Service de Biochimie, Institut National de la Santé et de la Recherche Médicale CJF 9402, Faculté de Médecine Paris-Ouest, Université René Descartes (Paris V), Centre Hospitalier de Poissy, 78303 Poissy Cedex, France
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ABSTRACT |
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Various clinical and epidemiological evidence strongly suggests a major role for sex steroid hormones in the determination of anatomical specificities of fat distribution in human. To date, no studies have examined the possible presence of androgen receptors (AR) in human adipocytes and preadipocytes. We have studied AR in preadipocytes from various anatomical locations (intra-abdominal and subcutaneous) in middle-aged men and women during the proliferation and differentiation processes (adipogenesis). Androgen binding sites quantified by [3H]R-1881-specific binding in whole cell extracts were twofold higher in intra-abdominal than in subcutaneous preadipocytes but identical for the same fat depots in men and women. Western blot analysis revealed 1) the presence of AR in the nuclear and cytosolic fractions of human preadipocytes, 2) a decrease of AR expression during adipogenesis, and 3) an upregulation of AR by androgens in vitro. RT-PCR experiments showed the presence of AR mRNA in human preadipocytes and adipocytes and also the regional specificity of AR distribution. However, AR mRNA expression was found to increase during adipogenesis. The same results were observed in rat preadipocytes. In conclusion, this study clearly demonstrates the presence of AR in human preadipocytes and adipocytes and suggests that androgens may contribute, through regulation of their own receptors, to the control of adipose tissue development.
adipogenesis; sex hormones; cultured preadipocytes
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INTRODUCTION |
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REGIONAL DISTRIBUTION of adipose tissue is of great importance with regard to the pathogenesis of cardiovascular diseases associated with obesity. Accumulation of adipose tissue in the intra-abdominal region (android type of obesity) but not in the femoral subcutaneous region (gynoid type) is indeed a major risk factor for cardiovascular diseases, non-insulin-dependent diabetes mellitus and stroke in both men and women. Adipose tissue deposition in different anatomical regions depends not only on the regional specificities of the metabolic activity of adipocytes (10, 18, 33) but also on the potency of local adipose precursor cells to differentiate into mature adipocytes.
There is now considerable evidence that sex steroids are involved in the site specificities of adipose tissue metabolism (19, 32, 33). However, the role that sex hormones could play in the regulation of the adipoconversion process is still poorly understood.
It is generally accepted that steroid hormone action results from steroid binding to specific intracellular receptors followed by the interaction of the hormone-receptor complex with specific DNA binding regions to finally regulate gene expression (reviewed in Refs. 1 and 38).
Androgen receptor (AR) expression has been investigated in various tissues and cells, such as the brain, pancreas, fibroblasts, and the human prostatic cell line LNCap (9, 14, 22, 24). In adipose tissue, only binding studies have suggested the presence of low levels of AR in human (25) and hamster (15) adipocytes and high levels of AR in male rat preadipocytes (7, 12). Furthermore, there is so far no immunologic or molecular data providing direct evidence for the existence of such receptors in adipocytes and preadipocytes.
This led us to conduct the present investigation in which the subcellular distribution of AR, the evolving pattern of AR expression during adipogenesis, and the possible regulation of AR expression by sex steroid hormones were investigated in both human and rat adipocytes and preadipocytes from various anatomical regions.
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MATERIALS AND METHODS |
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Materials.
DMEM, DMEM/Ham's F-12 (DMEM/F-12; phenol red free), HEPES, porcine
insulin, transferrin, triiodothyronine
(T3),
NADH(H+), dihydroxyacetone
phosphate, testosterone, 5-dihydrotestosterone, 17
-estradiol,
triamcinolone acetonide, and BSA were from Sigma (St. Louis, MO);
collagenase was from Boehringer (Mannheim, Germany); FCS and
Superscript II RNase H
RT
were from GIBCO BRL (Grand Island, NY); and polyclonal rabbit anti-AR
antiserum NCL Arp was from Novo Castra.
[3H]R-1881 (sp act 86 Ci/mmol) was obtained from NEN-Du Pont (Les Ulis, France),
Taq polymerase and Ready-to-go kit
from Pharmacia Biotechnology (Uppsala, Sweden), and nylon membrane
Hybond N+ and
[
-32P]dCTP from
Amersham (Les Ulis, France).
Subjects. Adipose tissue samples were obtained from deep (mesenteric) or subcutaneous deposits of men (51.8 ± 4.7 yr) and women (63.9 ± 16.3 yr) undergoing surgical intervention. Patients were moderately overweight with a body mass index ranging from 25 to 31 and had no endocrine diseases. This study was approved by the local ethics committee.
Animals. Male Sprague-Dawley rats (125-150 g) were kept under controlled lighting conditions (light: 6 AM, dark: 8 PM) and constant temperature (21°C). Animals were killed by decapitation, and epididymal and femoral subcutaneous adipose tissues were immediately removed under sterile conditions.
Cell culture. Preadipocytes were obtained by collagenase digestion as previously described (29). The floating adipocytes were discarded, and the infranatant containing the stromal vascular fraction was successively filtered through 150- and 25-µm nylon screens. The filtrate was centrifuged at 600 g for 10 min. After two washes, cells were plated into cell culture dishes at a density of 2-4 × 104 cells/cm2 with DMEM/F-12 (1:1) containing 10% charcoal-treated FCS, streptomycin (0.1 mg/ml), and penicillin (100 IU/ml) and maintained at 37°C under 5% CO2 atmosphere. After plating, cells were extensively washed and maintained under the same conditions as above until confluence (3-4 days after plating). To induce and maintain the adipose differentiation process, the following protocols were used: 1) for human preadipocytes, the cells were refed immediately after plating with a chemically defined medium consisting of DMEM/F-12 supplemented with insulin (5 µg/ml), transferrin (10 µg/ml), T3 (2 nM), and antibiotics (0.1 mg/ml streptomycin and 100 IU/ml penicillin) and for the first 3 days IBMX (100 µM) and dexamethasone (100 nM) (13); 2) for rat preadipocytes, the cells were maintained after confluence (3-4 days after plating) in ITT medium consisting of DMEM/F-12 (1:1) supplemented with insulin (5 µg/ml), transferrin (10 µg/ml), T3 (2 nM), and antibiotics (0.1 mg/ml streptomycin and 100 IU/ml penicillin) according to Deslex et al. (8).
When added to the medium, steroid hormones were dissolved in ethanol (final ethanol concn never exceeding 0.01%).Binding assays.
AR were studied by radioligand assays in human confluent preadipocytes.
After removal of the medium, cells were washed three times with
DMEM/F-12 and incubated (in triplicate) at 37°C, in DMEM/F-12
containing different concentrations of
[3H]R-1881
(methyltrienolone) and a fixed concentration (10 µM) of triamcinolone
acetonide. Nonspecific binding was measured under the same conditions
except that 5-dihydrotestosterone (1 µM) was added to the
incubations. After 60 min, incubation medium was discarded, and the
cells were washed three times to remove the free steroids. Whole cell
assay was then performed as described by De Pergola et al. (7).
Briefly, cells were solubilized with NaOH (0.2 M), and the
radioactivity was counted directly. An aliquot was removed for the
assay of total cellular protein.
Cellular extraction. Cellular extraction (cytosolic and nuclear fractions) was performed as described by Vornberger et al. (37). Cells were scraped on ice with buffer A, which contained 0.25 M sucrose, 5 mM EDTA, and protease inhibitors (58 µM phenylmethylsulfonyl fluoride, 10 mM benzamidine, 42 µM leupeptin, and 25 µM aprotinin). Next, cells were sonicated 1 min and centrifuged (10,000 g at 4°C) for 10 min, and the supernatant (cytosolic fraction) was collected and conserved for Western blot analysis in Laemmli buffer (vol/vol). The resulting pellet was resuspended in 1 ml of buffer A and pelleted again, and the resulting nuclear fraction was collected for Western blot analysis.
Western blot analysis. Equal amounts of cytosolic (100 µg) or nuclear (50 µg) proteins were resolved by SDS-PAGE (7.5% acrylamide). Proteins were transferred to polyvinylidene difluoride membrane overnight at 4°C and blocked for 2 h at room temperature in buffer B (137 mM NaCl, 20 mM Tris · HCl, and 0.1% Tween 20) with 5% nonfat milk. Polyclonal rabbit anti-AR antibody (1:250 dilution) was then added in buffer B, and, after overnight incubation at 4°C, the primary antiserum was removed and the blot was washed extensively with buffer B. Secondary antiserum (horseradish peroxidase-labeled anti-rabbit IgG) was then added (1:2,000 dilution), incubated with the blot for 1 h at room temperature, and washed. Finally, an enhanced chemiluminescence kit from Amersham was used to detect the signal. Control experiments with various protein amounts (10-100 µg) were performed to ensure that the densitometric signal intensity was proportional to the loaded amount of protein. To obtain a positive control, immunoblot analyses of rat prostate extracts were conducted in parallel.
RNA extraction. Total cellular RNA was extracted from mature adipocytes, confluent and/or differentiated preadipocytes, and from rat prostate and LNCap cells (as controls) by the procedure of Chomczynski and Sacchi (6). The yield and quality of the extracted RNA were assessed by the 260- to 280-nm optical density ratio and by electrophoresis under denaturating conditions on 1% agarose gels.
RT-PCR.
Total RNA (1 µg) was denatured for 10 min at 72°C and was reverse
transcribed to cDNA by incubating with 20 µl RT reaction mixture
containing 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT),
0.5 mM dNTPs, 450 mU RNAguard, 50 ng random hexamers, and 200 U
Superscript II RNase H RT.
Incubation was performed at 42°C for 60 min, heated to 95°C for
5 min, and then quickly chilled on ice.
Glycerol-3-phosphate dehydrogenase assay. Cell differentiation was assessed by following glycerol-3-phosphate dehydrogenase (GPDH) activity (12). After 10-12 days, ITT medium was removed, and cells were scraped in cold buffer containing 50 mM Tris · HCl (pH 7.4), 0.25 M sucrose, 1 mM EDTA, and 1 mM DTT. Cells were sonicated in the same buffer and centrifuged at 100,000 g for 20 min at 4°C. The GPDH activity was measured in the supernatant according to Wise and Green (39) and expressed in milliunits (nmol NAD+/min) per milligram of protein.
Other determinations. Protein concentration was measured according to Bradford (4) with BSA as standard. All results are expressed as means ± SE from at least three individual experiments. Statistical significance of these data was established by use of the Student's t-test.
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RESULTS |
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Whole cell [3H]R-1881 specific binding was used in an attempt to characterize the presence of AR in human preadipocytes from different anatomical locations (deep and superficial fat deposits) of both men and women. As shown in Table 1, numbers of androgen-specific binding sites are identical in human confluent preadipocytes (3-4 days after plating) from the same anatomical location in men and women. In both sexes, however, numbers of [3H]R-1881 binding sites determined at a fixed ligand concentration (10 nM) are higher in preadipocytes from intra-abdominal (2-fold) than from subcutaneous fat deposits. Characteristics of [3H]R-1881 whole cell binding were also determined in human confluent preadipocytes obtained from one subject. [3H]R-1881-specific binding at equilibrium was threefold higher in human confluent preadipocytes from deep (260 fmol/mg protein) than from superficial (90 fmol/mg protein) fat deposits, but the [3H]R-1881 binding affinity was similar in both deposits (1.2 and 1.52 nM in deep and subcutaneous adipose tissue, respectively). [3H]R-1881 binding studies were also performed in cytosolic extracts from mature adipocytes at a saturable ligand concentration (10 nM). However, high nonspecific binding made quantitative analysis of AR difficult. Values ranging from 3 to 15 fmol/mg protein were obtained on three samples, confirming previously published data (25).
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The finding that high-affinity androgen binding sites are present in cells is not sufficient to warrant the assumption that these sites are true AR and consequently cannot provide accurate information on AR expression and regulation. We have therefore also attempted to characterize the AR protein and its mRNA in human preadipocytes by using specific antibodies and molecular probes. Moreover, to allow comparison, the same experiments were repeated in rat preadipocytes.
An immunoreactive AR protein of ~110 kDa was detected in nuclear and cytosolic extracts from adipose tissue (rat and human) as well as in rat prostate extracts, which were used as a positive control. The immunospecificity of this signal was confirmed, since, in the absence of primary antiserum or in the presence of rabbit IgG (in place of anti-AR antibody), the signal was not detected (data not shown).
First, Western blot analysis confirmed the above-described binding data
by showing that, in rats, expression of immunoreactive AR protein in
nuclear extracts is higher in confluent and differentiated preadipocytes from deep intra-abdominal (epididymal) than from superficial (femoral subcutaneous) fat depots (Fig.
1). Second, changes occur in the expression
of the immunoreactive AR protein during adipogenesis. In both humans
and rats, the AR protein amount is lower in differentiated
preadipocytes and mature adipocytes than in confluent preadipocytes,
and this difference is particularly obvious in human preadipocytes from
deep intra-abdominal fat deposits (87%; Fig.
2). These quantitative differences in
immunoreactive AR protein parallel those obtained in binding
experiments (present study and Ref. 12). Third, because androgens
modulate in vivo (15) and in vitro (7, 12) the number of androgen
high-affinity binding sites in rodent (rat and hamster) preadipocytes
and adipocytes, we have also investigated by Western blot analysis the
direct influence of androgens and estrogens in vitro on AR expression in primary cultured confluent and differentiated human and rat preadipocytes. Confluent or differentiated monolayer cells were incubated for 24 h with 0.1 µM testosterone, dihydrotestosterone, or
17
-estradiol. This relatively high concentration of steroid hormone
was used because FCS present in the culture medium during the
proliferative phase decreases the free hormone concentration by a
factor of five (data not shown). At the end of the incubation, both the
nuclear and cytosolic fractions of preadipocytes were assayed. As shown
in Fig. 3, dihydrotestosterone increased AR protein levels in confluent or differentiated preadipocytes from intra-abdominal fat depot in men. Under the same experimental conditions, however, 17
-estradiol was without any effect. In confluent and differentiated preadipocytes from rat, a twofold upregulation of AR protein amount by testosterone and
dihydrotestosterone was also observed (Fig.
4) regardless of the anatomical origin of
the cells.
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Finally, we have analyzed the gene transcripts of AR. RT-PCR was performed with human and rat RNA samples prepared from confluent or differentiated preadipocytes as well as from mature adipocytes. To determine the conditions required for a semiquantitative analysis, graded amounts of human total RNA (0.1-2 µg) were reverse transcribed and amplified for 25, 30, and 35 cycles. At 35 cycles, the signal intensity did not further increase proportionally to the amount of total RNA (data not shown). On the basis of these results, we chose to subject cDNA obtained from 1 µg of total RNA to 30 PCR cycles. Amplified products for human and rat AR had the expected size (342 and 248 bp, respectively). Identical results were obtained with LNCap cells (Fig. 5, lane 5) and rat prostate (Fig. 5, lane 11) for human and rat AR, respectively. DNA contamination could be excluded for each amplified product, since larger fragments would have been seen. Negative controls without the reverse-transcription step were also included to exclude internal contamination (Fig. 5, lane 12). The specificity of these amplimers was also established by restriction enzyme analysis. As expected (5), amplified cDNA of human AR was digested to 303 and 39 bp with EcoR I and 193 bp and 149 bp with Pvu I (data not shown). As also expected (5), amplified cDNA of rat AR was digested to 196 and 52 bp with Hind I and 170 and 78 bp with Sac I (data not shown). Taken together, these results allow us to conclude that the observed 342- and 248-bp bands are the specific amplified cDNA of human and rat AR, respectively. Following this procedure, we have compared AR expression in human and rat preadipocytes (confluent and differentiated) as well as in mature adipocytes.
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As shown in Fig. 5, A and B, human AR mRNA levels in confluent (undifferentiated) preadipocytes (lanes 1 and 2) and mature adipocytes (lanes 3 and 4) were again found to be higher (2-fold) in intra-abdominal fat deposits than in subcutaneous fat deposits. During adipogenesis, however, levels of the human AR transcripts (lanes 6-8) increased and were maximal in mature adipocytes (5-fold), which is just the opposite of what was observed for the AR protein (Fig. 2). As the same picture emerged for rat epididymal confluent (undifferentiated) preadipocytes and adipocytes (lanes 9-10), it can be concluded from these data that the AR mRNA level increases during the course of adipogenesis.
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DISCUSSION |
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It appears that sex steroid hormones play an important role in the determination of body fat distribution and pattern of obesity (android or gynoid) (3). Android obesity is strongly associated with hypertension, dyslipidemia, hyperuricemia, insulin resistance, hyperinsulinemia, and non-insulin-dependent diabetes mellitus (30).
Steroid hormone actions are mediated by specific intracellular receptors in their target cells. Estrogen receptors have recently been identified in human preadipocytes and mature adipocytes (26, 31). Glucocorticoid receptors have also been characterized in human adipose tissue but not those for progestagens (3). Surprisingly, the presence of AR in human preadipocytes and isolated adipocytes has not yet been identified. Only one study (25) has described androgen binding sites in whole human adipose tissue. In the present study, we have demonstrated the presence of a true AR protein and the expression of its mRNA in human (middle-aged men and women) and rat preadipocytes and adipocytes. In isolated mature adipocytes, very low levels of AR binding sites were found, a situation similar to that described for estrogen receptors (26). Our results from binding studies also indicate that, in human confluent preadipocytes, AR are more abundant than in mature adipocytes and that, for a given fat depot, AR numbers are identical in men and women. In both sexes, however, the number of androgen binding sites is higher in confluent preadipocytes from intra-abdominal than from subcutaneous fat deposits. These results are in agreement with our previous study in rats (12), showing a lower density of nuclear or cytosolic androgen binding sites in subcutaneous than in epididymal preadipocytes. Moreover, the present experiments reveal that androgen binding sites are more abundant in human (3-fold) than in rat preadipocytes.
Using immunoblotting analysis, we demonstrated the presence of AR
protein in both the nuclear and cytosolic fractions of human preadipocytes and adipocytes. The nuclear location of AR in
preadipocytes suggests the existence of ligand-activated AR in adipose
tissue (38). Our RT-PCR experiments showed a higher expression of AR mRNA in human preadipocytes and adipocytes from deep than from superficial depots, thus confirming the ligand binding data. From immunoblots and RT-PCR experiments performed on rat preadipocytes and
adipocytes, identical results were obtained. These site-related differences in AR equipments may contribute, at least in part, to
explain the differences in metabolic responses observed already between
intra-abdominal and subcutaneous adipose tissues with regard to their
sensitivity to androgens. Indeed, androgens in vivo modulate adipocyte
responses to several stimuli, particularly concerning their lipolytic
activity (34), - and
2-adrenoreceptor equipment (28,
41), protein kinase C activities (21), and mitogen-activated protein
kinase cascade and c-fos signaling
pathways (20).
Because, in rats and humans, AR are more abundant in preadipocytes than
in mature adipocytes, AR protein and mRNA expressions were studied
during adipogenesis. Our present data obtained by RT-PCR and
immunoblotting analysis revealed an opposite evolution between AR
protein and mRNA expression during the course of adipogenesis in vitro.
Indeed, in both humans and rats, AR protein expression was found to be
higher in confluent than in differentiated preadipocytes or mature
adipocytes, whereas AR mRNA expression was lower in confluent
preadipocytes than in mature adipocytes. Such a situation has already
been reported in other cells and tissues (LNCap cell line, rat ventral
prostate), which are particularly sensitive to androgens (17, 27, 40).
In adipose tissue, the reduction of AR protein expression observed both
in vitro during adipogenesis (this study) and in vivo between
preadipocytes and mature adipocytes (15) could be related either to a
decrease in the AR mRNA translation or to a reduction of the AR protein
stability. Different arguments favor a decrease in AR protein stability
during adipogenesis in vitro. Indeed, it is generally accepted that, in
absence of their ligand, steroid receptors are relatively unstable (2,
16). Moreover, as presently shown, the addition of testosterone or 5-dihydrotestosterone to the culture medium during both the
proliferation and differentiation phases results in AR protein
upregulation in both rat and human adipose precursor cells. In this
study, however, addition of 17
-estradiol failed to affect the AR
protein level in human confluent preadipocytes. The latter finding,
which is consistent with our previous observations in rat preadipocytes (12), strongly suggests that the upregulatory effect of androgens on
their own receptors does not involve androgen conversion to estrogen.
In conclusion, this study shows that AR protein and mRNA are expressed
in human (and rat) preadipocytes and mature adipocytes with regional
specificities, with AR expression being lower in mature adipocytes than
in preadipocytes. Previous studies from our laboratory strongly
suggested the presence of functional AR in both adipocytes and
preadipocytes. We have clearly demonstrated 1) a direct positive transcriptional
effect of androgens on the expression of the
2A-adrenoreceptor subtype in
hamster mature adipocytes (11, 28) and
2) a direct negative effect of
androgens on the GPDH expression during the adipose conversion process
studied in rat intra-abdominal preadipocytes (unpublished results). It is worthwhile to note that these effects of androgens were completely prevented by cell exposure to AR antagonists, a finding consistent with
a physiological function of AR in adipose tissue. Finally, in addition
to estrogen receptors, which mediate mitogenic effects in human
preadipocytes (35), the characterization of AR in both human and rat
adipose tissues strengthens the concept that androgens may also play an
important regulatory role in the mechanisms underlying the sexual
dimorphism of fat distribution and in those more specifically involved
in the deep fat deposition characterizing abdominal obesity (23, 36).
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NOTE ADDED IN PROOF |
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During preparation of this paper, O'Brien et al. (J. Clin. Endocrinol. Metab. 83: 509-513, 1998) reported the identification of progesterone receptors in human subcutaneous adipose tissue.
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ACKNOWLEDGEMENTS |
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We are particularly indebted to the General Surgery Division, Centre Hospitalier de Poissy, for their courtesy in making human adipose tissue available.
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FOOTNOTES |
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This study was supported by Institut National de la Santé et de la Recherche Médicale (CJF 9402), Université R. Descartes (Paris V), and Comité des Yvelines de la Ligue contre le Cancer.
Address for reprint requests: Y. Giudicelli, Service de Biochimie, Centre Hospitalier, 78303 Poissy Cedex, France.
Received 25 September 1997; accepted in final form 9 February 1998.
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