTTCCCAGGCTG-3’) or higher in obese subjects (P = 0.006, Table 2).
mutant (5’-AGTTGAATCGACTTTCCCAGGCTG-3’) NF-κB
binding site into the unique Nhe-I and Xho-I restriction sites Proteomic Analysis
upstream of the SV40 minimal promoter of pGL3 vector
In this study, we compared the proteomic profile of PBMCs
(Promega, Madison, WI).
from lean and obese subjects using a label-free proteomic
approach. Samples were subjected to a reverse phase nano-
Cell culture, transfection and luciferase assays LC coupled to high resolution Orbitrap-Velos MS and protein
Human embryonic kidney (HEK-293) cell line was obtained identification was done by Proteome Discoverer software using
from American Type Culture Collection (Rockville, Baltimore, Sequest and Mascot search engines against the human
MD). Cells were cultured in Eagle’s Minimum Essential Medium international protein index (IPI) database. Relative changes in
(EMEM) supplemented with 10% fetal bovine serum and protein levels between different groups were calculated from
penicillin/streptomycin. For transient transfection assays, cells the obtained mass spectra using Sieve software. To account
at ~80% of confluence were transfected with 24 µg of DNA for the biological variability, PBMCs samples from lean and
using Lipofectamine method as recommended by the obese subjects (n=6 each) were pooled into pairs to make
manufacturer (Invitrogen, Carlsbad, CA). Following three different biological replicates from each group and each
transfection, cells were incubated in complete EMEM media for peptide should be identified at least in 2 out of 3 biological
24-36 hours and then, harvested for luciferase assays. replicates as illustrated in the flow chart (Figure S1). All
Luciferase assays were performed using the Dual Luciferase proteins were identified at 1% of false discovery rate. As shown
Assay kit (Promega, Madison, WI). To induce NF-κB, cells in Figure 1A, a total number of 1434 proteins were identified
were stimulated with 25 ng/ml of TNF-α (R & D Systems, from the combined MS runs, consisting of 1321 proteins from
Minneapolis, MN) overnight. Luciferase activity was measured lean and 1176 proteins from obese, of which, 1063 proteins
on Synergy Hybrid H4 plate reader (BioTek, Winooski, VT) and were found to be common between the two groups (Figure 1A).
Figure 1B indicates that the total proteins identified in this study
normalized according to protein concentration.
are involved in a wide range of cellular functions including
metabolism, regulation of biological process, response to
Statistical analysis
stimuli, transport, and cell organization and biogenesis. The
Statistical analyses were performed with SAS version 9.2 detailed lists of all identified peptides and proteins in both
(SAS Institute, Inc., Cary, NC). Unless otherwise stated, all groups are presented in the Tables S1, S2, S3 and S4. Using
descriptive statistics for the variables in the study were SIEVE software to analyze differential protein expression levels
reported as means ± standard deviation. Non parametric between the two groups, 47 proteins among the 1063 common
Mann-Whitney t-test was used to determine significance of proteins shown in Figure 1A were found to be differentially
difference in means between the two groups as indicated in the expressed (at least 1.5-fold changes) between lean and obese
Figure legends. Correlations between variables were subjects out of which, 18 proteins were overexpressed in
calculated with the Spearman’s rank correlation test. obese and 29 proteins were overexpressed in lean (Table 3). A
Differences were considered statistically significant at P-values detailed list of quantified proteins common in both groups is
less than 0.05. displayed in Table S5.

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Downregulation of HDAC4 by Obesity

Figure 1. Effect of obesity on protein expression in PBMCs. Venn diagram showing the number of unique and overlapping
proteins identified from lean (LN) and obese (OB) subjects (A). Distribution of all the identified proteins according to their biological
processes (B). Both panels were generated using ProteinCenter software (Thermo Scientific, Germany). qRT-PCR data carried out
on 10 genes to validate the observed differential protein expression at the mRNA levels using total RNA isolated from PBMCs of
lean and obese male subjects (n=5 each) and the data are presented as fold changes in obese compared to lean subjects (C). The
gene names and primer sequences are shown in Table S6. * P < 0.05 as determined using student’s t-test.
doi: 10.1371/journal.pone.0075342.g001

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Downregulation of HDAC4 by Obesity

Table 3. List of differentially expressed proteins identified in Table 3 (continued).


PBMCs collected from lean and obese subjects.

Fold
Fold IPI Number Protein Description Symbol Changes* SD
IPI Number Protein Description Symbol Changes* SD H_3q26 provirus ancestral Env
IPI00400838.3 HERV ↓1.62 0.51
IPI00296099.6 Thrombospondin-1 TSP1 ↑7.7 0.05 polyprotein
IPI00219757.13 Glutathione S-transferase P GSTP1 ↑2.9 0.17 Tyrosine 3-monooxygenase/
IPI00029166.4 Homeobox protein EMX2 ↑2.3 0.23 tryptophan 5-monooxygenase
IPI00640721.1 YWHAB ↓1.61 0.58
IPI00293423.3 Perforin-1 PRF1 ↑2.2 0.12 activation protein, beta
IPI00645645.1 T-complex 11 homolog TCP11 ↑2.1 0.16 polypeptide
IPI00152658.1 Serine/threonine-protein kinase NEK7 ↑1.9 0.16 IPI00005782.1 Protein phosphatase 1D PPM1D ↓1.61 0.61
cDNA FLJ56016, highly similar to Seven transmembrane helix
IPI00794900.3 MTHFD1 ↑1.9 0.24 IPI00386324.2 GPR142 ↓1.60 0.57
C-1-tetrahydrofolate synthase receptor
Cat eye syndrome critical region Isoform 2 of Ankyrin repeat and
IPI00010832.1 CECR6 ↑1.8 0.14 IPI00329245.8 ANKLE2 ↓1.56 0.43
protein 6 LEM domain-containing protein2
PERQ amino acid-rich with GYF Caspase recruitment domain-
IPI00021739.2 GIGYF1 ↑1.8 0.26 IPI00869011.2 CARD11 ↓1.55 0.70
domain-containing protein 1 containing protein 11
IPI00031016.1 JAK2 Tyrosine-protein kinase JAK2 ↑1.7 0.39 IPI00742823.1 Similar to DNA-binding protein LOC283547 ↓1.55 0.53
Cytochrome c oxidase assembly Isoform 1 of Testis-specific
IPI00413436.2 COX19 ↑1.7 0.19
protein IPI00069208.1 chromodomain protein Y-linked, CDY1B ↓1.53 0.33
IPI00003362.2 GRP78 protein GRP78 ↑1.7 0.36 1B
Putative uncharacterized protein Isoform 2 of Platelet glycoprotein
IPI00383810.2 PIAS3 ↑1.6 0.36 IPI00464990.1 GP1BB ↓1.52 0.56
DKFZp434O0617 1b beta chain
IPI00007800.1 Angiopoietin-related protein 2 ANGPTL2 ↑1.6 0.04 Isoform 1 of Stearoyl-CoA
IPI00465139.2 SCD5 ↓1.52 0.57
IPI00456750.2 Niban-like protein 1 FAM129B ↑1.6 0.16 desaturase 5
Myeloid/lymphoid or mixed-lineage Isoform 2 of Nuclear receptor
IPI00100630.6 MLLT1 ↑1.6 0.30 IPI00470491.3 NCOA1 ↓1.52 0.53
leukemia translocated to 1 coactivator 1
IPI00028438.4 c-Maf-inducing protein isoform CMIP ↑1.5 0.11 IPI00297550.8 Coagulation factor XIII A chain F13A1 ↓1.51 0.45
IPI00400922.5 Protein RRP5 homolog PDCD11 ↑1.5 0.24 * Protein levels are expressed as a fold increase (↑) or decrease (↓) in obese
IPI00022022.1 Polyphosphoinositide phosphatase Figure 4 ↓2.26 0.92 relative to lean group. SD is calculated from 3 independent experiments.
Isoform 2 of Parkin coregulated doi: 10.1371/journal.pone.0075342.t003
IPI00410380.2 PACRG ↓2.19 0.62
gene protein
IPI00219806.7 Protein S100-A7 S100A7 ↓2.18 0.79
Validation of the proteomics data by quantitative real-
IPI00005685.2 Paraneoplastic antigen Ma1 PNMA1 ↓2.14 0.60
time PCR
IPI00298497.3 Fibrinogen beta chain FGB ↓2.11 0.57
In order to validate the above proteomics data, we selected a
IPI00010088.2 Histone deacetylase 4 HDAC4 ↓2.11 0.66
set of 10 genes among the differentially expressed proteins
IPI00414661.7 La-related protein 6 LARP6 ↓1.95 0.79
Similar to Cathepsin D precursor
between lean and obese subjects before exercise and
LOC100287770;LOC100291562
analyzed their expression pattern between lean and obese
IPI00745814.2 ↓1.86 0.62 subjects by quantitative real-time PCR (qRT-PCR). As shown
hypothetical protein
XP_002343109 in Figure 1C, there was clear up- and down-regulation in the
Isoform 1 of Leucine-rich repeat- expression pattern at the mRNA levels between the two groups
IPI00427739.1 LRRC1 ↓1.85 0.73
containing protein 1 that was consistent with the proteomics data. Indeed, qRT-
Putative uncharacterized protein PCR data showed a significant reduction in the expression of
IPI00915861.1 NGEF ↓1.71 0.61
NGEF histone deacetylase 4 (HDAC4) and the angiogenic factor
Tumor necrosis factor, alpha- AGGF1 mRNA (P < 0.05) and a significant increase in the
IPI00009448.1 TNFAIP3 ↓1.69 0.79
induced protein 3 expression of thrombospondin 1 (TSP1) mRNA (P < 0.05).
IPI00290462.5 Carbonyl reductase [NADPH] 3 CBR3 ↓1.66 0.51 There was also a trend of increase in the expression of the
IPI00019502.3 Isoform 1 of Myosin-9 MYH9 ↓1.65 0.44 remaining genes in obese subjects; however, this change was
IPI00552787.4 RNA binding motif protein 20 RBM20 ↓1.65 0.47 not statistically significant (Figure 1C).
Telomere length regulation protein
IPI00016868.3 TELO2 ↓1.65 0.49
TEL2 homolog Effect of physical exercise on proteomic profile
IPI00295976.6 Isoform 1 of Integrin alpha-IIb ITGA2B ↓1.65 0.49
Physical exercise is an important component of healthy
Isoform 1 of Coiled-coil domain-
IPI00418774.3 CCDC73 ↓1.62 0.48 lifestyle that is highly prescribed as a part of the prophylactic
containing protein 73
treatment and management of obesity and the clinical
manifestations associated with it. These beneficial effects
prompted us to investigate if physical exercise has an effect on

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Downregulation of HDAC4 by Obesity

protein expression levels in obese subjects. For this purpose, More interestingly, qRT-PCR data shown on Figure 4C
differential proteomic expression was carried out on PBMCs indicated a significant decrease in the expression of HDAC4 (P
collected from obese subjects prior to physical exercise = 0.046) and increased expression of TSP1 in obese subjects
(baseline) and at the end of 3 months of physical exercise (P = 0.011). Conversely, the expression of HDAC4 was
program. As shown in Figure 2A, a total of 1402 proteins were significantly increased in obese subjects after physical exercise
identified from the combined MS runs obtained from both intervention (Figure 4E, P = 0.02) whereas that of TSP1 was
groups, of which 1025 proteins were found to be common significantly reduced by physical exercise in obese subjects
between the two groups. In addition, 151 proteins were found (Figure 4E, P = 0.001). Since our study population consisted of
exclusively in obese before exercise against 226 proteins that males only, we investigated whether there was a gender effect
were found exclusively in obese subjects that completed the on the expression of HDAC4 and TSP1 by qRT-PCR using
exercise program (Figure 2A). The detailed lists of all identified adipose tissue. Data obtained from 5 males and 5 females
peptides and proteins in obese after physical exercise are indicated that gender has no effect on differential expression of
presented in the Tables S7 and S8, respectively. Further HDAC4 and TSP1 in obese subjects (Figure S2). Taken
analysis indicated that out of the 1025 common proteins together, the initial quantitative proteomics data performed on
between the two groups shown in Figure 2A, 38 proteins were PBMCs for TSP1 and HDAC4 is in agreement with qRT-PCR
found to be differentially expressed with at least 1.5-fold and IHC data carried out on adipose tissue.
changes between obese subjects before and after exercise out
of which, 17 proteins were increased by exercise and 21
Correlation analysis of HDAC4 and TSP1 with clinical
proteins were decreased by exercise (Table 4) and 9 of these
profile, inflammatory and metabolic stress markers
differentially expressed proteins were subsequently validated
To understand the consequence of dysregulated expression
by qRT-PCR (Figure 2B). A detailed list of quantified proteins
of HDAC4 and TSP1 in obese subjects on the physical, clinical
common in both groups is displayed in Table S9.
and various biochemical parameters, we investigated whether
Among the proteins showing dysregulated expression by
these parameters correlated with the levels of HDAC4 and
obesity that was then corrected by physical exercise, we
TSP1 detected in lean and obese subjects before exercise. As
focused on TSP1 and HDAC4 proteins for further
investigations. The selection of these protein candidates was shown in Table 5, there were negative correlations that were
also based on their potential therapeutic role as anti-obesity highly significant between HDAC4 levels and the BMI (r2=-0.66;
targets. To further confirm the aberrant regulation of HDAC4 P < 0.0001), PBF (r2=-0.58; P = 0.0012) and RANTES (r2=-0.8;
and TSP1 in obese subjects, we investigated their endogenous P = 0.001) and a positive correlation with VO2 Max (r2=0.54; P =
expression by Western blotting using PBMCs extracts from 0.024). There was also a trend of negative correlation between
lean and obese subjects. In agreement with proteomic and HDAC4 and PAI-1 but it was not significant. No other
qRT-PCR data shown above, obese subjects exhibited correlations were found between HDAC4 levels and the
reduced expression of HDAC4 protein and increased remaining parameters measured (Table 5 and data not shown).
expression of TSP1 protein (Figure 3A and 3B). Given that By contrast to HDAC4, TSP1 positively correlated with the BMI
HDAC4 has been shown to shuttle between nucleus and (r2=0.54; P = 0.0014) and slightly with PBF and PAI-1 but
cytoplasm [27], we particularly analyzed whether obesity without reaching statistical significance. A trend of negative
triggers a change in its cellular localization. Consistent with the correlation was also found between TSP1 and VO2 Max, although
immunoblot data obtained from whole cell lysates, obese it was not significant. No correlation was found betweenTSP1
subjects showed a reduced expression of HDAC4 protein and levels and the other parameters (Table 5 and data not shown).
increased expression of TSP1 protein than lean subjects To elucidate whether the observed changes in the expression
(Figure 3C and 3D). Using Lamin B, Tubulin and HSF-1 as of HDAC4 and TSP1 in obese subjects after physical exercise
internal controls, HDAC4 was predominantly found in the were concomitant to improvement of physical, clinical and
cytoplasm in both groups suggesting that obesity does not biochemical parameters, we performed a pair wise comparison
trigger a change in the localization of HDAC4. on 15 obese subjects before and after 3-months exercise to
first assess the effectiveness of the exercise program on
TSP1 and HDAC4 are differentially regulated in the physical, clinical and biochemical parameters and if so,
adipose tissue of obese subjects whether they were consistent with the levels of HDAC4 and
Adipose tissue is known to play a central role in the TSP1 before and after exercise. Table 6 shows that although
development of obesity. We therefore investigated if the there was no significant change in the BMI after 3 months of
changes in the expression of TSP1 and HDAC4 observed in exercise, there was a significant reduction of PBF, SBP and
PBMCs occurred also in the adipose tissue. For this purpose, DBP (P <0.05). There was also an improvement of VO2 Max (P =
adipose tissue was collected from lean, obese subjects before 0.011) along with reduced insulin levels (P = 0.036) and
and after exercise and the relative expression of these proteins improved inflammatory response as indicated by reduced
was determined by immunohistochemisty (IHC) and qRT-PCR. levels of the pro-inflammatory IL-6 cytokine and increased
Consistent with the data observed in PBMCs, IHC data showed levels of the anti-inflammatory IL-10 cytokine (P = 0.04). Under
trends of reduced expression of HDAC4 (Figure 4A) and these conditions, HDAC4 expression increased significantly (P
increased expression of TSP1 (Figure 4B) in obese compared = 0.0092) and TSP1 was reduced significantly by physical
to lean subjects that were corrected by exercise (Figure 4D). exercise (P = 0.027) (Table 6).

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Downregulation of HDAC4 by Obesity

Figure 2. Effect of physical exercise on protein expression in PBMCs of obese subjects. Venn diagram showing the number
of unique and overlapping proteins identified from obese before and after 3 months of physical exercise (A). qRT-PCR data carried
out on 9 genes to validate the observed differential protein expression at the mRNA levels. Total RNA were isolated from PBMCs of
male obese before and after exercise obese (n=5 each) and the data are presented as fold changes in obese compared to lean
subjects (B). * P < 0.05 as determined using student’s t-test.
doi: 10.1371/journal.pone.0075342.g002

Network analysis HDAC4 and NF-κB; the master regulator of the immune
The chronic conditions associated with obesity such as low response pathway.
grade inflammation, hyperlipidemia and dysregulation of the
stress response promoted us to investigate whether proteins Ectopic expression of HDAC4 abolished TNF-α-
identified from this work are connected to each other within mediated NF-κB activation in functional assays
interaction networks of cellular systems. Using Ingenuity In order to complement the in vivo data shown above, we
Pathways Analysis software, HDAC4 and TSP1 (known also as undertook a series of in vitro experiments using cell lines. The
THBS1) were found to be indirectly interconnected and decrease of HDAC4 in obese and its restoration by physical
involved in important components of functional network (Figure exercise is suggestive of a protective role of HDAC4 against
S3). Of particular interest, we found a direct crosstalk between obesity. Based on the negative correlation between HDAC4

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Downregulation of HDAC4 by Obesity

Table 4. List of differentially expressed proteins identified Table 4 (continued).


from PBMCs collected from obese subjects at baseline and
after 3 months of physical exercise.
Fold
IPI Number Protein Description Symbol Changes* SD
Fold Isoform 1 of Collagen alpha-6
IPI00856012.1 COL6A6 ↓1.61 0.53
IPI Number Protein Description Symbol Changes* SD chain
IPI00432360.1 Ubiquitin associated protein 2 UBAP2 ↑3.8 0.13 Bone-derived growth factor
IPI00015916.1 QSOX1 ↓1.59 0.62
IPI00045478.1 Ras-related protein Rab-40C RAB40C ↑3.7 0.14 (Fragment)
IPI00008711.3 Wolframin WFS1 ↑2.9 0.19 IPI00910581.1 cDNA FLJ57960 SARM1 ↓1.59 0.47
NEDD8-conjugating enzyme IPI00298994.6 Talin-1 TLN1 ↓1.56 0.19
IPI00022597.1 UBE2M ↑2.5 0.23
Ubc12 IPI00010807.1 Frizzled-8 FZD8 ↓1.55 0.66
Isoform 1 of NF-X1-type zinc Isoform 1 of Arf-GAP with
IPI00442274.3 NFXL1 ↑2.4 0.27
finger protein NFXL1 IPI00217393.1 GTPase, ANK repeat and PH AGAP2 ↓1.5 0.46
Isoform 1 of Cyclin N-terminal domain-containing protein 2
IPI00167285.4 CNTD1 ↑2.4 0.17
domain-containing protein 1 Cholesterol 7-alpha-
IPI00305356.1 CYP7A1 ↓1.5 0.43
IPI00010706.1 Glutathione synthetase GSS ↑1.9 0.24 monooxygenase
IPI00855998.1 Centromere protein F CENPF ↑1.8 0.24 * Protein levels are expressed as a fold increase (↑) or decrease (↓) in obese
PSMG4 Chromosome 6 open after exercise relative to obese at baseline. SD is calculated from 3 independent
IPI00552375.2 ↑1.7 0.17
reading frame 86 experiments.
IPI00017704.3 Coactosin-like protein COTL1 ↑1.7 0.22 doi: 10.1371/journal.pone.0075342.t004
Meiosis-specific nuclear
IPI00549955.3 MNS1 ↑1.6 0.34
structural protein 1 levels and various metabolic and inflammatory markers (Table
IPI00169383.3 Phosphoglycerate kinase 1 PGK1 ↑1.6 0.26 5) and given the importance of stress kinases such as JNK,
Procollagen IKKβ in obesity and insulin resistance, we sought to determine
IPI00514694.1 GLT25D2 ↑1.6 0.16
galactosyltransferase 2 whether overexpression of HDAC4 has an effect on JNK and
IPI00010088.2 Histone deacetylase 4 HDAC4 ↑1.5 0.28 NF-κB activation. For this purpose, HEK-293 cells were
WD40 repeat-containing protein transfected with pCMV-HDAC4 and investigated in our initial
IPI00305833.3 SMU1 ↑1.5 0.18
SMU1 attempt the status of JNK phopshorylation following its
cDNA FLJ52253, highly similar activation by palmitate. Under our experimental conditions, we
IPI00909631.1 to Mus musculus syntaxin 3 STX3 ↑1.5 0.35 failed to obtain consistent results supporting the link between
transcript variant C, mRNA JNK and HDAC4. However, our data demonstrated that
IPI00375813.3 Putative uncharacterized protein C10ORF122 ↑1.5 0.22 overexpression of HDAC4 impaired NF-κB activation by TNF-α
Isoform 2 of Double-strand-
in luciferase assays (Figure 5). Our data are in line with the
IPI00878979.1 break repair protein rad21-like RAD21L1 ↓2.33 0.67
network analysis shown in Figure S3.
protein 1
IPI00793653.1 NCOR1 protein (Fragment) NCOR1 ↓2.27 0.80
Discussion
Isoform 1 of DnaJ homolog
IPI00022501.1 DNAJC27 ↓1.97 0.52
subfamily C member 27
The biological underpinnings of obesity are multiple and
IPI00296099.6 Thrombospondin-1 TSP1 ↓1.90 0.95
complex. Although, the contributing roles of several behavioral,
ADP-ribosylation factor-like
IPI00027971.2 ARL4D ↓1.85 0.51 environmental and genetic factors to obesity are well
protein 4D
established, the molecular mechanisms are still not fully
cDNA FLJ50382, moderately
IPI00910512.1 ITGA5 ↓1.78 0.62 elucidated as obesity and its co-morbidities associated are
similar to Integrin alpha-5
continuing to rise at an escalating rate. Previous proteomics
IPI00005565.2 Diacylglycerol kinase theta DGKQ ↓1.83 0.59
profiling studies using two major intra-abdominal fat depots,
IPI00385917.4 TPCN1 protein TPCN1 ↓1.75 0.61
namely the subcutaneous and omental fat tissues have led to
Isoform 1 of Probable
the identification of a limited number of differentially expressed
IPI00175151.7 methylcytosine dioxygenase TET2 ↓1.74 0.51
proteins in obese subjects in comparison to lean [22]. Although
TET2
these studies were pivotal in shedding initial light into the
Isoform 1 of Cytosolic acyl
IPI00010415.2 ACOT7 ↓1.67 0.48 mechanisms involved in obesity and its complications, the
coenzyme A thioester hydrolase
limited number of identified proteins was, in large part due to
Tyrosine-protein kinase
IPI00739386.4 SGK223 ↓1.65 0.56 the inherent limitation of the technology (2D gel combined to
(PRAGMIN)
Maldi-Tof) and the technical challenges to extract soluble
Caspase recruitment domain-
IPI00869011.2 CARD11 ↓1.64 0.48 proteins from fat adipose tissue. To complement these studies,
containing protein 11
we used peripheral blood mononuclear cells (PBMCs) as a
Isoform 6 of Xin actin-binding
IPI00893002.1 XIRP2 ↓1.61 0.55 surrogate tissue in our label-free LC-MS shotgun screening
repeat-containing protein 2
effort as it provides a large pool of expressed protein with the
IPI00741855.2 Keratin, type I cytoskeletal 39 KRT39 ↓1.61 0.61
potential to be differentially regulated in obese subjects. We

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Downregulation of HDAC4 by Obesity

Figure 3. Representative Western blot confirming differential expression of HDAC4 and TSP1 in obese subjects. (A and B)
Total proteins were extracted from PBMC of lean (n=7) and obese (n=7) non-diabetic participants and subjected to western blot
using the indicated antibodies. The bands were quantified as described in materials and methods and the relative intensity was
determined after correction with GAPDH that was used as internal control to monitor loading efficiency (A). The data are presented
in the form of graphs as fold changes compared to lean group (B). (C and D) Nuclear (N) and cytoplasmic (C) extracts were
prepared from PBMCs isolated from lean (LN) and obese (OB) male subjects and subjected to Western blot using the indicated
antibodies. The bands representing the cytoplasmic fractions were quantified as described in materials and methods and the
relative intensity was determined after correction with Tubulin from the cytoplasmic fraction that was used as internal control to
monitor loading efficiency. Lamin B was used as control to monitor nuclear localization (C). The data are presented as fold changes
in obese compared to lean subjects (D). The blots shown are representatives of at least two independent experiments with
consistent results.
doi: 10.1371/journal.pone.0075342.g003

report here the identification and quantification of 47 proteins on differential protein expression in obese subjects. Among the
that were differentially regulated between lean and obese differentially expressed protein between lean and obese
subjects. Furthermore, we investigated the expression profiling subjects, we focused on TSP1 and HDAC4 as they may
pattern in obese subjects before and after a defined physical represent potential targets against obesity. The selection of
exercise program and we identified and quantified 38 proteins these proteins was based on the fact that they were among the
that showed differential expression before and after physical proteins dysregulated in obese subjects and then, corrected by
exercise in obese subjects. To the best of our knowledge, this physical exercise.
study provides the first large scale quantitative proteomics TSP1 is a multifunctional adipokine that has been shown to
snapshot on human obesity from PBMCs between lean and be involved in the regulation of various pathways such as
obese subjects. It also describes the effect of physical exercise angiogenesis, cell proliferation as well as inflammation and

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Downregulation of HDAC4 by Obesity

Figure 4. HDAC4 and TSP1 are also differentially expressed in the adipose tissue of obese humans. Representative
immunohistochemical (IHC) staining using fat adipose biopsies from lean (n=4) and obese (n=8) male subjects illustrating the
expression pattern of HDAC4 (A) and TSP1 (B). Aperio software was used to quantify positive staining (indicated by arrows) and the
values are illustrated at the bottom as fold changes compared to lean. As negative control (NC) for the experiment, the primary
antibodies were omitted (A, B). qRT-PCR analysis confirming differential expression in of HDAC4 and TSP1 at the mRNA levels in
the adipose tissue. Total RNA was extracted from subcutaneous adipose tissue from lean and obese subjects (n=10 each) and
analyzed by qRT-PCR. The data are presented as fold changes in obese compared to lean subjects (C). Graphic illustration of IHC
quantification of HDAC4 and TSP1 proteins in subcutaneous adipose tissue collected from obese before and after exercise (n=8
each). Aperio software was used to quantify positive staining (D). qRT-PCR analysis showing the effect of physical exercise on the
expression of HDAC4 and TSP1 mRNA in the adipose tissue. Total RNA was extracted from subcutaneous adipose tissue from
obese subjects before and physical exercise (n=10 each) and analyzed by qRT-PCR. The data are presented as fold changes in
obese before exercise compared to obese after exercise (E). * p< 0.05 and ** p< 0.01 as determined using student’s t-test.
doi: 10.1371/journal.pone.0075342.g004

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Downregulation of HDAC4 by Obesity

Table 5. Correlation of HDAC4 and TSP1 mRNA TSP1 with obesity and insulin resistance [29,30,31]. In
expression levels with physical, clinical and biochemical agreement with previous studies [29,32], the observed increase
parameters of the study population. of TSP1 in our study population correlated also positively with
BMI and slightly with PBF. Furthermore, our data showed for
the first time that obesity-induced dysegulation of TSP1 mRNA
HDAC4 TSP1 and protein was corrected by regular physical exercise in both
PBMCs and adipose tissue. The significant attenuation
(r2) P-value (r2) P-value
observed in TSP1 levels following physical exercise and the
BMI -(0.66) <0.0001 0.54 0.0014
improvement of clinical outcomes associated with obesity for
PBF -(0.58) 0.0012 0.34 0.072
the participating group confirmed the hypotheses that physical
VO2 Max 0.54 0.024 -(0.42) 0.097
exercise can provide an effective approach for combating the
Leptin -(0.20) 0.29 0.27 0.148
PAI-1 -(0.33) 0.071 0.31 0.09
deleterious effects associated with obesity. The direct effect of
RANTES -(0.80) 0.001 0.15 0.923
TSP1 on the inflammatory and metabolic profiles was recently
The correlation was based on ΔΔCT method obtained from qRT-PCR and it was
reported using diet-induced obesity in TSP1 KO mice [30].
done on 32 subjects consisting of lean (n=13) and obese (n=19) at baseline.
Indeed, this study clearly demonstrated that, although TSP1
Correlation was assessed by using Spearman’s rank correlation coefficient.
deficiency does not prevent the development of high fat diet
doi: 10.1371/journal.pone.0075342.t005
induced obesity, those animals displayed reduced inflammatory
response and improved glucose and insulin homeostasis as
compared to obese wild type mice [30]. In their model, the
Table 6. Physical, clinical and biochemical characteristics improved glucose tolerance and insulin sensitivity were
of obese population (n=15) before and after exercise. associated with reduced accumulation of pro-inflammatory
macrophages in adipose tissue and decreased levels of IL-6
and TNF-α [30]. In addition, it has been suggested that TSP1
Before exercise After exercise P-value may contribute to obesity-induced metabolic inflammation by
Age (year) 49.47 ± 13.61 - - modulating other immune cells such as T cells through TGF-β
BMI (Kg/m2) 33.74 ± 3.04 33.37 ± 2.47 0.36 signaling pathway [30]. In our study, the increase of TSP1 in
PBF (%) 33.89 ± 2.98 33.11 ± 3.94 0.047 obese subject was concomitant with increased circulating
Resting HR (beats/min) 73.78 ± 7.60 77.33 ± 11.87 0.39 levels of PAI-1; a downstream target of TGF-β that was shown
SBP (mmHg) 131.67 ± 12.25 122.22 ± 8.83 0.04 in previous studies to be associated with obesity and
DBP (mmHg) 86.00 ± 9.66 78.89 ± 3.33 0.043 inflammation [33,34]. This suggests that TSP1 is contributing to
VO2 Max (ml/kg/min) 18.8 ± 5.35 21.37 ± 5.09 0.011 systemic and local inflammation driven by obesity. Taken
Cholesterol (mmol/l) 4.88 ± 0.99 4.84 ± 1.01 0.98 together, the increased expression of TSP1 in obese subjects,
HDL (mmol/l) 1.09 ± 0.24 1.03 ± 0.27 0.73 its restoration by physical exercise and its significant
LDL (mmol/l) 3.05 ± 0.84 3.14 ± 0.97 0.63 correlation with metabolic, inflammatory and stress markers
TG (mmol/l) 1.60 ± 0.83 1.49 ± 0.56 0.58 suggest that TSP1 may represent a potential target to
Glucose (mmol/l) 5.73 ± 1.12 5.78 ± 0.48 0.85 modulate the chronic inflammatory and metabolic abnormalities
HBA1C (%) 5.96 ± 0.56 5.92 ± 0.46 0.57 associated with obesity.
C-peptide (ng/ml) 3.17 ± 1.46 2.48 ± 0.73 0.30 HDAC4, a protein involved in histone acetylation and
GLP-1 (ng/ml) 2.22 ± 1.24 1.95 ± 0.40 0.59
chromatin remodeling was another potential target that we
Insulin (ng/ml) 2.88 ± 2.29 1.98 ± 1.79 0.036
identified and validated in the current study. Our data
Leptin (ng/ml) 7.03 ± 3.04 6.34 ± 3.47 0.9
demonstrated for the first time a reduced expression of HDAC4
PAI-1 (ng/ml) 4.01 ± 0.83 3.21 ± 1.38 0.31
mRNA and protein in human obese subjects both in PBMCs
TNF-α (pg/ml) 23.28 ± 8.93 19.20 ± 9.19 0.55
and adipose tissue. This is consistent with clinical data in
IL-6 (pg/ml) 4.38 ± 1.10 3.26 ± 1.02 0.04
humans that associated the haploinsufficiency of HDAC4 with
IL-10 (pg/ml) 1.87 ± 1.70 2.64 ± 1.67 0.04
obesity [35,36]. In our study, the decrease of HDAC4 mRNA
IP-10 (ng/ml) 0.48 ± 0.15 0.50 ± 0.21 0.19
correlated positively with VO2 Max but negatively with BMI, PBF
RANTES (ng/ml) 1.60 ± 0.65 1.81 ± 0.59 0.21
and circulating levels of the pro-inflammatory chemokine
TBARS (μM) 1.61 ± 0.56 1.45 ± 0.31 0.21
RANTES. Future studies using HDAC4 KO animals and/or
HDAC4 (ΔΔCT) 0.66 ± 0.28 1.19 ± 0.47 0.0092
pharmacological inhibitors are needed to confirm these initial
TSP1 (ΔΔCT) 1.48 ± 0.52 0.74 ± 0.29 0.027
observations. Another important finding of our study is the
Data are presented as mean ± SD. Paired t-test was used to compare differences
restoration of the normal expression of HDAC4 in obese
in obese before and after 3 months of physical exercise.
subjects after a physical exercise program even though no
doi: 10.1371/journal.pone.0075342.t006
major change in the BMI was observed. Taken together, the
significant decrease of HDAC4 in obese subjects, its negative
wound healing [28,29]. In our current investigation, we found correlation with anthropometric and metabolic markers and the
that TSP1 protein and mRNA are upregulated in obese restoration of its normal expression after a defined exercise
subjects in both PBMCs and adipose tissue. Our findings on program suggest that HDAC4 may play a protective role in
TSP1 are consistent with previous studies that associated obesity and most likely in insulin resistance and type 2

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Downregulation of HDAC4 by Obesity

Figure 5. Ectopic expression of HDAC4 impaired NF-κB activation by TNF-α in luciferase assays. Reporter and expression
vectors were used in transient transfection assays in HEK293 cells as indicated in materials and methods. Reporter vector in which
the firefly luciferase gene is under the control of 3 copies of wild type (3xwt-κB) or mutant (3xmut-κB) NF-κB binding was
cotransfected with HDAC4 expression vector. pCMV was used a control empty vector. 24 hours post-transfection, cells were treated
with 25 ng/ml of TNF-α for overnight and then, the luciferase activity was carried out as described in materials and methods. Protein
concentration was used for normalization.
doi: 10.1371/journal.pone.0075342.g005

diabetes. These findings provided further evidence that reported to be involved in regulation of GLUT4 during
approaches leading to enhanced expression of HDAC4 or its adipocyte differentiation [54] as well as promoting lypolysis
activity may be used to mitigate metabolic stress triggered by [55]. Likewise, preadipocytes from HDAC9 KO mice exhibited
obesity. There is a widespread interest in studying protein accelerated adipogenic differentiation and thus, demonstrating
acetylation because of their critical role in regulating gene its direct role as a negative regulator of adipogenesis [56]. The
expression and their active involvement in a number of ubiquitin-binding HDAC6 was reported to play a crucial role in
metabolically related disorders such as obesity, insulin protein homeostasis “proteostasis” following accumulation of
resistance, diabetes and cardiovascular diseases misfolded and aggregated proteins [57]. This role is executed,
[37,38,39,40]. The recent investigation that documented the at least in part, through interaction with components of the heat
existence of 3600 lysine acetylation sites on 1750 proteins shock response [58]. HDAC3 was also shown to display a
highlights the importance of this post translational modification protective role against shear stress-induced endothelial cells
[41]. HDAC proteins regulate histone acetylation in a and vasculature injury [59]. Impaired expression of HDAC2 was
sequence-specific manner to repress and in some associated with induction of cell senescence in a manner that
circumstances to activate various transcription programs depends on p53 [60].
[42,43,44]. HDACs are also known for their ability to modulate HDAC proteins are known to shuttle between nucleus and
various biological processes by targeting other non-histone cytoplasm by a mechanism that involves AMPK, PDK and
proteins such as NF-κB [45,46], HSF-1 [47], STAT3 [48] and CamKII [52,61,62]. In our case, HDAC4 was found mainly in
the insulin receptor substrate-1 (IRS-1) [49]. Pioneer studies on the cytoplasm of PBMCs isolated from both lean and obese
the role of HDACs proteins in modulating various cellular subjects, suggesting that obesity does not trigger any change
responses were made available by using HDAC KO animals or in cellular localization of HDAC4. Our data are in agreement
HDAC inhibitors. For instance, HDAC4 was shown to play an with previous studies that reported a cytoplasmic localization of
anti-stress role in response to calorie restriction [50,51] as well HDAC4 in endocrine cells [61] and retinal cells [52]. In the
in promoting cell survival [52] and interfering with apoptosis skeletal muscle however, HDAC4 was predominately found in
[53]. In the context of obesity, HDAC4 was also recently the nucleus but exported immediately after exercise in a

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Downregulation of HDAC4 by Obesity

manner that was concomitant with the activation of AMPK and (TIF)
CaMKII kinases [27]. A recent study reported that DNAJB5
cochaperone acts as a nucleocytoplasmic shuttling of HDAC4 Figure S2. Validation of the proteomics data by qRT-PCR.
during oxidative stress [58]. This role is consistent with the qRT-PCR analysis showing the difference in expression level
findings that HDAC4 interacts with various members of DNAJ of HDAC4 and TSP1 between male and female obese subjects
family including DNAJB5 and such interaction is important for at the mRNA levels in adipose tissue. Total RNA was extracted
maintaining proteostasis and protection against stressful from subcutaneous adipose tissue from 5 males (M) and 5
conditions [47,58,63,64]. The cytoplasmic role of HDAC4 in females (F) indicated that gender has no effect on differential
PBMCs, if any, remains to be elucidated, nevertheless, the pro- expression of HDAC4 and TSP1 in obese subjects. The data
survival role of HADC4 on retinal cells was mediated in part by are presented as fold changes in obese compared to lean
activation of hypoxia inducible factor 1α [52]. Our results subjects.
indicated that HDAC4 suppresses TNF-α-mediated NF-κB (TIF)
activation (Figure 5) and such effect is supported by the
predicted network analysis (Figure S3). Previous studies Figure S3. Network analysis. Protein network analysis
reported that obesity and insulin resistance are associated with showing the link between HDAC4 and other proteins including
impaired expression of various components of the heat shock TSP1 (known also as THBS1*) and NCOR1.
response, including members of DNAJ family [6,65,66]. It is (TIF)
also known that physical exercise induces the heat shock
response and improves the clinical parameters associated with
Table S1. List of peptides identified in lean subjects.
obesity and insulin resistance [13,67]. In our current
(XLSX)
investigation, we also showed that exercise induced the
expression of HDAC4. On the other hand, HADC4 regulates
Table S2. List of proteins identified in lean subjects.
proteostasis via interaction with and deacetylation of members
(XLSX)
of DNAJ family [58] through a mutual crosstalk between
HDAC4 and component of the heat shock response [58].
Future investigations focusing on the role HSF-1, a master Table S3. List of peptides identified in obese subjects at
regulator of the heat shock response on the expression of baseline.
HDAC4 should elucidate such relationships. Our finding on (XLSX)
HDAC4 raised a series of questions that needs to be
investigated in future follow-up studies. For instance, what is Table S4. List of proteins identified in obese subjects at
the status of histone acetylation in obese subjects? What are baseline.
the molecular mechanisms governing the observed (XLSX)
downregulation of HADC4 in obese subjects and how does
exercise restores the normal expression of HDAC4? In Table S5. List of proteins quantified by Sieve 1.3 software
addition, are HDAC4 KO mice more prone to obesity and and shown as ratios of protein expression between lean
insulin resistance? and obese subjects at baseline.
There are limitations in this study that deserve consideration. (XLSX)
The number of subjects used to carry out the proteomic
profiling was small. In addition, despite the fact that both male Table S6. List of primers used for qRT-PCR.
and female are prone to obesity, the study analyzed differential (DOCX)
protein expression in adult male subjects only and this may not
reflect the global changes in females due to hormonal Table S7. List of peptides identified in obese subjects
regulation and menopause as well as in younger population. after 3 months of physical exercise.
However, despite these limitations, the present study showed (XLSX)
clearly that obesity triggers differential regulation of TSP1 and
HDAC4 proteins at protein and mRNA levels in both PBMCs Table S8. List of proteins identified in obese subjects after
and adipose tissue. 3 months of physical exercise.
In summary, our proteomic approach performed on obese (XLSX)
humans before and after physical exercise revealed HDAC4 as
target that may have a potential therapeutic benefit for the Table S9. List of proteins quantified by Sieve 1.3 software
control and management of obesity and insulin resistance. Our and shown as ratios of protein expression between obese
study documented also the beneficial effect of exercise in before and after exercise.
modulating the expression of HDAC4 and TSP1. (XLSX)

Supporting Information Acknowledgements

Figure S1. Workflow of the proteomic analysis using We would like to thank the staff at the Tissue Bank and Clinical
PBMC samples from human subjects. Laboratory for their technical assistance throughout this study.

PLOS ONE | www.plosone.org 15 September 2013 | Volume 8 | Issue 9 | e75342


Downregulation of HDAC4 by Obesity

Author Contributions JJ SK AK. Analyzed the data: MA AT JA FA NE IA SW MD.


Contributed reagents/materials/analysis tools: MA AT JA IA EB
Conceived and designed the experiments: MA AT JA FA IA KB PC MH JJ SK AK SW. Wrote the manuscript: MA AT MD.
SD MD. Performed the experiments: MA AT JA IA EB PC MH

References
1. Dixon AE, Holguin F, Sood A, Salome CM, Pratley RE et al. (2010) An 20. Pérez-Pérez R, García-Santos E, Ortega-Delgado FJ, López JA,
official American Thoracic Society Workshop report: obesity and Camafeita E et al. (2012) Attenuated metabolism is a hallmark of
asthma. Proc Am Thorac Soc 7: 325-335. doi:10.1513/pats. obesity as revealed by comparative proteomic analysis of human
200903-013ST. PubMed: 20844291. omental adipose tissue. J Proteomics 75: 783-795. doi:10.1016/j.jprot.
2. Mitchell NS, Catenacci VA, Wyatt HR, Hill JO (2011) Obesity: overview 2011.09.016. PubMed: 21989264.
of an epidemic. Psychiatr Clin North Am 34: 717-732. doi:10.1016/j.psc. 21. Pérez-Pérez R, Ortega-Delgado FJ, García-Santos E, López JA,
2011.08.005. PubMed: 22098799. Camafeita E et al. (2009) Differential proteomics of omental and
3. Al Rashdan I, Al Nesef Y (2010) Prevalence of overweight, obesity, and subcutaneous adipose tissue reflects their unalike biochemical and
metabolic syndrome among adult Kuwaitis: results from community- metabolic properties. J Proteome Res 8: 1682-1693. doi:10.1021/
based national survey. Angiology 61: 42-48. doi: pr800942k. PubMed: 19714809.
10.1177/0003319709333226. PubMed: 19398424. 22. Pérez-Pérez R, López JA, García-Santos E, Camafeita E, Gómez-
4. McGinnes RA, Lowthian JA (2012) Why exercise is an important Serrano M et al. (2012) Uncovering suitable reference proteins for
component of risk reduction in obesity management. Med J Aust 196: expression studies in human adipose tissue with relevance to obesity.
567-568. doi:10.5694/mja12.10352. PubMed: 22621145. PLOS ONE 7: e30326. doi:10.1371/journal.pone.0030326. PubMed:
5. Ross R, Després JP (2009) Abdominal obesity, insulin resistance, and 22272336.
the metabolic syndrome: contribution of physical activity/exercise. 23. Zhou H, Wang F, Wang Y, Ning Z, Hou W et al. (2011) Improved
Obesity (Silver Spring) 17 Suppl 3: S1-S2. doi:10.1038/oby.2009.29. recovery and identification of membrane proteins from rat hepatic cells
PubMed: 19927139. using a centrifugal proteomic reactor. Mol Cell Proteomics 10: O111:
6. Bruce CR, Carey AL, Hawley JA, Febbraio MA (2003) Intramuscular 008425. PubMed: 21749988.
heat shock protein 72 and heme oxygenase-1 mRNA are reduced in 24. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression
patients with type 2 diabetes: evidence that insulin resistance is data using real-time quantitative PCR and the 2(-Delta Delta C(T))
associated with a disturbed antioxidant defense mechanism. Diabetes Method. Methods 25: 402-408. doi:10.1006/meth.2001.1262. PubMed:
52: 2338-2345. doi:10.2337/diabetes.52.9.2338. PubMed: 12941774. 11846609.
7. Kurucz I, Morva A, Vaag A, Eriksson KF, Huang X et al. (2002) 25. Ghebeh H, Tulbah A, Mohammed S, Elkum N, Amer Bin et al. (2007)
Decreased expression of heat shock protein 72 in skeletal muscle of Expression of B7-H1 in breast cancer patients is strongly associated
patients with type 2 diabetes correlates with insulin resistance. with high proliferative Ki-67-expressing tumor cells. Int J Cancer 121:
Diabetes 51: 1102-1109. doi:10.2337/diabetes.51.4.1102. PubMed: 751-758. doi:10.1002/ijc.22703. PubMed: 17415709.
11916932. 26. al-Haj L, Al-Ahmadi. W, Al-Saif M, Demirkaya O, Khabar KS (2009)
8. Castro-Chavez F, Yechoor VK, Saha PK, Martinez-Botas J, Wooten EC Cloning-free regulated monitoring of reporter and gene expression.
et al. (2003) Coordinated upregulation of oxidative pathways and BMC Mol Biol 10: 20
downregulation of lipid biosynthesis underlie obesity resistance in
27. McGee SL, Fairlie E, Garnham AP, Hargreaves M (2009) Exercise-
perilipin knockout mice: a microarray gene expression profile. Diabetes
induced histone modifications in human skeletal muscle. J Physiol 587:
5211: 2666–2674. PubMed: 14578284.
5951-5958. doi:10.1113/jphysiol.2009.181065. PubMed: 19884317.
9. Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y et al. (2004)
28. Esemuede N, Lee T, Pierre-Paul D, Sumpio BE, Gahtan V (2004) The
Increased oxidative stress in obesity and its impact on metabolic
role of thrombospondin-1 in human disease. J Surg Res 122: 135-142.
syndrome. J Clin Invest 11412: 1752–1761. PubMed: 15599400.
doi:10.1016/j.jss.2004.05.015. PubMed: 15522326.
10. Liu H, Wang T, Huang K (2009) Cholestane-3beta,5alpha,6beta-triol-
29. Varma V, Yao-Borengasser A, Bodles AM, Rasouli N, Phanavanh B et
induced reactive oxygen species production promotes mitochondrial
al. (2008) Thrombospondin-1 is an adipokine associated with obesity,
dysfunction in isolated mice liver mitochondria. Chem Biol Interact 179:
adipose inflammation, and insulin resistance. Diabetes 57: 432-439.
81-87. doi:10.1016/j.cbi.2008.12.003. PubMed: 19121293.
11. Ye J (2009) Emerging role of adipose tissue hypoxia in obesity and PubMed: 18057090.
insulin resistance. Int J Obes (Lond) 33: 54-66. doi:10.1038/ijo. 30. Li Y, Tong X, Rumala C, Clemons K, Wang S (2011) Thrombospondin1
2008.229. PubMed: 19050672. deficiency reduces obesity-associated inflammation and improves
12. Hotamisligil GS (2010) Endoplasmic reticulum stress and the insulin sensitivity in a diet-induced obese mouse model. PLOS ONE 6:
inflammatory basis of metabolic disease. Cell 140: 900-917. doi: e26656. doi:10.1371/journal.pone.0026656. PubMed: 22039525.
10.1016/j.cell.2010.02.034. PubMed: 20303879. 31. Ramis JM, Franssen-van Hal NL, Kramer E, Llado I, Bouillaud F et al.
13. Hooper PL (2009) Inflammation, heat shock proteins, and type 2 (2002) Carboxypeptidase E and thrombospondin-1 are differently
diabetes. Cell Stress Chaperones 14: 113-115. doi:10.1007/ expressed in subcutaneous and visceral fat of obese subjects. Cell Mol
s12192-008-0073-x. PubMed: 18720028. Life Sci 59: 1960-1971. doi:10.1007/PL00012518. PubMed: 12530526.
14. Boura-Halfon S, Zick Y (2009) Phosphorylation of IRS proteins, insulin 32. Chavez RJ, Haney RM, Cuadra RH, Ganguly R, Adapala RK et al.
action, and insulin resistance. Am J Physiol Endocrinol Metab 296: (2012) Upregulation of thrombospondin-1 expression by leptin in
E581-E591. doi:10.1152/ajpendo.90437.2008. PubMed: 18728222. vascular smooth muscle cells via JAK2- and MAPK-dependent
15. Vallerie SN, Hotamisligil GS (2010) The role of JNK proteins in pathways. Am J Physiol Cell Physiol 303: C179-C191. doi:10.1152/
metabolism. Sci Transl Med 2: 60rv65. PubMed: 21123811. ajpcell.00008.2012. PubMed: 22592401.
16. Gao Z, Hwang D, Bataille F, Lefevre M, York D et al. (2002) Serine 33. Alessi MC, Bastelica D, Morange P, Berthet B, Leduc I et al. (2000)
phosphorylation of insulin receptor substrate 1 by inhibitor kappa B Plasminogen activator inhibitor 1, transforming growth factor-beta1, and
kinase complex. J Biol Chem 277: 48115-48121. doi:10.1074/ BMI are closely associated in human adipose tissue during morbid
jbc.M209459200. PubMed: 12351658. obesity. Diabetes 49: 1374-1380. doi:10.2337/diabetes.49.8.1374.
17. Wang K, Li WD, Zhang CK, Wang Z, Glessner JT et al. (2011) A PubMed: 10923640.
genome-wide association study on obesity and obesity-related traits. 34. Samad F, Yamamoto K, Pandey M, Loskutoff DJ (1997) Elevated
PLOS ONE 6: e18939. doi:10.1371/journal.pone.0018939. PubMed: expression of transforming growth factor-beta in adipose tissue from
21552555. obese mice. Mol Med 3: 37-48. PubMed: 9132278.
18. Dong C, Beecham A, Slifer S, Wang L, McClendon MS et al. (2011) 35. Burns B, Schmidt K, Williams SR, Kim S, Girirajan S et al. (2010) Rai1
Genome-wide linkage and peak-wide association study of obesity- haploinsufficiency causes reduced Bdnf expression resulting in
related quantitative traits in Caribbean Hispanics. Hum Genet 129: hyperphagia, obesity and altered fat distribution in mice and humans
209-219. doi:10.1007/s00439-010-0916-2. PubMed: 21104097. with no evidence of metabolic syndrome. Hum Mol Genet 19:
19. Prior MJ, Larance M, Lawrence RT, Soul J, Humphrey S et al. (2011) 4026-4042. doi:10.1093/hmg/ddq317. PubMed: 20663924.
Quantitative proteomic analysis of the adipocyte plasma membrane. J 36. Williams SR, Aldred MA, Der Kaloustian VM, Halal F, Gowans G, et al
Proteome Res 10: 4970-4982. doi:10.1021/pr200446r. PubMed: (2010) Haploinsufficiency of HDAC4 causes brachydactyly mental
21928809. retardation syndrome, with brachydactyly type E, developmental

PLOS ONE | www.plosone.org 16 September 2013 | Volume 8 | Issue 9 | e75342


Downregulation of HDAC4 by Obesity

delays, and behavioral problems. Am J Hum Genet 87: 219-228. doi: 52. Chen B, Cepko CL (2009) HDAC4 regulates neuronal survival in
10.1016/j.ajhg.2010.07.011. PubMed: 20691407. normal and diseased retinas. Science 323: 256-259. doi:10.1126/
37. Iyer A, Fairlie DP, Brown L (2012) Lysine acetylation in obesity, science.1166226. PubMed: 19131628.
diabetes and metabolic Disease. Immunol Cell Biol 90: 39–46. doi: 53. Majdzadeh N, Wang L, Morrison BE, Bassel-Duby R, Olson EN et al.
10.1038/icb.2011.99. PubMed: 22083525. (2008) HDAC4 inhibits cell-cycle progression and protects neurons
38. Liu Z, Cao J, Gao X, Zhou Y, Wen L et al. (2011) CPLA 1.0: an from cell death. Dev Neurobiol 68: 1076-1092. doi:10.1002/dneu.
integrated database of protein lysine acetylation. Nucleic Acids Res 39: 20637. PubMed: 18498087.
D1029–D1034. doi:10.1093/nar/gkq939. PubMed: 21059677. 54. Weems JC, Griesel BA, Olson AL (2012) Class II histone deacetylases
39. Wang Q, Zhang Y, Yang C, Xiong H, Lin Y et al. (2010) Acetylation of downregulate GLUT4 transcription in response to increased cAMP
Metabolic Enzymes Coordinates Carbon Source Utilization and signaling in cultured adipocytes and fasting mice. Diabetes 61:
Metabolic Flux. Science 327: 1004-1007. doi:10.1126/science. 1404-1414. doi:10.2337/db11-0737. PubMed: 22403301.
1179687. PubMed: 20167787. 55. Wang B, Moya N, Niessen S, Hoover H, Mihaylova MM et al. (2011) A
40. Shakespear MR, Halili MA, Irvine KM, Fairlie DP, Sweet MJ (2011) hormone-dependent module regulating energy balance. Cell 145:
Histone deacetylases as regulators of inflammation and immunity. 596-606. doi:10.1016/j.cell.2011.04.013. PubMed: 21565616.
Trends Immunol 32: 335-343. doi:10.1016/j.it.2011.04.001. PubMed: 56. Chatterjee TK, Idelman G, Blanco V, Blomkalns AL, Piegore MG Jr. et
21570914. al.. (2011) Histone deacetylase 9 is a negative regulator of adipogenic
41. Choudhary C, Kumar C, Gnad F, Nielsen ML, Rehman M et al. (2009) differentiation. J Biol Chem 286: 27836-27847. doi:10.1074/
Lysine acetylation targets protein complexes and co-regulates major jbc.M111.262964. PubMed: 21680747.
cellular functions. Science 325: 834-840. doi:10.1126/science.1175371. 57. Lee JY, Koga H, Kawaguchi Y, Tang W, Wong E et al.. (2010) HDAC6
PubMed: 19608861. controls autophagosome maturation essential for ubiquitin-selective
42. Miska EA, Karlsson C, Langley E, Nielsen SJ, Pines J et al. (1999) quality-control autophagy. EMBO J 29: 969-980. doi:10.1038/emboj.
HDAC4 deacetylase associates with and represses the MEF2 2009.405. PubMed: 20075865.
transcription factor. EMBO J 18: 5099-5107. doi:10.1093/emboj/ 58. Hageman J, Rujano MA, van Waarde MA, Kakkar V, Dirks RP et al.
18.18.5099. PubMed: 10487761. (2010) A DNAJB chaperone subfamily with HDAC-dependent activities
43. Sun X, Wei L, Chen Q, Terek RM (2009) HDAC4 represses vascular suppresses toxic protein aggregation. Mol Cell 37: 355-369. doi:
endothelial growth factor expression in chondrosarcoma by modulating 10.1016/j.molcel.2010.01.001. PubMed: 20159555.
RUNX2 activity. J Biol Chem 284: 21881-21890. doi:10.1074/ 59. Zeng L, Xiao Q, Margariti A, Zhang Z, Zampetaki A et al. (2006)
jbc.M109.019091. PubMed: 19509297. HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation
44. Matsuoka H, Fujimura T, Hayashi M, Matsuda K, Ishii Y et al. (2007) toward endothelial cells. J Cell Biol 174: 1059-1069. doi:10.1083/jcb.
Disruption of HDAC4/N-CoR complex by histone deacetylase inhibitors 200605113. PubMed: 16982804.
leads to inhibition of IL-2 gene expression. Biochem Pharmacol 74: 60. Harms K, Nozell S, Chen X (2004) The common and distinct target
465-476. doi:10.1016/j.bcp.2007.05.002. PubMed: 17559812. genes of the p53 family transcription factors. Cell Mol Life Sci 61:
45. Bendinelli P, Matteucci E, Maroni P, Desiderio MA (2009) NF-kappaB 822-842. doi:10.1007/s00018-003-3304-4. PubMed: 15095006.
activation, dependent on acetylation/deacetylation, contributes to HIF-1 61. Lenoir O, Flosseau K, Ma FX, Blondeau B, Mai A et al. (2011) Specific
activity and migration of bone metastatic breast carcinoma cells. Mol control of pancreatic endocrine beta- and delta-cell mass by class IIa
Cancer Res 7: 1328-1341. doi:10.1158/1541-7786.MCR-08-0548. histone deacetylases HDAC4, HDAC5, and HDAC9. Diabetes 60:
PubMed: 19671685. 2861-2871. doi:10.2337/db11-0440. PubMed: 21953612.
46. Chen Y, Wang H, Yoon SO, Xu X, Hottiger MO et al. (2011) HDAC- 62. Martin M, Kettmann R, Dequiedt F (2007) Class IIa histone
mediated deacetylation of NF-kappaB is critical for Schwann cell deacetylases: regulating the regulators. Oncogene 26: 5450-5467. doi:
myelination. Nat Neurosci 14: 437-441. doi:10.1038/nn.2780. PubMed: 10.1038/sj.onc.1210613. PubMed: 17694086.
21423191. 63. Ago T, Liu T, Zhai P, Chen W, Li H et al. (2008) A redox-dependent
47. Westerheide SD, Anckar J, Stevens SM Jr, Sistonen L, Morimoto RI pathway for regulating class II HDACs and cardiac hypertrophy. Cell
(2009) Stress-inducible regulation of heat shock factor 1 by the 133: 978-993. doi:10.1016/j.cell.2008.04.041. PubMed: 18555775.
deacetylase SIRT1. Science 323: 1063-1066. doi:10.1126/science. 64. Pandeya N, Williams GM, Green AC, Webb PM, Whiteman DC (2009)
1165946. PubMed: 19229036. Do low control response rates always affect the findings? Assessments
48. Kimura K, Yamada T, Matsumoto M, Kido Y, Hosooka T et al. (2012) of smoking and obesity in two Australian case-control studies of cancer.
Endoplasmic reticulum stress inhibits STAT3-dependent suppression of Aust N Z J Public Health 33: 312-319. doi:10.1111/j.
hepatic gluconeogenesis via dephosphorylation and deacetylation. 1753-6405.2009.00401.x. PubMed: 19689590.
Diabetes 61: 61-73. doi:10.2337/db10-1684. PubMed: 22124464. 65. Huey KA, Meador BM (2008) Contribution of IL-6 to the Hsp72, Hsp25,
49. Kaiser C, James SR (2004) Acetylation of insulin receptor substrate-1 and alphaB-crystallin [corrected] responses to inflammation and
is permissive for tyrosine phosphorylation. BMC Biol 2: 23. doi: exercise training in mouse skeletal and cardiac muscle. J Appl Physiol
10.1186/1741-7007-2-23. PubMed: 15522123. 105: 1830-1836. doi:10.1152/japplphysiol.90955.2008. PubMed:
50. Chu F, Chou P, Mirkin BL, Mousa SA, Rebbaa A (2008) Cellular 18927263.
conditioning with trichostatin A enhances the anti-stress response 66. Abubaker J, Tiss A, Abu-Farha M, Al-Ghimlas F, Al-Khairi Iet al (2013)
through up-regulation of HDAC4 and down-regulation of the IGF/Akt DNAJB3/HSP-40 Cochaperone Is Downregulated in Obese Humans
pathway. Aging Cell 7: 516-525. doi:10.1111/j. and Is Restored by Physical Exercise. PLOS ONE, 8: e69217 doi:
1474-9726.2008.00403.x. PubMed: 18489729. 10.1371/journal.pone.0069217. PubMed: 23894433.
51. Pallavi R, Giorgio M, Pelicci PG (2012) Insights into the beneficial effect 67. Noble EG, Shen GX (2012) Impact of exercise and metabolic disorders
of caloric/ dietary restriction for a healthy and prolonged life. Front. on heat shock proteins and vascular inflammation. J Autoimmune Dis,
Physiol (Bethesda Md.) 3: 318. 2012: 2012: 836519. PubMed: 23304460

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