Androgen receptors in human preadipocytes and adipocytes: regional specificities and regulation by sex steroids

M. N. Dieudonné, R. Pecquery, A. Boumediene, M. C. Leneveu, and Y. Giudicelli

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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. DMEM, DMEM/Ham's F-12 (DMEM/F-12; phenol red free), HEPES, porcine insulin, transferrin, triiodothyronine (T3), NADH(H+), dihydroxyacetone phosphate, testosterone, 5alpha -dihydrotestosterone, 17beta -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 [alpha -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 5alpha -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.

Specific primers for the AR were selected. The sequences were as follows: sense 5'-CTCTCTCAAGAGTTTGGATGGCT-3' and antisense 5'-CACTTGCACAGAGATGATCTCTGC-3' for human AR (A5-A6) and sense 5'-CCCATCGACTATTACTTCCC-3' and antisense 5'-TTACGAGCTCCCAGAGTCAT-3' for rat AR (A3-A4).

PCR generated 342- and 248-bp fragments of human and rat AR, respectively. These primers were designed to produce amplimers spanning RNA splicing sites in the DNA binding domain of the AR and thus to control for genomic DNA contamination.

The PCR reaction mixture contained 2 µl cDNA, 0.2 mM dNTPs, 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 12.5 pM of each primer, and 2.5 U of Taq polymerase. One cycle of PCR consisted of 1 min at 95°C, 1 min at 54°C, and 1 min at 72°C, and 35 cycles of PCR were performed with a Cyclone Thermocycler (Integra Bioscience). The resulting PCR products were fractionated on a 2% agarose gel in 90 mM Tris-borate-2 mM EDTA buffer (pH 8) and visualized by staining with ethidium bromide. Controls without RT were systematically performed to detect any cDNA contamination.

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.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Whole cell [3H]R-1881-specific binding in human confluent adipose precursor cells from intra-abdominal and subcutaneous fat deposits

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 17beta -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, 17beta -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.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 1.   Comparison of immunoreactive androgen receptor (AR) protein levels in rat preadipocytes from epididymal and femoral subcutaneous fat deposits at 2 stages of adipogenesis. AR protein levels were studied by immunoblotting as described in MATERIALS AND METHODS in confluent and differentiated preadipocytes. A: densitometric analysis of AR Western blots. Data are means ± SE from 5-6 separate experiments and are expressed as percentage of densitometric value obtained for AR in epididymal preadipocytes (control). *** P < 0.002; ** P < 0.005. B: representative Western blot analysis of AR protein in nuclear preparations of rat confluent (Conf) or differentiated (Diff) preadipocytes from epididymal (lanes 1 and 3) and subcutaneous (lanes 2 and 4) fat deposits and of rat prostate (P).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2.   Immunoreactive AR protein levels during adipogenesis in both rat and human deep adipose tissues. AR protein levels were studied by immunoblotting as described in MATERIALS AND METHODS in confluent and differentiated preadipocytes and mature adipocytes (Adipo) from rat epididymal or human intra-abdominal fat depots. A: densitometric analysis of AR Western blots. Data are means ± SE from 3-4 separate experiments and are expressed as percentage of densitometric value obtained for AR in confluent preadipocytes. ** P < 0.002; * P < 0.05. B: representative Western blot analysis of AR protein in nuclear preparations of rat prostate, rat epididymal confluent (lane 1) and differentiated preadipocytes (lane 2), rat epididymal mature adipocytes (lane 3), human intra-abdominal confluent (lane 4) and differentiated preadipocytes (lane 5), and human intra-abominal mature adipocytes (lane 6).


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 3.   Influence of sex hormones on immunoreactive AR protein levels in confluent and differentiated preadipocytes from intra-abdominal fat in men. At confluence and differentiation steps, dihydrotestosterone (DHT) or 17beta -estradiol (Est) was added to culture medium (final concn 0.1 µM). After 24 h, preadipocytes were washed with physiological serum, and nuclear and cytosolic extractions were realized as described in MATERIALS AND METHODS. A: densitometric analysis of AR Western blots (nuclear fraction). Data are means ± SE from 3 separate experiments and are expressed as percentage of densitometric value obtained for AR in preadipocytes maintained 24 h without steroid (control). * P < 0.05. B: representative Western blot analysis of AR protein in nuclear and cytosolic preparations of rat prostate, human confluent or differentiated preadipocytes incubated without steroid (lane 1), with DHT (lane 2), or with Est (lane 3).


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 4.   Influence of androgens on immunoreactive AR protein levels in rat confluent and differentiated preadipocytes from epididymal and subcutaneous fat depots. At confluence and differentiated steps, dihydrotestosterone (DHT) or testosterone (Testo) was added to culture medium (final concn 0.1 µM). After 24 h, preadipocytes were washed with physiological serum and nuclear extractions were realized as described in MATERIALS AND METHODS. A: densitometric analysis of AR Western blots (nuclear fraction). Data are means ± SE from 3-4 separate experiments and are expressed as percentage of densitometric value obtained for AR in preadipocytes maintained 24 h without steroid (control). ** P = 0.007; * P < 0.05. B: representative Western blot analysis of AR protein in nuclear preparations of rat prostate and rat epididymal and subcutaneous preadipocytes incubated without steroid (lane 1), with testosterone (lane 2), or with DHT (lane 3).

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.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 5.   Detection of presence of gene transcripts of AR in human and rat preadipocytes and adipocytes by RT-PCR analysis. Optimal conditions of RT-PCR were obtained with 30-cycle amplification of cDNA reverse transcribed from 1 µg of total RNA. RT-PCR products were analyzed by 2% agarose gel electrophoresis and ethidium bromide staining. A: representative RT-PCR analysis of human AR cDNA (lanes 1-8) and rat AR cDNA (lanes 9-11). Intra-abdominal (lane 1) and subcutaneous (lane 2) confluent preadipocytes, intra-abdominal (lane 3) and subcutaneous (lane 4) mature adipocytes, LNCap cells (lane 5), intra-abdominal confluent preadipocytes (lane 6), differentiated preadipocytes (lane 7) and mature adipocytes (lane 8), epididymal confluent preadipocytes (lane 9), mature adipocytes (lane 10), rat prostate (lane 11), and negative control (lane 12). a 100-bp ladder size marker. B: densitometric analysis of AR RT-PCR. Data are means ± SE from 3 separate experiments and are expressed as densitometric intensities relative to values obtained for AR cDNA in subcutaneous confluent preadipocytes. Statistical analyses of these data indicate P < 0.05 when data in subcutaneous (SC) and intra-abdominal (INTRA) are compared with control values (subcutaneous confluent preadipocytes).

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.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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), beta - and alpha 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 5alpha -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 17beta -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 alpha 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).

    NOTE ADDED IN PROOF

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.

    ACKNOWLEDGEMENTS

We are particularly indebted to the General Surgery Division, Centre Hospitalier de Poissy, for their courtesy in making human adipose tissue available.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Beato, M. Gene regulation by steroid hormones. Cell 56: 335-344, 1989[Medline].

2.   Berthois, Y., T. Dong, F. Roux-Dossetom, and P. M. Martin. Expression of estrogen receptor and its messenger ribonucleic acid in the MCF-7 cell line: multiparametric analysis of its processing and regulation by estrogen. Mol. Cell. Endocrinol. 74: 11-20, 1990[Medline].

3.   Björntorp, P. The regulation of adipose tissue distribution in humans. Int. J. Obes. 20: 291-302, 1996[Medline].

4.   Bradford, A. M. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72: 248-254, 1976[Medline].

5.   Chang, C. S., J. Kokontis, and S. T. Liao. Molecular cloning of human and complementary DNA encoding androgen receptors. Science 240: 324-326, 1988[Medline].

6.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

7.   De Pergola, G., X. Xu, S. Yang, R. Giorgino, and P. Björntorp. Up-regulation of androgen receptor binding in male rat fat pad adipose precursor cells exposed to testosterone: study in a whole cell assay system. J. Steroid Biochem. Mol. Biol. 37: 553-558, 1990[Medline].

8.   Deslex, S., R. Negrel, and G. Ailhaud. Development of a chemically defined serum free medium for differentiation of rat adipose precursor cells. Exp. Cell Res. 168: 15-30, 1987[Medline].

9.   Diaz-Sanchez, V., S. Morimoto, A. Morales, G. Robles-Diaz, and M. Cerbon. Androgen receptor in the rat pancreas: genetic expression and steroid regulation. Pancreas 11: 241-245, 1995[Medline].

10.   Dieudonné, M. N., R. Pecquery, and Y. Giudicelli. Characteristics of the alpha 2/beta -adrenoceptor-coupled adenylate cyclase system and their relationship with adrenergic responsiveness in hamster fat cells from different anatomical sites. Eur. J. Biochem. 205: 867-873, 1992[Abstract].

11.   Dieudonné, M. N., R. Pecquery, M. C. Leneveu, J. P. Dausse, and Y. Giudicelli. In vitro up-regulatory effect of testosterone on alpha(2)-adrenoreceptor expression in adipose tissue. Endocrine 2: 567-570, 1994.

12.   Dieudonné, M. N., R. Pecquery, M. C. Leneveu, A. M. Jaubert, and Y. Giudicelli. Androgen receptors in cultured rat adipose precursor cells during proliferation and differentiation: regional specificities and regulation by testosterone. Endocrine 3: 537-541, 1995.

13.   Hauner, H., P. Schmid, and E. F. Pfeiffer. Glucocorticoids and insulin promote the differentiation of human adipocyte precursor cells into fat cells. J. Clin. Endocrinol. Metab. 64: 832-835, 1987[Abstract].

14.   Jacobson, W., J. Routledge, H. Davies, T. Saich, I. Hughes, A. Brinkmann, B. Brown, and P. Clarkson. Localisation of androgen receptors in dermal fibroblasts, grown in vitro, from normal subjects and from patients with androgen insensitivity syndrome. Horm. Res. 44: 75-84, 1995[Medline].

15.   Jaubert, A. M., R. Pecquery, I. Sichez, M. N. Dieudonné, J. F. Cloix, and Y. Giudicelli. Androgen receptors in hamster white adipocytes and their precursor cells: regional variations and modulation by androgens. Endocrine 1: 535-540, 1993.

16.   Kemppainen, J. A., M. V. Lane, M. Sar, and E. M. Wilson. Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation---specificity for steroids and antihormones. J. Biol. Chem. 267: 968-974, 1992[Abstract/Free Full Text].

17.   Krongrad, A., C. M. Wilson, J. D. Wilson, D. R. Allman, and M. J. McPhaul. Androgen increases androgen receptor protein while decreasing receptor mRNA in LNCap cells. Mol. Cell. Endocrinol. 76: 79-88, 1991[Medline].

18.   Krotkiewski, M., P. Björntorp, L. Sjöström, and U. Smith. Impact of obesity on metabolism in men and women. Importance of regional adipose tissue distribution. J. Clin. Invest. 72: 1150-1162, 1983[Medline].

19.   Lacasa, D., B. Agli, M. Mur, J. P. Dausse, and Y. Giudicelli. Influence of ovarian status and regional fat distribution on protein kinase C in rat fat cells. J. Endocrinol. 140: 275-282, 1994[Abstract].

20.   Lacasa, D., E. Garcia, D. Henriot, B. Agli, and Y. Giudicelli. Site-related specificities of the control by androgenic status of adipogenesis and mitogen-activated protein kinase cascade/c-fos signaling pathways in rat preadipocytes. Endocrinology 138: 3181-3186, 1997[Abstract/Free Full Text].

21.   Lacasa, D., M. Mur, B. Agli, and Y. Giudicelli. Protein kinase C in rat adipocytes is influenced by the androgenic status and the regional fat distribution. J. Endocrinol. 138: 493-501, 1993[Abstract].

22.   Lee, C., D. M. Sutkowski, J. A. Sensibar, D. Zelner, I. Kim, I. Amsel, N. Shaw, G. S. Prins, and J. M. Kozlowski. Regulation of proliferation and production of prostate-specific antigen in androgen-sensitive prostatic cancer cells, LNCap, by dihydrotestosterone. Endocrinology 136: 796-803, 1995[Abstract].

23.   Marin, P., L. Lönn, B. Andersson, B. Oden, L. Olbe, B. A. Bengtsson, and P. Björntorp. Assimilation of trigycerides in subcutaneous and intraabdominal adipose tissues in vivo in men: effects of testosterone. J. Clin. Endocrinol. Metab. 81: 1018-1022, 1996[Abstract].

24.   Menard, C. S., and R. E. Harlan. Up-regulation of androgen receptor immunoreactivity in the rat brain by androgenic-anabolic steroids. Brain Res. 622: 226-236, 1993[Medline].

25.   Miller, L. K., J. G. Kral, G. W. Strains, and B. Zumoff. Androgen binding to ammonium sulfate precipitates of human adipose tissue cytosols. Steroids 55: 410-415, 1990[Medline].

26.   Mizutani, T., Y. Nishikawa, H. Adachi, T. Enomoto, H. Ikegami, H. Kurachi, T. Nomura, and A. Miyake. Identification of estrogen receptors in human adipose tissue and adipocytes. J. Clin. Endocrinol. Metab. 78: 950-954, 1994[Abstract].

27.   Mora, G. R., G. S. Prins, and V. B. Mahesh. Autoregulation of androgen receptor protein and messenger RNA in rat ventral prostate is protein synthesis dependent. J. Steroid Biochem. Mol. Biol. 58: 539-549, 1996[Medline].

28.   Pecquery, R., M. N. Dieudonné, J. F. Cloix, M. C. Leneveu, J. P. Dausse, and Y. Giudicelli. Enhancement of the expression of the alpha 2-adrenoreceptor protein and mRNA by a direct effect of androgens in white adipocytes. Biochem. Biophys. Res. Commun. 206: 112-118, 1995[Medline].

29.   Pecquery, R., M. C. Leneveu, and Y. Giudicelli. Characterization of the beta -adrenergic receptors of hamster white fat cells. Evidence against an important role for the alpha 2-receptor subtypes in the adrenergic control of lipolysis. Biochim. Biophys. Acta 731: 397-405, 1983[Medline].

30.   Peiris, A. N., M. S. Sothman, R. G. Hoffman, M. I. Hensen, C. R. Wilson, A. B. Gustafson, and A. Kissebah. Adiposity, fat distribution and cardiovascular risk. Ann. Intern. Med. 110: 867-872, 1989[Medline].

31.   Price, T. M., and S. N. O'Brien. Determination of estrogen receptor messenger ribonucleic acid (messenger RNA) and cytochrome-P450 aromatase messenger RNA Levels in adipocytes and adipose stromal cells by competitive polymerase chain reaction amplification. J. Clin. Endocrinol. Metab. 77: 1041-1045, 1993[Abstract].

32.   Rebuffé-Scrive, M., L. Enk, N. Crona, P. Lönnroth, L. Abrahamsson, U. Smith, and P. Björntorp. Fat cell metabolism in different regions in women: effect of menstrual cycle, pregnancy and lactation. J. Clin. Invest. 75: 1973-1976, 1985[Medline].

33.   Rebuffé-Scrive, M., P. Lönnroth, P. Marin, C. Wesslau, P. Björntorp, and U. Smith. Regional adipose tissue metabolism in men and postmenopausal women. Int. J. Obes. 11: 347-355, 1987[Medline].

34.   Rebuffé-Scrive, M., P. Marin, and P. Björntorp. Effect of testosterone on abdominal adipose tissue in men. Int. J. Obes. 15: 791-795, 1991[Medline].

35.   Roncari, D. A. K., and R. L. R. Van. Promotion of human adipocyte precursor replication by 17-beta estradiol in culture. J. Clin. Invest. 62: 03-508, 1978.

36.   Vague, J., J. M. Meignen, and J. F. Negrin. Effects of testosterone and estrogens on deltoid and trochanter adipocytes in two cases of transsexualism. Horm. Metab. Res. 16: 380-381, 1984[Medline].

37.   Vornberger, W., G. Prins, N. A. Musto, and C. Suarez-Quian. Androgen receptor distribution in rat testis: new implications for androgen regulation of spermatogenesis. Endocrinology 134: 2307-2316, 1994[Abstract].

38.   Wahli, W., and M. Martinuzzo. Superfamily of steroid nuclear receptors: positive and negative regulators of gene expression. FASEB J. 5: 2243-2249, 1991[Abstract/Free Full Text].

39.   Wise, S., and H. Green. Participation of one isoenzyme of cytosolic glycero-P-dehydrogenase in adipose conversion of 3T3 cells. J. Biol. Chem. 254: 273-275, 1979[Abstract].

40.   Wolf, D. A., T. Herzinger, H. Hermeking, D. Blaschke, and W. Horz. Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgen. Mol. Endocrinol. 7: 924-936, 1993[Abstract].

41.   Xu, X., G. De Pergola, and P. Björntorp. The effect of androgens on the regulation of lipolysis in adipose precursor cells. Endocrinology 126: 1229-1234, 1990[Abstract].


Am J Physiol Cell Physiol 274(6):C1645-C1652
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society