Am J Physiol Cell Physiol 305: C355–C359, 2013.
First published May 29, 2013; doi:10.1152/ajpcell.00019.2013.
Testosterone induces cell proliferation and cell cycle gene overexpression in
human visceral preadipocytes
Anna Barbosa-Desongles, Cristina Hernández, Rafael Simó, and David M. Selva
Diabetes and Metabolism Research Unit, Vall Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona and
CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM; ISCIII), Barcelona, Spain
Submitted 18 January 2013; accepted in final form 21 May 2013
testosterone; human preadipocytes; cell cycle; proliferation; gene
expression
organ composed of adipocytes, a
connective tissue matrix, nerve tissue, stromovascular cells,
and immune cells (15). Apart from its major function as an
energy store lipid form, adipose tissue is well known to be an
active endocrine organ with a pivotal role in the control of
energy homeostasis (13).
An important sex difference in body fat distribution is
generally observed (22). Men are usually characterized by the
android type of obesity, with accumulation of fat in the abdominal region, whereas women often display the gynoid type
of obesity, with a greater proportion of their body fat in the
gluteal-femoral region. Accordingly, the amount of fat located
inside the abdominal cavity (intra-abdominal or visceral adipose tissue) is twice as high in men compared with women (4).
This sex difference has been shown to explain a major proportion of differing metabolic profiles and cardiovascular disease
risk in men and women (24). Given that men accumulate more
fat in the intra-abdominal depot than women, it seems reason-
ADIPOSE TISSUE IS A COMPLEX
Address for reprint requests and other correspondence: D. M. Selva, Diabetes
and Metabolism Research Unit, Vall d’Hebron Institut de Recerca (VHIR), Pg
Vall d’Hebron 119-129, 08035 Barcelona, Spain (e-mail: david.martinez.selva
@vhir.org).
http://www.ajpcell.org
able to postulate that testosterone plays an essential role in this
type of body fat distribution. In support of this concept, postmenopausal women accumulate more visceral fat than premenopausal women, and studies performed with women at
different stages of the menopausal transition have found that
bioavailable testosterone is associated with an increase of
visceral fat (12).
By contrast, there is clinical evidence that testosterone
administration induces a reduction of fat content mainly due to
a decrease in visceral adipose tissue (19, 20). However, it
should be noted that this effect has been observed in those
patients with a testosterone deficit or, in other words, when
testosterone was administered as a replacement treatment (1, 3,
8, 10, 11, 14, 18).
The mechanism by which testosterone regulates adipose
tissue mass in a depot-specific manner remains to be elucidated. White adipose tissue distribution in different anatomical
regions not only depends on the lipid synthesis and/or lipolysis
in mature adipocytes but also the site-specific modulation of
preadipocyte proliferation and/or differentiation (5). There is
significant in vitro information regarding the effects of testosterone in preadipocyte differentiation (4, 9), but little is known
about testosterone effects on human preadipocyte proliferation.
To shed light on this issue, we have studied the effect of
testosterone on human visceral preadipocyte proliferation.
In addition, differential expressed genes in preadipocytes
treated with testosterone or vehicle were explored by microarray analysis.
MATERIALS AND METHODS
Cell culture experiments. Human male preadipocytes obtained from
Tebu-Bio (cat. no. 088OP-F-3; Tebu-bio, Barcelona, Spain) were maintained in a humidified incubator with 5% CO2 at 37°C. Cells were
cultured in triplicates in preadipocyte commercial media OM-PM (Tebubio) for 3 days in the presence of testosterone (100 nM) or vehicle
(ethanol) to 80% confluence.
Proliferation and cell-cycle analyses. To evaluate the cell cycle in
human preadipocytes, we used the APC BrdU Flow Kit (BD Biosciences
PharMingen, San Diego, CA) according to the supplier’s instructions.
Briefly, cells were seeded on 150-mm plates with DMEM 15 mM
glucose supplemented with 10% FBS and antibiotics (100 U penicillin/ml and 100 ␮g streptomycin/ml). After 24 h, the medium was
changed to DMEM 15 mM glucose with testosterone 100 nM or with
ethanol, and it was changed every day for 3 days. During the last 3 or
6 h of the experiment, cells were labeled with bromodeoxyuridine
(BrdU) 10 ␮M in DPBS buffer. After that, cells were washed in DPBS
and were fixed with the Cytofix/Cytoperm provided buffer, treated
with DNAse for 1 h at 37°C, and incubated with the APC labeledBrdU antibody. The DNA was subsequently stained with 7-aminoactinomycin D (7-AAD), and cells were resuspended in staining
buffer for flow cytometric analysis. For cell cycle analysis, 7-AAD
and BrdU were measured simultaneously using a BD FACSCalibur
0363-6143/13 Copyright © 2013 the American Physiological Society
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Barbosa-Desongles A, Hernández C, Simó R, Selva DM. Testosterone induces cell proliferation and cell cycle gene overexpression in
human visceral preadipocytes. Am J Physiol Cell Physiol 305: C355–C359,
2013. First published May 29, 2013; doi:10.1152/ajpcell.00019.2013.—
Evidence from the literature suggests that testosterone plays an important
role in visceral fat accumulation since both men and women with
hyperandrogenism accumulate more adipose tissue in the abdominal
cavity than healthy women. However, the underlying mechanisms
remain to be elucidated. To shed light on this issue, we have used an
in vitro approach to examine the effect of testosterone on human
visceral preadipocyte proliferation. Our results showed that testosterone treatment significantly increased proliferation of human visceral
preadipocytes in proliferation assays using flow cytometric analysis.
We next performed a microarray gene expression analysis of human
visceral preadipocytes treated with testosterone or vehicle to identify
which genes were involved in the testosterone-induced increase in
preadipocyte proliferation. The results showed a total of 140 genes
differentially expressed between testosterone vs. vehicle. Among the
top 10 upregulated genes, 5 were involved in cellular cycle and
proliferation, and 3 (APOBEC3b, CCNA2, and PRC1) were significantly overexpressed by testosterone treatment when analyzed by
real-time PCR. We conclude that testosterone exerts a proliferative
effect on preadipocytes that may participate in the sex differences in
fat distribution and that it may explain visceral fat accumulation in
women with hyperandrogenism.
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TESTOSTERONE INCREASES HUMAN PREADIPOCYTE PROLIFERATION
Whitney). Values of statistical significance were set at P ⬍ 0.05
and 0.01. The statistical analyses corresponding to microarrays
were performed using the free statistical language R and the
libraries developed for microarray data analysis by the Bioconductor Project (www.bioconductor.org).
RESULTS
flow cytometer. Acquired multiparameter data were analyzed using
FCS Express version 3 software (De Novo Sotfware, Los Angeles,
CA). Flow cytometric analysis of cells stained with the reagents
allowed the discrimination of cell subsets that were in S phase, G0/G1
phase, G2 phase, and apoptotic. Fluorescence cut off was chosen at
1% positive events in a negative control for APC.
RNA isolation. Preadipocyte cells were scraped at the end of the
treatments and total RNA was isolated using an RNeasy Mini Kit
from Qiagen following the manufacturer’s instructions. Integrity and
concentration of RNA were analyzed by Bioanalizador 2100 (Agilent)
in the Scientific and Technical Support Unit of our Research Institute.
RNAs with RIN score above 7 were used in microarrays.
Microarray hybridization and analysis. The goal of the study was
to compare gene expression patterns between preadipocytes treated
with testosterone or vehicle. Microarrays were carried out using the
Affymetrix microarray platform and the Genechip Human Gene 1.0
ST Array (an array with 14,500 well-characterized human genes used
to explore human biology and disease processes). The arrays were
performed at the Scientific and Technical Support Unit of our Research Institute as described elsewhere (www.affymetrix.com). Data
obtained from the microarrays were analyzed by the Statistics and
Bioinformatics Unit of our research institute. To select differentially
expressed genes, they used a method described by Smyth GK (X20).
All the statistical analysis was done using the free statistical language
R and the libraries developed for microarray data analysis by the
Bioconductor Project (www.bioconductor.org).
Real-time PCR analysis. Reverse transcription (RT) was performed
at 42°C, for 50 min using 3 ␮g of total RNA and 200 U of Superscript
II together with an oligo-dT primer and reagents provided by Invitrogen. An aliquot of the RT product was amplified in a 25-␮l reaction
using SYBRGreen (Invitrogen SA) with appropriate oligonucleotide
primer pairs for hRRM2 (forward 5=-GCAGCAAGCGATGGCATAGT, reverse 5=-GGGCTTCTGTAATCTGAACTTC),
hCCNA2 (forward 5=-AGCTGCCTTTCATTTAGCACTCTAC, reverse 5=-TTAAGACTTTCCAGGGTA TATCCAGTC), hCDC20
(forward 5=-GCCCACCAAGAAGG AACATC, reverse 5=-TTTTCCACTGAGCCGAAGGA), hAPOBEC3B (forward 5=-CTATGGTCGGAGCTACACTT GG, reverse 5=-GGAAACACTTGTAAGCAGGCAG), hPRC1 (forward 5=-GAAAGCCCTA AATCACCTTCG, reverse 5=-CAATCATCATATCCAGGACTTCC), and
h18S (forward 5=-TAACGAACGAGACTCTGGCAT, reverse 5=CGGACATCTAAGGGCATCACAG). Amplification was done as
follows: one step at 50°C 2 min; one step at 95° 10 min; 40 cycles at
95°C 15 s, 60°C 1 min followed by dissociation curve (95°C 15 s,
60°C 20 s, and 95°C 15 s). For each condition, all reactions were run
in triplicate. Results were analyzed using the 7000 SDS program and
we used the 2⫺⌬⌬Ct method.
Statistical analyses. Data regarding cell proliferation and realtime PCR were analyzed using a nonparametric test (U-Mann-
Fig. 2. Analysis of BrdU incorporation. Graphics of the analysis of BrdU
incorporation in ethanol-treated cells (A) and testosterone-treated cells (B).
C: both ethanol and testosterone 3-day-treated preadipocytes were incubated with BrdU during 3 and 6 h and later the percentage of cells in S
phase incorporating BrdU was calculated. Experiment was performed 3
times, and data points are means ⫾ SD. *P ⬍ 0.05, compared with vehicle.
**P ⬍ 0.01, compared with vehicle.
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Fig. 1. Cell cycle and bromodeoxyuridine (BrdU) incorporation analysis.
Human preadipocytes were analyzed using flow cytometry to study the cell
cycle and cell proliferation through BrdU incorporation. A: 3-day ethanoltreated cells were incubated with 7-amino-actinomycin D (7-AAD) and their
DNA content was measured by flow cytometry. B: 3-day testosterone-treated
cells were incubated with 7-AAD and their DNA content was measured by
flow cytometry.
Cell cycle and proliferation analysis. Asynchronous cultured human visceral preadipocytes were treated with ethanol
or testosterone (100 nM). After treatment, cells were stained
with 7-AAD to study cell DNA content and the cell cycle.
After flow cytometric analysis, our results showed that both
ethanol- and testosterone-treated preadipocytes had divided
correctly, without any cell cycle arrest being produced by the
treatment, and with the correct peaks corresponding to the
proportion of DNA amount in each cell cycle phase (Fig. 1, A
and B).
We next quantified and analyzed the amount of BrdU incorporation to check if testosterone treated cells proliferated more
than ethanol-treated cells (Fig. 2, A and B). To confirm this, the
number of BrdU-positive cells in the S phase was used to
TESTOSTERONE INCREASES HUMAN PREADIPOCYTE PROLIFERATION
C357
DISCUSSION
Due to sex differences in body fat distribution, it can be
postulated that testosterone regulates adipose tissue mass in a
depot-specific manner. White adipose tissue distribution in
different anatomical regions not only depends on lipid synthesis and/or lipolysis in mature adipocytes but also on the
site-specific modulation of preadipocyte proliferation and/or
Fig. 3. Microarrays results and differentially expressed genes in the top biology
functions between ethanol and testosterone-treated preadipocytes. A: heat map
illustrating differentially expressed genes generated from DNA microarray
data, reflecting gene expression values in testosterone or ethanol treatment
conditions of the human visceral preadipocytes. Every condition was done in
triplicate. B: number of molecules differentially expressed from the comparison of testosterone and ethanol preadipocyte treatments in the top biology
functions.
quantify the proliferating index in human visceral preadipocytes treated with ethanol or testosterone. We observed that
treatment with testosterone induced an increase in cell proliferation (at 3 and 6 h of BrdU incorporation) since the percentage of cells in the S phase was significantly increased when
compared with ethanol-treated preadipocytes (Fig. 2C).
Differential expressed genes and pathway analysis. Heat
maps (color-coded graphs with samples in columns and genes
in rows) were used to visualize the differentially expressed
Fig. 4. Top 10 upregulated genes from the testosterone vs. ethanol microarray
data comparison and validation of cell cycle genes overexpression. A: 5 cell
cycle genes among the top 10 upregulated genes from the testosterone vs.
ethanol microarray data. B: overexpressed cell cycle genes validation by
RT-PCR. CDC20, APOBEC3b, CCNA2, PRC1, and RRM2 mRNA abundance was determined in relation to S18 RNA (means ⫾ SD) of vehicle-treated
preadipocytes. The experiment was performed 3 times and data points are
means ⫾ SD. *P ⬍ 0.05, compared with vehicle.
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genes between ethanol and testosterone (Fig. 3A). Microarray
analysis showed a total of 143 genes differentially expressed
(adjusted P ⬍ 0.05) between testosterone vs. ethanol. These
genes differentially expressed were classified in several top
biology functions, such as diseases and disorders (83), molecular and cellular functions (47), and physiological system
development and function (13) (Fig. 3B).
Validation of the top five cell cycle genes of microarray
data. Among the top 10 upregulated genes from the testosterone vs. ethanol microarray data comparison, we found five
genes involved in cell cycle regulation: CDC20, APOBEC3b,
CCNA2, PRC1, and RRM2 (Fig. 4A). We next analyzed and
validated the upregulated cell cycle genes by real-time PCR.
Although all five genes were upregulated, three out of five
genes (APOBEC3b, CCNA2, and PRC1) were significantly
overexpressed by testosterone treatment when compared with
vehicle-treated preadipocytes (Fig. 4B).
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TESTOSTERONE INCREASES HUMAN PREADIPOCYTE PROLIFERATION
should be contemplated only as a component of a multifactorial
event regulating body fat distribution.
In conclusion, testosterone increases preadipocyte number in
visceral fat. This finding could be one of the mechanisms by
which men exhibit more visceral fat than women and also
could be an underlying factor involved in the accumulation of
intra-abdominal adipose tissue observed in women with hyperandrogenism. However, further studies exploring the significance of this finding within the complex mechanisms involved in the regulation of body fat distribution are needed.
ACKNOWLEDGMENTS
We thank Lorena Ramos, Research Institute Hospital Vall d’Hebron for
technical assistance. D. M. Selva is the guarantor and takes full responsibility
for the article and its originality.
GRANTS
This work was supported by a Grant from the Instituto de Salud Carlos III
(to D. M. Selva) and CIBER de Diabetes y Enfermedades Metabólicas
Asociadas (CIBERDEM), an initiative of Instituto de Salud Carlos III (to A.
Barbosa-Desongles, C. Hernández, R. Simó, and D. M. Selva). D. M. Selva is
the recipient of a Miguel Servet contract.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: A.B.-D. performed experiments; A.B.-D., C.H.,
R.S., and D.M.S. analyzed data; A.B.-D., C.H., R.S., and D.M.S. interpreted results of experiments; A.B.-D. prepared figures; A.B.-D. and
D.M.S. drafted manuscript; A.B.-D., C.H., R.S., and D.M.S. edited and
revised manuscript; A.B.-D., C.H., R.S., and D.M.S. approved final version
of manuscript; R.S. and D.M.S. conception and design of research.
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testosterone exerts a proliferative effect on human visceral
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induces a reduction of fat content (19, 20). However, it should
be noted that whereas the reduction in fat content and the
change in body fat distribution has been clearly shown when
testosterone is administered as replacement treatment in men
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The effect of androgens on human preadipocyte proliferation
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The effect of testosterone on adipose tissue depends not only of
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local basis. In this regard, recent evidence suggests that enzymes
from the aldoketoreductase 1C family are very active and may be
important modulators of androgen action in adipose tissue. In
addition, the expression of steroidogenic and steroid-inactivating
enzymes by adipose tissue itself is crucial in accounting for body
fat distribution (4). Furthermore, the effect of estradiol on preadipocyte proliferation should also be considered. In this regard,
experiments performed in vitro in mouse and human preadipocytes showed that estradiol exerts different effects depending
on the concentration and the source of preadipocyte used (male or
female, and visceral or subcutaneous; Refs. 2, 17). Therefore, the
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