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Androgen inactivation and steroid-converting enzyme expression
in abdominal adipose tissue in men
Karine Blouin, Christian Richard, Gae
´
tan Brochu
1
,Fre
´
de
´
ric-Simon Hould
1
, Ste
´
fane Lebel
1
,
Simon Marceau
1
, Simon Biron
1
, Van Luu-The and Andre
´
Tchernof
Molecular Endocrinology and Oncology Research Center, Laval University Medical Research Center, Laval University, 2705 Laurier Boulevard (T3-67),
Que
´
bec, Que
´
bec, Canada G1V 4G2
1
Department of surgery, Laval University, 2705 Laurier Boulevard (T3-67), Que
´
bec, Que
´
bec, Canada G1V 4G2
(Requests for offprints should be addressed to A Tchernof; Email: andre.tchernof@crchul.ulaval.ca)
Abstract
We examined 5a-dihydrotestosterone (5a- DHT) inacti-
vation and the expression of several steroid-converting
enzymes with a focus on aldoketoreductases 1C (AKR1C),
especially AKR1C2, in abdominal adipose tissue in men.
AKR1C2 is mainly involved in the conversion of the potent
androgen 5a-DHT to its inactive forms 5a-androstane-
3a/b,17b-diol (3a/b-diol). Subcutaneous (s.c.) and omental
(Om) adipose tissue biopsies were obtained from 21 morbidly
obese men undergoing biliopancreatic derivation surgery and
11 lean to obese men undergoing general abdominal surgery.
AKR1C2 mRNA and 5a-DHT inactivation were detected
in both s.c. and Om adipose tissue. After incubation of
preadipocytes with 5a-DHT, both 3a-diol and 3b-diol were
produced through 3a/b-ketosteroid reductase (3a/b-HSD)
activity. In preadipocyte cultures, 3a-reductase activity was
significantly predominant over 3b-reductase activity in cells
from both the s.c. and Om compartments. Expression levels
of AKR1C1, AKR1C3 and of the androgen receptor were
significantly higher in s.c. versus Om adipose tissue while
mRNA levels of 17b-HSD-2 (hydroxysteroid dehydrogenase
type 2) and 3(a/b)-hydroxysteroid epimerase were signifi-
cantly higher in Om fat. 3a/b-HSD activity was mainly
detected in the cytosolic fraction, suggesting that AKR1C
may be responsible for this reaction. Experiments with
isoform-specific AKR1C inhibitors in preadipocytes showed
that AKR1C2 inhibition significantly decreased 3a-HSD and
3b-HSD activities (3a-HSD: 30G24% of control for s.c. and
32G9% of control for Om, 3b-HSD: 44G12% of control for
s.c.). When cells were incubated with both AKR1C2 and
AKR1C3 inhibitors, no significant additional inhibition was
observed. 5a-DHT inactivation was significantly higher in
mature adipocytes compared with preadipocyte cultures in
s.c. adipose tissue, as expressed per microgram total protein
(755G830 versus 245G151 fmol 3a/b-diol per mg protein
over 24 h, P!0
.
05 nZ10 cultures). 5a-DHT inactivation
measured in tissue homogenates was significantly higher in
the s.c. depot compared with Om fat (117G39 versus 79 G
38 fmol 3a/b-diol per mgprotover24h,P!0
.
0001). On the
other hand, Om 3a/b-HSD activity was significantly higher
in obese men (body mass index (BMI)R30 kg/m
2
) compared
with lean and overweight men (84G37 versus 52G30 fmol
3a/b-diol per mg protein over 24 h, P!0
.
03). No difference
was found in s.c. 3a/b-HSD activity between these groups.
Positive correlations were found between s.c. 5a-DHT
inactivation rate and circulating levels of the androgen
metabolites androsterone-glucuronide (rZ0
.
41, P!0
.
02)
and 3a-diol-glucuronide (rZ0
.
38, P!0
.
03) and with the
adrenal precursor androstenedione (rZ0
.
42, P!0
.
02). In
conclusion, androgen inactivation was detected in abdominal
adipose tissue in men, with higher 3a/b-HSD activity in the
s.c. versus Om depot. Higher Om 5a-DHT inactivation rates
were found in obese compared with lean men. Further studies
are required to elucidate whether local androgen inactivation
in abdominal adipose tissue is involved in the modulation of
adipocyte metabolism and regional fat distribution in men.
Journal of Endocrinology (2006) 191, 637–649
Introduction
The physiological impor tance of excess adipose tissue
accumulation in the etiology of type 2 diabetes and
cardiovascular disease is of g rowing interest in the context
of the obesity epidemic prevailing in affluent societies (Must
et al. 1999, Lewis et al. 2002, Ravussin & Smith 2002).
A predominantly abdominal fat distribution with increased fat
accumulation within the abdominal cavity (visceral obesity)
has been identified as a critical cor relate of obesity-related
metabolic alterations leading to adverse health outcomes
including insulin resistance, hyperinsulinemia, a dyslipidemic
state and proinflammatory/prothrombotic alterations (Despre
´
s
et al. 1990, Lemieux et al. 2001, Juhan-Vague et al. 2002).
Epidemiological evidence indicated that androgens may be
involved in the regulation of obesity and body fat distribution
637
Journal of Endocrinology (2006) 191, 637–649 DOI: 10.1677/joe.1.06365
0022–0795/06/0191–637 q 2006 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org
patterns (Khaw & Barrett-Connor 1992, Tchernof et al.
1995, Gapstur et al. 2002). Testosterone administration to
female-to-male transsexuals leading to supraphysiological
plasma hormone concentration induced a significant increase
in visceral adipose tissue area assessed by magnetic resonance
imag ing (Elbers et al. 1999, 2003). However, endogenous
plasma testosterone levels are negatively associated with
visceral f at accumulation in healthy men (Khaw &
Barrett-Connor 1992, Tchernof et al. 1995), and treatment
with physiological androgen doses improves the metabolic
profile possibly through reductions in visceral fat accumu-
lation (Ma
˚
rin et al. 1992, Boyanov et al. 2003). In addition,
plasma 5a-androstan-3a,17b-diol-glucuronide (3a-diol-glu-
curonide), a marker of androgen metabolism (inactivation) in
peripheral tissues (Labrie et al. 1997), was positively correlated
with total adiposity and visceral adipose tissue accumulation
in men, suggesting that local androgen conversion may be
related to adipose tissue metabolism (Tchernof et al. 1997).
The notion that adipose tissue is a complex and
metabolically active organ which possesses endocrine, para-
crine, and autocrine hormonal properties is increasingly
recognized (Mohamed-Ali et al. 1998, Kershaw & Flier
2004). In this regard, several steroid-converting enzymes
involved in local sex steroid metabolism were detected in
adipose tissue, including aromatase, 3b-hydroxysteroid
dehydrogenase (3b-HSD), type 1 11b-HSD, type 2 and type
317b-HSD, 7a-hydroxylase, 17a-hydroxylase, 5a-reductase,
and uridine diphosphate (UDP)-glucuronosyltransferase 2B15
(reviewed by Be
´
langer et al. 2002). Our group also described
the presence of three members of the aldoketoreductase 1C
(AKR1C) family, namely AKR1C1 (20a-HSD), AKR1C2
(type 3 3a-HSD), and AKR1C3 (type 5 17b-HSD) in whole
adipose tis sue samples and preadipocyte s obtained from
women (Blouin et al. 2003, Blanchette et al. 2005).
AKR1C1, AKR1C2 and AKR1C3 display ketosteroid
reductase activities (3a-HSD, 20a-HSD, and 17b-HSD), but
in different proportions. Experiments in intact cells have
shown that AKR1C2 is mainly involved in the inactivation of
the most potent androgen 5a-androstane-17b-ol-3-one (5a-
dihydrotestosterone, 5a-DHT) into the weak androgen 5a-
androstan-3a,17b-diol (3a-diol) through its 3a-HSD
activity. This enzyme also possesses a low 17b-HSD activity
(w2–3% of its 3a-HSD activity) and a moderate 20a-HSD
activity (w10% of its 3a-HSD activity) (Zhang et al. 2000,
Dufort et al. 2001). AKR1C1 mainly inactivates progesterone
into 20a-hydroxyprogesterone through its 20a-HSD activity,
but also forms testosterone from 4-androsten-3,17-dione
(D4-dione) (w3–4% of its 20a-HSD activity) and inactivates
5a-DHT into 3 a-diol (w8% of its 20a-HSD activity).
AKR1C3 also mainly inactivates progesterone into 20a-
hydroxyprogesterone, but also shows significant 17b-HSD
activity leading to testosterone formation from D4-dione
(w35% of its 20a-HSD activity), and 3a-HSD activity (w7–
8% of its 20a-HSD activity; Dufort et al. 1999, Zhang et al.
2000). Purified enzyme assays have shown that AKR1C1,
AKR1C2 and AKR1C3 also possess 3b-HSD activity leading
to the formation of 5a-androstan-3b,17b-diol (3b-diol) from
5a-DHT (Steckelbroeck et al. 2004).
We previously observed positive correlations between
visceral adipose tissue area assessed by computed tomography
and 3a/b-HSD and 20a-HSD activities as well as AKR1C
mRNA levels measured in Om adipose tissue homogenates in
women (Blouin et al. 2003, 2005, Blanchette et al. 2005). The
aim of the present study was to examine 3a/b-HSD activity
(5a-DHT inactivation) and the expression of several enzymes
involved in androgen metabolism in s.c. and omental (Om)
adipose tissue obtained from lean to morbidly obese men, and
to further investigate the relationship between 3a/b-HSD
activity and obesity. We tested the hypothesis that AKR1C
enzymes would be detected in abdominal adipose tissue
depots and 5a-DHT inactivation would be related to obesity
in men.
Materials and Methods
Subjects
Men of this study were recruited through the elective
surger y schedule of the Laval University Medical Center and
through the bariatric surgery schedule of the Laval Hospital.
The study included 12 men aged 38
.
6–57
.
1 years (body mass
index (BMI) 30
.
9G5
.
0 kg/m
2
, range 24
.
6–39
.
1kg/m
2
)
undergoing general abdominal surgery. Reasons for surgeries
were: umbilical hernia (nZ7), endocholecystectomia (nZ2),
giant parastomal hernia (nZ1), and sigmoid restriction
(nZ2). In the analysis, 22 men aged 22
.
6–61
.
2 years (BMI
51
.
8G9
.
2 kg/m
2
,40
.
6–79
.
1 kg/m
2
) undergoing biliopan-
creatic derivation surgery for morbid obesity were also
included. None of the subjects were taking hormonal
treatments except for thyroid hormones (nZ2). Excluding
these two subjects from the analyses did not alter the present
results. One subject was treated with domperidone and one
subject was taking an anti-obesity drug. Some subjects also
received medication for diabetes (nZ10), hypertension
(nZ16) and dyslipidemia (nZ6). BMI and waist circumfer-
ence were measured according to standardized procedures
(Lohman et al. 1988). Approbations by the medical ethics
committees of Laval University, Laval Hospital and Laval
University Medical Center were obtained. All subjects
provided written informed consent before their inclusion
in the study.
Sex steroid hormones, C
19
steroid precursors and androgen
metabolite measurements in plasma and culture media
Concentrations of D4-dione, testosterone, 5a-DHT, 5a-
androstan-3a-ol-17-one (androsterone), 3b-diol, estrone,
and 17b-estradiol were measured using high performance
gas chromatography and negative chemical ionization mass
spectrometry (GC–MS). The intra- and inter-assay coeffi-
cients of variation did not exceed 5
.
9% for these
K BLOUIN and others
.
Androgen inactivation in abdominal adipose tissue638
Journal of Endocrinology (2006) 191, 637–649 www.endocrinology-journals.org
measurements. Androsterone-glucuronide and 3a-diol-glu-
curonide levels were determined using liquid chromatog-
raphy and mass spectrometry (LC–MS) using a PE Sciex API
300 tandem mass spectrometer (Perkin-Elmer, Foster City,
CA, USA) equipped with a Turbo ionspray source. The intra-
and inter-assay coefficients of variation did not exceed 6
.
4%
for these measurements. HPLC was used for the identification
and the relative quantification of
14
C-5a-DHT metabolites
after 24-h incubations with Om preadipocytes. Briefly,
14
C-
labeled steroids were analysed using a Zorbax cyano normal-
phase HPLC column (4
.
6!250 mm, 5 mm). The mobile
phase was hexane/tetrahydrofuran (96/4 v/v), with a flow
rate of 1
.
5 ml/min. Radioactivity was monitored in the
eluent using a Beckman 171 HPLC Radioactivity Moni-
toring System.
14
C-steroids (5a-DHT, 3a-diol, 3b-diol, 5a-
androstan-3,17-dione (A-dione)), used as standards, were
HPLC-purified in the laboratory in the same conditions.
Adipose tissue sampling
Paired Om and s.c. adipose tissue samples were collected
during the surgical procedure and immediately carried to the
laboratory in 0
.
9% saline preheated at 37 8C. A portion of the
biopsy was used for adipocyte and preadipocyte isolation and
the remaining tissue was immediately frozen at K80 8C for
subsequent analyses.
Separation of cytosolic and membrane fractions
Separation of whole adipose tissue cytosolic and membrane
fractions was performed according to a previously published
method with some modifications (Cormont et al. 1993 ).
Briefly, frozen adipose tissue samples were homogenized 10
times in 10 mM Tris, 1 mM EDTA, 250 mM sucrose, and
1 mM phenylmethylsulphonyl fluoride, using a Potter device.
Homogenates were centrifuged at 288 g to remove lipids.
Following a 75 min ultracentrifugation at 210 000 g, cytosol
(supernatant) was harvested. The pellet was homogenized
again using the same procedure and ultracentrifuged for
75 min at 210 000 g. The pellet (total membrane fraction) was
resuspended in buffer. Isolated cytosolic and membrane
fractions were used for 3a/b-HSD activity determination.
Preadipocyte isolation and primary cultures
Tissue samples were digested with collagenase type I in Krebs–
Ringer–Henseleit (KRH) buffer for 45 min at 37 8C according
to a modified version of the Rodbell (1964) method. Adipocyte
suspensions were filtered through nylon mesh and washed thrice
with KRH buffer. Preadipocytes were isolated using a
modification of the method previously described by Hauner
(Hauner 1990, Hauner et al. 2001). Briefly, the residual KRH
buffer of the adipocyte isolation was centrifuged and the pellet
was washed in Dulbecco’s modified Eagle’s medium (DMEM)-
F12 supplemented with 10% fetal bovine serum, 0
.
25 mg/ml
amphotericin B, and 50 mg/ml gentamicin. The cells were
treated with erythrocyte lysis buffer (154 mM NH
4
Cl, 10 mM
K
2
HPO
4
, and 0
.
1mMEDTApH7
.
5) and DMEM-F12 w as
added. Preadipocytes were then subsequently filtered through
140 and 30 mm nylon mesh to remove endothelial cells, placed
in culture plates and cultured at 37 8Cundera5%CO
2
atmosphere. The medium was changed every 2–3 days.
Cell size measurements
Mature adipocyte suspensions were visualized using a contrast
microscope attached to a camera and computer interface.
Pictures were taken and the Scion Image software (Scion
Corporation, Frederick, MA, USA) was used to measure the
size of 250 adipocytes.
Lipolysis and lipoprotein lipase (LPL) activity
Basal lipolysis experiments were performed by incubating
isolated cell suspensions for 2 h at 37 8C. Glycerol release in
the medium was quantified by bioluminescence using the
nicotinamide adenine dinucleotide hydroxide-linked bacterial
luciferase assay (Kather et al. 1982), a Berthold Microlumat plus
bioluminometer (LB 96 V) and the WinGlow software (EG&G,
Bad Wildbad, Germany). The average coefficient of variation
for duplicate glycerol release measurements was 11
.
5%. Lipid
weight of the cell suspension was measured by performing Dole’s
extraction, and lipolysis results were expressed as a function of
adipocyte surface area (nanomoles glycerol/2 h!10
8
mm
2
).
Hepar in-releasable (HR)-LPL activity activity was
determined in 30–50-mg frozen adipose tissue samples by
the method of Taskinen et al. (1980). Tissue eluates were
obtained by incubating the sample in Krebs–Ringer
phosphate buffer and heparin at 28 8C for 90 min. The
eluates were then incubated with excess concentrations of
unlabeled and
14
C-labeled triolein in a Tris–albumin buffer
emulsified with ultrasound. The reaction was carried out at
37 8C for 60 min with agitation. The resulting free fatty acids
liberated from triolein by the LPL reaction were isolated by
the Belfrage extraction procedure. Porcine plasma was used as
a source of Apo -CII to stimulate LPL activity, a nd
unpasteurized cow’s milk was used as an internal LPL activity
standard for inter-assay variations. The activity results were
expressed in nanomoles oleate/10
6
cells/h.
Real-time RT-PCR
Total RNA was isolated using Rneasy kit (Qiagen). First
strand cDNA synthesis was accomplished using 2 mg isolated
RNA in a reaction containing 200 units of Superscript II
Rnase H-reverse transcriptase (Invitrogen), 300 ng oligo
dT18, 500 mM dNTP, 10 mM dithiothreitol, and 34 units
porcine RNase inhibitor (Amersham Pharmacia) in a final
volume of 50 ml incubated at 42 8C for 2 h. The resulting
products were then treated with 1 mg Rnase A for 30 min at
37 8C and purified thereafter with Qiaquick PCR purifi-
cation kits (Qiagen). A Light-Cycler PCR (Roche
Androgen inactivation in abdominal adipose tissue
.
K BLOUIN and others 639
www.endocrinology-journals.org Journal of Endocrinology (2006) 191, 637–649
Diagnostics) was used to measure quantitative expression
using sets of primers shown in Table 1. The FastStart DNA
Master SYBR green kit (Roche Diagnostics) was used in a
final reaction volume of 20 ml containing 4 mM MgCl
2
,
20 ng of each primer and 20 ng of the cDNA template. The
PCR was carried out according to the following conditions:
95 8C/10 min, 40 cycles (95 8C/10 s, 62 8C/5 s, 72 8C/
11 s, 81 8C/3 s), and temperature transition was 3 8C/s for all
reactions. PCR results were normalized according to subunit
O of ATP synthase expression levels. A universal standard
curve was generated with ATPase from an amplification with
perfect efficiency (i.e. efficiency coefficient EZ2
.
00), using
cDNA amounts of 0, 10
2
,10
3
,10
4
,10
5
, and 10
6
copies. The
crossing points (Cp) to calculate the amount of copies in
initial cDNA specimens were determined with the double-
derivative method (Luu-The et al. 2005). For each sample, the
Cp value of the gene quantified was divided by that of the
housekeeping gene. To further minimize inter-assay varia-
bility, this Cp ratio was then multiplied by the average Cp
generated for housekeeping gene amplifications of all the
samples examined in the present experiment (Luu-The et al.
2005). PCR data are expressed as normalized numbers of
copies per microgram total RNA.
Enzymatic activities
3a/b-HSD activity was measured in preadipocyte primary
cultures, in mature adipocytes, in whole tissue homogenates,
and in isolated cytosolic and membrane fractions. Preadipocyte
cultures were grown in 12-well culture plates. Culture medium
was changed for fresh medium containing 87 nM (or 100 nM,
depending on lot specific activity) 5a-DHT (Perkin-Elmer Life
Sciences Inc.) as substrate for 3a/b-HSD activity and cells were
incubated for 3, 6, 12, and 24 h. For experiments using
AKR1C-specific inhibitors, cultures were preincubated 2 h
with inhibitor(s) prior to the addition of radioactive 5a-DHT.
AKR1C2 was inhibited using 100 mM5b-cholanic acid-
3a,7a-diol (5b-chol) and AKR1C3 with 20 mM indomethacin
(Indo). For measures in adipose tissue homogenates, tissue
samples were homogenized with a Polytron in 50 mM sodium
phosphate buffer (pH 7
.
4),20%glycerol,1mMEDTA,and
1 mM NADPH. For measures in adipose tissue homogenates as
well as in cytosolic and membrane fractions,
14
C-labeled 5a-
DHTwas added and reactions were performed at 37 8Cinafinal
volume of 1 ml for 24 h. For mature adipocytes, incubation
with radiolabeled 5a-DHT was performed in BSA-free KRH
buffer at 37 8C in a final volume of 1 ml for 24 h. Steroids from
culture media and tissue homogenates were extracted twice with
one volume ether as described previously (Dufort et al. 2001).
For mature adipocytes, the steroid extraction with ether was
preceded by two extractions with three volumes ethanol:ace-
tone (1:1) to remove lipids. The organic phases were pooled and
evaporated to dryness. The steroids were solubilized in 50 ml
dichloromethane (reference standards were diluted in ethanol)
and applied to Silica Gel 60 thin layer chromatography (TLC)
plates (Merck) using 10 m l calibrated micropipets. The
separation was done either by migration in toluene-acetone
(4:1, does not allow for the separation of 3a-diol and 3b-diol) or
in ether:ethyl acetate (1:1) to separate 3a-diol and 3b-diol.
The radioactivity was detected using a Storm 860
Table 1 Oligonucleotides used in real-time RT-PCR quantification
Oligonucleotide sequence
Gene
AKR1C1 5
0
-CCTATAGTGCTCTGGGATCCCAC-3
0
5
0
-AGGACCACAACCCCACGCTGT-3
0
AKR1C2 5
0
-CCGTCAAATTGGCAATAGAAGCC-3
0
5
0
-CAACTCTGGTCGATGGGAATTGCT-3
0
AKR1C3 5
0
-CAACCAGGTAGAATGTCATCCGTAT-3
0
5
0
-ACCCATCGTTTGTCTCGTTGA-3
0
3b-HSD-1 5
0
-CTTCAACCGCCACATAGTCACATT-3
0
5
0
-AGGAGGGTGGAGCTTGATGACAT-3
0
17b-HSD-2 5
0
-GCGCCTCTCGGTGCTCCAAATG-3
0
5
0
-CGGCCATGCATTGTTTGTAGTCAGTCA-3
0
17b-HSD-3 5
0
-GCGATGGAATTGGGAAAGCGTACT-3
0
5
0
-CTCCCTGTAGTCCGCTCGATCTCTG-3
0
3(a/b)-Hydroxysteroid epimerase 5
0
-TTCGTGGGCCTGTACTACCTTCTGC-3
0
5
0
-CAGGTTCCCAAAGCCCGAGTCACA-3
0
11-cis-Retinol dehydrogenase (RDH5) 5
0
-CTGATCTGTGACCCGGACCTAA-3
0
5
0
-GGGGCAGAAATAAATCAAAGTCCTT-3
0
5a-Reductase-1 5
0
-TGGCGATTATGTTCTGTACCTGTA-3
0
5
0
-AACCACAAGCCAAAACCTATTAGA-3
0
P450 aromatase 5
0
-CGACAGGCTGGTACCGCATGCTC-3
0
5
0
-AAGAGGCAATAATAAAGGAAATCCAGAC-3
0
Androgen receptor 5
0
-AGCCATTGAGCCAGGTGTAGTGT-3
0
5
0
-CATCCTGGAGTTGACATTGGTGAA-3
0
ATP synthase O subunit 5
0
-ATTGAAGGTCGCTATGCCACAG-3
0
5
0
-AACGACTCCTTGGGTATTGCTTAA-3
0
K BLOUIN and others
.
Androgen inactivation in abdominal adipose tissue640
Journal of Endocrinology (2006) 191, 637–649 www.endocrinology-journals.org
PhosphorImager (Amersham Pharmacia Biotech Inc.) and
quantification was done using the ImageQuant software version
5.1 (Amersham Pharmacia Biotech Inc). Proteins were
quantified by the BCA method for the comparison between
preadipocytes and mature adipocytes or by the method of Lowry
for other experiments. Total proteins were u sed in the
calculation of activity values.
Statistical analyses
Apairedt-test procedure was used to compare enzyme activitiy
or expression in s.c. versus Om adipose tissue. Analyses were
performed on log
10
-transformed or Box Cox-transformed
values when variables were not normally distributed. When
variances were unequal based on the Levene test (P!0
.
05),
the Welch ANOVA was used to compare the means between the
groups. When normality could not be reached, a posteriori mean
contrasts were used for comparison. The Bonferroni correction
was used to adjust for multiple comparisons. The nonparametric
Wilcoxon rank-sum test was used to compare means between
lean and obese subjects. Cut-off for obesity (Fig. 9) was defined
as a BMIR30 kg/m
2
. Correlation analyses were performed
in the entire sample and also excluding men with class III obesity
(BMIR40 kg/m
2
; WHO guidelines). Spearman rank corre-
lation coefficients were computed to quantify associations.
The analyses were performed using the JMP statistical software
(SAS Institute, Cary, NC, USA).
Results
Steroid-converting enzyme mRNA expression in adipose tissue
Tabl e 2 shows the real-time RT-PCR quantification of several
enzymes involved in androgen metabolism, including
members of the aldoketoreductase 1C family, and the
androgen receptor. The expression of AKR1C1, AKR1C3,
and androgen receptor was significantly higher in s.c. versus
Om adipose tissue (P!0
.
05). The expression of 17b-HSD-2
and 3(a/b)-hydroxysteroid epimerase was higher in Om
versus s.c. adipose tissue (P!0
.
05). AKR1C enzymes were
strongly expre ssed in both adipose tissue depots when
compared with other mRNAs measured.
Androgen metabolites in preadipocyte primary cultures
Figure 1 shows a representative thin layer chromatogram of
steroid products obtained when incubating s.c. or Om
preadipocyte primary cultures with radiolabeled 5a-DHT
for 24 h. When incubating cells with 5a-DHT, 3a/b-diol
and A-dione were detected, with 3a-diol and 3b-diol being
the major reaction products (Fig. 1, lanes 1–4). Separation of
3a-diol and 3b-diol was not possible using this chromatog-
raphy method (Fig. 1, lanes 6–7). HPLC separation in Fig. 2
shows that, in addition to A-dione, both 3a-diol and 3b-diol
were produced when Om preadipocytes were incubated with
5a-DHT for 24 h. Figure 3 shows the GC/LC–MS
quantification of steroid metabolites produced after incu-
bation of preadipocytes with D4-dione (Fig. 3A) or 5a-DHT
(Fig. 3B). When cells were incubated with the adrenal
precursor D4-dione, testosterone, 5a-DHT and androsterone
were produced (Fig. 3A). When cells were incubated with
5a-DHT, high levels of 3b-diol were produced (Fig. 3B)
which represented approximately 25% of to tal steroids.
Quantification of 3a-diol was not possible using this method.
Figure 4 shows the quantification of 3a-diol and 3b-diol in
s.c. and Om preadipocytes. Average 3a-reductase activity was
significantly higher when compared with 3b-reductase
activity in both depots. 3b-reductase activity was not
significantly different between s.c. and Om preadipocytes.
However, there was a trend for higher 3a-reductase activity in
Table 2 Expression levels of steroid-converting enzymes and nuclear receptor involved in androgen
metabolism and action in subcutaneous and omental adipose tissue in 13 men. Data are meansG
S.D
mRNA levels (10
3
copies/mg total RNA)
s.c. Om P value
Aldoketoreductases
AKR1C1 1029
.
0G553
.
5 485
.
1G149
.
1 !0
.
05
AKR1C2 802
.
5G714
.
6 430
.
9G179
.
4 !0
.
09
AKR1C3 488
.
2G334
.
1 253
.
4G96
.
4 !0
.
05
Short-chain dehydrogenases
3b-HSD-1 0
.
2G0
.
20
.
2G0
.
4NS
17b-HSD-1 0
.
01G0
.
04 0
.
3G0
.
4 !0
.
05
17b-HSD-3 1
.
2G2
.
42
.
4G4
.
2NS
3(a/b)-Hydroxysteroid epimerase 0
.
2G0
.
213
.
4G12
.
9 !0
.
05
11-cis-Retinol dehydrogenase (RDH5) 152
.
3G108
.
8 130
.
2G105
.
2NS
Others
5a-Reductase-1 1
.
2G1
.
51
.
0G0
.
8NS
P450 aromatase 0
.
6G1
.
30
.
1G0
.
2NS
Androgen receptor 42
.
6G29
.
520
.
8G12
.
6 !0
.
05
NS, not significant.
Androgen inactivation in abdominal adipose tissue
.
K BLOUIN and others 641
www.endocrinology-journals.org Journal of Endocrinology (2006) 191, 637–649
s.c. preadipocytes. In order to evaluate whether the 3a/
b-HSD activity originated from AKR1C enzymes, which are
cytosolic, assays were performed in isolated cytosolic and
membrane fractions. Figure 5 shows that 3a/b-HSD activity
Figure 1 Thin layer chromatogram showing steroid products
obtained when incubating s.c. or Om preadipocyte primary
cultures with radiolabeled 5a-DHT. Results are representative of
experiments performed in at least seven cultures from each fat
depot, in duplicate.
Figure 2 Identification and relative quantification by HPLC of 5a-
DHT metabolites, namely 3a-diol, 3b-diol and A-dione.
14
C-5a-DHT
was incubated for 24 h in the presence of Om preadipocytes, in
duplicate. Percentage of steroid products are shown above each peak.
Figure 3 Quantification of steroid products by GC/LC–MS after
incubation of Om preadipocytes with (A) 0
.
35 mM D4-dione or (B)
5a-DHT. Experiments were performed in duplicate. MeansG
S.E.M.
are shown.
K BLOUIN and others
.
Androgen inactivation in abdominal adipose tissue642
Journal of Endocrinology (2006) 191, 637–649 www.endocrinology-journals.org
was mostly present in the cytosol (approximately fourfold).
Figure 6 shows the effects of specific AKR1C inhibitors on
the formation of 3a-diol and 3b-diol. The strongest
inhibition of 3a-diol and 3b-diol production was observed
when cells were incubated with 100 mM5b-cholanic acid-
3a,7a-diol (3a-HSD: 30G24% of control for s.c. and
32G9% of control for Om, 3b-HSD: 44G12% of control
for Om). When cells were incubated with both 5b-cholanic
acid-3a,7a-diol, and indomethacin, no significant additional
inhibition was observed. Figure 7 shows the results of time-
course and dose–response experiments in preadipocyte
cultures. Maximal stimulation of 3a/b-HSD activity was
not reached at 1 mM substrate (Fig. 7A). Time course
showed a linear 3a/b-diol formation over 24 h incubations
(Fig. 7B). Om and s.c. preadipocyte cultures were
not different.
3a/b-HSD activity in preadipo cytes and mature adipocytes
Figure 8 shows that 5a-DHT inactivation through 3a/b-diol
formation in the s.c. depot was significantly higher in mature
adipocytes compared with preadipocyte cultures, as expressed
in femtomoles of 3a/b-diol formed per microgram total
protein over 24 h (755G830 versus 245G151 fmol 3a/
b-diol per mg protein over 24 h, P!0
.
05 nZ10 cultures). No
significant difference was observed between preadipocytes
and mature adipocytes in Om adipose tissue (nZ11 mature
adipocyte cultures and nZ6 preadipocyte cultures) and
between the s.c. and Om depots for each given cell type.
Figure 6 The effects of the specific AKR1C inhibitors 5b-cholanic
acid-3a,7a-diol (5b-chol; inhibits AKR1C2) and indomethacin
(Indo; inhibits AKR1C3) on the conversion of 5a-DHT into 3a-diol
and 3b-diol after a 2-h preincubation period with the inhibitors in
s.c. (A) and Om (B) preadipocyte cultures. Results are expressed as
percentage of control. Data were obtained in s.c. cultures from four
subjects and in Om cultures from five subjects, in duplicate.
*Different from control (P!0
.
05) by a posteriori mean contrasts and
Bonferroni correction.
†
P!0
.
05 3a-diol versus 3b-diol formation
by paired t-test. MeansG
S.E.M. are shown.
Figure 4 Measurement of 3a-reductase and 3b-reductase activities
in s.c. and Om peadipocyte cultures using TLC in ether:ethyl acetate
(1:1). The results were obtained in s.c. cultures from four subjects and
in Om cultures from five subjects, in duplicate. MeansG
S.E.M. are
shown. *P!0.05, 3a-diol versus 3b-diol formation.
Figure 5 Quantification of 3a/b-HSD activity in adipose tissue
cytosolic and membrane fractions. Data were obtained from six s.c.
and five Om fat samples. *P!0
.
0001 membranes versus cytosol.
MeansG
S.E.M. are shown.
Androgen inactivation in abdominal adipose tissue
.
K BLOUIN and others 643
www.endocrinology-journals.org Journal of Endocrinology (2006) 191, 637–649
3a/b-HSD activity in adipose tissue homogenates
Table 3 shows characteristics of the sample of men in which
adipose tissue homogenate 3a/b-HSD activity measures were
performed. Subjects covered a wide range of BMI values
(24
.
6–79
.
1 kg/m
2
). Om HR-LPL activity was significantly
higher than s.c. HR-LPL activity (P!0
.
05). Basal lipolysis as
well as adipocyte diameter were not significantly different
between the two adipose tissue compartments.
3a/b-HSD activity measured in s.c. adipose tissue
homogenates was significantly higher than that of the Om
depot (117G39versus 79G38 fmol3ab-diol/mgprotein/24h,
P!0
.
0001) in the entire group. Figure 9 shows that 3a/b-HSD
activity measured in Om adipose tissue was significantly higher
in obese compared with lean or overweight men (84G37 versus
52G30 fmol 3a-diol/mg protein/24 h, P!0
.
05). 5a-DHT
inactivation in s.c. adipose tissue was not different between lean/
overweight and obese men.
No significant correlation was observed between 3a/b-
HSD activity in Om adipose tissue and Om adipocyte size as
well as LPL activity when all the subjects were considered.
When extremely obese subjects were excluded (class III
obesity, 40 kg/m
2
), a significant positive association was
observed between Om 3a/b-HSD activity and adipocyte
Figure 7 (A) Dose–response for 3a/b-HSD activity in preadipocyte
primary cultures. (B) Time-course for 3a/b-HSD enzymatic activity in
preadipocyte primary cultures. Experiments were performed in three
cultures from each depot, in duplicate. MeansG
S.E.M. are shown.
Figure 8 Comparison of 3a/b-HSD activity measured in s.c. and
Om mature adipocytes and preadipocyte cultures. Data obtained
from 10 s.c. and 11 Om samples for mature adipocyte cultures and
from 10 s.c. and 6 Om samples for preadipocyte cultures. Measures
were performed in triplicate for mature adipocytes and in duplicate
for preadipocytes. *P!0
.
05 preadipocytes versus mature adipo-
cytes by Welch ANOVA. MeansG
S.E.M. are shown.
Table 3 Physical characteristics of the sample of 34 men
MeanG
S.D. Range
Characteristic
Age (years) 43
.
8G10
.
022
.
6–61
.
2
Weight (kg) 138
.
7G46
.
970
.
4–265
.
0
BMI (kg/m
2
)44
.
4G12
.
924
.
6–79
.
1
Waist circumference (cm) 136
.
1G27
.
491
.
5–190
.
0
Subcutaneous
HR-LPL activity 41
.
9G19
.
714
.
5–110
.
8
Basal lipolysis 79
.
9G58
.
010
.
0–301
.
3
Adipocyte diameter (mm) 109
.
3G11
.
285
.
8–131
.
3
Omental
HR-LPL activity 52
.
3G24
.
022
.
4–102
.
2
Basal lipolysis 85
.
2G104
.
321
.
2–568
.
1
Adipocyte diameter (mm) 110
.
0G9
.
694
.
2–129
.
1
HR-LPL activity expressed as nmol FFA/h!10
6
cells. Lipolysis expressed as
nmol glycerol/2 h!10
8
mm
2
; nZ33 for waist circumference, nZ30 for
HR-LPL activity, nZ24 for s.c. lipolysis, nZ26 for Om lipolysis.
Figure 9 Comparison of 3a/b-HSD activity measured in s.c. and
Om adipose tissue homogenates in non-obese (BMI!30 kg/m
2
,
nZ6) or obese (BMIR30 kg/m
2
, nZ28) men. *P!0
.
05 non-obese
versus obese by Wilcoxon rank-sum test;
†
P!0
.
05,
‡
P!0
.
0005 s.c.
versus Om by paired t-test. MeansG
S.E.M. are shown.
K BLOUIN and others
.
Androgen inactivation in abdominal adipose tissue644
Journal of Endocrinology (2006) 191, 637–649 www.endocrinology-journals.org
diameter (rZ0
.
69, P!0
.
02). No significant association was
observed between s.c. 3a/b-HSD activity and s.c. adipocyte
diameter.
We examined associations between plasma steroid hor-
mones and 3a/b-HSD activity values in the two adipose
tissue depots. Plasma 5a-DHT and testosterone were not
related to 3a/b-HSD activity measured in any fat compart-
ment. Figure 10 shows significant positive correlations of s.c.
3a/b-HSD activity with androsterone-glucuronide (rZ0
.
41,
P!0
.
02, nZ34), 3a-diol-glucuronide (rZ0
.
38, P!0
.
03,
nZ34), and D4-dione (rZ0
.
42, P!0
.
02, nZ34) levels. No
association was observed between circulating steroid levels
and 3a/b-HSD activity measured in Om adipose tissue.
Discussion
The aim of the present study was to examine 5a-DHT
inactivation as well as the expression of enzymes involved in
androgen metabolism in s.c. and Om adipose tissue obtained
from normal weight t o morbidly obese men, and to
investigate the relationship between 5a-DHT inactivation
and obesity. We tested the hypothesis that AKR1C enzymes
would be detected in abdominal adipose tissue compartments
and 5a-DHT inactivation would be related to obesity in men.
AKR1C2 mRNA and 5a-DHT inactivation were, indeed,
detected in both s.c. and Om adipose tissues, with whole s.c.
adipose tissue having higher values of 5a-DHT inactivation
and a trend for higher AKR1C2 mRNA levels. Both 3a-diol
and 3b-diol were detected following incubation of pre-
adipocytes with 5a-DHT. The androgen receptor and several
enzymes involved in androg en metabolism were also
expressed in adipose tissue. Consistent with our hypothesis,
Om 3a/b-HSD activity in tissue homogenates was signi-
ficantly higher in obese men compared with men with
BMI!30 kg/m
2
.3a/b-HSD activity was detected mostly in
the cytosolic fraction of whole adipose tissue. 5a-DHT
inactivation was significantly higher in mature adipocytes
compared with preadipocytes in the s.c. depot. This is the first
study to report on the presence of AKR1C and 5a-DHT
inactivation in abdominal adipose tissue obtained in lean and
obese men. Future studies are needed to clarify the potential
physiological importance of local androgen inactivation in the
modulation of regional adipose tissue distribution.
This study in men was prompted by the recent results
obtained in women. We reported higher 5a-DHT inacti-
vation in female s.c. adipose tissue compared with Om
adipose tissue (Blouin et al. 2003, 2005). We also found that
women with high visceral adipose tissue area (assessed by
computed tomography) had higher AKR1C1 and AKR1C2
mRNA levels as well as higher 5a-DHT inactivation rates in
Om adipose tissue homogenates compared with women with
low visceral adipose tissue area (Blouin et al. 2003, 2005).
Significant positive correlations were also observed between
5a-DHT inactivation (and mRNA levels of AKR1C1 and
AKR1C2) and visceral adipose tissue accumulation or Om
adipocyte diameter (Blouin et al. 2003, 2005 ). Together, our
results show that 3a/b-reduction of 5a-DHT is detected in
abdominal fat compartments of both men and women, and
that Om reaction rates are positively associated with adiposity
Figure 10 Correlations between 3a/b-HSD activities measured in s.c.
adipose tissue homogenates and plasma measurements of D4-dione,
androsterone-glucuronide and 3a-diol-glucuronide levels. Analysis
performed on log
10
-transformed values for 3a/b-HSD activity.
Androgen inactivation in abdominal adipose tissue
.
K BLOUIN and others 645
www.endocrinology-journals.org Journal of Endocrinology (2006) 191, 637–649
measures in both sexes, at least in the lean to moderately obese
range. Mechanisms common to men and women may be
involved in the regulation of and rogen proces sing and
inactivation in abdominal adipose tissue.
The study of 5 a-DHT inactivation in adipose tissue is
relevant only in the context of the global pathway of androgen
metabolism and action in this tissue. Obviously, androgens
and androgen precursors must be present in adipose tissue,
which was shown by several groups (Feher & Bodrogi 1982,
Deslypere et al. 1985, Szymczak et al. 1998 ) including ours
(Be
´
langer et al. 2006). In the present study, the expression of
several en zymes i nvolved in and rogen met abolism was
detected in fat samples, including 5a-reductase-1, which is
necessary for 5a-DHT formation. Incubation of preadipo-
cytes with the adrenal precursor D4-dione led to the
formation of testosterone, 5a-DHT, and androsterone (or
epiandrosterone) as assessed by GC/LC–MS. The expression
of the androgen receptor was also detected and mRNA levels
were higher in s.c. versus Om adipose tissue. Together, these
data support the relevance to study 5a-DHT inactivation in
adipose tissue. Interestingly, the expression of P450 aromatase
was relatively low and neither 17b-estradiol nor estrone was
detected using GC–MS when D4-dione was incubated with
Om preadipocytes for 24 h (not shown). Accordingly, a
previous study has shown that real-time RT-PCR-measured
mRNA levels of P450 aromatase in mature adipocytes were
very low, and sometimes undetectable (Dieudonne
´
et al.
2006). We measured P450 aromatase mRNA levels in whole
adipose tissue samples of obese men, which were most likely
enriched in mature adipocytes. Thus, our finding of only
moderate aromatase expression in such tissue samples is not
surprising. Killinger et al. (1990) also demonstrated that
percent conversion of androstenedione to estrone ranged
between 0
.
01 and 0
.
8% in abdominal s.c. adipose stromal cells
and near 0
.
01% for Om preadipocytes. In the conditions of
the present study, the most sensitive method used (GC–MS)
would have allowed for the detection of approximately 0
.
25%
estrone formation. The low rates of estrone formation in
these cells (Killinger et al. 1990) could not have been detected
using our method. Altogether, our results do not seem to be
in disagreement with other previous studies regarding
aromatization in adipose tissue.
The expression of the 3 AKR1C enzymes was particularly
high compared with other enzymes measured in the present
study, with marked depot differences. We also report that
3a/b-HSD activity originated mainly from the cytosolic
fraction. Since AKR1C enzymes are known to be cytosolic
enzymes, and AKR1C are highly expressed in fat samples, we
can suggest that AKR1C, and especially AKR1C2, are
actually involved in androgen inactivation in adipose tissue in
men. To assess this issue, experiments were performed with
specific AKR1C inhibitors in preadipocyte cultures. Incu-
bation with a specific AKR1C2 inhibitor showed that this
enzyme seems to be responsible for the generation of
approximately 70% of the 3a-diol and 40–60% of the 3b-
diol. When the cells were incubated with both AKR1C2 and
AKR1C3 inhibitors, no significant additional inhibition was
observed indicating that AKR1C1 could be responsible for
most of the remaining 3a-HSD and 3b-HSD activities.
From the physiological standpoint, there is a growing
interest for the study of local androgen synthe sis or
inactivation in adipose tissue. For example, Corbould et al.
(2002) found that BMI and waist circumference were
positively associated with the ratio of type 3 17b-HSD to
aromatase mRNA measured in intra-abdominal adipose
tissue, suggesting that androgen formation may be higher
than its inactivation through aromatization in intra-abdominal
adipose tissue of abdominally obese women (Corbould et al.
2002). More recently, Quinkler et al. (2004) studied several
steroid-converting enzymes involved in adipose tissue local
androgen metabolism during the differentiation of preadipo-
cyte primary cultures. We hereby demonstrate that 5a-DHT
inactivation is an impor tant reaction that could also
contribute to limited exposure of adipose cells to 5a-DHT
in men through a pre-receptor regulatory mechanism. The
finding that 5a-DHT is produced locally at relatively low
rates, but is strongly and rapidly converted into other steroids
suppor ts this hypothesis.
Cross-sectional analyses cannot help in establishing the
cause and effect relationships. However, a few possibilities can
be raised to explain the higher Om 3a/b-HSD activity in
obesity. Androgen effects on adipose tissue are postulated to
be at least partly mediated through the androgen receptor, as
male knock-out mice for this receptor develop late onset
obesity (Sato et al. 2003). It is possible that increased androgen
inactivation in the omentum of obese men through AKR1C
enzymes represents a pathological condition underl ying
visceral obesity. According to this hypothesis, increased
androgen inactivation in visceral adipose tissue could have
led to decreased exposition to active androgens, which have
been shown to inhibit adipose tissue LPL activity and to
accelerate lipid turnover in men (Ma
˚
rin et al. 1995).
Androgens could also exert their effects directly through the
control of adipogenesis. Accordingly, androgens were found
to inhibit adipogenesis in C3H 10T1/2 mouse pluripotent
cells as well as in the mouse preadipocyte cell line 3T3-L1
(Singh et al. 2003, 2006). This hypothesis is also consistent
with higher AKR1C enzyme expression and 3a/b-HSD
activity in s.c. adipose tissue, since this depot is generally
larger than the Om/visceral fat depot. Consistent with this
hypothesis and the higher s.c. versus Om androgen
inactivation rates, we also found that adipose tissue 5a-
DHT concentrations are higher in the Om than in the s.c.
depot (Be
´
langer et al. 2006).
On the other hand, the regional depot difference in
androgen inactivation rates and androgen inactivating
enzymes could be a consequence of obesity, or other
characteristics of the adipose tissue compartments examined.
We found significantly higher 5a-DHT inactivation rates in
mature adipocytes compared with preadipocytes in s.c.
adipose tissue. Thus, depot or obesity-related differences in
stromal cell, preadipocyte, or mature adipocyte content could
K BLOUIN and others
.
Androgen inactivation in abdominal adipose tissue646
Journal of Endocrinology (2006) 191, 637–649 www.endocrinology-journals.org
explain t he reg ion al difference obser ved in androgen
inactivation. However, the question of preadipocyte number
in various fat depots and in obesity is not completely resolved,
a possible reason being that the assessment of preadipocyte
number is considered labor intensive and error prone (Bakker
et al. 2004 ). No significant depot difference was found in one
study, whereas an examination of breast adipose tissue
indicated that BMI was not correlated to the ratio of stromal
cells/mature adipocytes (van H armelen et al. 2003),
suggesting that the degree of obesity does not influence cell
population proportions at least in that depot. Further studies
are required to establish whether 3a/b-HSD activity and
androgen inactivation in adipose tissue is a consequence of
obesity-related characteristics of adipose tissue s uch as
differences in cell populations or is causally related to
abdominal obesity.
We have shown that appreciable amounts of both 3a-diol
and 3b-diol were produced when preadipocytes were
incubated with 5a-DHT. This finding could be of
physiological importance because 3b-diol is known to
stimulate the estrogen receptor b-1 (ERb-1) (Pak et al.
2005). Pedersen et al. have found previously that ERb-1
mRNA and protein were expressed in abdominal s.c. and
intra-abdominal adipose tissue in men and women with
significantly higher levels found in s.c. adipose tissue in both
sexes (Peder sen et al. 2001). Interestingly, we found that the
expression of 3(a/b)-hydroxysteroid epimerase was low,
but was dramatically different between s.c. and Om adipose
tissue. Further investigations are needed to evaluate the
relative importance of active androgens and their metabolites,
including 3b-d iol on adipose cell metabolism and
differentiation.
The higher 5a-DHT inactivation found in mature
adipocytes also suggests that 3a/b-HSD activity and local
5a-DHT concentration could be involved in the modulation
of preadipocyte differentiation, an increasing 5a-DHT
inactivation during differentiation preventing exposure to
5a-DHT in mature adiocytes. This hypothesis is supported by
the observed in vitro inhibition of mouse pluripotent cell and
3T3-L1 adipogenesis by 5a-DHT (Singh et al. 2003, 2006).
However, caution is needed when interpreting our compari-
son of preadipocytes and mature adipocytes because it is based
on a protein normalization. DNA normalization may have
been preferable because it may be more representative of the
number of cells examined. Moreover, differences in the lipid
content between cell types and the added lipid extraction step
in mature adipocytes may have interfered with the
determination of 3a/b-HSD activity and/or total protein
content. These results need further validation in differentiat-
ing adipocytes.
We observed no association between plasma steroid levels
and 3a/b-HSD activity measurements with the exception of
the 3a-reduced androgen metabolites androsterone-glucur-
onide and 3a-diol-glucuronide, and with D4-dione, which
were positively associated with s.c. 3a/b-HSD activity. Om
3a/b-HSD activity was not related to circulating androgen
metabolite levels. These data suggest that steroidogenesis
taking place in s.c. adipose tissue may contribute to circulating
steroid concentrations due to a mass action effect, whereas
Om local steroid conversion may have little or no impact on
circulating hormone levels given the relatively small size of
this compartment. Quinkler et al. (2004) also suggested that
s.c. adipose tissue may contribute significantly to systemic
androgen production based on their observation that s.c.
adipose tissue predominantly activated androgens, whereas
Om adipose tissue predominantly inactivated androgens.
However, other studies have shown that local steroid
metabolism in adipose tissue may not necessarily reflect
circulating steroid levels due to the fact that other tissues,
including the liver, also express steroidogenic and/or steroid-
inactivating enzymes (Dufort et al. 2001, Rask et al. 2002,
Blouin et al. 2005).
In conclusion, 5a -DHT inac tivation was detected in
abdominal adipose tissue in men, and higher Om adipose tissue
3a/b-HSD activity was found in obese men. The expression of
several enzymes involved in local androgen metabolism was also
detected, with AKR1C1, AKR1C2, and AKR1C3 having
especially high expression levels and large depot-differences
compared with other enzymes measured. Higher 5a-DHT
inactivation was found in mature adipocytes compared with
preadipocytes, and 5a-DHT inactivation rates were higher in
Om fat from obese men. Further studies are required to
elucidate whether increased local androgen inactivation in Om
adipose tissue is directly involved in the modulation of adipocyte
metabolism and regional fat distribution in men.
Acknowledgements
This study was supported by the Canadian Institutes of
Health Research (MOP-53195 to A T). Karine Blouin is the
recipient of a fellowship from the Canadian Institutes of
Health Research. Andre
´
Tchernof is the recipient of a
Scholarship from the Canadian Institutes of Health Research.
The authors would like to acknowledge the contribution of
Drs Picard Marceau and Odette Lescelleur to the recruitment
process of the study and to the collection of surgical biopsies.
The authors would like to thank Lucille Lacoste, Ve
´
ronique
Bellemare, Martin Perreault, and Ronald Maheux for their
technical assistance. The authors wish to thank all men who
participated in the study for their excellent collaboration. The
authors declare that there is no conflict of interest that would
prejudice the impartiality of this scientific work.
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Received in final form 8 September 2006
Accepted 22 September 2006
Made available online as an Accepted Preprint
3 October 2006
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